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Collagen: Structure, function, and metabolism in normal and fibrotic tissues
S eminars in Arthritis and Rheumatism AUGUST 1983
VOL. XIII, NO. 1
Collagen:
Structure,
Function, Fibrotic
and Metabolism Tissues
in Normal
and
By Marcel E. Nimni
C
OLLAGEN is the single most abundant animal protein in mammals accounting for about 30% of all proteins. The collagen molecules, after being secreted by the cells, assemble into characteristic fibers responsible for the functional integrity of tissues such as bone, cartilage, skin, and tendon (Fig. 1). They contribute a structural framework to other tissues such as blood vessels and most organs. Crosslinks between adjacent molecules are a prerequisite for the collagen fibers to withstand the physical stresses to which they are exposed. Significant progress has been made towards understanding the functional groups on the molecules that are involved in the formation of such crosslinks, their nature, and location. A variety of human conditions, normal and pathologic, involve the ability of tissues to repair and regenerate their collagenous framework. Some of these conditions are characterized by excessive deposition of collagen (e.g., cirrhosis, scleroderma, keloid, pulmonary fibrosis, diabetes, etc.). After trauma or surgery, abnormal deposition of collagen may impair function (adhesions following repair of long tendons, scar formation during healing, etc.). In addition, many disabling conditions result from changes in the nature and organization of collagen (heart-valve lesions, osteoarthritis, rheumatoid arthritis, and congenital collagen diseases such as Marfan’s and Ehlers-Danlos syndromes, osteogenesis imperfecta, etc.). In human tissues there are five different collagen types that have been well characterized. Many others are currently being studied. We know that collagen molecules in cartilage differ from those in cornea, tendon, bone matrix, dermis, the parenchyma of organs, periodontal ligaments, and many other locations within the organism. Why is cornea transparent, tendon tough, inelastic and able to sustain significant stresses, Seminars in Artbntis and Rheumatism. Vol. 13, No. 1 (August), 1983
and cartilage resilient and viscoelastic? What kind of uniqueness is there in the chemical structure of collagen that enables these tissues to perform such diverse biologic functions? What regulates fiber diameter, orientation, concentration of fibers in a particular volume of tissue, and packing of the fibers into larger bundles? How can we understand the problem of aging of connective tissues and changes related to pathology? It now seems as if mesenchymal cells that have acquired the ability to make a particular type of collagen can be influenced by environmental factors to change the rate and nature of the collagen molecules that they synthesize, and that in some instances these changes are reversible. This review will address itself to the various fundamental steps of the anabolism and catabolism of collagen as well as to the unique types of collagen present in skin, tendon, bone, blood vessels, cartilage and basement membranes and will focus on diseases associated with excessive accumulation of collagen, which leads to various forms of fibrosis. THE COLLAGEN MOLECULE
The arrangement of amino acids in the collagen molecule is shown schematically in Fig. 2. From the Departments of Biochemistry. Medicine and Orthopaedics, University of Southern California and Orthopaedic Hospital, Los Angeles. CA. Supported in part by NIH grams AMlO35X. AMl6404. and AG02.577, as well as funds from the Orthopaedic Hospital. Address reprint requests to Marcel E. Nimni. Ph.D., Professor of Biochemistry, Medicine & Orthopedics. Bone & Connective Tissue Research Laboratory, P.O. Box 60132 Terminal Annex, Los Angeles, CA 90060. 0 1983 by Grune & Stratton, Inc. 0049-0172/83/1301-0001$5.00/0
1
MARCEL E. NlMNl
Fig. 1.
Bundles
a Haversian
of collagen
canal in a human
prepared
by
detergent
for
microscope
cleaning 6 hr.
and
(original
Drs. A. Boyde
in
fibers
in the osteoid
long bone.
a
solution
observed
on
magnification
lining of
The specimen
was
containing
enzyme-
a scanning
electron
5,400
XI.
(Courtesy
of
and S. Reid.)
Every third residue is glycine. Proline and OHproline follow each other relatively frequently, and the (gly, pro, hyp) sequence makes up about 10% of the molecule. This triple helical structure generates a symmetrical pattern of three lefthanded helical chains that are, in turn, slightly displaced to the right, superimposing an additional “supercoil” with a pitch of approximately 86 8, units. The amino acids within each chain are displaced by a distance h = 2.91 8, with a relative twist of - 1 lO”C, making the number of residues per turn 3.27 and the distance between each third glycine 8.7 A. The individual residues are nearly fully extended in the collagen structure, since the maximum displacement within a
Schematic
Fig. 2. helix.
The
individual
approximately turn
around
The
hydrogen
shown)
form
twist. (not
three
coiled
chains
(interpeptide
quite
different
acids
located
from within
drawing (Y chains
showing are
residues each
per
other
bonds
turn.
a-helices
which
the same
polypeptide.
and
occur
chains
with are
in
right-handed
the
residues
bonding),
triple
helices a
stabilize
opposing
hydrogen
collagen
The
following
which
between
the
lee-handed
fully stretched chain would be approximately 3.6 A. This separation is nevertheless such that it will not allow intrachain bonds to form (as does occur in the alpha helix), and only interchain hydrogen bonds are possible.’ The exact number of hydrogen bonds that stabilize the triple helical structure has not been determined. The model that Ramachandran and Kartha described has two hydrogen bonds for every three amino acids, whereas the Rich and Crick’ version assumes one hydrogen bond for every three residues. In addition to these intrammolecular conformational patterns, there seems to exist a supermolecular coiling. Microfibrils, possibly representing intermediate stages of packing, may be present; such possibilities will be discussed later. Figure 3 shows a native collagen fiber with its repeating 680-A (68nm) periodicity alongside a schematic drawing of a collagen molecule (3000 8, length). The relationship between the length of the molecule and the periodicity that prompted the quarter-staggered theory of packing3 can be clearly seen. The stacking of collagen molecules to give rise to segment long spacing (SLS) crystallites (Fig. 4) has provided a useful tool for understanding the dimensions of the molecule, to order the peptides originating after CNBr cleavage of the molecule and to identify and characterize the various collagen types. This form of packing, which does not lead to normal fiber formation, will also be discussed in connection with the intracellular translocation of procollagen. The process of self-assembly that causes the collagen molecules to organize into fibers is shown schematically in Fig. 5. The thermody-
triple
helix
in different are therefore
between
amino-
Fig. tungstic matic
Native
3.
acid
collagen
showing
representation
approximately
300 nM.
the of
fiber 68
nM
collagen
(Courtesy
stained
with
periodicity molecules
phospho-
and
a sche-
measuring
of Dr. J. Petruska.)
COLLAGEN STRUCTURE AND METABOLISM
Fig. when
4.
Segment-long-spacing
ATP
(adenosine
of collagen. charge
The
density
negatively
around
positions their
can now of the
orientation.
these
have
which
transport
charged
observed
procollagen
The
by altering them
arrows
in the crystallite
It is of interest
been
ATP causes
form
to a solution the
to aggre-
in such a way that the individual
be visualized.
molecules
(SLSI
is added
the molecules
gate side by side in register molecules
crystallites
tryphosphate)
that
within
indicate
the
and represent
structures intracellular
similar
to
vesicles
to the cell surface.
namics of such a system involve changes in the state of the water molecules associated with the nonpolar regions of the collagen molecule. Also illustrated is the role of the polar groups on the surface of collagen, which we now know are distributed so as to effectively aid in the quarterstaggered packing that leads to the native fibrillar banded structure.4 ’
Fig.
The Procollagen
Molecule
In order for the organism to develop an extracellular network of collagen fibers, the cells involved in the biosynthetic process must first synthesize a precursor known as procollagen. This molecule is later enzymatically trimmed of its nonhelical ends, giving rise to a collagen molecule that spontaneously assembles into fibers in the extracellular space. Procollagen molecules have been identified as precursors of the three interstitial collagens (types I, II, and Ill). Several of the N- and C-terminal peptides (propeptides) have been characterized and, in some instances, the primary sequence determined.’ The N-terminal propeptides for both procY1(I) and pro 1(III) chains have a terminal globular domain of 77-86 amino acids, followed
to 37°C
native
fibers.
molecules overlap.
The
the
The
that
opposing signs.
collagen
by cold neutral
warmed
collagen
upper
have
alignment
water
these phobic
bonds
The driving system
that
since
greatly results
the release
state
process may
of
which
disorder
be associated
to operate
through
with
hydrogen-
clustered
system.
allows
expose these
of the fiber.
water water of
insolubilization
this phenomenon
~
rise to hydro-
of entropy
Part
of
around
and
the stability
into “random”
the lifespan
the
giving
an increase
the
+ and
“melt”
of “organized”
of
by the
of water
other,
leads to a gradual
represents
to interactions
37°C
circles),
enhance from
sites and its transformation the
drawing
due
collagen,
each
are into
in a quarter-staggered
Exclusion
with
most
solutions
reassemble
as depicted
(open
surfaces.
force
the
approaches of
from
If these
is primarily
regions
to interact
of
to align
charges
molecules
nonpolar
surfaces
be extracted
molecules
part
begun
electrostatic
hydrophobic
can
salt solutions.
As the temperature
bonded the
BIOSYNTHESIS
Soluble
5.
tissues
which
from
of the initial
increases the
aging
of collagen continues
of the individual.
by a collagen-like domain of about 40 amino acids. The collagen-like domain is joined to the chain by a short noncollagen sequence of 2-8 amino acids. The N-terminal propeptide of proLu2(1) has not been as well characterized and in some species may lack the N-terminal nonglobular domain.’ Type II procollagen isolated from chick embryos seems to contain a similar amino-propeptide but with a smaller N-terminal globular domain.’ The N-terminal propeptide of
4
type I collagen contains one residue of N-acetylglucosamine” and intrachain but no interchain disulfide links. On the other hand, interchain disulfide links seem to be present in the N-terminal propeptides of type III procollagII.12 en. The carboxyterminal propeptides of both procul and proa chains have molecular weights of 30,000-35,000 daltons and globular conformations without any collagen-like domain.13 These peptides contain asparagine-linked oligosaccharide units composed of N-acetylglucosamine and mannose.‘4,‘5 This carbohydrate sidechain is located in the carboxy-terminal half of the carboxyl propeptide in a region containing the sequence Asn-X-Thr,16 which is compatible with the structural requirement for glycosylation of asparaginyl residues by oligosaccharide transferases.” The functional importance of the carbohydrate in the carboxy-end of procollagen is unknown but may be part of a recognition mechanism for alignment, secretion or assembly into microfibrils. A diagram summarizing the major characteristics of procollagen type I is shown in Fig. 6. The central segment of the molecule is arranged in a triple helical conformation characteristic of collagen, and the globular extensions can be seen at both ends. Also shown in the diagram (indicated by arrows) are the sites of cleavage by specific procollagen peptidases. The extensions play a key role in the assembly of the tri-helical collagen molecule and their possible functions in this connection will be discussed later in this section. Once the molecule is completed and translocated to the cell surface the extensions are enzymatically removed (in the case of types I, II, and III collagens) and fibrillogenesis occurs. Enzymes that selectively remove these extensions can be found in a variety of
Procollagen molecule showing the nonhelical Fig. 6. terminal extensions. The N-terminal end contains a small helical domain and the C-terminal end is stabilized by interchain disulfide bonds. The sites of cleavage by procollagen peptidases are indicated by arrows.
MARCEL
connective tissues, and in the culture derived from collagen-secreting cells.15
E.
NIMNI
media
Intracellular Event Leading to the Synthesis of Procollagen Gene Expression. Since the discovery, about 12 years ago, of a distinct form of collagen in cartilage, labeled type II collagen, more than a dozen collagen chains have been characterized in different tissues of the same animal species. The best defined (types I-V) clearly represent unique amino acid sequences coded by different genes. By cell hybridization it has been shown that human chromosome number 17 contains the coding information for the cul and (~2 chains of type I collagen.‘8~‘9 Type III collagen seems to be encoded in another chromosome. Recent advances in recombinant DNA technology have opened the way for study of the structure and regulation of the collagen gene. In part this interest is generated by the fact that collagens constitute a family of closely related proteins, and are synthesized by specialized cells, that can be experimentally manipulated to synthesize different collagen types. The collagen gene is large, about 10 times the size of the functional mRNA.20 These messenger RNAs have a unique sequence of codons, called signal codons, to the right of the initiation codon. These are translated on a free ribosome to a unique sequence of about 15-30 amino acid residues (signal sequence) on the amino terminal of the nascent chain. In the case of collagen, the (~2(1) collagen gene from chick is 38 kilobases in length and contains at least 52 coding sequences (exons). Many of these exons are 54 base pairs in length and are separated from each other by large intervening sequences (introns) that range in size from about 80 to 2,000 base pairs.2’ The gene itself contains 38,000 base pairs, and is the most complex gene so far isolated.22 It was surprising to find that a molecule as uniform and regular as collagen should be coded by a gene of such complexity and divided into so many domains. In particular, the finding that most exons of the gene for the cy2 chain have identical lengths may have important implications in the understanding of evolution, since it suggests that the ancestral gene for collagen was assembled by multiple duplications of single genetic units containing an exon of 54 bp (Fig. 7). It is likely that
COLLAGEN STRUCTURE AND METABOLISM
-0
---etc.
c-2
54
54
34
The
Fig. 7.
54
collagen
containing
64
base
sequences
of
18
minimum tigators (redrawn
fashion,
interested with
54
54
gene
pairs,
amino
sequence,
an exacting
54
is made
each acids.
of The
and the fact that provides in the
valuable
process
up of multiple
which
units
corresponds
conservation it is repeated infomation
of evolution
of
to this
in such to inves-
of proteins
permission).”
a primordial exon this size could have encoded for a gly-pro-pro tripeptide repeated six times (3 x 3 x 6). Such a polypeptide of 18 amino acids probably had the minimum length needed to form a stable triple helical structure. The 54 bp exon structure may be common to all collagen genes. These observations generate interesting possibilities, and may allow us to better understand the nature of defects associated with some of the heritable collagen diseases. Translational, Cotranslational. and Early Posttranslational Events. After the gene is transcribed, it is spliced to yield a functional mRNA that contains about 3,000 bases. Specific mRNAs for each chain and collagen type are translocated to the cytoplasm and translated in the rough endoplasmic reticulum on membranebound polysomes (Fig. 8). A pre-pro cychain that contains an unusually large N-terminal hydrophobic signal or “leader’ sequence is the final translational product. The signal portion facilitates transfer of the chain into the lumen of the rough endoplasmic reticulum (RER) and is probably removed by intramembranous endopeptidase after it has served its function of orienting compatible procv chains in apposition. As the collagen polypeptide is synthesized in the rough ER, important cotranslational events accompany this process. It is a well known, extensively documented observation, that neither hydroxyproline nor hydroxylysine can be directly incorporated into proteins;23 it is only after peptide bond formation that hydroxylation of proline and lysinc can occur, mediated by two enzymes, prolyl and lysyl hydroxylases. These enzymes are specitic and require for their activity ferrous
Fig.
8.
collagen:
Sequence
procollagen
chains.
polysomes proline
of the
residues
hydroxylase
of
n-chains formation
molecules
of vesicles
the molecule nonhelical extensions
with
by specific
the
by
signal ribosomes.
4-proline (01. (4)
galactosyl-transferase peptide. (7) of
19) Intracellular and packaging by the removal
and part
on
of specific
(A)and
(6)
(13). (5) Release
Recognition
prepeptide
Folding
of
message
hydroxylase
the cell membrane
accompanied
extensions
of
by a glucosyltransferase
(8)
of a triple-helix.
biosynthesis
for the different
Hydroxylation
by lysyl
the C-terminal
crosslinks.
the procollagen Fusion
from
the
hydroxylase
N-terminal
through
disulfide
(3)
hydroxylysine
a-chains
in
mRNAs
Translation E.R.
of glucose
of the
completed
(2)
rough
and of lysine
(M) and addition Removal
events
of specific
by 3-proline
(A)
Glycosylation
of
of
(1) Synthesis
of of 3
and formation
the
molecule
and
translocation into vesicles.
of (10)
and extrusion
of
of the C-terminal
of the N-terminal
nonhelical
peptidases.
iron, ascorbate, and cu-ketoglutarate. They seem to recognize sequences surrounding the target imino or amino acids with differing affinities and thus selectively modify randomly alternating proline and lysine residues. The degree of hydroxylation differs from tissue to tissue and probably with availability of substrate, rates of synthesis, and turnover as well as the time that the molecule remains in the presence of the hydroxylating enzymes. As noted later, these
6
factors may also affect the position of the carbon atom in the peptide bound proline that is hydroxylated (e.g., the 3- or 4-carbon). The time required for the synthesis of a complete proa chain is about 6.7 min.24 The radioactive label, however, appears in fully aligned triple-helical chains after a further delay, a fact that may have significant physiologic implications as the time lapse between polypeptide synthesis and folding may affect the nature and extent of hydroxylation and glycosylation, another important posttranslational event. The enzyme prolyl hydroxylase (4-hydroxylase) has been isolated from several sources and extensively characterized.‘5,25,26 The active enzyme is a tetramer with a molecular weight of 240,000 daltons, and consists of two different types of monomers with molecular weights of about 64,000 and 60,000 daltons. Lysyl hydroxylase has been extensively purified and is apparently a dimer with two subunits, each having a molecular weight of approximately 90,000 daltons.*’ Prolyl 3-hydroxylase has been only partially purified and characterized.28 These three enzymes are specific and act only on cy chains in the nonhelical conformation, a reason why the time elapsed between peptide synthesis and folding may be so important. This time varies considerably, being approximately 10 min in cells synthesizing type I procollagen, 20 min in cells synthesizing type II procollagen, and 60 min in cells synthesizing basement membrane collagen.3’ After folding of the procollagen polypeptides, there is an additional time interval before the collagen molecule is secreted: this varies between 18 min for tendon cells,32 36 min for cartilage cells,33 and 60 min in parietal yolk sac tissue.34 Proline analogues, such as cis-hydroxyproline and azetidine 2-carboxylic acid, are known to be incorporated into collagen in the proline position and to inhibit triple helix formation within the cell. Using such an experimental tool it was shown that the glycosylation of hydroxylysine35 and the hydroxylation of proline at the 3 position2* are increased approximately twofold by inhibiting helix formation. These observations, coupled with the time factors noted above, may be linked to the increased hydroxylation of proline in the 3 position, the increased content of hydroxylysine and the greater degree of glycosyl-
MARCEL E. NIMNI
ation seen in basement membrane collagen, and may explain differences in hydroxylation seen in pathologic connective tissues. As lysyl residues in the newly synthesized proa chains are hydroxylated, sugar residues are added to the resulting hydroxylysyl groups. Glycosylations are catalyzed by two specific enzymes, a galactosyltransferase and a glucosyltransferase.36 The first of these enzymes adds galactose to the hydroxylysyl residues, and the second adds glucose to the galactosylhydroxylysine that is formed. The galactosyltransferase from chick embryo has been purified about I OOOfold and the glucosyltransferase has been isolated as a homogeneous protein. Both enzymes are glycoproteins and their activity requires the presence of sulfhydryl groups. The activity of partially purified galactosyltransferase is separated by gel filtration into three species with apparent molecular weights of 450,000,200,000, and 50,000 daltons. The purified glucosyltransferase has a molecular weight of 70,000 daltons. Both these transferases use sugar in a form of a uridine diphosphate glycoside, and require the presence of bivalent cations, preferably manganese.37 These enzymes, like the hydroxylases, require that the pro-o chains be in a nonhelical conformation. In intact cells, glycosylation is initiated while the polypeptides are still being assembled on the ribosomes, but probably continues after the release of complete proa chains in the cisternae of the rough ER; activity ceases when the chains acquire a triple-helical conformation. The oligosaccharides present in the extension peptides associated with the C-terminal region of collagen resemble those present in most other glycoproteins; they contain N-acetylglucosamine and mannose, and are attached to asparagine residues.38,39 Their composition suggests that they are added as intermediates via the dolichol phosphate pathway, and that final remodeling occurs in the Golgi after the helix has been formed.40 Once the translation, moditications and additions are completed it is essential that the individual prom chains become properly aligned for the triple-helix to form. We do not know if this alignment occurs while the polypeptides are still attached to the ribosome or if they have to detach, or if the N-terminal “signal” peptide plays a role in this connection. In any case, proper alignment should juxtapose the
COLLAGEN STRUCTURE AND METABOLISM
appropriate cysteine residues as a prerequisite for formation of the disulfide bridges that link the individual procv chains at the C-terminal end. Earlier studies involving subcellular fractionation suggested that the disulfide bridges could appear during translocation of procollagen from the ribosome to the smooth endoplasmic reticulum, probably in the cisternae of the ER.4’ More recently it has been proposed that disulfide bond formation occurs while the propeptides are still attached to the ribosome.4’ In any case, it seems clear that for assembly and secretion the Cterminal extensions must be present.43 ” Although helix formation seems to be a spontaneous entropy driven process, the possibility that an enzymatic system, similar to that which regulates the coiling of DNA and shifts it from one topologic form to another involving DNAgirases and topoisomerases4” could be associated with the rough ER, and which controls and monitors this step, cannot be discounted. The kinetics of triple helix formation of type I procollagen within hbroblasts have been measured in pulse-chase experiments followed by proteolytic digestion.4’ Production of triple-helical molecules requires X-9 min after completion of procY-chains.47 Disulfide bond formation precedes triple helix formation, and serves as a catalyst for it to occur. Similar observations and conclusions have been derived from studies on the biosynthesis of type I I I procollagen by chick embryo blood Vf2SXlS.44~4X In this case further stability may be added by interchain disulfide bond formation in the helical region as well as in the N-terminal nonhelical region, but the exact timing of these subsequent events remains difficult to ascertain. lntracellulur Translocation of Procollagen and Extrusion into the Extracellular Space. The synthesis of procollagen chains involves the translation of a specific messenger RNA on membranebound ribosomes similarly to secretory proteins in generalJy and core protein of proteoglycans.” The signal sequence, an unusually large one in the case of collagen,” may trigger the attachment of the ribosome that generated it to a specific binding site on the membrane. In most instances the signal sequence is removed from the nascent polypeptide chain by a specific endopeptidase before chain completion.c2,i’ It is not clear whether this is the case with collagen since before movement along the secretory pathways begins three separate specific
7
chains have to register to form the triple-helical structure. It is possible that the signal peptide, due to its hydrophobic character, guides the nascent cy chain to the interior of the rough membrane intracisternal space where the translational and posttranslational events occur. Once the procollagen molecule is assembled (devoid of the signal peptide) the secretory pathway seems to be similar to that followed by other glycoproteins. These experimental findings have been recently reviewed.54 Essentially the procollagen molecules, now detached from the ribosome, emerge from the endoplasmic reticulum and move towards the Golgi apparatus through the microsomal lumen (Fig. 9). In the Golgi its C-terminal mannose-rich carbohydrate extension is probably remodelled and the completed procollagen molecules packaged in Golgi-derived vessicles and carried towards the cellular membrane by cytoskeletal movements. During transit they sit side by side to form structures that sometimes may be identified with the electron microscope as SLS crystallites (see fibrillogenesis).
Fig. 9. of the
Movement
rough
transitional packaged
endoplasm into secretory
by exocytosis.
of procollagen
endoplasmic
reticulum (TE)
to
vesicle
the (SV)
through (RER) Golgi prior
the cisternae and
(G)
through where
to extrusion
a
it is IEX)
8 The small aggregates of oriented procollagen molecules are probably trimmed of their nonhelical amino and carboxyl extensions by specific peptidases when they reach the extracellular space. In the case of type I collagen, the first peptidase to act seems to be the amino protease; this is followed by a carboxyprotease. In type III collagen the sequence of removal may be reversed.54~ss Recent immunoelectron microscopic studies have shown that the thinner collagen fibers (200-400 A) contain procollagen molecules that still retain their NH,-terminal extensions;56 this is not the case for larger collagen fibers. Thus, certain fibers in normal human skin still possess, at least on their outer surface, procollagen or pN-collagen molecules. The latter possibility is most likely in view of earlier work” and because antibody reactivity could be eliminated by treating the tissues with procollagen NH,-terminal protease. These findings suggest that removal of extension aminopropeptides could be involved in the control of fiber growth. It is possible that selective removal of portions of the extensions may occur at the cell surface thus allowing for formation of 4-D staggered microfibrils, and that final removal of these propeptides occurs in the extracellular space. This process would allow for modulation of fiber growth by apposition and elongation as well as for fusion of thin fibers (20-40 mm) into larger diameter fibers (Fig. 10). Lysyl Oxidase Somewhere in the process of extrusion, recently formed microfibrils must be recognized by the enzyme lysyl oxydase, which converts certain peptide-bound lysines and hydroxylysines to aldehydes. This enzyme initiates the biosynthesis of cross-links in collagen. A recent review summarized most of our knowledge in this field.58 This enzyme, first described by Pinnell and Martin,59 catalyzes the oxidative deamination of lysine and hydroxylysine, as summarized in Fig. 1 1. The enzyme is an extracellular amine oxidase, and has been purified from a variety of connective tissues. The molecular weight in most species is 30,000 daltons, or a multiple thereof. It requires Cu+ + and probably pyridoxal as cofactors, and molecular oxygen seems to be the cosubstrate and hydrogen acceptor. It is irreversibly inhibited by the lathyrogen BAPN. This
MARCEL E. NlMNl
EXTRACELLULAR
PROCOLLAGEN
C”O;S&KED
Fig. 10. Fibrillogenesis: Microfibrils in a quarter-staggered configuration have lost their C-terminal nonhelical extensions and part of their N-terminal extensions. In this form they seem to organize readily into small-diameter fibers which retain part of the N-terminal nonhelical extensions. After being relieved of these peptides by a procollagen peptidase, fibers are able to grow in diameter by apposition of microfibrils or by merger with other small diameter fibers.
enzyme exhibits maximal activity when acting on reconstituted collagen fibers rather than upon monomeric collagen. Because of this it is felt that the enzyme is most likely to act upon microfibrillar aggregates of collagen molecules at the time of extrusion. It would be difficult to conceive how it could act otherwise on collagen, since once assembled into the quarter-staggered array and compacted into fibers, it would be difficult for the enzyme to gain access to the interior of the fiber. Fibrillogenesis During the preceding discussion, the tendency of collagen molecules to form macromolecular aggregates has been constantly emphasized. This tendency is common with most fibrous proteins that form filaments with helical symmetry and which occupy equivalent or quasiequivalent positions. Both electron micrographic and x-ray diffraction data support the view that within col-
iw
hi
PEPTIDE
BOUND LlSlNE
H
k
k .z-AMNOAOIPIC
L-SEMALOEHYOE
Fig. 11. The oxidative deamination of peptide-bound lysine by the enzyme lysyl oxidase generates the aldehydes associated with the collagen molecule.
COLLAGEN
STRUCTURE
AND
9
METABOLISM
lagen fibers there is a regular arrangement of molecules that could result from such an ordered assembly.60 ” A five-stranded microfibril was first suggested to account for such a substructure, one that would satisfy the condition that adjacent molecules were equivalently related by a quarterstagger, as suggested by Hodge and Petruska.3.63 The exact mode of organization, that is the mode in which the collagen molecules pack in the microfibril, their exact number, and the nature and location of crosslinks within and between microfibrils still remains a subject for speculation (Fig. 12). The five subunit assembly proposed earlier by Smith seems to be the most widely accepted basic unit. For detailed discussion the reader is referred to the reviews mentioned earlier as well as to recent detailed publications.‘” “) The earliest form of intracellular interaction is documented by the appearance of what seem like side to side nonstaggered arrangements of collagen molecules resembling SLS and end-overlapped SLS crystallites. These formations have been found in vacuoles within odontoblasts, epithelial cells, and fibroblasts.70~74 SLS crystallites normally do not form under physiologic conditions of pH and ionic strength but occur if negatively charged counterions (such as ATP) are added to the solution to mask and interfere with ionic interactions that normally cause collagen molecules to form quarter-staggered arrays (Fig. 4). What causes such associa-
tion to occur early within the cell is not known. but they could be due to the presence of the Cand N-terminal extensions of procollagen or to the existence of ionic species within the vacuoles that may favor lateral stacking. In any case, such a conformation may prove to be of advantage at the time of intracellular transport since it would inhibit normal fibrillogenesis and probably position the procollagen molecules favorably for the procollagen proteases to exert their enzymatic activities during the process of extrusion. After removal of the nonhelical extensions, the newly formed collagen molecules could then be free to organize into quarter-staggered microfibrils and proceed to form native fibers. Under pathologic conditions, cells that are actively synthesizing collagen and that have accumulated large concentrations of proteoglycans have been shown to exhibit intracellular deposits of collagen fibrils within cytoplasmic inclusions.74 The process of in vivo tibrillogenesis has been equated for a long time with the ability of monomeric collagen to form fibers in vitro (Figs. 5 and 13). When monomeric collagen is heated to 37°C it progressively polymerizes, generating a turbidity curve that reflects the presence of intermediate aggregates. This phenomenon has been the focus of intensive investigation, and it has also led to the assumption that analogues exist between in vitro and in vivo situations. The lag phase (monomers), the nucleation and appearance of turbidity (microfibrils), and the rapid increase in turbidity (fiber formation) have been equated while attempting to understand how the cell handles these processes.7s ”
7 Subumts
Fig. 12. tion
Microfibrils
of collagen
widely
accepted
five collagen the
degree
Also
shown
represent
on its way form
of microfibril
molecules. of
is a higher
degree
microfibrils
width
as en alternate
as well
ecules
7 collagen
to sit side-by-side
is that
measured
way
molecules without
The
involves
view
indicates
in D values where
in a unit
of packing allowing
lateral
most
which
of organization
are packed
of organiza-
a fiber.
The cross-sectional
displacement
five-stranded accommodate
early forms
to becoming
(O-4). four
cell 8 nm in which
could
for some
mol-
displacement.
Fig. 13. its potential fibers.
Formation
of the five-membered
for lateral
and end-to-end
microfibil
aggregation
and
to form
MARCEL E. NIMNI
The experimental approaches used to study the nature and size of the aggregates formed during early stages of self assembly have been recently reviewed.7’~80 The reverse process, the disassembly of recently synthesized rat tail tendon collagen fibers by 0.1 M acetic acid, has also led to the conclusion that dimers and higher molecular weight aggregates can be recognized, since, as long as stable covalent crosslinks are not formed, the process is reversible. Current evidence, therefore, seems to indicate that monomeric collagen does not float freely in the extracellular space, but polymerizes into microfibrills and probably even into fibers before leaving the surface of the cell from which it originates. Autoradiographic studies show that the site of matrix deposition is near the cell surface.70,8’,“2 Similar observations have been made for other tissues such as the developing chick embryo, tendon, cornea, and basement lamella of fish all of which provide good models for fiber growth and elongation.72,79383 Although we are still not able to completely understand the detailed nature of the microtillaments nor the mechanism by which collagen fibers are deposited in the connective tissues in an ordered and specific manner, the overall picture is becoming more clear. From the folding of the procollagen molecule, to its transport through the cisternae of the endoplasmic reticulum, its packaging into vacuoles in the Golgi to extrusion and filament elongation at the cell surface, the sequence seems logical and amenable to further investigation. Collagen
Metabolism
Collagen is the most abundant of all body proteins. It therefore has to be synthesized and accumulated in large amounts during periods of growth and at sites of injury or tissue repair. Its rate of turnover is also rapid at these times and locations. Tissues such as bone, which are involved in active remodelling, are responsible for the major turnover, while other less dynamic tissues in the full grown individual, such as skin and tendons, may exhibit slow and almost negligible turnover. Although some specific cellular metabolic products have been implicated in stimulating or inhibiting collagen synthesis,84 the mechanisms involved are not understood. Under normal circumstances of growth and activity,
mechanical stress, piezoelectric phenomena, cell density, and cell-cell interactions are likely to affect cell membranes and modulate the synthesis and secretion of collagen. This area of research is difficult to investigate and, therefore, has not received much attention and our knowledge is scanty. Some aspects of these events will be discussed when we review the synthesis of collagen by cultured cells and the effects of specific drugs and inhibitors on such systems. Control could be exerted at both the transcriptional and translational levels within the cells, as well as during the extracellular assembly and crosslinking. In general the activity of cells and tissues in regards to their ability to synthesize collagen are assessed by their ability to synthesize hydroxyproline or by measurement of the activities of specific enzymes, the proline and lysyl hydroxylases being the most commonly assayed. Endogenous
Modulators
Many endogenous factors produced by cells in culture or derived from tissues have been investigated for their ability to regulate collagen accumulation; these have been documented in a recent review.85 Peptides removed from the NH,terminal end of procollagen during conversion to collagen are able to inhibit collagen synthesis in a reticulocyte cell-free system86’87 as well as when added to the media of cultured cells.88 Castor’s group was the first to demonstrate the presence of a series of factors that were capable of inducing a state of hypermetabolic activity in cultured synovial cells.“’ This material, assigned the name connective tissue activating peptide (CTAP), is a peptide of lO,OOO-15,000 daltons that promotes synovial cell proliferation (suggesting a role in pannus formation) and stimulates hyaluronate synthesis, but does not influence collagen synthesis. The search for endogenous factors that stimulate collagen synthesis led to the isolation from homogenates of experimentally (Ccl,) injured liver” of four fractions that, upon elution from Sephadex G-25, increase collagen synthesis up to eightfold in fibroblast cultures. It was subsequently reported that cytosol extracts and the supernatants from cultured, isolated egg granulomas obtained from livers of mice with infection due to Schistosoma mansoni
COLLAGEN STRUCTURE AND METABOLISM
stimulated fibroblasts to incorporate tritiated thymidine and to proliferate in vitro.” Since this fibrotic lesion results from an inflammatory granulomatous response, these investigators examined the nondialyzable fractions derived from antigen-stimulated T-cells for their ability to stimulate fibroblast proliferation and collagen synthesis.92 These preparations stimulated thymidine incorporation and increased both collagen and noncollagen protein synthesis when added to fibroblast medium, thus confirming earlier reports that lymphokine-rich supernatants derived from phytohemagglutinin (PHA)stimulated mononuclear cells and promoted collagen accumulation in cultures of fibroblasts.93 In addition to lymphkines, the presence of macrophage Factors capable of stimulating fibroblast proliferation94 and collagen synthesis by libroblasts has also been reported.q5 In contrast, the extracts from unstimulated mononuclear cells produced no effect on collagen synthesis when added to cultured fibroblasts, while supernatants from PHA-stimulated cells produced a 90% inhibition of collagen synthesis.” On the other hand, media from nonstimulated lymphocytes was shown to inhibit collagen synthesis by chondrocytes while stimulating that by fibroblasts.” Such conflicting findings can probably be explained by the presence in the media of both stimulating and inhibiting factors, and possibly by the concentration dependence of such effects, which could, of course, vary from experiment to experiment. It is therefore possible that circulating metabolites can reach the connective tissue cells and modulate their biosynthetic activity. In addition to these factors, hormones and drugs can influence such a pattern of behavior, but their effects will be discussed separately.
The endocrine control of connective tissue metabolism has received little attention. Of all the hormones known, only the glucocorticoids have generated significant interest because of their ability to inhibit the production of collagen. This topic has been the subject of a recent review.9x The most conspicuous side-effect.of systemic glucocorticoid therapy is somatic growth inhibition. This problem is especially disturbing in
11
children chronically treated with relatively low doses of glucocorticoids. As little as 45 vs 6 mg of cortisone or prednisone (6 mg/m*/day), a dose corresponding to the average endogenous daily secretion of hydrocortisone from the adrenals, suppresses somatic growth.99 Multiple daily injections of cortisone to young rats decreases the newly synthesized collagen content of skin but not the pool of mature collagen”” and can decrease urinary hydroxyproline excretion.“’ In subcutaneous rat granulomas, a single injection of betamethasone decreases hydroxyproline formation to a greater extent than total Procollagen molecules did protein synthesis.“* not accumulate in the granuloma and were fully hydroxylated.“’ Other investigators have confirmed these observations.” The fluorinated glucocorticoids are the most potent in selectively decreasing collagen synthesis. Multiple pharmacologic injections of glucocorticoids were shown to decrease prolyl hydroxylase activity in liver, granuloma, skin, aorta, lung, and human skin.lo4 The growth of most diploid fibroblasts is inhibited by glucocorticoids;“’ however, human lung”’ and skin’“‘~ lo9 fibroblasts represent major exceptions to this observation since proliferation of these cells is enhanced by glucocorticoids. In contrast to rodent fibroblasts, the enhanced proliferation rate of human skin fibroblasts occurs only while the cells are in log-phase growt.t~.“0 In the case of human WI-38 cells, hydrocortisone is also capable of significantly increasing the number of population doublings that the cell strain can achieve prior to senescence.‘“’ When chick embryo leg tendon cells were treated with betamethasone, hydroxyproline formation was decreased to a greater extemt than was total protein synthesis.“’ This fluorinated glucocorticoid also markedly depressed prolyl hydroxylase activity. Glucocorticoid effects on collagen synthesis have also been studied in normal skin and keloid fibroblasts.“L-“4 Hydrocortisone inhibited collagen synthesis in normal fibroblasts by 60% while noncollagen protein synthesis was increased.“’ In keloid fibroblasts, hydrocortisone did not decrease collagen synthesis. However, the synthetic fluorinated glucocorticoid, triamcinolone, markedly decreased collagen synthesis in keloid-derived fibroblasts.“3 Although prolyl
MARCEL E. NIMNI
hydroxylase activity is decreased, the degree of prolyl hydroxylation of procollagen was not affected.“4”‘5 The glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures.“6 Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50%70% inhibition of collagen synthesis and accumulation in polyvinyl spongeinduced granulomas, whereas total protein synthesis was inhibited by only about 25%. Growth rates, total protein synthesis, and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3 x lo-’ M hydrocortisone to the culture medium caused a 30% 50% inhibition of both collagen and noncollagen protein synthesis in fibroblasts from either source. Prolyl hydroxylase activity was reduced in both sponge granulomas from glucocorticoidtreated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as was prolyl hydroxylase activity while there was no effect on peptidyl prolyl hydroxylation. When the synthesis of prolyl hydroxylase by polysomes isolated from the dermis of neonatal rats was investigated, it was found that polysomes derived from control and glucocorticoidtreated rats synthesized similar amounts of enzyme, even though this enzyme is decreased in the tissues of the treated animals.“’ On the other hand, the dermal polysomes from the glucocorticoid-treated rats synthesized significantly less collagen than controls in the wheat-germ lysate assay system. Polysomal m-RNA isolated from the steroid-treated dermal polysomes also synthesized less collagen in a nuclease-treated reticulocyte lysate system. The mechanism of this reported selective reduction of polysomal procollagen m-RNA may relate to a suppression of procollagen gene expression. Glucocorticoids have been reported to decrease ribonucleic acid content and impair ribonucleic acid precursor accumulation in bone also decrease ribonucleic cells.“8 Glucocorticoids acid synthesis in growing mouse fibroblasts conwith a decrease of transcriptional comitantly”’ activity in isolated nuclei.‘*’ The administration of pharmacologic doses of glucocorticoids to neonatal animals suppresses cartilage growth and bone formation, probably
by inhibiting DNA synthesis.‘*’ The marked reduction of thymidine incorporation observed in proliferating cartilage cells is associated with a decline in protein and RNA synthesis and a receptor-mediated event. Developing long bones show an initial retardation or inhibition of calcification, but this is followed by the appearance of more numerous and larger matrix vesicles which, in turn, seem to lead to enhanced calcification. ‘*‘,‘**These events may be relevant to the mode of induction of ectopic calcification processes that can lead to functional and structural abnormalities. It is widely believed that glucocorticoids initiate their action by binding to specific receptor proteins in the cytoplasm of target tissues.‘23-‘26 Locally injected glucocorticoids can improve osteoarthrosis or acute refractory rheumatoid arthritis, but they may also induce progressive joint destruction. Their effects on joints could be mediated through direct hormonal influence on chondrocytes, the constitutive cell of cartilage. Until recently, glucocorticoid receptors had been described only on embryonic chick growth cartilage.‘*’ Evidence exists that glucocorticoid receptors also exist in cultured articular chondrocytes obtained from shoulder and knee joints of twomonth-old rabbits.‘** Vitamin A has been shown to overcome the inhibitory effects of cortisone on tensile strength and collagen deposition in the early stages of wound healing ‘29.‘30and the reduction of inflammatory cell infiltration, fibroplasia, capillary budding, and deposition of collagen fibers in granulomas following glucocorticoid administration is reversed by concurrent vitamin A thera“I It was also shown that vitamin A blocked PY. the inhibitory effect of glucocorticoids on collagen, protein, deoxyribonucleic acid and lipid deposition in granuloma tissue.13* Besides vitamin A, bioflavinoids have also been shown to block the inhibitory effect of glucocorticoids on collagen synthesis and deposition.‘33 Anabolic steroids do not block glucocorticoid inhibition of fibroblast cell proliferation and collagen synthesis in cell cultures,“’ but do diminish glucocorticoid-mediated depression of collagen synthesis. DIFFERENT
TYPES
OF COLLAGEN
Over a decade has passed since we first realized that all collagen fibers within a particular organism are not made up of identical molecules.
13
COLLAGEN STRUCTURE AND METABOLISM
Since 1970 a great deal of experimental work has been devoted to understanding these various collagen types, their molecular structure, biosynthesis, cells of origin, distribution, and turnover. In spite of great advances it is still not clear how the structure of these molecules relates to their function and it is hoped some concepts may emerge from a review of the existing data. The different collagen types have been identified using Roman numerals, which have been assigned as they are purified and characterized (Fig. 14). In addition to these major types (I-V) many lesser, albeit well characterized, collagens have been described. These have been identified using a variety of capital letters and Greek symbols. Type I Collagen Before 1969 this was the only mammalian collagen known. The basic molecule was called “tropocollagen” or “collagen molecule”; the latter term has survived. It is composed of three chains, two identical, termed (~1 chains, and one different from the other two, called a2. Using the new terminology we now call the chains cul type I or al (I) and cr2 type I or (r2(1) or simply cu2, since there is no a2 chain among the other collagen types. This molecule has been characterized both physically and chemically (amino acid sequence, sedimentation, molecular dimensions, carbohydrate attachments, viscosity,
TYPE
I
etc.).13’ Type 1 collagen is most abundant in skin, tendon, ligament, bone, cornea, etc., where it comprises between 80% and 99% of the total collagen. Bone matrix is essentially all type I collagen. The proportion of type I collagen in a particular tissue can vary at different sites, during development, with age and pathology. The most common technique used to isolate this molecule, and distinguish it both qualitatively and quantitatively from the other collagens, involves the use of solvents of different ionic strength and pH followed by differential salting out13’ (Table I). This fractionation is usually followed by identification of the intact chains and CNBr-derived peptides using polyacrylamide gel electrophoresis.‘37 Such separations are shown below (Fig. l5), followed by a schematic diagram that illustrates the distribution of methionines along the (Y chains and the sites where peptide bonds are cleaved by CNBr and responsible for the generation of characteristic patterns (Fig. 16). A more detailed analysis of the CN Br-derived peptides can be accomplished when these are separated by two-dimensional electrophoresis.‘3x The first dimension relies on differences on isoelectric point and the second on molecular weight. A separation of peptides using this procedure is shown below (Fig. 17). The amino acid composition of the better characterized human collagen types are shown in Table 2. The amino acids that are notable by the fact that they differ significantly from that of type I collagen are indicated in bold numbers. On many occasions these differences have been used Table
1.
Collagen
Selective
Fractionation
by
Salt Precipitation Collagen(s1Preclpltatmg
From:
of N&l
Neutral
0.5 M
IM)
Salt Solution
Acetic Ac,d
COllCWltratlOll
Types I, II, Ill
0.7
1.2
Types IV. V 7s
TYPE IU 1.5-1.8
Type III Type IV
1.8 2.0 Fig. 14. Type
Diagram
I is present
cartilage.
and
and es a minor are differences sylation. gen.
type
of the 3 interstitial in
skin,
Ill in blood
component in chain
Disulfide
bone,
vessels.
are only
of collagen.
etc.,
type
developing
in skin and other
composition
crosslinks
types
tendon,
seen
in type
2.2-2.5 3.5
tissues,
tissues.
and degrees
II in There
of glycoIll colla-
4.0 b4.0)
Cys-Rich Aggregate 7s TYPO 1 TYPO I Type I-Trimer Type II Type I-Trimer Tvpe V
MARCEL
Z.
I
_ .__L
II
1411
I.I+IIIR
III
IIIR
I’IIIR
E. NIMNI
BAPN
I YYt= Characteristic
Fig. 15. to the
CNBr
collagen
peptides
chains
alone
has a diagrostic
value
peptide
derived
patterns
from
types
and as mixtures. for identifying
corresponding al(I).
II, and
This peptide collagen
III
pattern
types.‘37
to identify collagen types or their mixtures and to suspect the presence of new or abnormal collagen species.“’ Type II Collagen The major difficulty encountered by earlier investigators interested in isolating collagen from cartilage was associated with its extraction in a native form from fresh tissues. This problem has been overcome in a variety of ways, most of which rely on the prior removal of proteoglycans and of the nonhelical protease sensitive crosslinks to solubilize the collagen molecule, or on the
The distribution
Fig. 16. cross-bars collagen
types
peptides elution tion
along which
from
along
molecular ing from
the
a
I, II, and Ill. Cleavage are
numbered
CM-cellulose the
of methionines
individual
chain).
weight.
Amino
the N-terminal
with
following
columns Their
chains
acid residues end.
by up
make
CNBr
gives
their
sequence
(rather
length
is indicated that
reflect
than
rise to
their
their
are numbered
of
posi-
relative start-
Fig.
17.
2-dimensions first
dimension
tric
focusing)
weight.‘=
The
separation
enhances by virtue and
of
resolution.
in the
of their second
CNBr
derived
Peptides electrical dimension
peptides
separate charge by
in
in the
(isoelecmolecular
15
COLLAGEN STRUCTURE AND METABOLISM
Table 2. Amino Acid Comoosition of the Human Collaaen Chains (Residues11000 Amino Acld
al(l)
a210
nl(lll
al(lll) 0
CYlWl
n2lV)
UlW)
Total Residues) rr3Wl 1
1
2
3
5
108
93
97
125
133
106
110
91
98
93
Aspartic Actd
42
44
43
42
51
50
49
42
46
50
Threonine
16
19
23
13
20
29
21
19
17
25
SeWlt?
34
30
25
39
37
34
23
34
25
28
Glutamlc Acid
73
68
89
71
79
89
100
97
107
98
3-Hydroxyproline 4-Hydroxyproltne
1
7
ProlIne
124
113
120
107
65
107
130
98
109
119
Gl)WllW
333
338
333
350
328
331
332
330
334
327
AlaWla
115
102
103
96
37
64
39
49
54
49
0
0
0
2
O-l
0
0
21
35
18
14
28
27
17
5
10
8
13
11
6
14
9
13
29
15
19
30
26
22
52
37
4
2
3
2
2
12
12
13
8
29
Half-Cystlne Valine Methionlne ls0laWXle Leucine Tyros~ne Phenylalantne Hydroxylystne Lyslne Hlstldlne Argrwva
1
0
0
29
28
18
9
8
10
9
17
20
15
16
36
56
35
39
4
2
2
3
11
12
9
11
11
9
12
20
5
49
23
36
43
38
40
26
18
15
30
9
13
14
15
19
15
3
12
2
6
6
10
6
14
6
11
50
50
50
46
26
48
40
42
45
48
28
34
Gal-Hydroxylysine
1
4
2
3
5
7
Glc-Gal-Hydroxylyslne
2
12
30
5
29
17
administration of BAPN to animals before killdone in this connecing. ‘w’~~’The experiments tion and on the initial characterization of this molecule have been reviewed by Miller.‘4’ Isolation and analysis of collagens from a variety of cartilagenous structures have shown that cartilage collagen is made up primarily of molecules containing three identical LYchains.‘39-‘43 These chains have chromatographic and electrophoretic characteristics similar to the crl chains of type I collagen. Because of intrinsic differences suggesting that their synthesis is directed by a different gene, the cartilage chains are now designated as o(, (II). The most signiticant features of cartilagecollagen are its high hydroxylysine content and its glycosidically bound carbohydrates, since it can contain up to fivefold more of these residues than the homologous chain from skin. Proteolytic enzymes that cleave the protein backbone of the proteoglycans have been of great help in attempting to elucidate the chemistry of the ground substance. In particular, papain has shown a marked specificity for depolymerizing the proteoglycan complexes of cartilage. intravenous injection of crude papain causes a softening and eventual collapse of the normally erect ear of rabbits.lJ4 This is accompanied by a marked decrease in the basophilic and metachromatic
staining and a decrease of the chondromucoprotein content of cartilage.“S.‘46 The collagen matrix does not seem to be affected, and these changes are reversed within a few days. Purified papain will cause a rapid disintegration of bovine articular cartilage at physiologic pH and temperature.‘47 A similar effect of papain had been previously demonstrated using chondroprotein from pig laryngeal cartilage as a substratc.‘4X Due to the temperatures used in most of the experiments (37”-60°C), collagen became rapidly degraded. During the course of some of our experiments it appeared that the difficulties encountered in isolating collagen from cartilage were related to the presence of large amounts of glycosaminoglycans in the tissue. After removal of significant amounts of these substances by papain at 4°C the collagen could be solubilized by increasing the ionic strength of the media. The residual glycosaminoglycans in the crude extract tend to coprecipitate with collagen during high-speed centrifugation, and only after most of these are removed does collagen remain in solution. The purified collagen extracted from bovine articular cartilage exhibits all the characteristics of native soluble collagen, as judged by its high intrinsic viscosity, its optical rotation, and its melting-temperature profile.“’ After denatura-
16
MARCEL E. NlMNl
tion it gives rise to one single component having the same electrophoretic mobility, elution pattern from CM-cellulose, and sedimentation velocity as the (Y, chain from skin collagen. It contains 22 hexose residues per 1,000 amino acid residues, of which eight to nine are glucose and 13 galactose. All galactose residues are bound via 0-glycosidic bonds to hydroxylysine. After periodate oxidation the hydroxylysines with attached sugars were not degraded and could be recovered in the acid hydrolysate. Fig. 18 shows the elution patterns of collagen from bovine articular cartilage and skin collagen. The hexoses, which remain tightly bound to the purified collagen, were separated by paper chromatography and showed a glucose : galactose ratio of 1.6. Only trace amounts of &components were detected in the crude or purified extracts of cartilage collagen, as well as in the collagen extracted in lesser amounts by nondegradative procedures, such as 1M MgCl,, I M NaCl, or water. All of these fractions showed one single component that migrated like CZ~from bovine skin. Limited proteolysis with pepsin has also been used in the isolation of type II collagen from pig LA
0.8-
CARTILAGE al (II)
0.4z no (u 5 go.8 b in d”
200
400
600 SKIN
al
_
0.4
-
0
200 Effluent
400
600
(ml)
Fig. 18. Chromatagraphic patterns of type I from bovine skin (bottom) and Type II from bovine articular cartilage collegens (top) fractionated on CM-cellulose.
laryngeal cartilage’49 and infant articular cartilage.“’ It should be noted that collagen extracted from hyaline cartilage as the result of limited proteolysis has been found to contain little, if any, type I collagen. On the other hand, cartilagenous tissues such as chick sternum and human intervertebral discs contain variable mixtures of types I and II co11agen.‘5’x’52 Limited proteolysis using pepsin or papain has proven to be a valuable method to prepare large quantities of type II collagen. It is of interest in this connection that whereas cartilage collagen from young animals, when solubilized with papain (or pepsin), gives rise primarily to (Y chains upon denaturation, collagen from the skin of the same species but of older age exposed to similar mild proteolytic degradation in the cold, gives rise to large amounts of polymeric material and fl chains upon denaturation.‘39 This reflects the greater susceptibility of the terminal extension of young cartilage collagen to proteolytic cleavage. As the cartilage ages, probably because of the presence of new or more stable crosslinks, it becomes more resistant to solubilization following proteolysis of the tissue. This observation may be worthy of further investigation since it may provide insight into why mature cartilage is more likely to develop degenerative changes. Although type 11 collagen is the major collagen type present in cartilge, it is also present in significant amounts in other connective tissues. The intervertebral disc contains a central gel-like region, the nucleus pulposus, which is surrounded by concentric rings of highly ordered dense collagen fibers known as the annulus fibrosus. The nucleus pulposus is a highly hydrated gel of collagen fibers enveloping glycosaminoglycan molecules similar to those found in hyaline cartilage. The cartilaginous end plate of the intervertebral disc has a composition similar to hyaline cartilage.‘5’-‘54 In contrast, the nucleus pulposus and annulus fibrosus represent a mixed connective tissue matrix of at least two collagen types. The principal collagen type found in the nucleus pulposus is type II collagen. This collagen type represents greater than 85% of the total collagen present, the remainder being type 1 collagen. The structure of the annulus fibrosus is morphologically more complicated than that of the nucleus. Two distinct fiber forms can be seen
COLLAGEN STRUCTURE AND METABOLISM
ultrastructurally in the annulus. Most prominent are the dense parallel fiber bundles forming the concentric ringed pattern radiating from the nucleus pulposus. Surrounding these rings is an amorphous matrix that is rich in proteoglycans. In the human, type I collagen accounts for approximately 50% of the total collagens identified in the annulus at five years of age.ls3 The amount of type I collagen increases with age and represents approximately 66% of the total collagens from year 16 onwards. In addition, the distribution of type I and type II collagens is not uniform. The predominant collagen of the transition zone between the nucleus and the annulus is clearly type II collagen, but the amount of type I collagen increases dramatically as one examines samples radially towards the outer rings of the annulus, where the predominant collagen is type I. It would seem as if the dense fiber bundles are composed of type I collagen, while the amorphous surrounding matrix is richer in type II collagen and proteoglycans. This tissue will be discussed in more detail in connection with degenerative disc disease. Another tissue that contains appreciable amounts of type II collagen is the vitreous.‘55-‘58 Comparisons of collagens extracted from cartilage and vitreous by pepsin treatment show differenccs in amino acid composition, carbohydrate content, and the presence of additional N chains that migrate more slowly than GUI(II). It has recently been reported that rabbit vitreous in addition to cul (II) collagen contains one of the newer collagen species present in hyaline cartilage,3.‘5x to be discussed later. Type II collagen is synthesized during the chondrogenic stages of development of the mesoderm.‘59.‘h” During amphibian limb regeneration the tissues undergo three distinct stages of development during the transition from mesenthyme to cartilage and bone. The synthesis and presence of type II collagen correlate with chondrogenic activity. A similar developmental pattern has been observed in the bone matrix induced endochondral calcification system.‘h’ Type I11 Collagen When the residue of human skin remaining after prolonged extraction with neutral salt and dilute acetic acid solutions is subjected to digestion with CNBr, a series of peptides appear that do not correspond to any of the known peptides
17
derived from types I or II collagens.‘62.‘h3 Because of the similarities of two of these peptides to well characterized regions of the LY’(I) and O(~(II) chains it seemed related to the l-family of chains and was therefore designated o(, (III). When human dermis was digested with pepsin under conditions that allowed the collagen molecules to maintain their helical conformation, it was found that type I molecules could be separated from type III by differential salt precipitation at pH 7.5.‘64 The type III molecules were composed of three identical chains, cy, (I) and 01~. The pepsin-resistant portion of the new chains is similar in size to the previously isolated collagen chains and can migrate with CX,(1) and LY,(III) on conventional sodium dodecyl sulfateepolyacrylamide gels during electrophoresis, although in some systems it can be seen to migrate slightly slower.‘h’ Characteristic of this collagen is the relatively high degree of hydroxylation of proline, its higher glycine content (more than 33% of the residues) and above all the presence of intramolecular disulfide bonds involving two cysteine residues close to the C-terminal region of the triple helix. Because the ratio of type I to type 111 collagen changes with age, type 111 being predominant in fetal skin, this type of collagen is many times referred to as fetal or embryonic collagen. Some interesting conclusions can be derived from observing Epstein’s data on the ratio of types I to III collagen present in the dermis of people of various ages as well as in the insoluble residue remaining after the soluble collagen has been removed. In early fetal life (16 weeks) type III collagen is more abundant than type I collagen. This coincides with the rapid rate of synthesis seen in the developing fetus.lhh At time of birth the ratio of I to III is 2.6 in the total de:rmis but 1.7 in the insoluble dermis. In fetat fife type III collagen seems to enhance significantly strength of the dermis, since insolubility and crosslinks go hand-in-hand. The possibility that type III collagen can become instantly crosslinked through intermolecular disulfide bridge formation is supported by the recent findings of Cheung et al.16’ The ability of this collagen to rapidly crosslink through this mechanism could be of great advantage during early developrnent and wound healing, where collagen is deposited at a rapid rate in an area where no previous connective tissue existed.
18
The distribution of types 1 and II1 collagen in human skin has been assayed by biochemical and immunohistochemical techniques.‘68-‘7’ Whereas biochemical determinations of collagens failed to show relative changes in dermis cut with a dermatome, immunohistochemical studies seemed to indicate that type III collagen is present primarily in the papillary dermis, just beneath the epidermis and around dermal blood vessels and appendages. The reasons for discrepancies are not clear but age-related changes in solubility or preferential masking of the antigenic determinants could be responsible. Type III collagen, when reconstituted in vitro, gives rise to thinner fibers than type I collagen.‘72 The physiologic significance of these findings is not yet clear. Although the denaturation temperatures of type III and type I collagen molecules do not differ, renaturation is more rapid and complete with the type III preparations, due to the presence of interchain disulfide bonds.‘73 This property has been used to further purify type III collagen which still retains type I collagen as a contaminant.‘74 Separation and quantitation of types I and III (Y, chains has most recently been accomplished using HPLC and HEMA 1000 Glc (a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate covalently coated with glucose).‘75 Conversion of type I procollagen to collagen is more efficient than that of type III procollaThese findings are consistent with gen. ‘37*‘76,‘77 the greater yields of type III procollagens extracted by neutral salt solutions from tissues and cell cultures, and with the estimated halflives determined in growing rabbit skin: 26 min for type I procollagen and 3.9 hr for type II1.‘78 The amino acid sequence of the (Y’ (III) chain, consisting of 1028 residues, was determined by Fietzek et a1.‘79 Following the demonstration that type III collagen was a normal constituent of skin (lO%20% of the total collagen), it has been found in many other connective tissues.“’ Normal bone matrix may be the only tissue containing type I collagen that lacks type III. It is present in variable amounts associated with type I collagen in lung, heart muscle, heart valves, uterus, nerves, liver, placenta, unbilical cord, blood vessels, spleen, gingiva, kidney, lymph nodes, sclera, and other eye structures. Blood vessels are partic-
MARCEL E. NIMNI
ularly rich in type III collagen. The actual amounts determined vary with the method of assay, but 20%-30% of the total collagen in human aorta seems to be type III.‘8’,‘82 The media seems to contain the highest proportion of type III collagen and the atherosclerotic plaque the least. Contrary to what had been observed in earlier studies,‘83 where atherosclerotic plaque was found to have more type I than III collagen, more recent work’84 has failed to substantiate such a dramatic reversal even though a small shift in favor of type 1 collagen was observed. In the latter studies, cyanogen bromide treatment solubilized approximately 80% of the total collagen in the intima and plaque and approximately 65% of that in the media. Solubilization with pepsin was much less, generally under 20%. In vitro biosynthetic studies with arterial tissues have also yielded conflicting data since both an increase and a decrease in the relative proportions of types I and III have been reported in diseased tissue.i85 It is conceivable that, as in dermal wound repair, there is in the early stages of plaque formation a preponderance of type III and later a reversion in the III : I ratio towards normal. It is of significant interest with this type of collagen that cells in fibrous joints, such as these present in the sagital sutures in young rabbit calvaria, which normally synthesize only type I collagen can be stimulated to synthesize significant quantities of type III collagen when tensile mechanical stresses are applied to this structure.‘86 Further studies are required to elucidate the sequence of events and the implications of these findings. Type I Trimer Collagen In addition to the genetically distinct types of interstitial collagens described, another molecular form of collagen, al(I)-trimer or type Itrimer has been demonstrated.‘87-‘9’ The LYI(I)trimer consists of three polymers that are genetically identical to the al-chains of type I collagen. It was originally isolated from a virus-induced tumor and its synthesis was observed in cultures of polyoma virus-induced mouse tumors.‘92 Human skin has also been shown to contain al (I)-trimer.“’ Its amino acid composition is, in general, similar to that of al(I) from type I collagen, except that hydroxylysine is increased
19
COLLAGEN STRUCTURE AND METABOLISM
and lysine is decreased. The content of 3-hydroxyproline was also increased, suggesting that both chains are derived from the same gene, but that the cotranslational or posttranslational hydroxylation of lysyl residues is more extensive. This could be a result of a slower recognition and folding of the cul (I) chains in the trimer molecule in the cisternae of the rough RER. Since the synthesis of type I trimer is an early event in the dedifferentiation of chondrocytes in culture,“” and accompanies a variety of unusual circumstances associated with cultured cells, its potential for monitoring abnormalities in the collagen biosynthetic pathway may be of significant interest.“’ IL)’ Type IV Collagen
and Other Basement
Membrane-Associated
Macromolecules
Type IV collagen is the major component of basement membranes and is generally regarded as the most characteristic of a large number of macromolecules that comprise these structures. Basement membranes are specialized connective tissue structures that underlie epithelia and endothelia and perform a multiplicity of structural and functional roles. For instance, they are involved in cell differentiation and orientation, membrane polarization, selective permeability to macromolecules, and are a target for a large number of diseases. The thickening of glomerular basement membrane in diabetics and the deposition of antibodies and complement in bullous pemphigoid, Goodpasture syndrome, and dermatitis herpetiformis are some of the associated pathologic processes. The literature on basement membrane is extensive and the reader is referred to recent reviews on the subject.‘y4~‘y8 In addition to collagen, a large number of other macromolecules, which include glycoproteins such as laminin, fibronectin, proteoglycans, and other less welldefined structures, participate in the formation of basement membranes. This discussion will focus on some of the better documented aspects of basement-membrane research: it will discuss individually the most relevant characteristics of the macromolecules that have been isolated from basement membranes while trying to provide the reader with an overview of the molecular structurc and organization of this vital connective tissue matrix. For a more detailed discussion of
the macromolecules involved, the Timpl and Martin is suggested.ly4
review
by
Type IV Collagen
Kidney glomeruli and lens capsules have been frequently used as a source of basement membranes since they can be readily isolated from other tissue elements.‘99 By treating these tissues with pepsin, Kefalides’“’ was able to solubilize and characterize a collagenous protein, now called type IV. that contains a single type of chain, the cul (IV) chain. It was suggested that basement membranes were composed of type IV collagen linked to noncollagenous glycoproteins by disulfide bonds.‘“x,‘O’ Spiro et al.,‘0’.‘04 using denaturing solvents and reducing agents, were able to extract collagenous proteins ranging in size from 20,000 to over 200,000 daltons from isolated glomeruli. These investigators proposed that glomerular basement membrane was composed of a number of dissimilar peptide subunits with collagenous and noncollagenous sequences of variable length. Type IV collagen differs from interstitial collagens in its amino acid composition (Table 2). In comparison to the interstitial collagens, it contains higher amounts of hydroxylated amino acids (including 3-hydroxyproline) and a lower content of alanine and arginine. The glycine content is less than 3370, indicating the presence of noncollagenous segments. Most of the hydroxylysine residues are substituted by glucosyl-cu( I-2)-galactosyl$ groups linked to thse hydroxyl group. Heteropolysaccharide side chains consisting of glucosamine, mannose, galactose, fucose, and sialic acid have also been identiiied as part of the type IV collagen molecules.20c~2”h Carbohydrate accounts for 10% of the mass of type IV collagen, a higher level than that found in most other collagens. A schematic diagram of type IV is shown in Fig. 19. It consists of a large triple helical domain and noncollagenous extensions that make it resemble procollagen. There are frequent interruptions of the triplet sequence Gly-X-Y within the triple-helical domain with glycine replaced by other amino acids.“‘7 Soluble forms of type IV collagen can be extracted with acidic solvents (usually dilute acetic acid) from the matrix of the EHS tumor20x and from bovine lens capsu1e.20’~“” When electrophoresed the reduced tumor col-
20
MARCEL E. NIMNI
,
-
Fig. lagen
lg.
Basement
is a major
characteristically droxyproline of
contains
and sugar
its nonhelical
ment.
A protease
region insoluble
which
membrane
component
of
significant
residues
extensions sensitive
complicates
collagen: basement
amounts
and seems
in the
problem
IV
col3-hy-
to retain
most
within of
It
of
extracellular
area is located the
type
membrane.
environthe helical
isolation
from
matrices.
lagen migrates as two chains al(lV) and ~u2(lV), with apparent molecular weights of about 160,000 and 140,000.210~2” The chains are not separated by molecular sieve chromatography and their resolution on electrophoresis may be due to compositional differences more than to differences in molecular weight. Evidence exists for a third constituent polypeptide chain. Ultrastructural studies suggested that the pepsin-solubilized collagens contain globular structures at each end of the molecule.2’2~2’3 Pepsin and other proteases are used to dissolve otherwise insoluble collagen. Considerable heterogeneity is observed in the type IV collagen solubilized by pepsin from various tissues such as glomeruli or kidney cortices, lens capsule, placenta, muscle and the EHS tumor, with polypeptides that ranged from M, = 15,000 to 140,000.‘94 Type IV collagen contains nonhelical regions interrupting the helical domain which are sensitive to various proteases, resembling that found in the triple helical domain, of the complement component Clq where it is associated with a bend in the triple helical strands.2’4 These breaks in the helix of type IV collagen may impart a similar flexibility to these molecules. The chains in the triple-stranded molecules of type IV collagen are connected by disulfide bonds. In the al(IV) chain, the disulfides are located at the N-terminal end of the triplehelical domain.2’5 In spite of the sequence peculiarities indicating breaks in the helix, type IV collagen has a thermal stability similar to that of interstitial collagens which is not altered by the presence or absence of disulfide bonds. Ultracentrifugal analysis showed a molecular weight of 450,000 t
50,000 for acid-soluble mouse type IV collagen, consistent with molecules composed of three 150,000-dalton chains. Type IV collagen solubilized by limited proteolysis precipitates when warmed but the precipitate is composed of randomly oriented fibrils lacking periodicity.2’2 Based on such observations, it was proposed that in tissues type IV collagen is arranged as a random meshwork of single molecules connected by covalent bonds.2’3 The size of the polypeptides comprising the triple helix of type IV collagen was estimated by chemical analyses to be in the range of 11 O,OOO145,000, although more recent work on type IV collagen obtained from EHS sarcoma would suggest that anything smaller than 180,000 is a degradation product, and that type IV procollagen (molecular weight around 180,000 daltons per chain) is incorporated as such into basement membrane without processing.2’5 Considering a content of about 10% carbohydrate, these estimates indicated that the triple helix is at least as long as that of interstitial collagens (290-300 nm) but could be as much as 20%-30% longer. ,94.208,216.211 Type V Collagen
Type V collagen was discovered in pepsin digests of placental membranes2’s~2’8~2’9and other tissues. Type V collagen is more soluble than other collagens, particularly at high concentrations of NaCl (333.5 M) and at neutral pH, conditions that readily precipitate the interstitial collagens. Its amino acid composition in large part resembles interstitial collagens except for a high ratio of hydroxylysine to lysine and a low content of alanine. Similar to interstitial collagens, the hydroxylysines are only partially glycosylated with glucosylgalactose or galactosyl groups. Three chains al(V), cu2(V), and &(V) formerly named B, A and C2”) have been obtained to date from type V collagen. They are similar in size, based on their behavior on molecular sieve chromatography, to the 01 chains of interstitial collagens except that the al(V) chains are larger.220.22’The chains of type V collagen show a slightly lower electrophoretic mobility compared to the chains of type I collagen.2’8~222-22sThis may be due to a higher carbohydrate content or anomalous behavior, since their SLS crystallites
21
COLLAGEN STRUCTURE AND METABOLISM
are similar in length to those formed by interstitial collagen and resemble more those of the interstitial collagens than they do basementmembrane collagen.“‘.“’ In addition to the type V chains described, cartilage contains two additional chains that have been labeled E and F.226They are similar to the other chains in their CNBr and limited V8 protease profiles, indicating nonidentity but significant homology. Table 2 summarizes the amino acid composition of the various chains isolated from human hyaline cartilage. The E and F chains together comprise about 10%-l 5% of the total cartilage collagens, and are present in approximately equal amounts, suggesting that they may be part of two different molecules. Recently a series of disulfide-bonded macromolecular aggregates have been isolated from calf skin,“’ placental tissue,228 articular cartilage,“’ chicken hyaline carti1age,230 bovine nasal cartilage vitreous, and human intervertebral discs.“’ In some respects these materials resemble type IV collagen but, on the other hand, they are distributed in many tissues that lack basement membranes. Their association with fractions which extract with type V collagen suggests that they could be extensions of this collagen. Heterotrimeric disulfide-bridged procollagen molecules corresponding to (Y1(V) and c~2(V) (B and A chains) have been described recently.23’ The peptides remaining after collagenase regions are removed with bacterial collagenase are large, and in contrast to the ones present in the interstitial collagens (types I, II, and Ill), may be retained in tissues. The relationship of these procollagen extensions to the disulfide-bonded materials to be described later (7-S collagen) is not known, and further work is required to establish any connections. Type V collagen seems to be particularly abundant in vascular tissues, where it appears to be synthesized by smooth muscle cells, although it is also present in relatively large amounts within the avascular cornea1 stroma. Antibodies specific for type V collagen are closely associated with cell surfaces. In muscle it surrounds the individual myotubules functioning as a boundary, not only between cells but between cells and connective tissue elements of the peri- and epimysium.‘34 In differentiated cartilage, antibodies to type V collagen localize around the
pericellular matrix within the chondrocyte lacunae.235 Electron immunohistochemical localization of type V collagen in rat kidney shows that it is present in the renal interstitium as individual fibers in close apposition to interstitial collagens and vascular basement membranes.23h These observations suggest that type V collagen may be a unique form of collagen that contributes to cell shape by localizing on the surface of the cells and to the formation of an exocytoskeleton, as well as to binding to other connective tissue components. 7-S Collagen This molecule was first identified in a bacterial collagenase digest of the EHS tumor matrix2” and, although collagenase resistant, it has a composition that resembles type IV collagen. Chromatographic and physical studies indicated that it is a relatively homogeneous species. A large form of 7-S collagen (M, = 360,000) can be obtained when the digestion with bacterial collagenase is carried out at 20°C whereas a shorter variant with M, = 225,000 is produced from the long form by digestion with collagenase at 37°C. Electron microscopy shows the short form as compact, rod-like particles, with a length of 29 nm; the long form shows, in addition, four thinner strands (length, 30 nm) extending from both ends of the central core in a symmetric fashion.“” Both forms of 7-S collagen have a highly stable triple-helical domain that melts at 70°C compared to 38”-41°C for other collagens. Reduction of disulfide bonds under nondenaturing conditions produces a single melting profile with T,,, = 48%. This indicates that cysteine (40-45 residues per 1,000) are located within the more stable domain. Both domains also show other differences in chemical and immunologic properties which, in part, may be due to short noncollagenous structural elements. Common to both domains is a high carbohydrate content (20%25’%), including glucosylgalactosyl groups bond to hydroxylysine, and heteropolysaccharide chains containing mainly glucosamine and mannose.237 The reduced protein yields a mixture of peptides ranging from 15,000 to greater than 150,000 daltons, reflecting proteolytic cleavage during the preparation. The data available so far seem to suggest that 7-S collagen is a unique
22
MARCEL
structure that may result from segments of type IV collagen crosslinked by disulfide bonds. Noncollagenous Basement
Proteins Associated
with
Membranes
Basement membranes also contain significant amounts of noncollagenous glycoproteins that presumably account for their positive periodateSchiff reaction.‘38
Laminin Laminin comprises almost 50% of the matrix proteins of the EHS tumor, most of which can be extracted in neutral buffers of moderate ionic strength.239 It has also been isolated from cultured endodermal and teratocarcinoma cells, in some instances in native form after collagenase treatment.‘94 Immunohistochemical localization of laminin indicates that it is abundant in all basement membranes.240 The amino acid composition of laminin distinguishes it from fibronectin.24’ It contains approximately 1295-l 5% carbohydrate.242 While the native protein migrates in electrophoresis as a narrow band, reduction of disulfide bonds produces two broad, faster migrating bands with M, = 200-220,000 and 400-440,000. Ultracentrifugal analyses indicate that laminin has a molecular weight in the range of 800,0001,000,000 daltons both in neutral buffer and under dissociating conditions (6 M guanidine). Laminin interacts with heparin, heparan sulfate,242 and type IV collagen, and links endothelial cells to basement membranes.243 Fibronectin Fibronectin is a major biosynthetic product of cultured fibroblasts.244 It is similar to the coldunsoluble globulin of human plasma described earlier.245,246 It is also produced by a variety of cells, 247including endothelial and smooth muscle cells and some epithelial cells. Immunohistology studies have shown that fibronectin is produced early in development and is associated with most embryonic basement membranes, but is not always detectable in fully developed basement membranes.248v249 It can be found as fibrillar deposits crosslinked via disulfide bonds,250 presumably involving the free sulfhydryls in the molecule, and by yglutamyl crosslinks formed from glutamyl and lysyl residues by transglutaminase. Such cross-
E. NIMNI
links can occur between fibronectin and collagen.25’ Most chemical studies have been carried out with the plasma form of fibronectin. Fibronectin from the matrix or cell surface may be slightly different from the plasma form, particularly with respect to its hydrophobic nature, tendency to aggregate and protease sensitivity.252.253 Fibronectin has a molecular weight of 450,000 daltons and is thought to be composed of two identical polypeptide chains. Each chain of plasma fibronectin contains three to four carbohydrate side chains that are linked N-glycosidically to asparagine and consist of glucosamine, mannose, galactose, and sialic acid.254,255 Fibronectin binds to a variety of macromolecules, including several types of collagens and denatured co11agen.256~258One of the major binding sites for fibronectin on the collagen molecule has a hydrophobic nature and resides in cyanogen bromide peptide al(I)-CB7, near or including the cleavage site for animal collagenase.257~259 Denatured collagen competes with native collagen in bonding fibronectin260 and, since fibronectin inhibits fibrillogenesis, it may play a role in regulating this process as well as fiber size and distribution. The region on fibronectin responsible for its collagen-binding activity is located in the NH,terminal third of each chain,26’ and since there are two such sites, it may enable the molecule to interact with collagen to form extended polymers. The collagen-like region of Clq, a subcomponent of the first complement component, can also bind to this region and coexist as complexes.2h2 Fibroblasts adhere to collagen substrates through attachment to fibronectin.260,2h3 Fibronectin has been visualized by electron microscopy of shadowed specimens as long, thin flexible strands13’ that have recently been shown to bind to actin and that have many features in common with actin-binding protein and laminin.265’266 Since the points of attachment of fibronectin to the cell surface correspond to that of the actin cables in the cytoplasm, one may speculate on its involvement in establishing a continuum between the intracellular and extracellular domains. Fibronectin also binds several glycosaminoglycans, particularly heparin,2673268 an affinity that has facilitated its extraction from tissues.269
COLLAGEN STRUCTURE AND METABOLISM
Proteoglycans
Glycosaminoglycans were first detected in basement membranes of embryonic tissues.270~27’ Polyanionic sites in native basement membranes observed by staining the glomerulus with ruthenium red were shown to disappear after treatment with heparitinase or heparinase*“.*‘* as did the permeability of the kidney and the basement membrane of the glomerulus.272~273 Heparin sulfate and hyaluronic acid were isolated after proteolytic digestion from purified glomerular basement membranes, where they accounted for approximately 1% of the dry weight of the material.“’ A heparin sulfate containing proteoglycan, BM- 1 proteoglycan, (M, = 500,000-l ,OOO,OOO) containing equal amounts of protein and heparan sulfate has been extracted from the EHS tumor matrix.275 Antibodies prepared against these proteoglycans react mainly with the protein core and localize in various basement membranes. In addition to the glycoproteins discussed above, a variety of other macromolecules have been detected in basement membranes; these are discussed in the reviews cited. lmmunochemical Reactivity of Basement-Membrane Components
Considerable interest in this subject arose when it was first discovered that individuals suffering from a variety of autoimmune diseases produced antibodies against basement-membrane components. Bullous pemphigus, chronic glomerulonephritis, and Goodpasture glomerular syndrome have attracted most attention in this connection.27h~277 Patients with the Goodpasture syndrome produce antibodies that react exclusively with alveolar and renal basement membranes, causing lung hemorrhages and renal failure. The antigenic component has been associated with noncollagenous proteins*” and the carbohydrate region of basement membrane collagen.279 In contrast, patients with bullous pemphigoid, a blistering disease of the skin, have circulating antibodies that react primarily with epidermal basement membrane. A variety of animal models have been developed to produce renal damage by injecting antigens such as intact basement membranes or glomerular extracts. These are strongly
23
immunogenic agents capable of eliciting autoimmune responses even when administered in insoluble form.280,‘8’ Rabbit antisera against type IV collagen or laminin injected into mice localizes on the renal and alveolar basement membranes and produces lesions resembling those in Goodpasture syndrome.282 Antigenic determinants to type IV collagen have been identified on the noncollagenous segments of the molecule and on its triple-helical portion.‘94 Rabbit antibodies usually recognize helical antigenic determinants. Laminin and fibronectin both elicit strong immunologic responses.239.283 The major antigenie determinants of laminin are localized in the center of the native molecule. Other antigenic determinants have been identified on a pepsin fragment, and on the reduced and alk:ylated chains. The various basement membrane proteins seem to behave as unique antigens and usually do not share antigenic determinants. In general, comparison of the specific components by absorption or immunochemical assays seems to indicate that type IV collagen, 7-S collagen, or laminin whether obtained from tumors, endodermal cells, or from authentic basement membranes are similar. Cross-reactivity was also found for fibronectins obtained from a variety of sources. Well characterized antibodies to basement-membrane components have been purified by affinity chromatograph:y and used to localize these proteins in tissues and cultured cells. At the iight microscope level, complete codistribution is found with few exceptions for laminin, type IV collagen and the basement-membrane proteoglycan. Type V collagen usually codistributes with the basement membrane proteins and also stained areas that lack basement membranes. Radioimmunoassays and enzyme immunoassays have been developed for the detection of type IV collagen, 7-S collagen, fibronectin, laminin, and laminin fragments in the nanogram range. (For a more extensive discussion the reader is referred to the review by Timpl and Martin.‘94) These immunologic techniques are becoming powerful tools that are certain to add to our understanding of the biochemistry and metabolism of normal basement membranes and their pathogenesis in a number of autoimmune, degenerative, and endocrine disorders.
24
MARCEL E. NIMNI
A CONTINUUM
BETWEEN CYTOPLASM AND
EXTRACELLULAR
MATRIX
The role of the extracellular matrix in determining cell shape, orientation, movement, and metabolic activity has generated considerable interest.284 Cornea1 fibroblasts, which have the capacity to migrate through extracellular matrices, can provide a good model to understand this phenomenon.285 The migrating cornea1 fibroblast is an elongate, bipolar cell, possessing a leading pseudopodium and a trailing cell process that, by successive attachments and retractions, seems able to change shape and location. Using antibodies to various fibrillar proteins, it was observed that fibroblasts grown in collagen gels exhibit significant ultrastructural differences from those attached to glass surfaces.285 The fibroblasts grown on glass exhibit a network of well-defined fibers, “stress fibers” that stain for actin, a-actinin, and myosin. These fibers seem to traverse the cell in all directions radiating from the cell surface along lines of stress. In contrast, the cells grown in collagen gels fail to show such patterns but, rather, show diffuse cytoplasmic staining, and actin and a-actinin but not myosin, and seem to concentrate in the cell cortex and in filipodia. It would seem reasonable to visualize how developing and adult living tissues could possess both of these characteristics at different times, and in different regions of the cell depending on their degree of activity. Even though such stress fibers have not been detected in vivo,286 it is conceivable that such structures could be found in areas of cell attachment. In many ways the phenomena described resembles the organization of microfilaments in smooth muscle cells at the time of contraction. The association of myosin to actin and a-actinin in the stress filaments, and the absence of myosin in the cortical microfilaments of the “nonstressed” cell supports this analogy (Fig. 20). The possible penetration of fibronectin into the cell membrane, its affinity for actin, its general characteristics and distribution, and the fact that the chemotaxis of the fibroblast requires a cytoskeletal organization that seems to be associated with the presence of fibronectin2” suggests that a network must exist which connects the extracellular matrix with the cell cytoplasm (and probably the cell nucleus). This
Fig.
20.
A fibronectin
cell to the surface molecule bonds collagen ment
composed
located and
located
Fibronectin sulfated
may present
cell (stress
link”4 allow
to the
disulfide
via
is attached
period.
a fibrin
binding
fibronectin
to
forms
or tangentially
attachment and
binding
clearly to
is close
a to
is a “trans-
identified actin
of organization oriented
area.’
site* and
site3 which
attach
a site on
cleavage
in this diagram
has not been
to
arrange-
The
collagenase
site. Shown which
linked ends,
site on fibronectin
in addition
in different fibers
nm
a
a dimeric
in a quarter-staggered
glycoseminoglycan
membrane
attaching
Fibronectin,
subunits
by a 68
close
is shown
fiber.
C-terminal
binding
contains
the cell attachment
(AF)
two
the placed
separated
a specific
collagen
which
of
near
molecules
involves
molecule
of a collagen
and
filaments within
the
microfilaments).
network could link the collagen-proteoglycan extracellular matrix via fibronectin, laminin, or chondronectin (or combinations of these molecules with other basement-membrane components discussed earlier) to the actin-binding protein and the microfilamentous network within the cell. The transmembrane mode of communication is not known, but transmethylation reactions involved in chemotaxis of fibroblasts supports membrane involvement.287 Epithelial cells may also be part of such a communication network, but less information is available. Evidence exists for the presence of a receptor for type I collagen on the cell surface of fibroblasts, but not on the apical surface of epithelial cells grown in culture.288 Epithelial cells do not bind directly to collagen but seem to do so through laminin.289 On the other hand, the basal cell surface of cornea1 epithelial cells seems to respond directly to collagen molecules added to the media, without the presence of intermediates, suggesting that this surface may contain a collagen receptor that interacts across the plasmalemma with the actin-rich cortical cytoskeleton.“’ This modality of interaction seems also to hold true at a more macrostructural level. The electron microscopic analysis of the extracted mus-
COLLAGEN STRUCTURE AND METABOLISM
cle-tendon junctions shows that the relationship between the terminal myofilaments and the lamina densa of the basal lamina is retained, despite the extensive extraction of the plasma membrane by nonionic detergentsz9’ Fine filaments (2-7 nm) are seen to connect the lamina densa with an electron-dense intracellular layer into which terminal actin filaments appear to insert. These fine hlaments are considered to represent an important component of the structural linkage between myofilaments and connective tissue and hence to be a significant component of the tension transmitting mechanism. Their precise nature is not known, but some part of the filaments must pass through the hydrophobic compartment of the plasma membrane and thus must be a transmembrane component of considerable tensile strength. These studies suggest that detergentextractable membrane lipids play no significant role in the transmission of tension at the muscleand that fine filaments are tendon junction, responsible for transmitting tension from myofilaments, through the plasma membrane, to the lamina densa of the basal lamina. These studies, among others, strongly support the existence of a well-defined network that links the cytoplasm with the extracellular space, a network that may be able to relay information between the intracellular and extracellular compartments, and probably from cell to cell by means of a series of interactions with components of the extracellular matrix. COLLAGEN
DEGRADATION
The changing patterns of the connective tissue matrix during growth, development, and repair following injury, require a delicate balance between synthesis and degradation of collagen and proteoglycans. Under normal circumstances this balance is maintained, while in many diseased states it is altered, leading to an excessive deposition of collagen or to a loss of functional tissue. The first animal enzyme capable of degrading collagen at neutral pH was isolated from the culture fluid of tadpole tissue.2” This was shown to cleave the native molecule into two fragments in a highly specific fashion at a temperature below that of denaturation of the substrate.“’ These fragments were characterized by electron microscopy and shown to reflect the cleavage of a native collagen molecule at a
25
specific site closer to the C-terminal end of the molecule, yielding segments of 25% and 75% the length of the native collagen molecule; the larger fragment was termed TC, and the smaller fragment TC,. Collagenolytic enzymes have been obtained following cell and organ culture from a wide range of tissues from animal species in Iwhich collagen is present.294m299 In general, these enzymes have a number of fundamental properties in common: they all have a neutral pH optima; they are not stored within the cell but, rather, are secreted either in an inactive form or bound to inhibitors. Figure 21 summarizes schematically its fundamental aspects enzyme and its mode of action. They appear to be zinc metalloenzymes requiring calcium, and are not inhibited by agents that block serine or sulphydryltype proteinases.300 They are inhibited by kelating agents such as EDTA, 1,lO-o-phenanthroline and cysteine, which may inactivate zinc and perhaps other metals required for enzymatic activity, and the zinc in the latent enzyme can be replaced by other divalent cations such as Co. Mn, Mg, and Cu.“’ Nearly all the collagenases studied so far have a molecular mass that rianges from 25,000 to 60,000 daltons. The enzymes are usually present in a latent or inactive form. In some instances they seem to be associated with the presence of a zymogen, but in most cases are bound to an inhibitory protein component that can be removed to form the active enzyme; this step is accompanied by a decrease in molecular weight. Although proteolytic enzymes have been mostly used for activation, some latent collagenases can be activated by nonproteolytic agents, such as cheotropic salts or organic mercurial compounds, suggesting that the collagenase and inhibitors, though forming a tight complex, might not be peptide-linked as in a proenzyme. Chelating agents can inhibit a metalloenzyme either by binding to the enzyme through the metal-ion to form an inactive complex, or by detaching the functional metal-ion to produce an inactive apoenzyme. The collagenase produced by rabbit bone cultures generates an inactive apoenzyme upon removal of zinc.“’ The apocollagenase is unstable and undergoes a slow transition to become irreversibly inactivated if zinc is not readily restored. The latent collagenase with the bound inhibitor is much more resistant to
26
MARCEL
E.
NIMNI
Table 3. Substances Which Have Been Shown to Modulate Collagenase Activity ACtWatOrS
lnhtitors
Tryps18’*
NEUTRAL
EDTA3”
Plasmin
COLLAGENASE
Lisosomal
PROTEAYS
I
Proteinase?
Metallo-Proteinase305 Mast
I-lo-o-Phenanthroline3” a 2-Macroglobulin3’z~3’3
Cell-Proteinase306
Senne
Proteinase”07
Cartilage
Protein”‘4
Nal”’
Platelet
NaSCN”’
Rheumatoid Bone
4-Aminophenyl
Mercuric
Acetatesw
Factor
Culture
Human
43’s
Synovwm3’6 Facto?”
Skin Fibroblasts””
Tumours3’9.320
Chloromercuribenzoate, Mersaly13” Cystww.
Oxidized
Gluta-
Human
Tendor?’
throne”’ Relaxin3’5
Cysteinezg’ Reduced
Glutathionezg7
Fibronectir? Bovine
/==r;‘O& ____.--.._. @ I’
:. -,
Fig.
21.
degradation
Sequence
of
of collagen
,
. . . . . .._..s
I
J
events
fibers
,f
_
- : d .
,
which
by the
,’
I
can
enzyme
lead
to
the
collagenase.
(1 I A variety of factors have been described which stimulate connective tissue cells to synthesize collagenase. glycosydases and neutral proteases. (2) The proteoglycan degrading enzymes remove the mucopolysaccharides which surround collagen fibers and expose it to collagenase. (31 Inactive collagenase is secreted. (4) The enzyme is usually found in the extracellular space bound to an inhibitor. 15) An activating enzyme removes the inhibitor. 16) Glycosidases complete the degradation of the proteoglycans. (7) The active collagenase binds to fibrillar collagen. (8) Collagenase splits the first collagen molecule into two fragments (TC, and TC.) which denature and begins to unfold at body temperature. The enzyme now moves on to an adjacent molecule. (9) The denatured collagen fragments specific
are
now
neutral
susceptible proteases
to
other
proteases.
degrade the
collagen
(10)
Non-
polypep-
tides.
irreversible inactivation than apocollagenase, indicating that the inhibitory portion is a stabilizing factor. There seems to be critical dependence of collagenase activity on the concentration of free zinc (Zn+ +). In excess, zinc can act as an enzyme inhibitor. Table 3 lists some of the agents and conditions that have been shown to modulate
Aorta323
the activity of collagenase by shifting the equilibrium between active and latent forms, by altering the availability of cations essential for enzymatic activity, or by modifying the redox potential around the enzyme inhibitor complex. The cells that synthesize collagenase are influenced to a great extent by the environment in which they live. This includes the permanent resident cells of the connective tissues and adjacent structures and the migratory cells that accumulate as a result of injury, inflammation or immune phenomena, as well as the products secreted by these cells. Epithelial cells and factors secreted by such cells may also play a significant role in the development and remodeling of connective tissues by virtue of their ability to regulate collagenase production by the mesenchymal cells.324x325 Media conditioned by epithelial cells from the adult rabbit cornea is capable of both stimulating and inhibiting the production of latent collagenase by stromal cells from the same source.326 Cytochalasin B is necessary for these effects to be manifested, and optimal stimulation is engendered by low density epithelial cell cultures. A large number of soluble factors have been shown to stimulate collagenase production by mesenchymal cells in culture, among them the prostaglandins (PGEY~),~~~ particularly when combined with endotoxins and proteolytic enzymes’** such as plasmin, trypsin, chymotryp-
27
COLLAGEN STRUCTURE AND METABOLISM
sin, pancreatic elastase, papain, bromelain, thermolysin or cu-protease, but not thrombin. As mentioned earlier, proteases may act not only as stimulators but may activate latent collagenase by acting on the zymogen or specifically degrading the inhibitor bound to the enzyme. Other stimulators of collagenase production are heparin,32y CAMP,“’ collagen,33’.332 type II collagen-derived CNBr peptides,33’ and cytocholasin B.‘-‘4 The stimulatory effects of soluble collagen on collagenase production by cultured fibroblasts is greatly enhanced by a human blood mononuclear cell factor (interleukin 1)332 that has recently been shown to be a potent stimulator of fibroblast proliferation.335 In addition, phorbol ester,j3’ thyroxine,337 colchicine,33x latex beads,33y and hydroxyapatite and calcium pyrophosphate crystals340 have been shown to stimulate collagenase production by cultured cells. Glucocorticoids34’ and progesterone342 inhibit collagenase production, whereas nonsteroidal antiinflammatory drugs inhibit prostaglandin Ez formation but do not significantly affect synthesis of collagenase.343.344 Retinoids that are known to enhance epithelial cell differentiation and decrease proliferation of such cells345.346inhibit collagenase production by rheumatoid synovial cells.‘47 Both the naturally occurring all-transretinoic acid and the synthetic compound 13cis-retinoic acid completely inhibited collagenase production and reduced prostaglandin E, synthesis by monolayer cultures of human rheumatoid synovial cells, at concentrations which were nontoxic to the cells. Enzyme
Specificity
Mammalian collagenases display a great deal of specificity by hydrolizing a single polypeptide bond on each (Y chain of the native triplestranded collagen helix. A significant amount of work has been devoted to understanding the unique characteristics of the cleavage site. The cleavage site of the (Y 1 chain was identified by electron microscopy34x and later by sequence analysis.324.349.35n
Although no conclusive physico-chemical explanation is available to describe the specificity of the cleavage, there are indications that the helix in that region is thermodynamically less stable. The actual bond cleaved in all species studied is a Gly-Leu or Gly-lle link (Fig. 22).
Fig. genase tide
22.
bond
action 775-776
Cleavage
showing
(Gly-lie)
of the
which
enzyme.
is highly
site of chick-skin
the amino The
conserved
acid sequence is selectively sequence from
collagen around
by collathe
hydrolyzed around
species
peptide
pep-
by the bond
to species.
There are slight differences in the amino acid sequence surrounding the scision site: these may account for the differences in the rates at which various collagens are degraded.“’ “’ Type I and III collagens seem to be digested at approximately similar rates, while type II collagen exhibits the slowest rate of hydrolysis. Purified mammalian collagenases do not substantially degrade type LV collagen.3s4 “’ On the other hand, a collagenase derived from a metastatic murine tumor cleaves type IV collagen but not type I, II. 111, or V.‘“’ Proteinase from human polymorphonuclear leukocytes35x,‘59 from mast cells360 and a metalloproteinase from the culture fluid of a metastatic mouse tumor”’ can also cleave type IV collagen. Type V collagen can be degraded by a metalloproteinase secretesd by activated macrophages3h2 as well as enzymes from mast cells35y and tumor cells.‘“” From the culture media of rabbit bone explants, several metalloproteinases have been identified with abilities to degrade proteoglycans. laminin, fibronectin, pepsin-solubilized type IV collagen, and insoluble type IV collagen. A fraction capable of degrading gelatin was also able to solubilize type V and the la, 2n, and 3tr cartilage collagens.3”4 The presence of such a large variety of enzymes in a single preparation support:j the idea that normal connective tissue cells are able to produce a family of metalloproteinases with the combined abilities to digest all the native components of the extracellular matrix. The binding of human skin fibroblast col#agenase to reconstituted collagen has been recently studied in detail.“’ The enzyme interacts tightly with the collagen fiber and appears to remain bound to the macromolecular aggregate during the degradation process. Approximately 10% of the collagen molecules in the reconstituted col-
28
MARCEL E. NIMNI
lagen appears accessible for binding, in close agreement with the theoretical number of molecules estimated to be present on the surface of the fiber. The in vitro data obtained seemed to indicate that digestion proceeds until completion without the enzyme returning to the solution but, rather, hopping from one molecule to another. These observations also would explain why collagenase activity is enhanced when the enzyme is exposed to dilute suspensions of reconstituted polymeric collagen rather than compact fibers of large diameter.366 This mode of action of the enzyme, and its modality of handling the substrate, may explain the low turnover numbers observed (25 molecules of collagen cleaved per enzyme per hour), one of the lowest turnover numbers associated with an enzymatic reaction. It is possible that in vivo the rates could be even slower, due to the presence of enzyme inhibitors and because the active enzyme may have to compete with the latent enzyme for binding sites on the collagen fiber.367 Another factor that seems to slow down the breakdown of collagen is the presence of crosslinks. The introduction of artificial methylenebridges with formaldehyde368 or of native-crosslinks by the use of purified lysyl oxidase369 increases the resistance of collagen to collagenase degradation. Native collagen fibers crosslinked by glutaraldehyde cannot be digested even by bacterial collagenase.370 Collagen from individuals of increasing age becomes more resistant to enzymatic digestion, suggesting that an agerelated accumulation of crosslinks may be responsible.37’ It is therefore possible that crosslinking of collagen plays a role not only in generating mechanical stability to the fibers but also in the regulation of collagen turnover in vivo. Cell to Cell Interactions Collagenase Activity
and the Modulation
of
Tissue culture techniques are of paramount value in understanding the nature of the enzymes involved in connective tissue degradation. The interactions of a variety of cells seem to play a major role in this connection and to be part of a major regulatory mechanism.372.373 Numerous types of cells are present within the connective tissues, including, in inflammatory situations, the invading polymorphonuclear leukocytes and
macrophages. Each of them might participate in the production of the enzymes responsible for collagen and proteoglycan degradation or be the source of soluble factors that may activate the resident cells to produce such catabolic enzymes. Multinuclear giant cells are found frequently in fixed sections of rheumatoid synovium.374 These are usually located in the synovial cell layers near the surface, and not in the deeper layers. Ln addition, when rheumatoid cells are put into monolayer culture, multinucleate cells are observed to form in vitro.375 Many dendritic cells in rheumatoid synovial cell cultures have more than one nucleus. A lymphokine, a soluble factor produced by activated lymphocyte/monocytes, has been characterized that induces multinucleate cell production in cell culture. Because multinucleate cells often are found in areas of connective tissue turnover, Brinckerhoff and Harris376 explored the possibility that these cells could have functional capabilities for increased proteolytic enzyme secretion. Using a one minute exposure to polyethylene glycol (PEG), rabbit synovial fibroblasts growing in monolayer culture could be induced to fuse with one or more neighboring cells, producing a final count of up to 40% polykaryons (cells with two or more nuclei) per culture. After this time, and for up to 28 days, culture in the absence of serum, collagenase in latent form was released from cultures containing giant cells in quantities tenfold that of control cultures. The cooperation between lymphocytes, macrophages and fibroblasts to cause the degradation of connective tissue matrices has been reviewed by Vaes et al.“’ It has also been reported that mononuclear cells isolated from whole human peripheral blood and fractionated by erythrocyte rosetting into T- and B-lymphocytes can secrete collagenase into the media. Removal of the macrophages from the “non-T cells” did not seem to affect collagenase production by the lymphocytes3’* The complexity of these interactions and an excellent chronological review of the development of the different findings that contribute to our understanding of this field is provided by Krane.379 The stimulation of adherent fibroblasts derived from human synovium to produce collagenase can be modulated by a variety of soluble
29
COLLAGEN STRUCTURE AND METABOLISM
cell factors, among which a monocyte-macrophage-derived substance of a molecular weight in the range of 14,000-24,000 daltons termed MCF (mononuclear cell factor) seems to be the most active.“’ Human MCF has many properties in common with murine interleukin 1 (lymphocyte activating factor). Interactions of monocytemacrophages with other cells, such as T-lymphocytes or with immune complexes (or Fc fragments of IgG) or with macromolecules of the extracellular matrix, such as collagen (type II and III), further modulate cell function by stimulation of MCF. This results in changes in PGE, and collagenase production, cell proliferation, and collagen and fibronectin synthesis. Cultured chondrocytes can be stimulated to produce collagenase by a protein fraction (corresponding to a molecular weight of about 14,000 daltons) obtained from endotoxin38’ and lipopolysaccharide-stimulated382 macrophages. Human blood mononuclear cells in short-term culture secrete a factor that induces degradation of matrix proteoglycans and collagen from cartilage explants in culture.383 This matrix degrading activity seems to be associated with monocytes, is stimulated by PHA, and seems to possess characteristics similar to factors described earThe relationships of these factors to lier. ‘7x.38’.384 MCF and interleukin I have not yet been defined. Using another experimental approach, thioglycollate-elicited mouse peritoneal macrophages were cultured in contact with an extracellular matrix produced by rat smooth muscle cells. Both the live macrophages and their conditioned media hydrolyzed glycoproteins, elastin and collagen.3x5 The glycoproteins in the matrix markedly inhibited the rate of digestion of collagen and elastin. When plasminogen was added to the media, activation of plasminogen to plasmim resulted in hydrolysis of the glycoprotein components which then allowed enhanced degradation of the fibrous protein to occur.38h Degradation
of Cartilage
Since degradation of the proteoglycans in the cartilage matrix seems to precede the breakdown of collagen, it is important to understand this process before focusing on the collagenolytic events. A recent symposium devoted to osteoarthritis, the proceedings of which were published
in this journal, devoted a significant effort to this problem.“’ Although earlier work had focused on the participation of lysozomal enzymes or acid hydrolases, 388~390it is now apparent that these enzymes cannot have direct access and be effective in the in situ degradation of the extracellular proteoglycan matrix, particularly in tissues such as cartilage, where the chondrocytes have limited mobility. Currently most studies are focusing on neutral proteases.39’.392 Sapolsky et a1.“j3 extracted from human articular cartilage a metaldependent neutral protease that digested proteoglycans at pH 7.25; similar enzymes are present in cultures of articular chondrocytes394m3q’7 and polymorphonuclear cells.398 Another approach to understanding the degradation of cartilage matrix is illustrated by the observation of Fell and Jubb399 that synovial tissue in organ culture can degrade living cartilage. It was later shown that the synovial tissue was producing an acidic protein of about 17,000 daltons that was stimulating the chondrocytes to resorb the matrix proteoglycans.400,40’ Other connective tissues, such as the sclera, also produce this protein, which has been termed catabolin. It is a heat stable protein (70°C), pI = 4.6, and its synthesis can be suppressed by cortisol. The relationship of catabolin to other factors or to cytokins, proposed to regulate connective tissue catabolism, is not yet clear and there is yet no direct evidence that catabolin causes matrix resorption by stimulating the chondrocytes to secrete proteinases. Cellular
Events in the Degradation
of
Cartilage
Mononuclear phagocytes generated by the in vitro differentiation of rabbit bone marrow cells, when cultivated in a serum-free medium with cartilage, are able to degrade the proteoglycan matrix in a matter of a few days402 This proteoglycan degradation is associated with secretion by the cells of a metal-dependent neutral proteinase. The enzyme is not stored in the macrophages but, rather, is synthesized just before secretion. Under these conditions, no significant degradation of collagen was observed. Rabbit skin or synovial fibroblasts in culture are also able to degrade cartilage proteoglycans under similar circumstances due to the secretion of a rnetaldependent neutral proteinase similar to that produced by the macrophage, but after a few days
30
will degrade the collagen as well, by secretion of collagenase.403.404 Wahl et al.4o5 observed that soluble lymphocyte products (lymphokines) secreted by mitogenor antigen-stimulated spleen cells, enhanced the production of collagenase by macrophages. Since macrophages produce both collagenase and substances that will activate other mesenchymal cells to produce this enzyme, the sequence of events remains uncertain. Hauser and Vaes406 observed a further activation of the macrophage-fibroblast collaboration in the degradation of cartilage; they noted that when rabbit spleen cells are stimulated with either antigens or mitogens they release soluble products that, in turn, stimulate macrophages.4073408These lymphocyte factors have two effects on the macrophages: first, they stimulate the secretion of proteoglycan-degrading neutral proteinases and of collagenase by the macrophages themselves; second, they enhance the production by macrophages of factors that enhance the rate of collagen degradation achieved by fibroblasts. These observations illustrate how macrophages and fibroblasts may successfully collaborate in the degradation of cartilage or other connective tissue matrices made of collagen and proteoglycans and how this activity can be initiated or maintained by properly stimulated lymphocytes. This concept supports the view that macrophages and fibroblasts may be among the main effector cells responsible for an immunologically controlled destruction of chemically altered connective tissues. Chondrocytes do not usually synthesize collagenase in tissue culture, but may be able to do so in the presence of a factor secreted by endotoxin-treated peritoThese observations, couneal macrophages.38’ pled with the fact that human osteoarthritic cartilage in culture may release collagenase,409 indicate that chondrocytes have the capacity to degrade matrix collagen. Nevertheless, the mechanism that causes cartilage destruction in joints is not clearly understood. Removal of proteoglycans seems to be an essential prerequisite for collagen to be actively degraded. While neutral proteases made by the chondrocytes are linked to this initial event, the collagenolytic pathway remains more elusive. The presence of various cell types that can manufacture collagenase, as well as the abundance of inhibitors that can modulate the activity of this
MARCEL E. NIMNI
enzyme, makes this pathway to understand.
URINARY
EXCRETION
DEGRADATION
even more difficult
OF COLLAGEN PRODUCTS
Because of the relatively large amounts of hydroxyproline (12%14%) present in collagen, the assay of this amino acid in body fluids has raised considerable interest. Hydroxyproline is excreted in a peptide-bound form: less than 5% is excreted as free hydroxyproline.4’0,4” Studies using radioactive gelatin and the synthetic dipeptide L-prolyl-L-hydroxyproline showed that the major routes of excretion of collagen metabolites are via lungs and kidneys. Approximately 75% of the peptides released by the degradation of collagen are hydrolyzed to its constituent amino acids and catabolized to carbon dioxide with excretion by the lung; the remaining 25% of the peptides released are excreted in the urine. The prolyl-hydroxyproline peptide appears to be excreted almost quantitatively in the urine, presumably because of its resistance to peptidase activity.4’2 The normal values for urinary hydroxyproline excretion in humans depend on age, sex, body size, and dietary intake of hydroxyproline. When the values are determined on gelatin-free diets and corrected for body surface area, the effect of these variables can be minimized and normal values, reported by a number of laboratories using adequate methods for assays of urinary hydroxyproline, are in agreement413 Altered urinary hydroxyproline values are found in conditions affecting collagen metabolism systemically, such as endocrine disorders, or in conditions accompanied by relatively extensive involvement of bone collagen. Small local changes in connective tissue do not usually cause significant changes in hydroxyproline excretion.4’4 In addition, urinary excretion of hydroxyproline has been reported to be increased in growing children and in many pathologic conditions such as acromegaly, Paget disease of bone, hyperthyroidism, Marfan syndrome, hyperparathyroidism, and primary and metastatic bone tumors. Other conditions that may be accompanied with altered hydroxyproline values include certain skin diseases, thermal burns, some conditions associated with changes in hormonal levels, and administration of certain drugs.4’5
31
COLLAGEN STRUCTURE AND METABOLISM
In most of the above-mentioned conditions, the changes in the excretion of free hydroxyproline and peptide-bound hydroxyproline are of the same magnitude, and the ratio of free hydroxyproline to total urinary hydroxyproline changes only slightly. However, during early infancy, and in hydroxyprolinemia, hyperparathyroidism, severe uremia, and some aminoacidurias the ratio of free hydroxyproline to total urinary hydroxyproline is significantly elevated. A comment should be made regarding the presence of hydroxyproline in Clq, one of the components of the complement system.4’h This compound, a glycorprotein with a molecular weight of approximately 400,000, daltons contains I9 residues of glycine and two to three residues of hydroxyproline per 100 amino acids. The plasma concentration of Clq in normal individuals is around 6.8 mg/kg, and the daily plasma-pool turnover rate is around 4.5 mg/kg. In certain diseases, however, the turnover of Clq can increase threefold to ninefold over normal. In any case, one should be aware of this additional source of hydroxyproline in body fluids. If the hydroxyproline-containing peptides derived from Clq are metabolized in the same fashion as gelatin,“7,4’x one would find each day in urine about 2 mg of OH-proline originating from Clq, which could represent about 15% of the amount of daily hydroxyproline excreted at the lower end of the normal spectrum. Clq would therefore contribute much less to the total output of younger people or those with connective-tissue diseases, whose excretion is elevated. Nevertheless, under circumstances where Clq is turning over rapidly, the implications of such a contribution should be kept in mind. The glycosides, galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine that are characteristic moieties of the collagen molecule, have been identified in urine and used as criteria of collagen degradation.4’“~422 The usefulness of this technique is based on the fact that these hydroxylysine bound glycosides are not metabolized and their total body pools are small relative to their daily renal clearance.42’ Since different collagens contain various proportions of these two glycosides, attempts have been made to quantitate their ratios in the urine in an attempt to identify their tissue of origin. It should be kept in mind that Clq has a high proportion of its hydroxylysine glycosylated4’4 and may contribute up to
50% of the total Hyl-Gal-Glu found in the urine.423 Recently, desmosine, the lysyl-derived crosslink is elastin, and dehydrohydroxylysinonorleutine and 3_hydroxypyridinium, the collagen crosslinks, have been identified in human urine and in patients with Paget disease.4’s The amounts of 3-hydroxypridinium excreted in 24 hr may vary with age; 3-hydroxypyridinium values of leucine equivalents per 24 hr in a 13year-old female and 22-year-old male were 55 1 and 85 nmoles, respectively. The fact that a significant amount of the collagen synthesized by cells may be degraded intracellularly before secretion, and that such degradation products will also appear in the urine,42h further complicates the phenomenon. It has been estimated that between 10%6OQl of the newly synthesized collagen can be degraded intracellularly by a variety of cell types.“‘!’ This pathway can serve as a regulatory role to determine the rates, types, and distribution of the collagen secreted. Abnormalities in the control of such intracellular degradation should be investigated in conditions which affect the quality and quantity of collagen deposited in the extracellular matrix, such as fibrosis, diabetes mellitus, and scurvy. CROSSLINKING Crosslinking renders the collagen fibers stable, and provides them with an adequate degree of tensile strength and visco-elasticity to perform their structural role. The degree of crosslinking, the number and density of the fibers in a particular tissue, as well as their orientation and diameter, combine to provide this function. Crosslinking begins with the conversion to peptide-bound aldehydes of specific lysine and hydroxylysine residues in collagen, a reaction that was illustrated earlier (Fig. 10). It involves the oxidative deamination of the t-carbon of lysine or hydroxylysine to yield the corresponding semialdehydes (allysine or hydroxyallisine) and is mediated by lysyloxidase.42x4” Since this enzyme remains tightly bound to collagen, purified by conventional precipitative methods, incubation of such collagen at 37°C neutral pH and physiologic ionic strength, will cause additional aldehydes to form on the molecule.4”’ This effect can be enhanced by tissue extracts that possess lysyl oxidase activity or by the purified enzyme. This
MARCEL
32
strong affinity of lysyl oxidase for collagen has been used for purifying the enzyme by affinity adsorption.432 Enzymatic activity is inhibited by P-aminopropionitrile, chelating agents such as EDTA and D-penicillamine, and isonicotinic acid hydrazide and other carbonyl reagents. Lysyl oxidase exhibits particular affinity for the lysines and hydroxylysines present in the nonhelical extensions of collagen, but can, at a slower pace, also alter residues located in the helical region of the molecule.433 It has been proposed that it binds initially to the carboxyl-terminal nonhelical end of the fibrillar collagen since it has been observed that the t-NH, groups in this region are the first to be converted to aldehydes.434
marized in Fig. 24. Alkaline hydrolysis of the reduced collagen yielded the reduced form of the aldol-condensation product that generates intramolecular crosslinks (Fig. 25). Although these experiments provided us with basic information on the crosslinking precursors and their early reaction products, they did not allow all of the intricacies of the crosslinking process to unfold. Simple Schiff bases are acid labile, which means that dilute organic acids, such as acetic acid should be able to cleave them. Yet we know that a significant amount of collagen is insoluble in this reagent, particularly when the tissue originates from older individuals. Crosslinking
Intramolecular
and Intermolecular
Crosslinks
Inhibitors of crosslinking, such as BAPN and D-penicillamine, made it clear that aldehydic groups were essential for formation of crosslinks (Fig. 23). Additional progress was made when it was discovered that agents that can add across double bonds or stabilize Schiff bases, such as CN-, and NaBH,, could decrease the solubility of collagen and increase its mechanical the use of tritiated strength.435-‘38 In particular, NaBH, became useful to identify and characterize reducible crosslinks. Analysis of the radioactive compounds showed the presence of aldehydes that have been converted to the parent alcohols (e.g., hydroxynorleucine) and reduced Schiff bases (lysinonorleucine, hydroxylysinonorleucine, and dihydroxylysinonorleucine).439 These fundamental chemical reactions are sum-
Formation
In Vitro
Further insight into the mechanism linking has been provided by studies
of crossin which
R
Y q=o
4=--o - C= 0 H
bH-(CH,),
/I/H
NH,- CH,-(CH,),-
+
k
k
f
I
?
F=O
c=o
L;H-OfzkCH=N-CH,-(CHzk+H
SCMFF
YH R
BASE
YH R
Y
c=o
c=o
~H-/cH~)~-C~
-NH-CH,-(CH,/!!_
tH iH
i/H k
k L Ysbvo/vo~L
Fig. Formation
lar crosslinks occur sation
groups
I collagen.
in the nonhelical reaction
aldehydes links
in type
of intramolecular
on
between
within the
regions
a single
other
of lysine
hand
present
intermolecu-
Intramolecular
and involve
lysine
and
or
molecule. involve
in different
crosslinks
an aldol conden-
hydroxylysine Intermolecular aldehydes molecules.
and
derived crossc-amino
$H NH
F
Fig. 23.
E. NIMNI
24.
Schiff
lysine-derived This interaction
stabilization stable.
The
reaction
and
is primarily
intermolecular hydoxylysine.
base
aldehyde
EUCIM
crosslinks
responsible and
It is acid-labile to
render
reduced
lated derivatives)
the
forms
occurring
an unmodified can
and seems
can be isolated
after
lysine
to require
fiber
(lysinonorleucine
either heat-
of of
further
and
acid-
or its hydroxy-
reduction.
a
group.
for the formation
involve
collagen
between
t-amino
33
COLLAGEN STRUCTURE AND METABOLISM
R
R
&O
;T=o
k-:-CH,I,-C=O
;vH
+
O--C-(CH,I,-hH H
H
k
I
NH R
1
9.
c=o
R
?=a
c-rcH,,,-C=CH-CH,-(~~-~H f?=o H
;vH K
NH k
2
,
4
6
8
Incubation Period (Weeks)
ALDOL Fig. 25.
kx-j¶ UNSATURATED The
aldol
aldehydes
located
molecule.
This product
reaction
involves
in the nonhelical generates
ALDEHYDE) two
extensions
lysine-derived of a collagen
the intramolecular
cross-
link.
Fig.
The
collagen
NaBsH,
to identify
insoluble
reduced
the
bases
amine,
coincides insoluble
crosslinks
bases
obtained
at 37°C.
aldehydes
Schiff
hydroxylysinonorleucine to become
fibers,
is incubated
sors. The precursor the
Schiff
neutral salt-soluble collagen obtained from rat skin was aged in vitro. The collagen fibers become increasingly insoluble as a function of time; within the first two weeks some of the collagen can be redissolved by simply cooling the preparation to 4OC, but later the fibers become insoluble. Following this period, and for another two weeks, some of the collagen can be solubilized by 0.5-M acetic acid or 0.2-M cysteamine, pH 7.0,440 but later these agents are unable to solubilize collagen. In order to interpret these findings, the various collagen fractions were reduced with NaB3H4 and analyzed on an amino acid analyzer for tritium incorporation. After removal of the uncrosslinked collagen by cooling to 4”C, the residue, which remained insoluble, was treated with NaB3H4, various amounts of Schiff bases, in addition to reduced aldehydes and aldol condensation products, could be detected.43’,44’ The Schiff bases identified earlier as lysinonorleucine44’ and hydroxylysinonorleucine?4’ Increase rapidly during the first two weeks of incubation (Fig. 26). In collagens from soft tissues there is a good correlation between the amount of /3 components seen after denaturation and the concentration of cu-P-unsaturated aldol. In bone, this point is less clear, and it is possible that some fl components arise from intermolecular crosslinks of the Schiff base type. Removal of the N-terminal nonhelical peptides by limited
26.
soluble
and
appear
as
(HLNL).
when
The
M acetic
native
be labeled
crosslinking
decrease
with
precur-
as OH-norleucine
lysinonorleucine
with the ability in 0.5
can
ILNL)
and and
in reducible
of the maturing
fibers
acid and 0.2 M cyste-
pH 7.0.
cleavage with CNBr or by enzymes depletes soluble collagen of the nonhelical aldol crosslinks and generates slightly shortened (Ychains, which migrate as do the corresponding a(! and cy2 chains in the parent molecule. The exact significance of intramolecular crosslinking (aldol condensation product) is not known but, as we shall see later, it may proceed to become part of a tetra functional inter-intra-molecular crosslink such as His-OHmerodesmosine. After reaching a maxima, the concentration of reducible crosslinks begins to decline, reaching almost zero after eight weeks of incubation. This decline in reducible crosslinks correlates with increasing insolubility of the collagen fibers, indicating that acid stable and nonreducible crosslinks are formed. In general, lysine-derived crosslinks seem to predominate in soft connective tissues such as skin and tendon, whereas hydroxylysine-derived crosslinks are prevalent in the harder connective tissues such as bone, cartilage, and dentine, which are less prone to yield soluble collagens. In addition to lysinonorleucine (Fig. 24), a compound first identified in elastin, other Schiff bases have been identified (Fig. 27). These are hydroxylysinonorleucine, which results from the association of a lysine-derived
34
A.
B.
MARCEL E. NIMNI
....CH,-
CH,-
r
N- CH-CH, .,..
lminium form
Y
lminium form
..__CH,-$-Ci$-NH-CH,-?i-CH, 0 OH
. ...
Keto - imina
Fig. 27. Two types of crosslinks are illustrated. (A) A crosslink between a lysine from one collagen polypeptide and a lysine derived aldehyde from another. The Schiff base formed is in the iminium configuration and the double bond is susceptible to cleavage by acetic acid and HCI. (B) A crosslink formed between a hydtoxylysine from one molecule and a hydroxylysine derived aldehyde from another molecule. This crosslink is prevalent in bone and in collagens rich in hydtoxylysine. The initial imminium form contains a hydroxyl group conjugated with the N=C bond. This can form the enamine which readily tautomerizes to the ketoimine. The tautomeric form is stable to acid hydrolysis and explains the insolubility of bone collagen.
aldehyde with a hydroxylysine residue, and dihydroxylysinonorleucine, which originates from a hydroxylysine-derived aldehyde and an unmodified hydroxylysine residue. These hydroxylysinecontaining crosslinks seem to be the most prevalent intermolecular crosslinks in native insoluble collagen. The chemical structures of the principal-lysine and hydroxylysine-derived crosslinks identified in collagens are shown in Fig. 28. Recently, several other crosslinks have been identified and their location established. These more complex polyfunctional crosslinks can con-
tain histidine, or can result in the formation of pyridinium ring structures. The earlier work in this connection has been reviewed and many of the possible crosslinking entities discussed in great detail.444 A “post-histidine” peak443 that relates to the aldol-condensation complex in a product: precursor fashion turned out to be the polyfunctional crosslinking compound histidinohydroxy-merodesmosine44s (Fig. 29). This compound seems to result from a Michael addition of histidine N to the 0 carbon of the /3 unsaturated aldehyde (aldol) followed by condensation with the e-amino group of hydroxylysine to form the iminium compound dehydro-His-OHMerDes.446 Reduction with NaBH, converts the Schiff base to the secondary amine, His-OHMerDes, which further stabilizes the crosslink. A recent detailed study of this crosslink has shed significant light on its modality of formation and its location within the collagen fiber.447 Following cleavage of this crosslink (at pH 4.3) into its precursors, its formation was investigated by re-equilibrating collagen fibers at pH 7.4 and 37%. The pepsin digest of the insoluble collagen, previously iodinated to destroy all the histidines except the one involved in formation of the crosslink, can give rise to CNBr peptides containing the crosslink that can then be isolated using chromatographic procedures. The structure of this compound, indicating the relative position of the four amino acids that combine to form it, is shown in Fig. 29. r-----------
;NHy$H-COOH
1
NH,-CH-COW :I 1’ (CH,), : : CHOH ’ , CH; ;mt “1” CH,
,
$H~H
WC), NH;-LH-C&H HYDROXYLYSINONORLEUCINE
MHYDROXYLYSlNONORLEUCCHE
Intermolecular crosslinks involving one lysine Fig. 28. and one hydroxylysine residue (hydtoxylysinonotleucine) and two hydtoxylysine residue (dihydtoxylysinonotleucine). The dotted lines enclose the contributing amino acids and the asterisk denotes the insertion of radioactive ‘H atoms used for the purpose of reducing and stabilizing the double bond as well as for identification.
HISTIDINO-
HYDROXYMERODESMOSINE
A tettafunctional covalent crosslink which Fig. 29. bridges three different molecules. Two of the residues are part of an aldol condensation product (intramolecular crosslink) and therefore associated with one single molecule.
35
COLLAGEN STRUCTURE AND METABOLISM
The peptides that contained the dehydro-HisOHMerDes, retained the histidine and had a composition similar to that of LYlCB5-8 located close to the N-terminus of the 01 chain. A diagram showing the location of the crosslink dehydro-histidinohydroxymerodesmosine in bovine skin insoluble collagen is shown herein (Fig. 30). Two molecules in a quarter-staggered arrangement displaced by 4 D periods show how 2 carboxyl-terminal nonhelical peptides can condense via an aldol reaction to form a Michael adduct with histidine in position 89. The aldol hi&dine, through its unreacted aldehyde group, is free to react with a hydroxylysine residue to complete the crosslink. More recent work emphasizes the need for the amino acids in question to be properly positioned for this tetrafunctional crosslink to form. A genetic variant of type I collagen, which contains a sequence transposition adjacent to histidine, is present in small amounts in calf skin. The collagen fibers that possess this microheterogeneity are not able to complete the formation of this crosslink, but can only form a trifunctional crosslink hydroxyaldolhistidine.448 Because of the pos-
sible significance of the histidine-containing crosslinks, more work is necessary to define its biosynthesis, distribution, and age-dependency. Following the observation in 1977 that achilles tendon collagen contained a naturally fluorescent crosslinking amino acid (subsequentl:y identified as a 3-hydroxypyridinium derivative), considerable interest has arisen in connection with this compound.449~5’ This crosslink is particularly abundant in hyaline cartilage, which may be the richest source of hydroxypyridinium crosslinks, since it contains about one residue per collagen moleculc,4i’ an amount that represents more than 40 times the dehydro-dihidroxylysineonorleucine present, the main reducible Schiff base type crosslink. The structure of this p:yridinium compound is shown in Fig. 3 I. It seems to arise from the condensation of two hydroxylysyl aldehydes and a hydroxylysine residue. Nevertheless, its biosynthesis may proceed. as will be discussed, through the interaction of pre-existing bifunctional crosslinks, explaining the observar-----------l
i HN L_:,\CH
,COOH .-J
1 I------
-l
/’ &j,J,____----+JH,
/’
riiq ,&$H,CH,-CH r-------l I if’;! I II I Hei+ ICH ’ L___+--&__ J INI
1
‘COOH i L_____l
..a, 3 D SHIFT WTERMINh.L Fig.
30.
molecules known
Schematic aligned
intermolecular residues
the
first
C-terminal
sites
are
the
hydroxylysyl
residue a
gN of
an
927 crosslinks
2 chain.
The
an a 1 chain of another
also
form
similar
an
residue
C-terminal
aldol
type of
crosslink
via a Michael
crosslink
between
helical
region
observation
an
that
1
chain
residue 17’
can
gN. The
residue
residue
Histidine
87
with
89 from adds
to
a
the this
an intermolecular 927
and bone
end
220
supporting
in the collagen
PYRIDINOLINE
17’may
crosslink
molecule
or
hydroxylysyl
The residue
Recently,
do form
are The
to 9” of an a 1 chain
a 1 chain.
in dentin
aldehydes
a
to hydroxyllysyl
adjacent
hydroxylsyl
was found
of
molecule.
addition.
of a 1 chains
through
intramolecular
of the other
region
The
hydroxylysine
be recognized.
C-terminal
of the 1 chain crosslinks
from
to 927
CI 1 chains
The
by (-----------I.
region
sites
I collagen
positions.
between
residue
to one or two
to
indicated
N-terminal
crosslinking
of type
staggered
formed
hydroxylysyl
crosslink
residue
and 4-D
crosslink
gN from
among
representation
in 3-D
crosslinking
C-TERMINAL
in the the
helix.“’
Fig. which
31.
This
joins
three
generated sine-derived
newly
described
adjacent
by one hydroxylysine aldehydes
or
trifunctional
collagen by
residue
molecules
hydroxylysine-5-keto-morleucine
from
a hydroxylysine
and a hydroxylysine
can
be
and two hydroxyly-
spontaneous
two
crosslink
interaction
residues
of
formed
aldehyde.4s*
MARCEL E. NIMNI
tion that the reducible, keto-imine crosslinks disappear from skeletal connective tissue with age. Fibrocartilage of the knee meniscus, a collagen that is essentially all type I, in contrast to the type 11 of hyaline cartilage, also has a high ratio of hydroxypyridinium to reducible crosslinks. Adult bone collagen contains fewer hydroxypyridinium residues than does cartilage and more reducible ketoimines,45’ which presumably provide this tissue with relatively stable, yet more readily biodegradable crosslinks necessary for it to withstand continuous remodeling. Quantitative analyses of crosslinking residues in various skeletal connective tissues suggest a precursor-product relationship between dihydroxylysinonorleucine and the hydroxypyridinium compound. Recently, a pyridinium crosslink has been located in dentine collagen. The peptides linked are alCB-5 and (cxICB-6),, reflecting the presence of a collagen microfibril with its molecules packed in a hexagonal array454 (Fig. 32). The study of collagen crosslinking has advanced steadily and, even though hindered by the difficulty in dealing with an insoluble threedimensional matrix composed of quarter staggered molecules, many crosslinking regions, primarily those involving the nonhelical extension peptides, have been identified.455-457 It is interesting in this connection that covalent crosslinks between type I and type III molecules have been recently identified.45R In addition to these aldehyde-derived crosslinks, intermolecular disulfide crosslinks have been recently identified between type III col-
A sequence
Fig. 32. molecules rable
is shown
set of molecules.
between dotted
molecules lines
poly-CB6
are
adjacent
disulfide
crosslinked
side-to-side
solid
lines
molecules.
crosslinks
and
collagen a compacrosslinks
subunits.
subunits
relevant
peptides
with
represent
microfibrillar
between
particularly
crosslinked
intermolecular
The
within
crosslinks
quarter-staggered crosslinks
of end-to-end
crosslinked
and
involving
the non
The
latter
to
the
existence
of
to
the
discovery
of
in type
set
Ill collegen.‘B7
of
lagen molecules, suggesting a rapid and efficient modality of crosslinking within fibers in rapidly proliferating and newly developing connective tissues.‘67 COLLAGEN
AND
AGING
The physical, as well as the chemical properties of collagen change with age. The content of soluble collagen of skin decreases with age, whereas the tensile strength and the insoluble collagen become progressively greater.459.460 The earlier reports are reviewed by Harkness46’ and Nimni.23.462
The age-dependence of the turnover of collagen was studied in growing rats.463 At different time intervals over a 11%day period, skin biopsies were performed in growing rats which had received an initial tracer dose of 14C-glycine. Specific-activity values for the different fractions of skin collagen isolated were corrected for growth, taking into account changes in surface area, skin thickness, and pool size of the particular fraction. The 0.15-M and 0.5-M NaCl fractions in the normally growing rats decayed almost exponentially, with half-lives of 17 and 20 days. The curve for the insoluble collagen did not reflect the presence of a single component, but seemed to indicate a progressive insolubilization. A half-life of 28 days was evident during the period of rapid growth, whereas a value of 300 days could be extrapolated toward the end of the experiment. Corticosteroid treatment (0.11 and 0.36 mg per day) decreased the turnover rate of all fractions and caused a decrease in the amount of collagen extractable by neutral salt. These values are only relative, since absolute determinations would require knowledge of precursor pool sizes as well as isolation of the different collagen types present, which are likely to differ in their individual turnover rates.464466 Transformation of skin collagen with age can, at present, best be described in terms of intramolecular and intermolecular crosslinks, but direct evidence of an increase in the number of crosslinks is scanty. Using stress-strain measurements, an increase in the number of crosslinks in rat skin with age was shown to occur, but the nature of the crosslinks remained unclear.4”7 Increased crosslinking of collagen chains with age has been reported,468.469 an it has been found
37
COLLAGEN STRUCTURE AND METABOLISM
that the isometric tension produced during thermal contraction increases with age.47M73 Changes in the physicochemical properties of collagen with age have been attributed to the formation of both covalent and noncovalent crosslinks. Neutral salt-soluble collagen, which has a low concentration of p components, will generate intramolecular bonds if gelled at 37’C. These intramolecular bonds seem to precede the formation of stable intermolecular crosslinks, since these gels can redissolve when cooled, to yield a soluble collagen with a higher content of /I components of intramolecular origin.474.475 Soluble collagen from rat tail tendons contained increasing amounts of /II and y components, indicating slow formation of crosslinks between collagen components with advancing age. The fact that the slowing down of animal metabolism by cooling prolongs life in poikilothermic species has raised the question of whether similar findings would occur in warmblooded animals, and in what way collagen might be implicated in such a phenomenon. Using the criteria of chemical contraction and relaxation of collagen as an indication of age, the effect of cooling on the speed of aging of collagen in vitro and of hibernation in the fat dormouse was investigated.47h Findings by these investigators suggest that a decrease in temperature slows the maturation of collagen. Although it is attractive, the crosslinking theory of aging awaits further experimental support. Obviously, stabilization of collagen fibers may occur by other means than by formation of new covalent crosslinks. An increase in the number of weaker forces that stabilize macromolecules and their aggregates, such as Van der Waals bonds, ionic interactions, hydrophobic bonds, and combinations of such forces, could account for changes in the physical and chemical properties of the collagen fibers. For instance, a slow-time dependent exclusion of intermolecular water could lead not only to an increase in hydrophobic contacts but could strengthen existing ionic bonds by placing them in an environment of decreased dielectric constant (Fig. 5). Changes in the concentration and composition of proteoglycans surrounding collagen may also play a major role. It is therefore necessary to contemplate many factors in attempting to understand
the age-related changes extracellular matrix. INHIBITION
that
OF COLLAGEN
take place in the
CROSSLINKING
Lathyrism
In connective tissue disorders lathyrism is used to describe primarily a defect in collagen metabolism associated with the ingestion or injection of BAPN (P-amino propionitrile and its chemical analogues) or extracts of the sweet pea or others members of the lathyrus family. The syndrome comprises two entities that have been comprehensively reviewed.477 The terms neurolathyrism and osteolathyrism were coined by Selye4” to describe the two seemingly unrelated metabolic effects. Neurolathyrism, occurs in a variety of animals, including man; thus far three distinct chemical agents have been isolated from the aforementioned plants that seem to be responsible for this condition. Clinical manifestations include spastic paraplegia, and degenerative changes are noted in histopathologic sections of the spinal cord. Epidemics of neurolathyrism have been described over the centuries and a historical review is provided in the reference cited above. Osteolathyrism
Osteolathyrism relates to the more recently described479 connective tissue form of the disease that is observed mainly in rats, turkeys. chicks, and other animals; it has not been described in humans. In this entity the skeletal changes observed differ from species to species, and vary with age, being much more pronounced in younger animals. The epiphyseal plate is a. prime of target.48” Typically one sees a proliferation cartilage cells with an irregular zone of calcified cartilage, retardation of endochondral ossification, loss of orientation of chondrocytes and of cohesion of cartilage, and loosening and detachment of the tendinous and ligamentous insertions. Exostoses occur in the long bones and mandible in the areas of muscle insertion, probably from the mechanical distortion of the weakened matrix. Vascular lesions are common and are frequently manifested in the form of aneurisms (angiolathyrism). In addition to BAPN, several other compounds have been described as having lathyrogenic activity.4x’ These include
38
MARCEL E. NIMNI
organic nitriles and ureides, such as semicarbazide and hydrazines.477 Several compounds have been shown to potentiate the effects of BAPN (inhibitors of monoamineoxidase, copper deficiency) and to inhibit the toxicity (Ca, Zn, Cu, reserpine, propanolol).477 The connective tissue abnormalities appear to be associated with crosslinking defects in collagen and elastin reflected by an increased solubility of these macromolecules in hypertonic neutral salt solutions,482 and to inhibition of lysyly oxidase activity.483 Since Cu deficiency also inhibits this enzymatic activity, the similarities of the defects induced by these two mechanisms are readily explainable. Dermalathyrism
Induced
by Penicillamine
Administration of peniciilamine to animals and humans causes an accumulation of neutral salt-soluble collagen in skin and various soft tissues.48ti87 Two of the more characteristic properties of penicillamine, namely the ability to trap carbonyl compounds and to chelate heavy metals, are of primary significance in impairing collagen crosslinking. The former property manifests itself in all effective dose ranges studied, whereas the latter occurs only at high dosages, far beyond those administered to humans.4s8 The collagen extracted from tissues of animals treated with D-penicillamine is able to form stable fibers in vitro and is not deficient in aldehydes, as is that from BAP-treated animals. In fact, its aldehyde content is even higher than normal, suggesting that the mechanisms of action of BAPN and penicillamine are different (Fig. 33).
Fig. 33. action
Schematic
of penicillamine.
vitro,
will
them
from
interact
intramolecular bility
of the
peptide-bound
with
subsequently and collagen
apy is discontinued thiazolidine
drawing This
complex
summarizing
compound,
aldehydes
on collagen
participating
intermolecular defect
seen
formed
aldehydes.
in the
crosslinks. when
can be explained between
the
mode
in viva as well
of
as in
preventing formation The
penicillamine
by the instability penicillemine
of
reversitherof the and the
Using the method of equilibrium dialysis, it was observed that cysteine, penicillamine, and other analogues bind specifically to free aldehydes on collagen. Both the free amino and sulphydryl groups are necessary for binding to occur. The collagen-mercapto-ethylamine product is in equilibrium with its constituents and can be completely dissociated by exhaustive dialysis.489 The relationship between the dose of penicillamine and the aldehyde content of collagen has been documented.48s+490 Collagen from the skin of young rats contains an average of 0.87 residues of aldehydes per 1,000 amino acid residues. D-peniciiiamine caused the concentration of aldehydes to increase, reaching a maximum of 200 mg/kg of body weight per day. As the dose was increased, however, the aldehyde concentration declined, until at the highest dose it resembled lathyrism induced by BAPN. Although all collagen preparations are able to form native collagen fibers with native 64OA periodicity, only those containing significant amounts of aldehydes form stable crosslinks. Thus collagen from animals treated with high levels of penicillamine, where lysyl oxidase is inhibited by virtue of the Cu being chelated, is incapable of forming stable crosslinks when reconstituted in vitro, and in this connection behaves like lathyritic collagen.49’ From these experiments one can conclude that penicillamine has the ability to block aldehydes and to inhibit lysyl oxidase activity. At low dosage it acts primarily by blocking the aldehyde residues present on the collagen molecule, whereas at a higher dose it also affects the activity of lysyl oxidase.488 This latter observation agrees with the fact that 10m4 M penicillamine partially blocks lysyl oxidase activity in vitro while lo-’ M inhibits it completely.492 Although the mechanism of this activity has not been defined, it may be related to the ability of penicillamine to chelate divalent cations or to interact with other cofactors. Depolymerization of Incompletely Crosslinked insoluble Collagen by Penicillamine
Fractionation of collagen according to its solubility has proven to be of value in disclosing the process of collagen maturation and crosslinking. After rat skin has been extracted exhaustively with 0.45 M NaCl, an additional amount of
39
COLLAGEN STRUCTURE AND METABOLISM
collagen can be dispersed by either dilute organic acids (citric, acetic) or by compounds such as cysteamine or penici11amine.493 In rats less than 60 days old, almost all the skin collagen can be dispersed by amino-thiols to yield a collagen that is soluble in neutral salt. The intrinsic viscosity of this material, 20-25 dl/g (I 3 dl/g), reflects a mixture of collagen molecules with polymeric aggregates. This collagen will withstand centrifugation at forces greater than 100,000 g for several hours without appreciable precipitation.494 In older animals, however, the amount of that is collagen soluble in dilute organic acids and amino-thiols becomes progressively less. In addition to preventing the crosslinking of newly synthesized collagen, peniciilamine seems to enhance in neutral salt solutions the solubility of a collagen fraction that otherwise would remain insoluble, this particular collagen fraction, rendered soluble by administration of penicillamine, is equivalent to that able to be dissolved in vitro by amino-thiols and by 0.5N acetic acid. The fact that both in vivo and in vitro one is able to generate a collagen enriched in aldehydes suggests that penicillamine can also enhance the solubility of collagen by labilizing Schiff base type crosslinks.49’.496 The size of this degradable pool is inversely proportional to the age of the animal; thus penicillamine exhibits maximum effect when given to young animals. Because of the rapid rate of collagen synthesis and turnover, the pool of thiol-soluble material is large at this time.497 By using total skin mass as an index of absolute changes caused by penicillamine, a decline in the amount of total insoluble collagen during the first two weeks of treatment was observed. Continued administration of D-penicillamine failed to affect the remaining insoluble collagen. The soluble collagen that continues to accumulate during treatment may be a part of a rapidly turning over pool or may correspond to the more recently synthesized material that has not been allowed to mature.498.499 Taking into account the variability in rates of collagen synthesis with age, it is possible to estimate that the half-life of this form of collagen-containing labile Schiff bases is about IO days in two-month-old rats.4y7 This period reflects the time required for the Schiff base to be transformed into one of the
more stable forms of covalent crosslinks, discussed previously. Subsequent studies have shown how penicillamine inhibits collagen crosslinking.50” I’enicillamine selectively inhibits the crosslinking of soft-tissue collagen and is much less elective than fi-aminopropionitrile in affecting bone collagen. This collagen, with a high hydroxylysine content, is relatively unaffected in experimental animals fed penicillamine. Contrary to expectations, it was shown that the synthesis of bifunctional crosslinks involving hydroxylysine, :such as N6:6’-dehydro-5.5’-dihydroxylysinonorleucine, increases in those animals treated with penicillamine. The thiazolidine ring interactions may not be sufficiently stable to block the synthesis of these bifunctional crosslinks involving hydroxylysine intermediates highly favored by the geometry of the fibril. On the other hand periarticular collagen from immobilized rabbits treated with penicillamine to decrease contractures reveal an almost complete inhibition of crosslinks formation.50’ These findings explain why the effects of penicillamine are significantly greater on soft tissues than on bone, since the former involve lysine-derived aldehyde intermediates. which will more readily form thiazolidine structures with penicillamine. The stability of the ketoimine form was first demonstrated during the determination of the reduced compound in bone and dentin collagen.“’ The stability of such a crosslink derived from two hydroxylysine residues, explains how it can still form in the presence of physiologic levels of penicillamine. Such a crosslink would be favored over the thiazolidine structure which form when lysine derived aldehydes are present. Figure 27, discussed earlier, illustrates the differences in behavior of these crosslinks; from these experiments it should become apparent why the action of penicillamine varies from tissue to tissue. Its ability to block crosslinking depends on the types of crosslinks that stabilize that particular collagen fiber, skin and soft tissues being more susceptible to the action of this drug, and bone being much less affected. WOUND
HEALING
There is an inherent tendency of tissues to repair after they have been damaged, with the nature of the repair process varying with the site
40
and mode of injury. Connective tissues repair by regeneration of their extracellular matrices. Therefore, a major part of the process involves the deposition of newly synthesized collagen and proteoglycans. Healing of experimentallyinduced skin lesions has been studied extensively in animal models. The cellular population of healing wounds changes from one associated with the initial inflammatory response, (i.e., granulocytes and macrophages) to that associated with a proliferative and anabolic response (fibroblasts). Immediately after wounding, no obvious fibroblasts are present. Before recognizable fibroblasts enter the wound, cells that resemble immature fibroblasts are seen in the perivascular connective tissue. The development of these cells into mature fibroblasts has been followed through the different stages of wound repair.’ 503Collagen synthesis has been reported to increase significantly by 24 hr in open skin wounds of the rat.‘04 A high proportion of type III collagen is produced in response to turpentine injection or to sponge implantationSo and in the initial phase of wound repair in humans.506 More quantitative studies on the nature of the collagen synthesized during the early phase of wound healing5” show an increase in synthesis of type III collagen 10 hr after infliction of the wound; by 24 hr, the percentage of type III collagen synthesized return to a normal value. The early increase in type III collagen observed by these investigators is probably derived from local fibroblasts that are activated by the wounding process, and the early type III collagen deposited may be important in establishing the initial wound structure and in providing a basic lattice for subsequent healing events. The ability of type III collagen to crosslink via disulfide bands may greatly assist in this connection.16’ However, it is unlikely that type III collagen contributes significantly to wound tensile strength as the greatest increase in tensile strength is not observed until later phases of wound repair. In young guinea pigs the rate of collagen synthesis in dermal scars is initially high, but gradually approaches that of the surrounding dermis after a period of about six months.508 Concomitant with the increased rate of collagen synthesis, the extent of hydroxylation of lysine in the early wound is significantly elevated, but declines to approach normal values about three weeks after wounding.
MARCEL
E. NIMNI
Dihidroxylysinonorleucine, derived from two residues of hydroxylysine, is the major reducible crosslink detectable in the early wounded tissue,5o9 but this is subsequently replaced by hydroxylysinonorleucine.5’o In addition to this increase in hydroxylation of lysine and hydroxylysine-derived crosslinks, the amounts of type III collagen is also increased in guinea pig dermal scars compared to uninvolved normal skin. It is noteworthy that the sequence described resembles that seen during early phases of growth and development. Further studies in the quantitative and qualitative distribution of collagen types in a variety of normal and injured tissues might thus provide further insight into the specific role that the different collagen types play in relationship to the normal structure and function of the connective tissues. VITAMIN
C, COLLAGEN WOUND
SYNTHESIS,
AND
HEALING
Well-healed, mature scars in man can disrupt because of asorbic acid deficiency. This phenomenon was recognized in ancient times when sailors on extended sea voyages, living on diets devoid of fresh fruits and vegetables, noted a breakdown in skin scars that had been healed for years. The participation of ascorbic acid in the hydroxylation of peptide-bound proline has been described.23,462 The collagen synthesized in scorbutic guinea pig tissue and in several fibroblast cell lines5”,5’2 in the absence of ascorbic acid contains reduced amounts of hydroxyproline, and the extensively underhydroxylated collagen produced by fibroblasts in culture accumulates within the cell. This raises the question of whether such accumulated collagen may further inhibit collagen synthesis by translational repression. Several earlier observations substantiate this possibility. Electron micrographs of wounds of normal and scorbutic guinea pigs show that vitamin C deficiency caused the cisternae of the endoplasmic reticulum to become dilated and polysomes to become detached.5’3 Using a sucrose gradient it was shown that the heavy collagen-synthesizing polysomes were reduced by vitamin C deficiency, giving rise to 80s monosomes.5’4 Other experiments using scorbutic guinea pigs failed to show a marked accumulation of underhydroxylated collagen, but, rather, decreased collagen synthesis as well as larger amounts of
41
COLLAGEN STRUCTURE AND METABOLISM
diffusible hydroxyproline were noted.5’5 These observations could be due to an increased degree of degradation of partially hydroxylated collagen. It is possible that translational inhibition due to intracellular accumulation as well as increased susceptibility to degradation, are both part of the basic connective tissue defect seen in scurvy. These observations are supported by more recent findings suggesting that the regulatory role of ascorbic acid goes beyond that of acting as a cofactor for hydroxylation.5’6 In addition it stimulates the synthesis of DNA5” and of proteoglycans”” by articular cartilage cells in organ or cell culture. A decrease in the biosynthesis of collagen alone would not be sufficient to account for the dramatic way in which the deficiency manifests itself in old wounds, an event which more likely reflects a combination of anabolic and catabolic events at the wound site.5’9 Metabolic activity in general remains increased, even after the healing process is completed. When the rate of collagen synthesis is reduced because of ascorbate deficiency, the state of equilibrium is altered, and the persisting collagenase activity results in a loss of collagen from the healed wound, and the old scar disrupts as collagen is resorbed. TISSUE
SPECIFIC
FIBROBLASTS
A tendency to investigate the biochemical activities of fibroblasts has been evident without regard to their site of origin, thus assuming that all fibroblasts are alikes. This may lead to errors in interpretation and prevent us from gaining insight into the pathogenesis of various circumscribed diseases. As an example, serum from patients with pretibial myxedema can stimulate fibroblasts to synthesize increased amounts of collagen and hyaluronic acid.52n In this study skin fibroblasts from the shoulder and lower extremities of normal individuals, and from patients with pretibial myxedema (PTM) were grown in culture. When the cells reached the monolayer stage, they were labeled with ‘H-glucosamine and tested for hyaluronic acid synthesis in the presence of either serum from PTM patients or normal human serum. It was of interest that the fibroblasts derived from the pretibial area of normal as well as affected individuals synthesized two to three times more hyaluronic acid when incubated with PTM sera than when incubated with normal
human serum. Fibroblasts cultured from skin of the back or prepuce, on the other hand, did not respond to PTM sera. This heat-stable, proteasesensitive, dialyzable fibroblasts-stimulating factor was not a 7Sy-globulin. The enhanced sensitivity to PTM sera exhibited by fibroblasts from the lower extremities could explain why the lesions in this disease are restricted primarily to a particular area of the lower extremities. Although controversy exists as to whether the serum from patients with scleroderma could specifically stimulate fibroblasts to produce collagen s2’~52sit is clear that serum factors, whether specifically or nonspecifically, stimulate collagen synthesis by fibroblasts originating from patients with this disease. The selective pattern of distribution of many fibrotic diseases may be tied directly to such a selectivity or may result from secondary adaptations of cells at various locations to differences in environmental factors (biochemical or mechanical) which, in turn, sensitizes them to such circulating factors. There is no doubt that fibroblasts from different regions of the dermis exhibit different abilities to synthesize collagen. Cultures from the lower dermis (reticular fibroblasts) accumulate significantly more collagen than do those from the upper dermis (papillary fibrob,asts),“23,524.S26 both in culture and in vivo. Numerous observations support the view that mesenchymal cells, including fibroblasts, from different locations are endowed with unique specificity and characteristics.5’7.5’” For example, estrogens that have been shown to significantly influence the metabolism of connective tissues associated with endocrine control arc also capable of affecting the metabolism of many connective tissues with seemingly non sex-related functions (bones, blood vessels, etc.) Estrogens are able to affect differently the ability of dermaland lung-derived fibroblasts to synthesize collagen,‘28 supporting the view that fibroblasts develop differently from organ to organ. The modality in which this specificity is acquired is not known and could provide an area for fruitful investigation. COLLAGEN
SYNTHESIS
BY CELLS
IN CULTURE
The uniqueness of fibroblasts and their importance in the development of the extracellular matrices of most connective tissues is widely recognized.529 The morphologic and functional
42
characteristics of these cells can vary according to the circumstances under which they are observed. They are widely used as experimental models because they seem to retain their phenotype in culture and express many of the characteristic patterns of behavior seen in vivo, thereby providing a valuable tool to the researcher interested in replicating in vitro abnormalities of the connective tissues seen in a variety of diseases. While normal tissue fibroblasts differ morphologically from fibroblasts grown in culture, many features of the cultured cells resemble those of fibroblasts involved in active metabolic processes (e.g., granulation tissues). Human fibroblasts in culture synthesize both types I and III collagens3 with type I accounting for 70%-90% of the tota1.532 In culture, the rates at which these proteins are synthesized is constant and apparently rigidly controlled.533 However, the proportions differ if cells are cultured with larger amounts of serum (increased type III/I ration).534 Cells obtained from patients with certain diseases such as the Ehlers-Danlos type IV syndrome make little or no type III collagen,535*536 whereas cells from patients with osteogenesis imperfecta exhibit an increased type III/I ratio.537l538 Cell density also alters the ratio of collagen types synthesized by fibroblasts.539 Normal fibroblasts from human and guinea pig skin produce proportionately more type III collagen at high cell density, probably as a result of a reduction in the synthesis of type I collagen. Similar findings are observed with human lung fibroblasts, which synthesize 26%-68% more type III collagen at confluency than at low cell density.540 In vivo, fibroblasts appear as nondividing cells that can, however, be induced to divide by the presence of stimulating factors, such as those associated with tissue damage.54’*542 In culture, conditions can also be altered so as to stimulate their proliferation. Fibroblasts of low and high population doubling potentials grow to confluency when plated on a plastic surface but cease to divide within four days when incorporated into collagen lattices.543 While division is arrested in such a lattice, this state can be reversed if cells are permitted to leave the lattice and again populate a plastic substrate. It thus appears that fibroblasts in tissue-like lattices may be responsive to controls similar to those experienced by connective tissues cells.
MARCEL
E. NIMNI
In order to maximize the information that can be obtained from fibroblasts in culture, many efforts have been made to standardize their growth544 and to assay quantitatively the amounts of procollagen produced.545 Under such optimal conditions, 80% of the procollagen in the media is type 1 and the remaining 20% is type III. CHONDROCYTES, PHENOTIPIC LABILITY, AND OSTEOARTHRITIS
Chondrocytes are highly differentiated mesenchymal cells that synthesize a unique blend of collagen and proteoglycans. These cells are embedded in the metachromatic matrix they produce, a mixture of chondroitin-4 and six sulfates, small amounts of other glycosaminoglycans, and significant amounts of type II collagen. 546~547 The nature of type II collagen has been discussed and earlier work on the lability of the chondrogenic phenotype that causes chondrocytes in culture to “dedifferentiate” and produce other collagen types has been reviewed.548 For their proper function, and for the maintenance of the differentiated phenotype, chondrocytes seem to require a carefully controlled, highly specific microenvironment. Alterations of this environment, either in vivo or in vitro can cause significant changes in cellular metabolism, leading to an alteration of the extracellular matrix. The possibility that such changes could predispose and be ultimately responsible for cartilage degeneration and accelerated wear and tear, have stimulated significant amounts of work to further understand the factors and the mechanisms involved. Early biochemical studies suggested that articular cartilage cells present in areas of degeneration could be switching their synthetic capacity from producing type II collagen to type 1 collagen.549 These findings were supported by immunohistochemical observations that some chondrocytes were surrounded by a fluorescent halo when reacted with type I collagen antibodies.550 Other studies55’,552 have demonstrated a higher solubility of collagen in osteoarthritic cartilage as compared to normal, but no type I collagen could be detected in the soluble fraction. In other instances no a 2 chain was seen in the pepsin solubilized collagen from osteoarthritic collagen,553 while a similar degree of hydroxylation of lysine in control and osteoarthritic-insoluble collagen was observed.554 This
COLLAGEN STRUCTURE AND METABOLISM
was taken as an indication of no major shift in the type of collagen synthesized by osteoarthritic cartilage. Studies using animal models for osteoarthritis have confirmed these observations,iiF.C(h These discrepancies can now be explained as a result of detailed biochemical and histologic analysis of human osteoarthritic cartilage in various stages of degeneration.557 From this study it seems that type I collagen is present in sizable amounts only when histologically identifiable fibrocartilage is formed. In other areas, where hyaline cartilage still persists, only type 11 collagen can be detected biochemically. Nevertheless, in the hypermetabolic areas of degenerating hyaline cartilage, it is possible to clearly see by immunofluorescence small amounts of type I collagen, which is probably present in ins&icicnt amounts (less than 3%) to be detected chemically.‘“” The ultimate significance of this alteration is not known. It would seem as if the small amounts of type I collagen synthesized by the chondrocytes in actively degenerating hyaline cartilage are a reflection of an altered metabolic activity, rather than being directly responsible for the formation of an unsuitable matrix that more readily degenerates. The fibrocartilage, on the other hand, can be interpreted as a healing response by cells that originate most likely from the subchondral bone. The healing response in question may be similar to that observed when full thickness lesions of the articular cartilage, those extending down to the subchondral bone, are produced.558 Such lesions rapidly fill with fibrocartilage,s59 predominantly type 1 collagen in the early stages of formation,“’ but which becomes mostly type II after eight weeks (Fig. 34).‘“’ The induction of cartilage formation at unusual locations has been reported5*’ (vascular lesions, a calcific heart valves, pseudoarthritis, etc.). An example of such a phenomenon is illustrated in Fig. 35. In this case the cruciate ligament of a rabbits knee was cut, and allowed to hang loose in the synovial space. After several months cartilage cells can be detected in the degenerating ligament, probably originating from fibrocytes which have responded to changes in mechanical stresses or to chondrogenic factors present in the joint space. The chondrogenic phenotype is labile and cultured chondrocytes tend to readily “dedifferentiate” when grown in
Fig.
Regeneration
34.
wounding
with
a drill
dral area.
After
eight
nous never hyaline
matrix
ment
was
therefore
This able
the subchondral
the hole filled with of type
fashion
progenitor
the
a poor cells
subchon-
a cartilage-
II collagen,
with
illustrates
area to differentiate
knee after
into the
generating
observation
to cause
in a rabbit
penetrated
primarily
in a continuous
cartilage,
ing surface.
weeks
consisting
blends
of cartilage
which
how
but
preexisting
weight the
bear-
environ-
originating
from
into chondroblasts.=
monolayer cultures,“’ and switch from s:ynthesizing type II to type I collagen. This process can be accelerated, as discussed earlier, by addition to the media of BrdU562 or embryo extracts Viral transformation of chick embryo-derived cultured chondrocytes caused a decrease in the amounts of type II collagen synthesized and increase in the amounts of fibronectin in the media, but did not cause the cells to produce type I collagen.“’ The normal pattern of proteoglycan and collagen synthesis by chondrocytes can also be altered by an excess of vitamin A in the media.“h4 The changes in phenotypic expression occur concomitantly with changes in cell shape, from polygonal to spindle or fibroblastic-like. Immunofluorescence experiments with antibodies to various collagen types5” showed that most fibroblast-like cells among chondrocytes stained positively for type I collagen but not for type II. However, type I collagen synthesis is also found among polygonal chondrocytes, suggesting that
MARCEL E. NIMNI
Fig. 35. Anterior cruciate ligament of a rabbit cut and allowed to dangle freely, attached from one end, in the knee-joint space. As a result of change in mechanical stresses to which the cells were exposed many cells within the ligament differentiated into chondrocytes and produced a characteristic metachromatic matrix. (Courtesy of Dr. E. Gendler.)
no absolute correlation exists between cell shape and collagen phenotype. It is also important to emphasize that the majority of the chondrocytes stain positively for either type I or type II collagen, and less than 1% stain simultaneously for both types. This could mean that the loss of the cartilage phenotype is not a gradual process but occurs rather abruptly5@ for any one particular cell. As discussed earlier attachment of fibroblasts and epithelial cells to collagenous matrices is mediated by the glycoproteins fibronectin and laminin, respectively, while chondrocytes are attached to type II collagen by a distinct factor, known as chondronectin.567 Antibodies to chondronectin, a glycoprotein of approximately 180,000 daltons, localize on the cell surface of the chondrocytes rather than in the matrix, and can inhibit their attachment to type II collagen.567 Although chondrocytes in culture can become metabolically active, in adult cartilage their metabolic activity is relatively low and mitosis has rarely been demonstrated.568 When degenerative changes occur in the joint cartilage, chondrocytes recover their ability to divide.569 Local traumatization of adult rabbit joint cartilage induces mitosis of chondrocytes not only in the traumatized joint but also in the normal cartilage of sham-operated or unoperated control joints.570 All cartilage of the patella groove of the femur of adult rabbits was excised while the other knee underwent arthrotomy or was left intact.
Radioactive thymidine and autoradiographic techniques demonstrated that labeled chondrocytes appeared not only around the excised cartilage but also scattered in the tibia and femoral condyles. Whether this stimulation of chondrocytes is due to a decrease in cell growth-inhibiting factor or to a liberation of stimulating substances is not known, but the mechanisms of this activation represents a significant question.57’ In this connection, much attention is being given to the role of somatomedin, growth factors and other metabolites that may stimulate the metabolic activity of chondrocytes. It has been recently shown that prostaglandin E2 stimulates cyclic AMP levels in cultured chondrocytes. The relative abundance of prostaglandin E2 in these cells suggests that it may have an important physiologic role in controlling cyclic AMP synthesis in chondrocytes.572 Stimulation of prostaglandin E, biosynthesis by a Ca++ ionophore is also accompanied by increased levels of cyclic AMP. Sodium meclofenamate and indomethatin, both potent inhibitors of prostaglandin cyclooxygenase, inhibited the ionophore-stimulated prostaglandin E2 production, which correlated with a reduction in cyclic AMP. How increased intracellular cyclic AMP concentrations affect chondrocyte metabolism are unknown at the present time. The state of differentation of chondrocytes can be monitored in a more quantitative fashion by determining the collagen types they produce. Rabbit articular chondrocytes grown in monolayer culture synthesize collagens other than type II, and the proportion of these collagen species changes during subculture.573 These additional collagens were identified by direct cyanogen bromide peptide analysis as types I, I-trimer, and III; partial characterization of two new collagen chains, X and Y, has been completed.574 Further evidence suggested that during primary chondrocyte culture, the synthesis of two collagen species, types 11 and III, increased at the same time and to the same degree. In addition, X,Y synthesis was stimulated prior to these changes, implying that this collagen may be regulated independently.575 The relative changes in collagen types produced by chondrocytes subcultured through five different passages is shown in Fig. 36. In order to further understand the factors that contribute to the stability (or instability) of the chondrogenic phenotype, cultured cells were
45
COLLAGEN STRUCTURE AND METABOLISM
ItA
4
\
. 3
; : \
I
2
p”
1
WEEKS
CULTURE Fig. 36.
Synthesis
subculture:
radioactive
acrylamide.
The
for
the
of collagen
of total
sample,
the
was
phenotype
41%
trimer.
type
of chains of fifth
I, 25%
13% type
were
sample
X,Y
fractionated peak
analyzed,
was the
during
during
was corrected
on 5%
chains
corrected number
Collagen
nine weeks
of
in
types
IN
synthesized
in culture. collagen
by cartilage
The radioactivity
for cell number
native
CULTURE slices
of each peak
and for the stoichiometry
molecules.
ND
not
of
detect-
able.57*
of al (1) and al (II) chains, in the native
culture (type
Ill and 1% type
Fig. 37.
by chondrocytes
of each
proportion
and the stoichiometry collagen
collagens
radioactivity
fraction
cells per
NO.
collagens.
chondrocyte
V collagen), II. ND=not
The
progeny 20%
type
detectabla.676
compared to chondrocytes in organ culture, which are able to retain their original shape, as well as those in a more physiologic environment (Fig. 37). Similarly to cultured cells during early stages of primary culture, transfer of fresh tissue to organ culture enhanced the metabolic activity of these cells although they did not show significant changes in collagen phenotype. This degree of stimulation has subsequently been shown to correlate with the concentrations of fetal calf serum present in the media. These experiments demonstrate both the sensitivity of the chondrogenic phenotype to environmental manipulation and the presence of metabolic stimulating factors in the tissue culture media. It is apparent from these studies that the differentiated phenotype of rabbit articular
I
chondrocytes, which consists primarily of type 11 collagen and cartilage-specific proteoglycane, is lost during serial monolayer culture and replaced by a complex collagen phenotype consisting predominately of type 1 collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes are able to re-express their differentiated phenotype during suspension culture in firm gels of 0.5% low T, agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains.‘77.“78 Collagen gels are also able to provide a suitable matrix in which embryonic chick chondrocytes proliferate and deposit a territorial matrix that resembles a chondrocytic lacuane.57x~i7” Figure 38 (phase contrast photograph) shows the growth characteristics of dedilferentiated chondrocytes after they have been transferred to
MARCEL E. NIMNI
collagen /:
SYNTHESIS
/
-I
DISH
L 4O
1
2
4
DAYS Fig. cyte
39.
IN
during
collagen,
as counts
per
suspended
in 0.5% f3sS0,)
CTAB-precipitable isolated
Phase
Fig. 38. drocytes ture
contrast
passage)
and subsequent
Exactly ited
(fifth the
same
proliferation
domains matrix indicate
(MD) fusion
culture was
(PI,
limited
around (MF)
both
between
release
in agarose
field
sites where
the culture
micrographs
after
of cultured
from
single
death
Cultures (CD),
and
exhibmatrix
cells and aggregates,
neighboring
examples
cul-
for 1.7, and 13 days.
photographed. cell
chon-
monolayer
of these
and
cells. Arrowheads events
occurred
as
developed.578
agarose. Also shown are the changes in metabolic activity that accompany the transition. Most marked is the increase in synthesis of collagen which are an indication of re-expression of the differentiated chondrocyte phenotype (Fig. 39). A more specific and quantitative way of docu-
12
were
14
CULTURE differentiated
culture.
correction
the
being
number
released
f3H-thymidine)
analyzed
radioactivity.
of synthesis
are expressed
for
(4”) or after DNA
chondro-
Rates
and proteoglycan
after
agarose.
as ‘H-leucine collagen
of the
agarose
cultures
teogycans
10
AGAROSE
protein
minute
cells in monolayer
measured
8
Reexpression
phenotype
for DNA,
6
as Protein
incorporated.
and
of and pro-
pronase-soluble, synthesis
Radioactivity
was in the
was also measured.“’
menting this phenomenon of reversion involves analysis of the collagen phenotypes produced. Figure 40 shows the individual collagen chains synthesized by dedifferentiated chondrocytes (4th subculture) grown as monolayered cells, and of similar cells which were placed in agarose in suspension and maintained in this condition for 14 days. Also shown are a series of dedifferentiated chondrocytes exposed to different number of subcultures (3-6) and transferred to agarose for 10 days. In all instances reversion to normal phenotype can be clearly seen (Fig. 41). These studies, particularly those dealing with the reversibility of the dedifferentiated state, emphasize the usefulness of the tissue culture approach to understand the more subtle changes that occur in the in vivo systems. As we are able to further identify the factors (physical and chemical) that contribute to this reversible pattern, we should be able to apply our findings to the development of new modalities of treatment of diseases where chondrocytes fail to properly remodel or repair damaged cartilage.
47
COLLAGEN STRUCTURE AND METABOLISM
4” 1
(days)
4”
;gar;se
1
1 5’
10
14
I
I
Fig. 40. tiated
Time
collagen
(100,000
(4”).
agarose
the
rose) on
course
chondrocyte
cpm)
a 3.5%-10% for
(l-10 trimer
(I
l(l)
hydroxyproline
The
from
known
type
bands
containing,
from
gel
duration
culture
culture
in SDS
and
of agarose collagen
labeled
CPl
collagenous
interstitial
BIOMECHANICAL
I
laga-
fractionated pro-
culture
for each lane. Also indicated
derived
l(H).
this
(5”) were
gradient
differen-
radioactive
monolayer
from
culture
The
is indicated
and
derived
monolayer acrylamide
of a
of the
Purified
the fourth
derived
fluorography.
days)
positions
phenotype.
from
cultures
and the fifth
cessed
of reexpression
collagen
are the
or type
and
CP2
peptides
I
are not
collagens.“’
PROPERTIES
CONNECTIVE
OF THE
TISSUES
While discussing the biology and chemistry of collagen we should always keep in mind that this macromolecule, by virtue of its becoming organized into fibers and in conjunction with other components of the connective tissue, is providing
3”
I
4*
1
1
5’
6O
MAMAMAMAA
Ill-
lo-day ated.
Effects
differentiated
dioactive
Collagen medium
of subculture chondrocyte
collagen agarose
deficient
o-
-
Fig. 41. the
CF
from culture
from
on the collagen
each monolayer (A)
derived
agarose
(CF) were
phenotype.
culture
from
cultures
reexpression
(M) and the
it were grown
also analyzed.“’
of Ra-
fractionin
calcium
organs with a mechanical function, either of a structural nature or as a selective barrier. So much information is available on this subject that any attempt to discuss it in any detail would be impractical, but some of the fundamental principles which emanate out of the unique physicochemical characteristics of connective tissues shall be reviewed, more to challenge the imagination of the reader than to provide him with the new data in the field. For detail several excellent reviews on the subject are available in journals in biomechanics and medical devices.58”~5xh To understand the elastic, viscous, and frictional properties of the connective tissues, one should look at the physicochemical properties of the individual components and the compartments to which they are restricted. The primary function of collagen, which can be associated with its fibrous conformation, is that of resisting or transmitting forces. Why do we have so many different types of collagens? We cannot answer this question now, but by observing the molecular structure of the various collagen types. their mode of organization at the ultrastructural level, their distribution, the diameter of such fibers, or the lack of hbrillar organization (such as in the basement membranes) one may intuitively begin to relate structure to function. Quantitation of these functions will undoubtedly come. Stress-Strain Behavior of Collagen Fibers Aligned Parallel to Each Other The biomechanical properties of collagen fibers have mostly been studied primarily in tissues where these fibers are oriented in a parallel fashion (i.e., tendons, ligaments). The stressstrain characteristics are well described in the literature, SXZ.iX7.58X A stress-strain curve for tendon, and the kind of information that can be derived from it is illustrated in Fig. 42. Quantitative interpretation of such data is difficult, however, because so many variables (e.g., water content of the specimen, measurement of cross-section, collagen content, presence of various collagen types, heterogeneous population of molecules and crosslinks, presence of other connective tissue components such as elastin, proteoglycans) significantly affect behavior. Which is the most meaningful part of the stressstain curve? The toe region (a) reflects the slack that is associated with elimination of the crimp-
48
MARCEL
E. NIMNI
skin with ageing. This would be difficult to do using stress-strain particularly in a tissue that has fibers oriented in a nonparallel fashion. The thermal shrinkage of collagen and the role of the intermolecular crosslinks in providing for the contractile force is shown in Fig. 43. Interactions STRAIN Fig. 42. nous
Changes
tissues
during
(A) Elimination aligned
fibers.
in the
internal
the stretching
of crimpling. (C) Failure
structure
of a tendon
(B) Resistance
of collegeor ligament.
displayed
by the
point.
ling of the fibers; during this phase the elastic modulus increases steadily. The linear part of the curve is where stress increases rapidly (b). This is the area from which the modulus is usually calculated and reflects the actual contribution of collagen molecules aligned parallel to each other and stretching of the crystalline network. Nevertheless, one still has to distinguish at this stage between intrafibrillar distortion and the slipping of fibers side by side. The increase in modulus reaches a plateau during the linear part of the curve; it is this value that is usually used to determine the “stiffness” of the specimen. When the point of maximum stress is reached, the tissue breaks (c); this is the failure point, and can occur suddenly, in bone, or can be preceded by a leveling off towards the strain axis, as in skin, ligaments, and tendons. Detailed analysis of the various stages of the viscoelastic behavior of such tissues is provided in the reviews previously cited. Thermal
of Collagen with Proteoglycans
(0)
Denaturation
In order to understand the physical properties of connective tissues such as cartilage and intervertebral disks, it is important to have an understanding of the salient features of the proeoglycan molecules59@594 (Fig. 44). There are essentially two types of glycosaminoglycans, those with weak negative changes, such as hyaluranic acid, and those with strong negative charges such as the chondoitin sulfates, heparins, and dermatan sulfate, the latter comprising the largest of proteoglycan species. Their distribution and physicochemical characteristics, which contribute to distinct functions, are also unique. Hyaluronic acid, with weak negative charges associated with the carboxylic acid residues present in glucuronic acid, has a tendency to form hydrated gels, and can therefore contribute signifiA
C
B
HEAT 1
of a Collagen Network
Since the stiffness of the stretched fibers relates to the degree of crosslinking of molecules within a fiber, other more sensitive methods have been used to evaluate this parameter. Denaturation of collagen by heat or chemicals can produce a collapse of the helical structure and a shrinkage in the direction of the longitudinal axis of the fiber. The isometric tensions developed during thermal shrinkage, as well as the hydrothermal swelling that accompanies this event, have been used to evaluate the physical properties of the collagen networks.589 Such an approach, particularly measurements of hydrothermal swelling, may allow us to quantitate differences in the degree of crosslinking of collagen that occur in
Thermal
Fig. 43. direction the
oriented
become those
The native
found
to
heat,
crosslinks
provided. there
are
when
the
If native
normally are
shrinkage
The degree
held
in magnitude
no thermal
are no bridges
between
together
On
the
such a
shrinkage the collapsing
rat
are
in (A).
by
these
If in addition by the
dialdehyde stability
when
it does
other
treated
hand
(C).
if
case
with
because
molecules
to
such as
as is the
occurs
a
such
tissues
by the greater
(B).
of
into
as shown
of contraction,
from
go
are introduced,
whatsoever,
originates
penicillamine,
and
crosslinks
“shrinks.”
is delayed
The
axis
molecules
adult
are generated
and force
no crosslinks tissue
in occurs
new crosslinks
crosslinks
collagen
collapse
and the tissue
is greater
the
of the tissue
crosslinks
artificial
As they
occur
network.
to the longitudinal
(gelatin).
molecules
“collapsing”
glutaraldehyde, occur,
by
contraction
the native when
fibers.
coil conformation
present,
of a collagen
is parallel
collagen
denatured
random as
shrinkage
of shrinkage
D-
there
COLLAGEN
STRUCTURE
AND
METABOLISM
49
Fig. 45.
negative
fluid)
and
positive Fig. 44. are usually
Collagen closely
ground
substance.
cule
cartilage,
in
containing (PGS)
and
fibers
This
to
acid
proteins
(A)
structure.
The
PGS
consists
which
negatively
charged
the
chondroitin
sulfate
(CSj
with
diagram
adjacent
hyaluronic link
do not exist
associated
in a vacuunt.
the proteoglycans depicts
a collagen
a proteoglycan
(HA), which
help
to
and keratan
sulfate
subunits
stabilize
core
glycosaminoglycan
mole-
aggregate
proteoglycan,
of a protein
They of the
(PC)
the from
chains
of
(KS) radiate.
cantly to the viscoelastic fluidity of synovial fluid and to the turgency of the skin of an infant. On the other hand, the negatively charged polysaccharides that contain sulfonic acid residues are able to develop strong ionic bonds with the positively charged amino acids on the surface of the collagen fibers, particularly lysine, hydroxylysine, and arginine. Such tissues are more compact, resilient, less hydrated, and exhibit the viscoelastic behavior typified by hyaline articular cartilage. They are also more collagenous than their fluid counterparts. Synovial fluid has no collagen, the vitreous has only small amounts of type 11 collagen, and the skin of a newborn rabbit has less than 2% collagen; this in contrast to the adult rabbit, which has more than 1.5% collagen.” Figure 45 illustrates these two extremes, the hyaluronic acid gel (A) and compact connective tissue (B) containing collagen and proteoglycans with strong negative charges such as chondroitin sulfate. The physicochemical properties of these two types of connective tissues, their viscoelastic properties, diffusion of macromolecules and of small ions through their midst and exclusion of molecules of various molecular weights (such as immunoglobulins) are, understandably, different Hydrodynamics
and Viscoelasticity
Articular cartilage is characterized by a high degree of hydration and owes its shock-absorbing
(A)
weak
fibers. to
shows
charges
surface. droitin
tendency
on
the
salt
collagen.
cations
other
with
affinity
divalent
cations
compact
structure
hand.
in hyaline
react
with
the
(+ +) or
due
to
by
the
group, the
assocation
the
collagen it can link
to
unstable.
charged
enhanced This
synnovral
of
by the sulfate
with
(e.g..
collagen
positively is
as shown. seen
on
are relatively
generated
linkages This
the
to
surface
such as calcium
groups
bonds
on
charges
gels
no
charged
sulfate,
a glycosaminoglycan
viscous
almost
but these
negative
acid,
forms
present
Via divalent
negatively
tight
Hyaluronic charges
the
cell
(Bj Chonstronger will form
residues presence generates
on of the
cartilage.
capacity to its ability to shift the water molecules from one domain to another. Water is not evenly distributed through the extracellular space,59h rather the upper 25% of articular cartilage contains approximately 85% water, but as the depth increases, the degree of hydration decreases in a linear manner to about 70%. However, these data differ with age, site of biopsy, and patholog:y. It is hypothesized that one of the major forces causing destruction of cartilage in osteoarthritis is hydration, which, through a process of swelling, causes the structure to collapse.597.598 Ultrastructural studies of the cartilage of dogs in which traumatic osteoarthritis is developing showed a disorganization of the perilacunar collagen associated with an increased volume of the tissue.s99 The rupture of the collagenous framework seems to occur as a result of osmotic pressure imbalance within the tissue that is caused by a dilution of the proteoglycan fixed charged density.600 Although most of the water in cartilage is located within the proteoglycan domain, the collagenous network may also play a role in the hemodynamic shift. When cartilagenous tissue is in contact with a saline solution or a physiologic fluid, the proteoglycans within the matrix exert an osmotic pressure r which depends on their concentration,60’ across the interphase. It is this osmotic pressure that is able to counteract an externally applied force and enables cartilage to remain hydrated under load. For cartilage to be in thermodynamic
50
MARCEL E. NIMNI
equilibrium with the synovial fluid, it must be balanced by a combination of tensile stresses exerted by the collagen network of the tissue (PC) and that coming from outside of the network, or applied pressure (Pa): (1) r = Pa + PC, or, expressed in another way, (2) Pa = Ps = ?r - PC, which means that at equilibrium the applied pressure has to be equal to the net swelling pressure of cartilage.600 The osmotic pressure of proteoglycans in solution has been measured at physiologic concentrations and is associated with the excess of ions present as a consequence of the fixed negative charges (FNC) of chondroitin and keratan sulphates. The molecular size or degree of aggregation of proteoglycans does not affect osmotic pressure.60’ Figure 46 shows a network of collagen fibers that contain part of a proteoglycan subunit. It represents resting cartilage where the osmotic pressure of the proteoglycan domain is neutralized by the built-in stresses exerted by the collagen network, which in turn prevents the tissue from swelling. If outside pressure is applied, the collagen pressure will be released as the cartilage begins to decrease in volume. The applied load will then compensate for the decreased contribution by collagen. The graph shows how the swelling pressure of cartilage (Ps) increases as a function of the FCD contributed by the sulfated glycosaminoglycans. The collagen in cartilage consists almost entirely of type II collagen, a collagen with unique characteristics. X-ray diffraction studies of collagen from human intervertebral discs showed differences in the intermolecular spacing of the collagen molecules within the fibers. Whereas the molecules in type I collagen are
Fig. 46.
Collagen
fibers
a proteoglycan
subunit
conditions
collagen
swelling sure
(PSI
is proportional
generated tional
the presure
by the
to their
are shown
within
their
pressure
of cartilage. to
the
fixed
proteoglycans,
concentration
constraining midst.
Under
part
(PC)
is neutralizing
The
net
charged which
the
swelling
pres-
density
(FCD)
in turn
in the tissue.MO
of
resting
is propor-
separated by a distance of 14 A, collagen type II molecules are separated by a distance of 16-17 A. No differences were noted after the specimens were dried.602 The intermolecular volume of fully hydrated collaged fibrils from a number of mineralized and nonmineralized tissues of adult rats has been determined by an exclusion technique and by monitoring specific x-ray diffraction parameters.603 Measurements made on wet rat tail tendon show that 55% of the volume of a fibril is occupied by collagen molecules and 45% by water.604 Assuming a cylindrical geometry for the fibers, one can estimate that the minimum measured increase in the lateral separation between type II molecules compared to type I is approximately 2A, which translates into a 30% intrafibrillar volume increase.602 From these estimates it can be calculated that a wet fibril of type II collagen may contain 50%-100% more water than does a fiber of type I collagen that has the same number of molecules. One can easily envision how additional water in type II fibers can be advantageous to tissues such as cartilage and nucleous pulposus, whose main functions are to absorb or distribute compressive loads, in contrast to type I fibers present in tendon and skin, which transmit tension. Such hydrated fibers can act as hydraulic shock absorbers, and in this way contribute to one of the major functions of the type II collagencontaining tissues. The etiology of this increased hydration is not clear, but could result in part from the higher degree of glycosylation of hydroxylysine, a characteristic of this “sugar coated” molecule. Proteoglycans surround the collagen fibers, but never penetrate into the interfibrillar space (Fig. 47).6o5 During the early stages of osteoarthritis, cartilage exhibits a loss of metachromasia due to a diminution of its glycosaminoglycan content.606 In vitro studies on the wear and tear of articular cartilage showed that removal of divalent cations (primarily calcium and, to a lesser extent, magnesium) by EDTA caused the tissue to exhibit a faster rate of wear.6o7 The cation binding properties of cartilage proteoglycans, particularly to calcium, have been long recognized6’* and it is possible that the loss of glycosaminoglycans seen in osteoarthritis and the decreased resistance to wear of affected cartilage may be associated with the loss of intermolecular stabilizing cations, such as calcium.
COLLAGEN
STRUCTURE
AND
METABOLISM
Figure 47 summarizes in a schematic fashion the mechanism that may further contribute to the changes in hydrostatic pressure within the various cartilage domains, and includes the contribution of intrafibrillar water to the overall picture. Role of Mechanical Factors in Determining the Organization of Collagen and Proteoglycans The normal mechanical stresses that act upon the connective tissues seem to be essential for maintenance of the cellular activities required for the synthesis, turnover, and organization of macromolecules of the extracellular matrix (Fig. 48). Reduction of such stresses by inactivity or immobilization results in loss of muscle mass (atrophy of disuse), bone mass (osteoporosis), and cartilage. The effects of immobility on the periarticular connective tissues is more difficult to recognize but just as significant, as it often leads to contracture, which is the principal complication of fracture treatment using cast immobilization. The largest change found in the composition of the stress-deprived periarticular connective tissues is reduction in the concentration of glycosaminoglycans and water.609 Consistent with such changes is the observation that the immobilized connective tissues show significant changes in the collagen crosslinking profile.6’0 The increase in sodium borohydridereducible intermolecular crosslinks must reflect
Fig. 47.
Water
and small
gen-proteoglycan among
the
various
pressure.
In this
domains
are shown
movement pressure
network
changes.
domains
diagram
of water
ions of
the
as distinct molecules
move
through
cartilage in
and
response
proteoglycan while
to
changes and
the arrows
shifting
the collaredistribute in
collagen
depict
to compensate
the for
Fig. 48.
Healing
ligaments
in rabbits.
(Courtesy
of Drs. W.
of surgically (A)
repaired
Mobilized
Akeson
joint.
and S. Woo.
anterior IB)
cruciate
Immobilized.
and D. Amiel).
the rapid turnover of the immobilized tissue. Hydroxylysinonorleucine and histidinohydroxymerodismosine are the major crosslinks contributing to this increase. This remodeling of the periarticular connective tissues, illustrated by an increased abundance of newly synthesized unorganized collagen, leads to a mechanically less stable ligament that is more susceptible to mechanical damage. On the other hand, the larger number of crosslinks could cause stiffness and are probably responsible for the development, of contractures. Administration of 17 fl-estradiol appears to exert a protective effect against such contractures by minimizing the loss of water and glycosaminoglycans from such tissues.“’ Cartilage tissue responds in a similar way. Immobilization by casting the knee of a normal dog results in rapid degeneration of its articular surface.6’2 This defect is accompanied by decreases in thickness of the cartilage, uranic acid and proteoglycan content, and by a defective aggregation of the newly synthesized proteoglycans. When the period of immobilization is less
52
MARCEL E. NIMNI
than six weeks, the defects are promptly reversed by ad libitum ambulation. On the other hand, vigorous exercise inhibits reversal of the atrophic changes induced by immobilization.6’3 Changes in knee cartilage after amputation of the ipsilateral paw resemble those produced by immobilization.6’4 From the examples cited, involving immobilization of ligaments and cartilage, the potential for additional research to lead to a greater understanding of the way in which mechanical factors affect the metabolism and structure of connective tissues becomes obvious.
FIBROSIS
Accumulation of collagen in excessive amounts is a major pathologic event that underlies several clinical conditions, including pulmonary fibrosis, liver cirrhosis, retrocorneal fibrous membrane formation, as well as various forms of dermal fibrosis, such as scleroderma, keloids, and familial cutaneous hypertrophic scars, collagenoma. Although in many of these diseases the terminal fibrotic lesion is considered to be the sequela of cellular injury, the cell populations injured and the endogenous mediators responsible for the postinjury fibrotic response vary from organ to organ. In many instances we seem to be dealing with an uncontrolled repair mechanism, where less organized and less specific connective tissue replaces a previously functional and carefully constructed matrix. In other instances we see an imbalance in the homeostasis of the extracellular matrix, where synthesis of macromolecules exceeds breakdown, the end result being an excessive accumulation of collagen. In order to understand some of the general features of fibrosis, we shall look at a few of the better investigated fibrotic diseases and experimental animal models.
Fibrosis and Cirrhosis
of the Liver
Although hepatic fibrosis is only one distinctive feature of liver cirrhosis, understanding of its pathophysiology has progressed rapidly and current efforts are directed at unveiling the mechanisms that connect cell injury with collagen deposition and at developing methods for early detection and designing new forms of thera615.616
PY.
In human liver disease and in animal models with liver fibrosis, proline has been found to increase in the liver. Although normal liver is capable of producing proline from glutamic acid, the amount synthesized is small. In Ccl,induced liver fibrosi?” or after ethanol administration6’* the formation of proline from glutamic acid is increased. In rats treated with Ccl,, inhibition of proline oxidase, the mitochondrial enzyme involved in proline degradation, contributes to the elevation of free proline.6’9 Proline concentration is critical for collagen biosynthesis and its incorporation into liver collagen in vitro is directly proportional to its concentration in the incubation medium.6’5 Normal human liver contains 5.5 mg of collagen per gram of fresh tissue6*‘; in rat liver the amount is much smaller (0.91 -t 0.15 mg/g).6’9 However, both human and rat liver contains the same types of collagen. In human liver, 33% is type I collagen, 33% is type III collagen, and the remainder is a mixture of type IV and type V collagens.h20 In a rat model of reversible Ccl,-induced liver fibrosis in the ratio of type I/III collagens remained constant throughout all stages of the disease.“2’ In human cirrhotic liver containing less than 20 mg of collagen per gram of fresh tissue, the ratio of type I/III collagen was also normal.620 However, in another study, higher concentrations of type III collagen were found in normal human liver (47%) and the ratio of type I/III was elevated in cirrhotic liver.622 A similar increase in type I collagen was observed in advanced cirrhotic liver containing more than 20 mg of collagen per gram of tissue.620 The distribution of liver collagens has been examined with the use of monospecific fluorescent-labeled collagen antibodies.623m625 The heavy collagen bundles of the liver correspond to type I collagen, the fibrous septa, the projections of the septa and the perivascular areas stain strongly with anti-type 1 collagen antibodies. Type III collagen corresponds to some but not all of the reticulin fibers. Antibodies directed against the procollagen terminal peptides also localize on liver type III collagen. Type IV collagen has been localized in sites containing basement membranes623 and type V collagen in the portal triads and the terminal venule areas. A marked increase of type 111 collagen within the parenchyma is observed in
53
COLLAGEN STRUCTURE AND METABOLISM
iver fibrosis. Septum formation is due to collapse of necrotizing hepatocytes as well as collagen The final cirrhotic stage is charleosynthesis. ,Icterized by thick collagen fiber bundles derived Yom type I collagen molecules. A distinct pattern of collagen neosynthesis is seen in liver fibrosis that is characterized first by ‘3asement membrane collagen deposition, second ‘3~ type 111 collagen deposition, and third by type I collagen deposition.
LIVER
CONTRACTION
One of the striking features of terminal cases of liver cirrhosis is the decrease in liver size. Since collagen per se has no contractile capacity, cellular elements that possess a contractile system may play a role in liver shrinkage. In this connection it has been shown that human626 and animalh” livers with fibrosis contain myofibroblasts that can be grown from explants of fibrotic tissue.h2s The presence of contractile myofibroblasts, and their positive response to drugs, such as herotonin and epinephrine,627 suggest that these cells are responsible for scar contraction and liver shrinkage. The increase within cirrhotic liver of all collagen types, with predominance of type I collagen, further supports the notion that the process of scar formation in liver is similar to that in skin and other tissues.
DIAGNOSIS
AND
TREATMENT
OF LIVER
FIBROSIS
Some novel approaches that may aid in the early diagnosis of liver fibrogenesis have been recently reviewed.h’5.628,629These include assay of the specific enzymes involved in posttranslational modification of collagen, and metabolites used in the synthesis of collagen and degradation products, including the procollagen peptides specific for the various collagen types. The problem with these assays is that they are not tissuespecific and the values obtained usually reflect changes in the overall metabolism of tissue collagen. Regarding treatment, two compounds with .mtifibrogenic activity, but which act by difrerent mechanisms, are proving to be of value. These are penicillamine for the treatment of 3rimary biliary cirrhosis6i0 and of colchicine in
other types of cirrhosis.6’5 The results obtained seem to warrant trolled use of these compounds. PULMONARY
encouraging further con-
FIBROSIS
Collagen is a major component of lung tissue and its concentration and distribution is affected by a number of adverse systemic and environmental factors. Pulmonary fibrosis is frequently the end result of such events. Regardless of the etiology, these diseases are usually characterized by a progressive loss of lung volume and functioning alveolar-capillary units. In order to understand the factors that may modulate the bisynthesis of collagen and lead to fibrosis of the lung, we shall discuss two situations that have received particular attention. These are idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis. 1PF is grouped with the fibrotic lung disorders because patients with this disease have characteristic restrictive physiologic findings as well as increased fibrous tissue within the alveolar interstitium.63’ By electron microscopy, one can visualize at least three morphologic forms of collagen in the normal alveolar interstitium632: (1) most prominent are the parallel arrays of 500~1,000 Athick, crossbanded fibers; (2) the randomly arrayed 160-350 A-thick fibrils, which differ from the 1OO- 150 A-thick microfibrillar component of elastic fibers (the former are larger, randomly arrayed, and show crossbanding, whereas the latter are smaller, usually found on the periphery of amorphous elastin, and are not crossbanded); (3) basement-membrane collagen, which usually appears amorphous or finely lilamentous. In the normal adult lung, more than 90% of the collagen is type I and IIIh3* normally found in a ratio of approximately 1.5-2.5 to 1.“’ Morphologic evaluation of the connective tissue in IPF has demonstrated thickening of alveolar interstitium with fibrous tissue, as well as basement membrane disruption and thickening. On the basis of collagen type fluorescence. two distinctive patterns of fibrosis were recognized while observing a variety of lung diseases.h34 Areas of mature collagen surrounding vessels and bronchi and in established scar tissue, for example in asbestotic pleural plaques, are ,virtually exclusively type I collagen. By contrast,
54
areas of early active fibrosis like sarcoid nodules and organizing pneumonia, which usually contained variable numbers of fibroblasts and chronic inflammatory cells, are characterized by an increased proportion of type III collagen and a greater intensity of both types I and III collagen fluorescence.634 It has been suggested that IPF is a disease in which there is a shift in ratio of interstitial collagens toward more type I collagen; in IPF, type I collagen represents approximately 80% of the lung collagen.633 Such a shift characterized by an increase in the thick, cross-banded fibers is seen on light and electron microscopy. Because type I collagen fibers are less elastic than those of other collagen types, the parenchyma would be less compliant compared with that of normal lung. Thus, a shift in collagen types without a change in total collagen content would be consistent with the morphologic and physiologic alterations found in IPF. In agreement with this, there was no significant differences in the collagen content between patients with IPF compared with that of control subjects.635 In addition, there was no correlation between collagen content and the morphologic assessment of the degree of fibrosis. Furthermore, the rates of collagen and noncollagen protein synthesis in explants of lung from patients with IPF demonstrated no significant difference compared with those of the control subjects. The results of this study are consistent with the concept that IPF is a disease of an alteration in quality, form, and location of collagen rather than simply a disease of increased interstitial collagen. Another study636 suggests that patients with IPF have active collagenase activity in their lower respiratory tract, thus providing a mechanism by which the initial disordering of interstitial collagen might occur. Thus, even though the rate of collagen production by lung cells may be normal in IPF, if cellular order is disrupted, as is the case in this disease, one would expect deposition of collagen in abnormal areas, and the morphologic finding of focal accumulations of collagen seen in IPF. An interesting experimental model that may also help us to understand the development of fibrosis relies on the use of bleomycin. This drug causes pulmonary fibrosis as an undesirable side effect,637 and has been used to produce fibrosis
MARCEL E. NIMNI
experimentally in mice,638 rats,639*640 hamsters 64’3642 and baboons.643 The baboons display all the physiologic features of interstitial lung disease, including edema and infiltration of the alveolar septums by inflammatory cells. The lobar content of collagen in the biopsy specimens increased 48% over that of the controls, and content of elastin nearly doubled.643 Following cessation of treatment the excess connective tissue proteins persist despite a resolution of inflammation, and their accumulation due to increased rates of synthesis also continues. The net rate of collagen synthesis in shortterm lung explant cultures from rats examined after administration of a single intratracheal dose of bleomycin showed that after a delay of three to four days, the net rate of collagen synthesis was significantly increased over that in untreated control animals.640 This increase was sustained during approximately 10 days and subsequently returned to normal or below normal values. The increased rate of collagen synthesis could not be explained by increased free radioactive proline pools or by age differences among the animals. The period of increased rate of collagen synthesis preceded the detectable increases in lung collagen content. In hamsters, collagen accumulation is also increased as a result of the enhanced rates of collagen synthesis. When lung fibroblasts are cultured in the presence of conditioned media obtained from explants of normal and bleomytin-exposed hamsters, it was observed that the conditioned medium decreased fibroblast proliferation and collagen production in a dose-dependent manner.642 This observation may help to explain why, after a period of enhanced collagen production, the synthesis of this protein is restored to normal in the experimental model. Exogenous PGE, and c-AMP also suppressed fibroblast proliferation and collagen production. The suppressive activity factor present in lungconditioned medium has an apparent molecular weight of 15,000-20,000 daltons, is heat stable, and may represent a means for limiting collagen accumulation following injury. To evaluate how tissue fibroblasts control collagen production, HFL-1, a diploid human lung cell strain, was studied during periods of rapid cell growth and relatively slow growth over 25 population doublings.644 Although the specific activity of intracellular free proline and the per-
55
COLLAGEN STRUCTURE AND METABOLISM
centage hydroxylation of proline in collagen varied considerably depending on the growth rate of the cells, collagen production by HFL-1 was constant, even during periods of rapid cell growth. Thus, under conditions of a stable environment, populations of soft tissue fibroblasts rigidly control collagen production, probably by internally modulating their levels of procollagen mRNA.645 This intrinsic homeostasis is probably disturbed by locally produced or systemic factors that arise as a result of the various forms of injury that are able to induce lung fibrosis. Further understanding of the control mechanisms involved should increase our ability to prevent, arrest, and reverse this damage. KELOIDS
AND
HYPERTROPHIC
SCARS
Keloids can be defined as abnormal scars that extend beyond the confines of the original wound and rarely regress, while hypertrophic scars are raised sears within the confines of the original wound and frequently regress spontaneously.646 Whereas hypertropic scars develop soon after keloids may not appear for many surgery, months. Their distribution, relationship to the degree of injury, and response to motion and to surgery also differ significantly.647,648 Both these entities are characterized by excessive deposition of collagen in healed skin wounds,649 and underlying abnormalities in both the synthesis and degradation of collagen have been suggested in their pathogenesis. The rates iof collagen synthesis, the level of proline hydroxylase6” and several other enzymes, as well as the number of fibroblastP’ are all increased in keioids. The growth kinetics for fibroblasts derived from normal skin, normal scar and keloid are similar; however, the rate of collagen synthesis per fibroblast is greater for the keloid derived cells6” The reason for this difference is not known although several hypotheses have been advanced.h52 The levels of collagenase in keloids have been reported to be increased,653 decreased,654 and unchanged655 when compared to normal skin.655 It has long been known that scars maintain a higher metabolic activity and rate of collagen turnover than the adjacent normal tissue6j6 and, in spite of variable levels of collagenase, collagen may not be removed because of the presence of inhibitors to this enzyme, such as cY-globulins, which are deposited more heavily in keloids and
hypertrophic scars than in normal scars or skin.6s7 There seem to be no significant differences in the ratio of types I and 111 collagen produced by fresh biopsies and fibroblasts from keloids compared to normal human skin controls.658 This is surprising since both keloids and hypertrophic scars seem to contain larger amounts of type III collagen than does normal skin6s9 and also exhibit changes in lysine and hydroxylysine-derived crosslinks resembling those seen in experimentally induced wounds. The fact that under culture conditions the fibroblasts derived from keloids do not differ from normal in the types of collagen produced may reflect the presence in vivo of local factors of a biochemical or mechanical nature that could be responsible for the abnormal cell behavior and induction of the defect. The recently described trifunctional crosslink, pyridinoline, has been reported to be present in hypertrophic scars.66” Since hydroxylysine is a precursor of such a crosslink, its presence is not surprising, and it is possible that such a stable crosslink may contribute to the unusual accumulation of collagen in such tissues. Because of the inhibitory effects of corticosteroids on protein synthesis and of collagen in particular, these agents have been used to treat these conditions. Injection of corticosteroids, particularly triamcinolone, into the lesion will cause keloids to soften and regress in size.h6’ When the effects of triamcinolone acetonide were tested on fibroblasts isolated from normal dermis and keloids, 662 it was observed that the DNA content and cell division of normal fibroblasts was inhibited to a greater extent than that of keloid fibroblasts. On the other hand, the drug enhanced the production of TCA-soluble 14Chydroxyproline in both cell types, suggesting that collagen degradation could be increased by this corticosteroid. FIBROTIC
RESPONSE FOREIGN
TO IMPLANTATION
OF
MATERIALS
Most biomaterials cause a host tissue inflammatory response resulting in collagen synthesis and deposition around the implanted material. In some instances, marked inflammation and collagen synthesis, is desired in order to produce a firm fibrous capsule of collagen, as around the sewing ring of a valve implant to secure it in place. Similarly, the repair of a mandibular
56
defect is best corrected by a biomaterial which not only fills the defect, but also stimulates collagen production to allow a strong fibrous connection between bone and the biomaterial. In other instances, however, minimal collagen deposition is desirable. For example, breast augmentation requires a material with minimal collagen response, thereby producing a soft, natural breast. The granulomatous response that results from the implantation of clay correlates well with the significantly increased relative rates of collagen synthesis. Sand, on the other hand, which causes minimal histologic reactivity, does not increase the relative rate of collagen synthesis compared to control wounds.663 Implantation of soft silicone rubber prostheses is followed by growth of an enclosing connective tissue capsule that may undergo a process of contracture that results in pain, tissue deformity, and extrusion of the prosthesis.664,665 Capsules developing in animals are histologically and ultrastructurally similar to those seen in humans, and also show contracture.666’667 The total collagen content of experimental capsules implanted in rats reaches a plateau with little change after 60 days.668 When fibrous tissue capsules around siliconegel and saline-filled breast implants were examined by light, transmission, and scanning elecregularly arranged dense tron microscopy, connective tissue capsules were seen.667 This tissue contains bundles of collagenous fibers that are densely packed and lie parallel to each other, forming structures of great tensile strength. The outer surface of the connective tissue capsule contains reticulum fibers, small in diameter, that branch to form a net-like framework which supports the collagenous material. At the inner, surface fibrocytes and histiocytes are present in single layers and form an epithelial-like structure. The connective tissue of the capsules also contains capillaries and fibrocytes, which are usually deployed along bundles of collagen fibers and appear as fusiform elements with long processes. Contractile fibroblasts (myofibroblasts) are also found in these fibrous capsules. However, deformation of the augmented breast does not seem to be caused by the action of the myofibroblasts, because their number is too small. Rather it is more likely caused by the inelastic arrangement of large amounts of collag-
MARCEL E. NIMNI
enous material layered around the capsule. Contracting capsules seem to present elevated levels of total glycosaminoglycans and chondroitin-4sulphate compared to noncontracted capsules.668 There is evidence that the ultrastructure of contracted capsules is similar to that of hypertrophic scars.665,667-669The kinetics of collagen deposition and of proteoglycan accumulation in fibrous capsules surrounding implants are similar to those of normal wound healing. DEGENERATION INTERVERTEBRAL
OF THE DISC
The intervertebral disc consists of three parts: the annulus fibrosus, the cartilage plates above and below, and the nucleus pulposus in the center. During adult life the intervertebral disc undergoes a series of progressive morphologic and physicochemical changes, that begin in the teens and become significant in the fourth and fifth decades of life.67o,67’ The two major components responsible for these changes are the annulus fibrosus and the nucleus pulposus. The annulus in the adult lumbar spine is formed by a series of concentric encircling lamellae, which are thicker anteriorly than posteriorly. The peripheral lamellae attach themselves to the bony edge of the vertebral body in the way of Sharpey fibers, while the remaining lamellae continue into the cartilagenous plates. The nucleus pulposus, centrally located, consists of a three-dimensional network of collagen fibrils enmeshed in proteoglycan gel. It occupies about 30%-50% of the disc cross-sectional area of the disc. It contains few cells resembling chondrocytes. The water content of the disc is highest at birth, being around 90% in the nucleous and 78% in the annulus; with age, these values decrease to about 70% in both areas. This change in hydration is coupled to changes in the chemical composition and mechanical properties. After childhood the nucleus is no longer sharply demarcated from the annulus, and the collagen fibers become gradually coarser and merge.672 The proportion of mucopolysaccharides decreases and with age changes its nature, the ratio of keratan sulphate to chondroitin sulfate becoming increased.673 In mature rabbits the administration of androgens, estrogens, and growth hormone is able to reverse these changes.674 The proteoglycans become smaller in molecular size and form fewer aggregates with
57
COLLAGEN STRUCTURE AND METABOLISM
hyaluronic acid.675 These changes seem to impair the ability of the nucleus to resist and redistribute loads, and the greater stresses imposed on the annulus fibrosus can lead to its eventual mechanical failure. Early studies showed that the collagen in the intervertebral disc had a higher content of hydroxylysine than was expected from the total collagen content.676 The collagen is not distributed homogenously in this tissue, as the outer regions of the annulus contain the largest amount of collagen.677 The collagen is heterogeneous and consists of a mixture of type I and II collagen (Fig. 49).678 Serial section from the outside towards the center of the nucleus pulposus reveals variable ratios of type I to type II collagen. The outer fibers in the pig are exclusively type I collagen and the inner mostly (95%) type II collagen. In the human these numbers are somewhat different since the annulus contains consistently larger proportions of type II collagen.679 As in all other connective tissues, the mechanical strength of this structure is related to the degree of crosslinking between the individual
TYPE
Fig.
49.
Distribution
disc. The outer collagen with
while
varying
(adapted
from
part
of collagen
of the annulus
the inner proportions
nucleus in
the data of Eyre
in the
fibrosus pulposus
the et al.).
I
intervertebral is mostly
is mostly
intermediate
type type zones
I II
collagen molecules, and the diameter and distribution of the fibers. Marked differences are observed in the reducible crosslinks of discs progressing down in young lumbar spines. 680The content of reducible crosslinks declines between Ll-L2 and L5-Sl in both nucleus and annulus collagens. Also, when spines with a grossly degenerated disc were studied, the disc above the degenerated one, but not the degenerated one itself, was rich in reducible crosslinks, normally absent from discs at that age. This was interpreted as a sign of recent synthesis of new collagen, suggesting that the normal disc had compensated in some way for the defective disc below. The new collagen was predominantly type I in both annulus and nucleus, based on an enrichment of hydroxylysinonorleucine and histidino-hydroxymerodesmosine, and an analysis of LYchains labeled radioisotopically during incubation of disc tissue in vitro.680 The relative contributions of types I and II collagens of the normal annulus to the crosslinking profiles are difficult to assess, though the profiles loosely fit a combination of those for type 1 collagen of insoluble tendon and type II of hyaline cartilage. The identification of elastic fibers within, the disc6*’ raises the possibility that such tissue makes a contribution to disc elastic function, however, the proportion present is not high. Immature elastic fibers, similar to those seen in other developing tissues, have been observe:d in the human fetal disc.6”2 The presence of elastic fibers within the annulus fibrosus could be of importance in supporting collagen fiber recovery after deformation, as has been found in other tissues. There is some evidence to suggest that both the collagen and glycosaminoglycans of some connective tissues are abnormal in scoliosis.6x’ The stability of the polymeric collagen of the skin from adolescents with idiopathic scoliosis was found to be significantly less than that of control subjects, except for two patients with mature skeletons, in whom the polymeric collagen was normal. One of three patients with congenital scoliosis also had polymeric collagen of low stability. In an attempt to further resolve these questions, the collagen extractability and distribution across normal and scoliotic discs was investigated.684v685 The collagen content of the scoliotic nucleus pulposus was found to be higher
58
MARCEL E. NIMNI
than normal, particularly at the apex of the curve, but no consistent correlation was found with the spinal mobility or degree of curvature.685 The collagen content and type of the annulus fibrosus from scoliotic discs was shown to be abnormally distributed, again only in those discs encompassed by the curve.6843685These results contrast with the pepsin extractability of collagen in the annulus fibrosus, which was abnormal in all the scoliotic discs examined, independent of location. While the precise interpretation of these latter findings is complex, it would seem that a subtle defect in collagen does exist within the scoliotic disc which, coupled with extraspinal influences, may play an important role in progression of the scoliotic curve. Attempts have been made to alter the properties of these discs by infiltration with proteolytic enzymes that will remove collagen and proteoglycans, but results are speculative.686 Degenerating discs, particularly in the elderly, tend to calcify687 as mineral is deposited in the annulus and in the perilacunar regions of the end plates. Other conditions that can lead to degeneration and calcification have been discussed.688 Because of its physiologic significance, the clearly age-related chemical changes observed, the unusual distribution of collagen types relative to the shape and function of this tissue, and the clinical implications associated with the high degree of dissability associated with the degeneration of the intervertebral disc, this structure deserves to be further studied.
SCLERODERMA (PROGRESSIVE SYSTEMIC SCLEROSIS)
Progressive systemic sclerosis is considered to be one of the most classical collagen diseases. It is characterized by extensive fibrotic infiltration of many organs with a concomitant accumulation of collagen. Most commonly involved is the skin, but other internal organs such as the lung, heart, kidney, and gastrointestinal tract are frequently affected. Two stages are evident in the development of this disease: a cellular stage characterized by perivascular or diffuse cellular infiltration of the dermis and subcutaneous tissues and a fibrotic stage that is associated with deposition of collagen. Most determinations of collagen in scleroderma have been done on skin, where it was observed that the most characteris-
tic feature consisted of replacement of subcutaneous tissue by dense collagen fibers.689*69a The etiology of the disease is not known although several recent reviews and symposia have addressed themselves to this question.69’*692 Here we shall briefly address the question of collagen deposition in an attempt to relate it to other forms of fibrosis. In general, fibroblasts from the skin of patients with scleroderma seem to produce more collagen in tissue culture than do their normal counterparts, although again this is not a universal finding.69’ The serum from scleroderma patients may enhance this effect, particularly if the fibroblasts are derived from the reticular dermis.693 As discussed earlier, normal skin contains primarily type I collagen (approximately 80%) and type III collagen (approximately 20%). Deviations from this ratio in skin of patients with scleroderma have been found by many investigators who studied this disease,69’ and such findings seem to correlate with the state of activity of the tissue. In general, the findings seem to parallel those of wound healing and other situations where there is a sudden burst of collagen synthesis. It appears that in the early stages of the disease there is an increased synthesis of type III collagen over that of type I, but this ratio reverts to normal when the disease activity subsides and the ultimately formed fibrotic tissue contains normal proportions of type I and III collagens. By immunofluorescent microscopy using specific antibodies against types I and III collagen, 694it was observed that in the cellular stage the dermis and adipose tissue contained perivascular or diffuse cellular infiltrates accompanied by enhanced deposition of type III collagen. The lower dermis also showed an increase in type III collagen. In the fibrotic stage the papillary layer shows a reduction and clumping of type III collagen and the lower dermis show compact collagen, mostly of the type I type. Although these observations are not quantitative, they reflect the general pattern of fibrosis that begins with enhanced production of type III collagen and leads to an ultimately normal proportion of type III (approximately 20%). These observations are confirmed by chemical analysis of scleroderma lungs, which show types 1 and III collagens to be present in the same proportions as in immunormal 1ung.695 Using the enzyme-linked nosorbent assay method antibodies to types I and
59
COLLAGEN STRUCTURE AND METABOLISM
IV collagens were detected in the sera of patients with scleroderma.696 Antibodies of type IV CO]Iagen are present in significant amounts and correlated with the presence of abnormal pulmonary diffusion capacity, so did antibodies to type 1 collagen, suggesting that autoantibodies to basement membrane and interstitial collagens may participate in the pathogenesis of this disease. Further support for the involvement of an immunologic component in this disease is provided by the fact that lymphocytes from scleroderma patients, when compared to those from normal donors were found to produce soluble factors that induce fibroblasts to increase synthesis of collagen without enhancing cell proliferation.697 The role of other cell types (platelets, endotheha1 cells) and their metabolic products,69’ collagen or procollagen degradation products,698 as well as fibronectin699 and other connective tissue macromolecules on the control of collagen synthesis and deposition remains a subject of interest. DlJPUYl-REN
CONTRACTURE
AND
MYOFIBROBLASTS
This is a disease that results in flexion contracture of one or more fingers. The process is not limited to the fingers, however, since it can also involve the soles of the feet and penis. The changes are usually progressive and irreversible and are associated with characteristic changes in the longitudinal fasciculi of the palmar aponeurosis. Histologic observation reveals a proliferation of connective tissue cells leading to the formation of characteristic nodules and contracted longitudinal bands of collagen. The nodules appear in the early phases of the disease and the collagen deposition reflects the more advanced stages.“’ For many years it was assumed that the contractile effect was associated with the intrinsic ability of the collagen fibers to shrink when (denatured, but this concept cannot be substantiated. A more feasible hypothesis arose when it was observed that many of the cells present in the involved area had characteristics similar to smooth muscle cells and exhibited a contractile apparatus: such cells have been termed myofii~rob1asts.70’~702The fibrillar system that develops
in these cells consists of parallel bundles of fibers, averaging 60 A in diameter, usually oriented along the long axis of the cell. Strips of granulation tissue containing such cells respond to a variety of pharmacologic agents in a manner similar to smooth muscle cells.“* It recently has been shown that fibroblasts exert forces larger than those needed for locomotion and that this excess traction may function in the rearrangement of extracellular matrices.‘03 Fibroblasts in vitro are capable of contracting hydrated threedimensional collagen gels into tissue-like structures, the extent of contraction varying directly with the cell concentration and inversely with the collagen concentration.704 Recent observations have been made which support the view that the fibroblast can modulate between the contractile and “normal” phenotype. Periodontal ligament fibroblasts contracting collagen gels can change their phenotype in accordance with the stages of gel compaction.705 In the early and active phase of gel compaction, the fibroblasts exhibited numerous cell processes containing microfilamentous material and few organelles, little RER and few Golgi membranes, but many free ribosomes, numerous structures resembling gap junctions, and a rich cell coat. Fibroblasts changed to a more rounded, relaxed state after 24 hr when the gel was compacted. At this stage, extensive RER and Golgi membranes, small peripheral microfilament bundles, little cell coat, and few, if any, gap junctions were observed. On the other hand, fibroblasts plated on plastic exhibited prominent RER and Golgi membranes, peripheral microfilament bundles, and junctions of the adherent type. The results suggest that the myofibroblasts could represent a reversible phenotypic modulation of fibroblasttype cells to a contractile state. When compared to age-matched control aponeurosis, lesions of Dupuytren disease contain larger amounts of water, collagen, and chondroitin-sulphate, as well as increased proportions of soluble collagen and reducible cross links.7”h The lesions show also increased amounts of type III collagen and an increased hydroxylation and glycosylation of the reducible crosslinks. All these parameters are characteristic of granulation and scar tissues. Type 111 collagen was located by immunofluorescence on thin argyrophilic fibers and within large fiber bundles that appeared to be disrupted into microbundles. The
60
MARCEL E. NlMNl
increase in type III collagen and the presence of myofibroblasts in the apparently unaffected aponeurosis suggest that the disease could be initiated within the aponeurosis and propagated by cells migrating along the collagen bundles. In general, the chemical changes observed reflect a situation of rapid synthesis and turnover of collagen since, compared to normal tissues, the involved fascia shows elevated levels of hydroxylysine, reducible aldimine intermolecular crosslinks and the presence of hydroxylysinohydroxynorleucine, virtually absent from normal adult palmar fascia.“’ The nature of the disturbance that leads to the proliferation of myofibroblasts and to the accumulation of abundant connective tissue of an immature nature is not known. The disease process can progress only as long as there are nodules present which have not yet undergone complete resolution. The nodules completely disappear, marking an end to the involutional (contraction) stage and the establishment of the residual stage (final stage) in which no further progression occurs. But the contractures previously incurred during the involutional stage, if not treated surgically persist without alterpostoperation ation.“’ Frequency of recurrence depends upon the stage of the disease at the time the operation is performed. Operations performed during the residual stages rarely recur. In a late involutional stage, complete excision of the nodules is followed by recurrence in 20% or less. In the proliferative early formative stage of the nodules, the recurrence rate is higher and especially so if parts of the nodules are not resected. CORNEAL
ical and thermal burns, and cornea1 graft failure. Diverse terms are used to describe cornea1 fibrosis, the most common of which are retrocorneal fibrous membrane (RCFM),“* or posterior collagenous layer (PCL).‘09 This abnormal tissue appears between Descemet membrane and the cornea1 endothelium. Histopathologic studies show a multifibrillar layer posterior to the normal Descemet membrane, the thickness varying in different regions of the cornea. Ultrastructural studies show that the RCFM is composed of fibrils, ground substances and cells (Fig. 50). The cells have a characteristic fibroblastic morphology in contrast to the polygonal shape of cornea1 endothelial cells. The origin of the fibroblast-like cells in RFCM remains controversial. While they could be derived from stromal fibroblasts7” or from transformed cornea1 endothelial cells, 7’1m7’3the endothelium is the most likely source, as substantiated in numerous experimental models. In culture, fibroblastic cells isolated from
FIBROSIS
Disease and damage of the cornea1 endothebum and its basement membrane (Descemet membrane) frequently cause loss of function of the cornea1 endothelium, cornea1 edema, and progressive cornea1 opacity and scarring (cornea1 fibrosis). Such fibrosis is a common final outcome for a number of cornea1 diseases and is a leading cause of blindness. The various ocular and cornea1 disorders that have been associated with such cornea1 fibrosis include Peter anomaly, congenital hereditary cornea1 dystrophy, hereditary cornea1 edema, macular dystrophy, posterior polymorphous dystrophy, Fuch dystrophy, interstitial keratitis, herpes simplex keratouveitis, postoperative retrocorneal membrane, chem-
Electron micrograph showing the changes Fig. 60. associated with the development of a retrocorneal fibrous membrane (RCFM). Cornea1 stroma (St) Descemets’ men brane (Dm) fibrocyte (f) and endothelial cell (Endo).
61
COLLAGEN STRUCTURE AND METABOLISM
RCFM synthesize predominantly type I procollagen and secrete it into the medium. The CO!lagen molecules extracted from the cellular fraction and RCFM in organ culture demonstrate a-size chains, the final product of processing. These findings suggest that procollagen peptidase activities are associated with cell surfaces and/or the extracellular matrix associated cell surface. The stoichiometry of type I collagen shows two CY,and one LY*chains, indicating that type 1 trimer is not present on this tissue. On the other hand, the major collagenous peptide synthesized by rabbit cornea! endothelial cells is basement membrane collagen.7’437’5 However, type ITT collagen is synthesized by bovine709 but not by rabbit cornea! endothelial cells. Other components of the RCFM such as proteoglycans and glycoproteins, have not yet been studied. Little is known about how transformation of one cell type to another occurs. It has been suggested that endothelial cells are pluripotent and become either epithelial-like7” or fibroblastextracts from leukocytes like; 7”x~7”9lysosomal have been reported to induce endothelium to acquire an epithelial-like pattern.7’” However, little is known about what causes such transformation. When injury is caused to the cornea! endothelium, either directly or indirectly, polymorphonuclear leukocytes migrate to the wound (Fig. 5 I ). Under such conditions, collagenase released by the leukocytes is activated and damages the exposed Descemet membrane. Leukocytic lysosomal enzymes can also cause cell damage.
~nwation wn byr d DM,
GWMOI ibrolb (RCFM)
TmrfmnDd ndomllum
Fig. 51. Schematic diagram showing the sequence of events following the application of a frozen probe to the cornea of a rabbit which leads to retrocarneal fibrosis.
Therefore, the cells participating in the repair process are faced with an abnormal extracellular matrix (which may negatively modulate their behavior) and may suffer damage. Consequently, the endothelial cells could redifferentiate into fibroblast-like cells which then deposit an abnormal extracellular matrix and generate a barrier to light transmittance.7’7 The scar remains permanently in situ and the regenerating endothelium may resume production of a physiologic collagen type. For more detailed information on RCFM, the review by Waring et al. 709is recommended. NERVE REGENERATION AND SCAR: FORMATION
The factors that modulate growth and regeneration of nervous tissue are part of a most interesting biologic and clinical problem. Studies done both in vivo and in vitro have shed significant light on the active role played by collagen in enhancing or inhibiting this process. The classic observations by Key and Retzius7” ancl Ranvier719 have laid the foundation for subsequent studies on the microscopic anatomy of the peripheral nerves. The former suggested the presence of an epineurium, perineuriurn, and endoneurium, which correspond to the perifascicular tissue, the lamellated sheath and the intrafascicular tissue described by Ranvier.‘19 Electron microscopists have examined the nature of the connective tissue that surrounds peripheral nerve fibers.720,72’ The Schwann cells of myelinated nerve fibers are ensheathed by a basement membrane that is continuous across the nodes of Ranvier from one internodal segment to the next. These, in turn, are surrounded by a condensation of endoneural collagen fibers. The structural organization of peripheral nerves includes within the perineurium two tissue constituents: the population of nerve fibers and the endoneural connective tissue. The fine structure of cross-section of a peripheral nerve is consistent with this concept since it shows a background of fairly evenly distributed collagen fibrils, running para.llel to the axis of the nerve fibers, the latter appearing to be suspended between them. An entirely different pattern of organization of collagen fibrils becomes apparent when isolated nerve fibers are studied, particularly with the scanning electron microscope.722
62
MARCEL E. NIMNI
Observation of the isolated nerve fibers of rat sciatic nerve show that the majority of the collagen fibers form a tightly woven cuff around individual nerve fibers. A relatively small fraction of the fibrils form an extremely loose, cobweb-like reticulum spun between the individual nerve fibers that holds them within a wide-mesh net. This continuum manifests itself along the entire nerve fiber, running parallel to the axis and often taking a slightly sinusoid curse (Fig. 52). These findings clearly support the preelectron microscope concept of Key and Retzius, generated more than 100 years ago, proposing a distinct membranous sheath around each individual nerve fiber, and are further supported by the work of Thomas, who observed (using the electron microscope) a condensation of collagen around nerve fibers. Following surgical transection of a peripheral nerve, regeneration proceeds from the stump by axonal sprouting and cell proliferation, causing the connection between the proximal and distal stumps to become reestablished. These cells proliferate and migrate to form a union scar.723 Regenerating axons invade the scar and cytologic differentiation towards a Schwann cell
Fig.
52.
Peripheral
old child during fibers
aligned
magnification
nerve
autopsy.
obtained
Showing
from
a three-year-
cross sections
parallel
to the direction
12,500
x; courtesy
of collagen
of the nerve.
of Dr. Mei
Liu.)
(Original
seems to occur from undifferentiated mesenchyma1 cells. The direction and course of the regenerating axon seems to be determined by the primitive mesenchymal cells of the granulation tissue, which exhibit a high degree of affinity and provide mechanical support for the regenerating axon. It has often been postulated that the central nervous system is capable of regeneration, but that its efforts are defeated by other processes, such as mesenchymal scar formation, which by depositing collagen creates a barrier that physically blocks the advance of regenerating axons into and across the wound. Chemical analysis showed that little collagen is present in unwounded spinal cord compared with surrounding mesenchymal membranes.724 Nevertheless, significant collagen synthetic potential was found within the spinal cord, a tissue of neurectodermal origin. The rate of collagen synthesis per unit of collagen content in the unwounded spinal cord is high and is equivalent to that of wounds at their maximum collagen synthetic rates. Wounding the spinal cord, pia mater, and dura mater caused substantial elevations in rates of collagen synthesis and deposition in these tissues. These synthetic rates remained at maximum levels throughout the eight-week study, a prolonged period when compared with other wounded tissues. Prolyl hydroxylase activity is extremely high in unwounded spinal cord, and after wounding a marked accumulation of collagen is seen to occur.724 The cell type responsible for this collagen synthetic activity has not been identified. In order to further understand the interactions between granulation tissue and nerve growth several experimental approaches have been used, one of which involves study of the migration of peripheral nerves through a preformed tissue space.725 For this purpose a tubular channel was created by implanting a silastic rod. When the rod was removed, a pseudosynovial tube was created that was then used to guide the growth of a resected sciatic nerve. Although growth of the nerve occurred within the tubular space it did not seem to be guided by the walls of the tube. In the process of regeneration, a perineural connective tissue structure was formed which was similar to that which surrounds the normal peripheral nerve. This experiment strongly indicates that the regenerating nerve fibers are responsible for
COLLAGEN STRUCTURE AND METABOLISM
the development and maintenance of the structural organization of the perineurium, mediated, at least in part, by the production of neurotropic substances that may play a key role in guiding axonal growth during normal development and regeneration of the peripheral nerves.726,727 The process by which axons regenerate following freeze injury to the optical nerve of the newt has been investigated.728 It seems as if the basal lamina which persists inside the optic sheath provides continuity across the site of injury and orients the axon sprouts in an orderly process of axon regeneration without neuroma formation. Types I and III collagen have been detected in human femoral nerve.729 Observation with the electron microscope shows two different populations of collagen fibers, based on their diameters:“” the collagen fibers in the endo- and perineurium have distinctly smaller diameters than those of the epineurium. These investigators have developed a method that relies on changes in birefringency observed in tissue sections stained with Picrosirius and that allows type I collagen to appear as thick, bright (strongly birefringent) red or yellow fibers, whereas type III collagen shows up as thin, pale (weakly birefringent) greenish fibers. When applied to nerve sections, this method localized collagen type III in the endoneurium and type 1 in the epineurium. Leprous lesions, which are known to induce proliferation of collagen in nerves, do so by enhancing the deposition of type III collagen in the peri- and endoneurium and deposition of type I in the epinerium.‘j’ This polymorphism of collagen, coupled with the different modalities of organization that it can acquire, are probably important in dictating the growth of neurons. The proper orientation and type of collagen present seem to be a must, while massive amounts of dissorganized collagen pose a strong hindrance to regeneration. Collagen, which has frequently been used as a substrate in tissue culture because of its ability to provide favorable conditions for cell adhesion, growth and differentiation, is required for the ensheathment and myelination of nerve fibers by Schwann cells.7’2 The capacity of collagen substrates to bind and grow neurons differs markedly with the method of preparation and the amount of collagen plated per unit area.733 It has been shown recently that human plasma fibronectin, when absorbed to a variety of conven-
63
tional culture substrata, promotes rapid neurite outgrowth from aggregates of embryonic chick neural retina cells.734 A clonal cell line (RN-2) obtained from a chemically-induced rat Schwann cell tumor has been described.735 This cell line produces unusual collagenous polypeptides736 and has been examined as a model for the demyelinization induced by viruses.737 This same cell line synthesizes procollagen of an unknown type and fibronectin.73” In peripheral nerves, fibronectin has been detected in the basement membrane around the Schwann cells, and is especially abundant in the nodes of Ranvier, suggesting that the tibronectin in this area could be produced by Schwann cells. Another interesting approach that has been used in the last few years to culture cells in general and neurons in particular involves the use of hydrogels, a class of polymeric materials that are swollen extensively in water but that do not dissolve in it. Collagen-hydroxyethylmethacrylate hydrogels can be prepared by polymerizing monomeric hydroxyethylmethacrylate in the presence of various concentrations of soluble native collagen.739 The resulting transparent hydrogels have been evaluated as substrata for growth of IMR-90 human embryonic lung fibroblasts. Without collagen, no significant growth occurred, whereas a dose-response curve expressing maximal cell growth against collagen concentration could be constructed quite readily by the use of appropriate hydrogels. Because of the potential of this method to probe into the mechanisms of cell adhesion and differentiation, it has been used to investigate nerve fiber growth.740 Cultured neurons become attached to hydrogels substrates prepared from 2-hydroxyethylmethacrylate but grow few nerve fibers unless fibronectin, collagen, or nerve growth factor is incorporated into the hydrogel. Antibodies to fibronectin inhibit nerve fiber growth on hydrogels containing fibronectin, which suggests that growing neurons interact directly with proteins trapped in the hydrogel. The adhesive requirements for attachment of neurons appear distinct and possibly less specific than those for fiber growth. It therefore seems as if defined hydrogel substrates offer a controlled method for analyzing complex substrates that support nerve fiber growth and neuronal differentiation. Further studies along these lines should lead
64
MARCEL E. NIMNI
us to further understanding of the role the connective tissue macromolecules play in modulating the process of nerve growth under normal and pathologic conditions. PHARMACOLOGY
OF THE FIBROTIC REACTION
A variety of chemicals have been investigated for the purpose of inhibiting fibrosis and accelerating the removal of collagen. The inhibitors of crosslinking, the lathyrogens and penicillamine, have been discussed earlier. Since collagen with lesser crosslinks is more readily degraded by collagenase, it was expected that these agents would facilitate its removal. The potential of /3-aminopropionitrile (BAPN) in the prevention and treatment of wound scar contractures, peritendineous and perineural adhesions, and other pathologic defects resulting from the polymerization of collagenous structures, were recognized several years ago.74’*742Although human trials showed promise, wide clinical use of this drug is lacking, due to toxic side-effects,743*744 the mechanism of which is not clearly understood. In animals this could be associated with the decreased food consumption induced by the drug, either via monoamineoxidase inhibition leading to alterations in the serotonin content of the brain, or via depletion of Zn associated with regulation of acuity of the taste buds. The latter hypothesis of serum Zn seem unlikely,745 since reduction levels seems secondary to an inhibition of protein synthesis by the liver. Zn levels can be restored by administering Zn while reducing the lathyrogenic effects of BAPN without correcting the body weight loss. Because of these toxic effects, the clinical use of BAPN has been limited. In view of this systemic toxicity, the topical route of administration has been explored.746 It was shown that 14C-BAPN can be absorbed readily across the skin of rats, the free base more so than the fumarate salt. The lysyl oxidase activity of a subcutaneously induced granuloma was significantly inhibited by a single small dose of BAPN applied to the intact skin over the implant, and this was accompanied by increased local extractability of collagen. This route of administration may thus prove of value in reducing scar contracture and in decreasing tendon adhesions and joint stiffness while minimizing systemic toxic effects.
Penicillamine, on the other hand, in addition to displaying considerable side effects at the dose required to inhibit collagen crosslinking, generates a reversible effect. Once the drug is discontinued or the dose lowered the aldehydes on collagen blocked by the presence of the drug will become free and normal crosslinks will form. (Fig. 33). So this compound has to be used with this limitation in mind, and may only prove useful in limited situations, such as early or acute stages of scleroderma and transient fibrotic episodes. Its apparent effectiveness in the treatment of alcoholic liver cirrhosis may be associated with this mode of action. As a corollary to the studies on the synthesis and structure of collagen a variety of chemical agents have emerged that have the ability to interfere with many of the biosynthetic steps involved. Many of these have a significant potential for the treatment of fibrosis, many are toxic, and still others are nonspecific for collagen and would interfere with other biosynthetic process. Rojkind, as a result of his interest in the treatment of liver fibrosis, has studied these compounds in a systematic fashion and grouped them into six categories.747.748 A brief description of their mode of action and possible toxic effects follows. Proline Analogues (L-azetidine-2-carboxylic acid, cis-hydroxyproline, cis-fluoro proline, 3,4-Dehydro-DL-proline, cis-Bromoproline) This group includes several compounds that are similar but not identical to proline and that compete with this imino acid for its functions. L-azetidine-2-carboxylic acid competes with proline for transport749 and acylation of tRNA.“O It is incorporated into collagen and is not hydroxylated by collagen prolylhydroxylase. Tendon cells incubated with cis-hydroxylproline can incorporate it into collagen to the extent that it can occupy up to 19% of the imino acid positions. With cis-fluoroproline the degree of replacement colcan reach 27%.“’ The under-hydroxylated lagen chains are unable to form triple helical structures and are not secreted but they are digested inside the cell by nonspecific proteolytic enzymes.749 Even though proline analogues are incorporated efficiently into collagen molecules, they are also incorporated into other proteins and could
COLLAGEN STRUCTURE AND METABOLISM
alter their function. Although these compounds are highly effective in vitro and in experimental studies are animals,748 detailed pharmacologic still lacking, and thus they can not be used for the treatment of human disease. Iron Chelators [cY,a-dipyridyl, 9,9-dihydroxy-7-methylbenzo bromide
quinolizinium
(GPA 1734)/
These have been used in many systems in vitro but are not suitable for use in vivo because of their toxicity. They remove ferrous ion required by the two hydroxylating enzymes, thus inhibiting hydroxylation of prolyl and lysyl residues.752 The nonhydroxylated collagen remains inside the cell where it is degraded. These compounds are used in the preparation of under-hydroxylated collagen, as a substrate for the hydroxylating enzymes. Lathyrogens acetonitrile.
(P-amino propionitrile,
amino
etc.)
These compounds, which inhibit at relatively low concentration the activity of lysyl oxidase and the subsequent formation of intramolecular and intermolecular crosslinks, have been discussed in detail. Aldehyde
Trapping
Cysteamine,
Agents (Penicillamine,
etc.)
At low doses, penicillamine combines with free aidehyde groups in collagen, thus preventing the formation of intra- and intermolecular crosslinks.753 The bond formed, a thiazolidine ring, is unstable and dissociation occurs when the concentration of penicillamine in a given tissues decreases. Upon discontinuing treatment, many aldehyde groups are re-exposed and formation of crosslinks quickly occurs.753 At high doses (toxic for the animals) penicillamine inhibits the activity of Iysyloxidase.488,754 Penicillamine combines very readily with pyridoxal phosphate, thus producing a deficiency of this vitamin: this toxic effect can be avoided by daily administration of vitamin B,. Antimicrotubular Related
Agents
(Colchicine
and
Compounds)
Colchicine inhibits microtubule assembly and the transcellular movement of collagen.755~757 It
induces the production of collagenase in synorelease vium75x and enhances protease-activated decreases colof procollagenase.759 Colchicine lagen deposition and breaking strength of wounds in rats but only at a dose that produces systemic side effects. 760Colchicine has been used in the treatment of human liver fibrosis in a group of severely ill patients. After several weeks of treatment with a dose of 1 mg per day, these patients showed a striking clinical improvement. Long-term treated patients showed normalization of biochemical parameters (serum albumin and proline levels)76’ and side effects were minimal. Nonspecijc
Compounds
Corticosteroids can be included in this group and their effects on collagen metabolism have been described in detail earlier. Another compound that has attracted attention of individuals working in cell culture is the monovalent ionophore monensin, that interferes with a variety of eukaryotic cell functions. Monensin inhibits secretion of macromolecules,762~763 its major effect on secretion appears to be in the Golgi complex, which becomes markedly distended in cells treated by this drug.762-763Secretion of types I and I1 collagen, fibronectin, and cartilage proteoglycan are also inhibited in cultured cells treated with monensin.765m766 These macromolecules also accumulate within the treated Ce,,s.765-767
The macromolecules secreted by monensintreated cells have abnormalities in some of their posttranslational modifications. Chicken embryo chondrocytes treated with monensin secrete undersulfated cartilage proteoglycans.76x Normal intracellular processing of the N-asparagine-linked oligosaccharides on fibronectin”” and other macromolecules76x is also disrupted by monensin. Although obviously nonspecific for collagen, such a drug may help us understand factors involved in the intracellular translocation of collagen and its secretion into the extracellular space. It is obvious that there is a great need to further investigate drugs that will inhibit excessive deposition of collagen, alter the phenotypes expressed by cells at the site of injury, and modulate its synthesis and turnover. Combinations of drug therapy, surgery and physical
66
MARCEL E. NIMNI
manipulation should make it possible to regulate cell behavior in such a way that the biosynthesis of the connective tissue macromolecules proceeds in a normal and controlled fashion. Maintenance of a normal extracellular matrix is essential for the overall homeostasis of the organism.
ACKNOWLEDGMENT
I thank Drs. David Cheung, Paul Benya, E. P. Kay, Ken Nishimoto, and J. Vernon Luck, Sr. for their helpful suggestions and discussions, as well as the many colleagues who provided me with preprints of their unpublished work.
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VOL. XIII, NO. 1
Collagen:
Structure,
Function, Fibrotic
and Metabolism Tissues
in Normal
and
By Marcel E. Nimni
C
OLLAGEN is the single most abundant animal protein in mammals accounting for about 30% of all proteins. The collagen molecules, after being secreted by the cells, assemble into characteristic fibers responsible for the functional integrity of tissues such as bone, cartilage, skin, and tendon (Fig. 1). They contribute a structural framework to other tissues such as blood vessels and most organs. Crosslinks between adjacent molecules are a prerequisite for the collagen fibers to withstand the physical stresses to which they are exposed. Significant progress has been made towards understanding the functional groups on the molecules that are involved in the formation of such crosslinks, their nature, and location. A variety of human conditions, normal and pathologic, involve the ability of tissues to repair and regenerate their collagenous framework. Some of these conditions are characterized by excessive deposition of collagen (e.g., cirrhosis, scleroderma, keloid, pulmonary fibrosis, diabetes, etc.). After trauma or surgery, abnormal deposition of collagen may impair function (adhesions following repair of long tendons, scar formation during healing, etc.). In addition, many disabling conditions result from changes in the nature and organization of collagen (heart-valve lesions, osteoarthritis, rheumatoid arthritis, and congenital collagen diseases such as Marfan’s and Ehlers-Danlos syndromes, osteogenesis imperfecta, etc.). In human tissues there are five different collagen types that have been well characterized. Many others are currently being studied. We know that collagen molecules in cartilage differ from those in cornea, tendon, bone matrix, dermis, the parenchyma of organs, periodontal ligaments, and many other locations within the organism. Why is cornea transparent, tendon tough, inelastic and able to sustain significant stresses, Seminars in Artbntis and Rheumatism. Vol. 13, No. 1 (August), 1983
and cartilage resilient and viscoelastic? What kind of uniqueness is there in the chemical structure of collagen that enables these tissues to perform such diverse biologic functions? What regulates fiber diameter, orientation, concentration of fibers in a particular volume of tissue, and packing of the fibers into larger bundles? How can we understand the problem of aging of connective tissues and changes related to pathology? It now seems as if mesenchymal cells that have acquired the ability to make a particular type of collagen can be influenced by environmental factors to change the rate and nature of the collagen molecules that they synthesize, and that in some instances these changes are reversible. This review will address itself to the various fundamental steps of the anabolism and catabolism of collagen as well as to the unique types of collagen present in skin, tendon, bone, blood vessels, cartilage and basement membranes and will focus on diseases associated with excessive accumulation of collagen, which leads to various forms of fibrosis. THE COLLAGEN MOLECULE
The arrangement of amino acids in the collagen molecule is shown schematically in Fig. 2. From the Departments of Biochemistry. Medicine and Orthopaedics, University of Southern California and Orthopaedic Hospital, Los Angeles. CA. Supported in part by NIH grams AMlO35X. AMl6404. and AG02.577, as well as funds from the Orthopaedic Hospital. Address reprint requests to Marcel E. Nimni. Ph.D., Professor of Biochemistry, Medicine & Orthopedics. Bone & Connective Tissue Research Laboratory, P.O. Box 60132 Terminal Annex, Los Angeles, CA 90060. 0 1983 by Grune & Stratton, Inc. 0049-0172/83/1301-0001$5.00/0
1
MARCEL E. NlMNl
Fig. 1.
Bundles
a Haversian
of collagen
canal in a human
prepared
by
detergent
for
microscope
cleaning 6 hr.
and
(original
Drs. A. Boyde
in
fibers
in the osteoid
long bone.
a
solution
observed
on
magnification
lining of
The specimen
was
containing
enzyme-
a scanning
electron
5,400
XI.
(Courtesy
of
and S. Reid.)
Every third residue is glycine. Proline and OHproline follow each other relatively frequently, and the (gly, pro, hyp) sequence makes up about 10% of the molecule. This triple helical structure generates a symmetrical pattern of three lefthanded helical chains that are, in turn, slightly displaced to the right, superimposing an additional “supercoil” with a pitch of approximately 86 8, units. The amino acids within each chain are displaced by a distance h = 2.91 8, with a relative twist of - 1 lO”C, making the number of residues per turn 3.27 and the distance between each third glycine 8.7 A. The individual residues are nearly fully extended in the collagen structure, since the maximum displacement within a
Schematic
Fig. 2. helix.
The
individual
approximately turn
around
The
hydrogen
shown)
form
twist. (not
three
coiled
chains
(interpeptide
quite
different
acids
located
from within
drawing (Y chains
showing are
residues each
per
other
bonds
turn.
a-helices
which
the same
polypeptide.
and
occur
chains
with are
in
right-handed
the
residues
bonding),
triple
helices a
stabilize
opposing
hydrogen
collagen
The
following
which
between
the
lee-handed
fully stretched chain would be approximately 3.6 A. This separation is nevertheless such that it will not allow intrachain bonds to form (as does occur in the alpha helix), and only interchain hydrogen bonds are possible.’ The exact number of hydrogen bonds that stabilize the triple helical structure has not been determined. The model that Ramachandran and Kartha described has two hydrogen bonds for every three amino acids, whereas the Rich and Crick’ version assumes one hydrogen bond for every three residues. In addition to these intrammolecular conformational patterns, there seems to exist a supermolecular coiling. Microfibrils, possibly representing intermediate stages of packing, may be present; such possibilities will be discussed later. Figure 3 shows a native collagen fiber with its repeating 680-A (68nm) periodicity alongside a schematic drawing of a collagen molecule (3000 8, length). The relationship between the length of the molecule and the periodicity that prompted the quarter-staggered theory of packing3 can be clearly seen. The stacking of collagen molecules to give rise to segment long spacing (SLS) crystallites (Fig. 4) has provided a useful tool for understanding the dimensions of the molecule, to order the peptides originating after CNBr cleavage of the molecule and to identify and characterize the various collagen types. This form of packing, which does not lead to normal fiber formation, will also be discussed in connection with the intracellular translocation of procollagen. The process of self-assembly that causes the collagen molecules to organize into fibers is shown schematically in Fig. 5. The thermody-
triple
helix
in different are therefore
between
amino-
Fig. tungstic matic
Native
3.
acid
collagen
showing
representation
approximately
300 nM.
the of
fiber 68
nM
collagen
(Courtesy
stained
with
periodicity molecules
phospho-
and
a sche-
measuring
of Dr. J. Petruska.)
COLLAGEN STRUCTURE AND METABOLISM
Fig. when
4.
Segment-long-spacing
ATP
(adenosine
of collagen. charge
The
density
negatively
around
positions their
can now of the
orientation.
these
have
which
transport
charged
observed
procollagen
The
by altering them
arrows
in the crystallite
It is of interest
been
ATP causes
form
to a solution the
to aggre-
in such a way that the individual
be visualized.
molecules
(SLSI
is added
the molecules
gate side by side in register molecules
crystallites
tryphosphate)
that
within
indicate
the
and represent
structures intracellular
similar
to
vesicles
to the cell surface.
namics of such a system involve changes in the state of the water molecules associated with the nonpolar regions of the collagen molecule. Also illustrated is the role of the polar groups on the surface of collagen, which we now know are distributed so as to effectively aid in the quarterstaggered packing that leads to the native fibrillar banded structure.4 ’
Fig.
The Procollagen
Molecule
In order for the organism to develop an extracellular network of collagen fibers, the cells involved in the biosynthetic process must first synthesize a precursor known as procollagen. This molecule is later enzymatically trimmed of its nonhelical ends, giving rise to a collagen molecule that spontaneously assembles into fibers in the extracellular space. Procollagen molecules have been identified as precursors of the three interstitial collagens (types I, II, and Ill). Several of the N- and C-terminal peptides (propeptides) have been characterized and, in some instances, the primary sequence determined.’ The N-terminal propeptides for both procY1(I) and pro 1(III) chains have a terminal globular domain of 77-86 amino acids, followed
to 37°C
native
fibers.
molecules overlap.
The
the
The
that
opposing signs.
collagen
by cold neutral
warmed
collagen
upper
have
alignment
water
these phobic
bonds
The driving system
that
since
greatly results
the release
state
process may
of
which
disorder
be associated
to operate
through
with
hydrogen-
clustered
system.
allows
expose these
of the fiber.
water water of
insolubilization
this phenomenon
~
rise to hydro-
of entropy
Part
of
around
and
the stability
into “random”
the lifespan
the
giving
an increase
the
+ and
“melt”
of “organized”
of
by the
of water
other,
leads to a gradual
represents
to interactions
37°C
circles),
enhance from
sites and its transformation the
drawing
due
collagen,
each
are into
in a quarter-staggered
Exclusion
with
most
solutions
reassemble
as depicted
(open
surfaces.
force
the
approaches of
from
If these
is primarily
regions
to interact
of
to align
charges
molecules
nonpolar
surfaces
be extracted
molecules
part
begun
electrostatic
hydrophobic
can
salt solutions.
As the temperature
bonded the
BIOSYNTHESIS
Soluble
5.
tissues
which
from
of the initial
increases the
aging
of collagen continues
of the individual.
by a collagen-like domain of about 40 amino acids. The collagen-like domain is joined to the chain by a short noncollagen sequence of 2-8 amino acids. The N-terminal propeptide of proLu2(1) has not been as well characterized and in some species may lack the N-terminal nonglobular domain.’ Type II procollagen isolated from chick embryos seems to contain a similar amino-propeptide but with a smaller N-terminal globular domain.’ The N-terminal propeptide of
4
type I collagen contains one residue of N-acetylglucosamine” and intrachain but no interchain disulfide links. On the other hand, interchain disulfide links seem to be present in the N-terminal propeptides of type III procollagII.12 en. The carboxyterminal propeptides of both procul and proa chains have molecular weights of 30,000-35,000 daltons and globular conformations without any collagen-like domain.13 These peptides contain asparagine-linked oligosaccharide units composed of N-acetylglucosamine and mannose.‘4,‘5 This carbohydrate sidechain is located in the carboxy-terminal half of the carboxyl propeptide in a region containing the sequence Asn-X-Thr,16 which is compatible with the structural requirement for glycosylation of asparaginyl residues by oligosaccharide transferases.” The functional importance of the carbohydrate in the carboxy-end of procollagen is unknown but may be part of a recognition mechanism for alignment, secretion or assembly into microfibrils. A diagram summarizing the major characteristics of procollagen type I is shown in Fig. 6. The central segment of the molecule is arranged in a triple helical conformation characteristic of collagen, and the globular extensions can be seen at both ends. Also shown in the diagram (indicated by arrows) are the sites of cleavage by specific procollagen peptidases. The extensions play a key role in the assembly of the tri-helical collagen molecule and their possible functions in this connection will be discussed later in this section. Once the molecule is completed and translocated to the cell surface the extensions are enzymatically removed (in the case of types I, II, and III collagens) and fibrillogenesis occurs. Enzymes that selectively remove these extensions can be found in a variety of
Procollagen molecule showing the nonhelical Fig. 6. terminal extensions. The N-terminal end contains a small helical domain and the C-terminal end is stabilized by interchain disulfide bonds. The sites of cleavage by procollagen peptidases are indicated by arrows.
MARCEL
connective tissues, and in the culture derived from collagen-secreting cells.15
E.
NIMNI
media
Intracellular Event Leading to the Synthesis of Procollagen Gene Expression. Since the discovery, about 12 years ago, of a distinct form of collagen in cartilage, labeled type II collagen, more than a dozen collagen chains have been characterized in different tissues of the same animal species. The best defined (types I-V) clearly represent unique amino acid sequences coded by different genes. By cell hybridization it has been shown that human chromosome number 17 contains the coding information for the cul and (~2 chains of type I collagen.‘8~‘9 Type III collagen seems to be encoded in another chromosome. Recent advances in recombinant DNA technology have opened the way for study of the structure and regulation of the collagen gene. In part this interest is generated by the fact that collagens constitute a family of closely related proteins, and are synthesized by specialized cells, that can be experimentally manipulated to synthesize different collagen types. The collagen gene is large, about 10 times the size of the functional mRNA.20 These messenger RNAs have a unique sequence of codons, called signal codons, to the right of the initiation codon. These are translated on a free ribosome to a unique sequence of about 15-30 amino acid residues (signal sequence) on the amino terminal of the nascent chain. In the case of collagen, the (~2(1) collagen gene from chick is 38 kilobases in length and contains at least 52 coding sequences (exons). Many of these exons are 54 base pairs in length and are separated from each other by large intervening sequences (introns) that range in size from about 80 to 2,000 base pairs.2’ The gene itself contains 38,000 base pairs, and is the most complex gene so far isolated.22 It was surprising to find that a molecule as uniform and regular as collagen should be coded by a gene of such complexity and divided into so many domains. In particular, the finding that most exons of the gene for the cy2 chain have identical lengths may have important implications in the understanding of evolution, since it suggests that the ancestral gene for collagen was assembled by multiple duplications of single genetic units containing an exon of 54 bp (Fig. 7). It is likely that
COLLAGEN STRUCTURE AND METABOLISM
-0
---etc.
c-2
54
54
34
The
Fig. 7.
54
collagen
containing
64
base
sequences
of
18
minimum tigators (redrawn
fashion,
interested with
54
54
gene
pairs,
amino
sequence,
an exacting
54
is made
each acids.
of The
and the fact that provides in the
valuable
process
up of multiple
which
units
corresponds
conservation it is repeated infomation
of evolution
of
to this
in such to inves-
of proteins
permission).”
a primordial exon this size could have encoded for a gly-pro-pro tripeptide repeated six times (3 x 3 x 6). Such a polypeptide of 18 amino acids probably had the minimum length needed to form a stable triple helical structure. The 54 bp exon structure may be common to all collagen genes. These observations generate interesting possibilities, and may allow us to better understand the nature of defects associated with some of the heritable collagen diseases. Translational, Cotranslational. and Early Posttranslational Events. After the gene is transcribed, it is spliced to yield a functional mRNA that contains about 3,000 bases. Specific mRNAs for each chain and collagen type are translocated to the cytoplasm and translated in the rough endoplasmic reticulum on membranebound polysomes (Fig. 8). A pre-pro cychain that contains an unusually large N-terminal hydrophobic signal or “leader’ sequence is the final translational product. The signal portion facilitates transfer of the chain into the lumen of the rough endoplasmic reticulum (RER) and is probably removed by intramembranous endopeptidase after it has served its function of orienting compatible procv chains in apposition. As the collagen polypeptide is synthesized in the rough ER, important cotranslational events accompany this process. It is a well known, extensively documented observation, that neither hydroxyproline nor hydroxylysine can be directly incorporated into proteins;23 it is only after peptide bond formation that hydroxylation of proline and lysinc can occur, mediated by two enzymes, prolyl and lysyl hydroxylases. These enzymes are specitic and require for their activity ferrous
Fig.
8.
collagen:
Sequence
procollagen
chains.
polysomes proline
of the
residues
hydroxylase
of
n-chains formation
molecules
of vesicles
the molecule nonhelical extensions
with
by specific
the
by
signal ribosomes.
4-proline (01. (4)
galactosyl-transferase peptide. (7) of
19) Intracellular and packaging by the removal
and part
on
of specific
(A)and
(6)
(13). (5) Release
Recognition
prepeptide
Folding
of
message
hydroxylase
the cell membrane
accompanied
extensions
of
by a glucosyltransferase
(8)
of a triple-helix.
biosynthesis
for the different
Hydroxylation
by lysyl
the C-terminal
crosslinks.
the procollagen Fusion
from
the
hydroxylase
N-terminal
through
disulfide
(3)
hydroxylysine
a-chains
in
mRNAs
Translation E.R.
of glucose
of the
completed
(2)
rough
and of lysine
(M) and addition Removal
events
of specific
by 3-proline
(A)
Glycosylation
of
of
(1) Synthesis
of of 3
and formation
the
molecule
and
translocation into vesicles.
of (10)
and extrusion
of
of the C-terminal
of the N-terminal
nonhelical
peptidases.
iron, ascorbate, and cu-ketoglutarate. They seem to recognize sequences surrounding the target imino or amino acids with differing affinities and thus selectively modify randomly alternating proline and lysine residues. The degree of hydroxylation differs from tissue to tissue and probably with availability of substrate, rates of synthesis, and turnover as well as the time that the molecule remains in the presence of the hydroxylating enzymes. As noted later, these
6
factors may also affect the position of the carbon atom in the peptide bound proline that is hydroxylated (e.g., the 3- or 4-carbon). The time required for the synthesis of a complete proa chain is about 6.7 min.24 The radioactive label, however, appears in fully aligned triple-helical chains after a further delay, a fact that may have significant physiologic implications as the time lapse between polypeptide synthesis and folding may affect the nature and extent of hydroxylation and glycosylation, another important posttranslational event. The enzyme prolyl hydroxylase (4-hydroxylase) has been isolated from several sources and extensively characterized.‘5,25,26 The active enzyme is a tetramer with a molecular weight of 240,000 daltons, and consists of two different types of monomers with molecular weights of about 64,000 and 60,000 daltons. Lysyl hydroxylase has been extensively purified and is apparently a dimer with two subunits, each having a molecular weight of approximately 90,000 daltons.*’ Prolyl 3-hydroxylase has been only partially purified and characterized.28 These three enzymes are specific and act only on cy chains in the nonhelical conformation, a reason why the time elapsed between peptide synthesis and folding may be so important. This time varies considerably, being approximately 10 min in cells synthesizing type I procollagen, 20 min in cells synthesizing type II procollagen, and 60 min in cells synthesizing basement membrane collagen.3’ After folding of the procollagen polypeptides, there is an additional time interval before the collagen molecule is secreted: this varies between 18 min for tendon cells,32 36 min for cartilage cells,33 and 60 min in parietal yolk sac tissue.34 Proline analogues, such as cis-hydroxyproline and azetidine 2-carboxylic acid, are known to be incorporated into collagen in the proline position and to inhibit triple helix formation within the cell. Using such an experimental tool it was shown that the glycosylation of hydroxylysine35 and the hydroxylation of proline at the 3 position2* are increased approximately twofold by inhibiting helix formation. These observations, coupled with the time factors noted above, may be linked to the increased hydroxylation of proline in the 3 position, the increased content of hydroxylysine and the greater degree of glycosyl-
MARCEL E. NIMNI
ation seen in basement membrane collagen, and may explain differences in hydroxylation seen in pathologic connective tissues. As lysyl residues in the newly synthesized proa chains are hydroxylated, sugar residues are added to the resulting hydroxylysyl groups. Glycosylations are catalyzed by two specific enzymes, a galactosyltransferase and a glucosyltransferase.36 The first of these enzymes adds galactose to the hydroxylysyl residues, and the second adds glucose to the galactosylhydroxylysine that is formed. The galactosyltransferase from chick embryo has been purified about I OOOfold and the glucosyltransferase has been isolated as a homogeneous protein. Both enzymes are glycoproteins and their activity requires the presence of sulfhydryl groups. The activity of partially purified galactosyltransferase is separated by gel filtration into three species with apparent molecular weights of 450,000,200,000, and 50,000 daltons. The purified glucosyltransferase has a molecular weight of 70,000 daltons. Both these transferases use sugar in a form of a uridine diphosphate glycoside, and require the presence of bivalent cations, preferably manganese.37 These enzymes, like the hydroxylases, require that the pro-o chains be in a nonhelical conformation. In intact cells, glycosylation is initiated while the polypeptides are still being assembled on the ribosomes, but probably continues after the release of complete proa chains in the cisternae of the rough ER; activity ceases when the chains acquire a triple-helical conformation. The oligosaccharides present in the extension peptides associated with the C-terminal region of collagen resemble those present in most other glycoproteins; they contain N-acetylglucosamine and mannose, and are attached to asparagine residues.38,39 Their composition suggests that they are added as intermediates via the dolichol phosphate pathway, and that final remodeling occurs in the Golgi after the helix has been formed.40 Once the translation, moditications and additions are completed it is essential that the individual prom chains become properly aligned for the triple-helix to form. We do not know if this alignment occurs while the polypeptides are still attached to the ribosome or if they have to detach, or if the N-terminal “signal” peptide plays a role in this connection. In any case, proper alignment should juxtapose the
COLLAGEN STRUCTURE AND METABOLISM
appropriate cysteine residues as a prerequisite for formation of the disulfide bridges that link the individual procv chains at the C-terminal end. Earlier studies involving subcellular fractionation suggested that the disulfide bridges could appear during translocation of procollagen from the ribosome to the smooth endoplasmic reticulum, probably in the cisternae of the ER.4’ More recently it has been proposed that disulfide bond formation occurs while the propeptides are still attached to the ribosome.4’ In any case, it seems clear that for assembly and secretion the Cterminal extensions must be present.43 ” Although helix formation seems to be a spontaneous entropy driven process, the possibility that an enzymatic system, similar to that which regulates the coiling of DNA and shifts it from one topologic form to another involving DNAgirases and topoisomerases4” could be associated with the rough ER, and which controls and monitors this step, cannot be discounted. The kinetics of triple helix formation of type I procollagen within hbroblasts have been measured in pulse-chase experiments followed by proteolytic digestion.4’ Production of triple-helical molecules requires X-9 min after completion of procY-chains.47 Disulfide bond formation precedes triple helix formation, and serves as a catalyst for it to occur. Similar observations and conclusions have been derived from studies on the biosynthesis of type I I I procollagen by chick embryo blood Vf2SXlS.44~4X In this case further stability may be added by interchain disulfide bond formation in the helical region as well as in the N-terminal nonhelical region, but the exact timing of these subsequent events remains difficult to ascertain. lntracellulur Translocation of Procollagen and Extrusion into the Extracellular Space. The synthesis of procollagen chains involves the translation of a specific messenger RNA on membranebound ribosomes similarly to secretory proteins in generalJy and core protein of proteoglycans.” The signal sequence, an unusually large one in the case of collagen,” may trigger the attachment of the ribosome that generated it to a specific binding site on the membrane. In most instances the signal sequence is removed from the nascent polypeptide chain by a specific endopeptidase before chain completion.c2,i’ It is not clear whether this is the case with collagen since before movement along the secretory pathways begins three separate specific
7
chains have to register to form the triple-helical structure. It is possible that the signal peptide, due to its hydrophobic character, guides the nascent cy chain to the interior of the rough membrane intracisternal space where the translational and posttranslational events occur. Once the procollagen molecule is assembled (devoid of the signal peptide) the secretory pathway seems to be similar to that followed by other glycoproteins. These experimental findings have been recently reviewed.54 Essentially the procollagen molecules, now detached from the ribosome, emerge from the endoplasmic reticulum and move towards the Golgi apparatus through the microsomal lumen (Fig. 9). In the Golgi its C-terminal mannose-rich carbohydrate extension is probably remodelled and the completed procollagen molecules packaged in Golgi-derived vessicles and carried towards the cellular membrane by cytoskeletal movements. During transit they sit side by side to form structures that sometimes may be identified with the electron microscope as SLS crystallites (see fibrillogenesis).
Fig. 9. of the
Movement
rough
transitional packaged
endoplasm into secretory
by exocytosis.
of procollagen
endoplasmic
reticulum (TE)
to
vesicle
the (SV)
through (RER) Golgi prior
the cisternae and
(G)
through where
to extrusion
a
it is IEX)
8 The small aggregates of oriented procollagen molecules are probably trimmed of their nonhelical amino and carboxyl extensions by specific peptidases when they reach the extracellular space. In the case of type I collagen, the first peptidase to act seems to be the amino protease; this is followed by a carboxyprotease. In type III collagen the sequence of removal may be reversed.54~ss Recent immunoelectron microscopic studies have shown that the thinner collagen fibers (200-400 A) contain procollagen molecules that still retain their NH,-terminal extensions;56 this is not the case for larger collagen fibers. Thus, certain fibers in normal human skin still possess, at least on their outer surface, procollagen or pN-collagen molecules. The latter possibility is most likely in view of earlier work” and because antibody reactivity could be eliminated by treating the tissues with procollagen NH,-terminal protease. These findings suggest that removal of extension aminopropeptides could be involved in the control of fiber growth. It is possible that selective removal of portions of the extensions may occur at the cell surface thus allowing for formation of 4-D staggered microfibrils, and that final removal of these propeptides occurs in the extracellular space. This process would allow for modulation of fiber growth by apposition and elongation as well as for fusion of thin fibers (20-40 mm) into larger diameter fibers (Fig. 10). Lysyl Oxidase Somewhere in the process of extrusion, recently formed microfibrils must be recognized by the enzyme lysyl oxydase, which converts certain peptide-bound lysines and hydroxylysines to aldehydes. This enzyme initiates the biosynthesis of cross-links in collagen. A recent review summarized most of our knowledge in this field.58 This enzyme, first described by Pinnell and Martin,59 catalyzes the oxidative deamination of lysine and hydroxylysine, as summarized in Fig. 1 1. The enzyme is an extracellular amine oxidase, and has been purified from a variety of connective tissues. The molecular weight in most species is 30,000 daltons, or a multiple thereof. It requires Cu+ + and probably pyridoxal as cofactors, and molecular oxygen seems to be the cosubstrate and hydrogen acceptor. It is irreversibly inhibited by the lathyrogen BAPN. This
MARCEL E. NlMNl
EXTRACELLULAR
PROCOLLAGEN
C”O;S&KED
Fig. 10. Fibrillogenesis: Microfibrils in a quarter-staggered configuration have lost their C-terminal nonhelical extensions and part of their N-terminal extensions. In this form they seem to organize readily into small-diameter fibers which retain part of the N-terminal nonhelical extensions. After being relieved of these peptides by a procollagen peptidase, fibers are able to grow in diameter by apposition of microfibrils or by merger with other small diameter fibers.
enzyme exhibits maximal activity when acting on reconstituted collagen fibers rather than upon monomeric collagen. Because of this it is felt that the enzyme is most likely to act upon microfibrillar aggregates of collagen molecules at the time of extrusion. It would be difficult to conceive how it could act otherwise on collagen, since once assembled into the quarter-staggered array and compacted into fibers, it would be difficult for the enzyme to gain access to the interior of the fiber. Fibrillogenesis During the preceding discussion, the tendency of collagen molecules to form macromolecular aggregates has been constantly emphasized. This tendency is common with most fibrous proteins that form filaments with helical symmetry and which occupy equivalent or quasiequivalent positions. Both electron micrographic and x-ray diffraction data support the view that within col-
iw
hi
PEPTIDE
BOUND LlSlNE
H
k
k .z-AMNOAOIPIC
L-SEMALOEHYOE
Fig. 11. The oxidative deamination of peptide-bound lysine by the enzyme lysyl oxidase generates the aldehydes associated with the collagen molecule.
COLLAGEN
STRUCTURE
AND
9
METABOLISM
lagen fibers there is a regular arrangement of molecules that could result from such an ordered assembly.60 ” A five-stranded microfibril was first suggested to account for such a substructure, one that would satisfy the condition that adjacent molecules were equivalently related by a quarterstagger, as suggested by Hodge and Petruska.3.63 The exact mode of organization, that is the mode in which the collagen molecules pack in the microfibril, their exact number, and the nature and location of crosslinks within and between microfibrils still remains a subject for speculation (Fig. 12). The five subunit assembly proposed earlier by Smith seems to be the most widely accepted basic unit. For detailed discussion the reader is referred to the reviews mentioned earlier as well as to recent detailed publications.‘” “) The earliest form of intracellular interaction is documented by the appearance of what seem like side to side nonstaggered arrangements of collagen molecules resembling SLS and end-overlapped SLS crystallites. These formations have been found in vacuoles within odontoblasts, epithelial cells, and fibroblasts.70~74 SLS crystallites normally do not form under physiologic conditions of pH and ionic strength but occur if negatively charged counterions (such as ATP) are added to the solution to mask and interfere with ionic interactions that normally cause collagen molecules to form quarter-staggered arrays (Fig. 4). What causes such associa-
tion to occur early within the cell is not known. but they could be due to the presence of the Cand N-terminal extensions of procollagen or to the existence of ionic species within the vacuoles that may favor lateral stacking. In any case, such a conformation may prove to be of advantage at the time of intracellular transport since it would inhibit normal fibrillogenesis and probably position the procollagen molecules favorably for the procollagen proteases to exert their enzymatic activities during the process of extrusion. After removal of the nonhelical extensions, the newly formed collagen molecules could then be free to organize into quarter-staggered microfibrils and proceed to form native fibers. Under pathologic conditions, cells that are actively synthesizing collagen and that have accumulated large concentrations of proteoglycans have been shown to exhibit intracellular deposits of collagen fibrils within cytoplasmic inclusions.74 The process of in vivo tibrillogenesis has been equated for a long time with the ability of monomeric collagen to form fibers in vitro (Figs. 5 and 13). When monomeric collagen is heated to 37°C it progressively polymerizes, generating a turbidity curve that reflects the presence of intermediate aggregates. This phenomenon has been the focus of intensive investigation, and it has also led to the assumption that analogues exist between in vitro and in vivo situations. The lag phase (monomers), the nucleation and appearance of turbidity (microfibrils), and the rapid increase in turbidity (fiber formation) have been equated while attempting to understand how the cell handles these processes.7s ”
7 Subumts
Fig. 12. tion
Microfibrils
of collagen
widely
accepted
five collagen the
degree
Also
shown
represent
on its way form
of microfibril
molecules. of
is a higher
degree
microfibrils
width
as en alternate
as well
ecules
7 collagen
to sit side-by-side
is that
measured
way
molecules without
The
involves
view
indicates
in D values where
in a unit
of packing allowing
lateral
most
which
of organization
are packed
of organiza-
a fiber.
The cross-sectional
displacement
five-stranded accommodate
early forms
to becoming
(O-4). four
cell 8 nm in which
could
for some
mol-
displacement.
Fig. 13. its potential fibers.
Formation
of the five-membered
for lateral
and end-to-end
microfibil
aggregation
and
to form
MARCEL E. NIMNI
The experimental approaches used to study the nature and size of the aggregates formed during early stages of self assembly have been recently reviewed.7’~80 The reverse process, the disassembly of recently synthesized rat tail tendon collagen fibers by 0.1 M acetic acid, has also led to the conclusion that dimers and higher molecular weight aggregates can be recognized, since, as long as stable covalent crosslinks are not formed, the process is reversible. Current evidence, therefore, seems to indicate that monomeric collagen does not float freely in the extracellular space, but polymerizes into microfibrills and probably even into fibers before leaving the surface of the cell from which it originates. Autoradiographic studies show that the site of matrix deposition is near the cell surface.70,8’,“2 Similar observations have been made for other tissues such as the developing chick embryo, tendon, cornea, and basement lamella of fish all of which provide good models for fiber growth and elongation.72,79383 Although we are still not able to completely understand the detailed nature of the microtillaments nor the mechanism by which collagen fibers are deposited in the connective tissues in an ordered and specific manner, the overall picture is becoming more clear. From the folding of the procollagen molecule, to its transport through the cisternae of the endoplasmic reticulum, its packaging into vacuoles in the Golgi to extrusion and filament elongation at the cell surface, the sequence seems logical and amenable to further investigation. Collagen
Metabolism
Collagen is the most abundant of all body proteins. It therefore has to be synthesized and accumulated in large amounts during periods of growth and at sites of injury or tissue repair. Its rate of turnover is also rapid at these times and locations. Tissues such as bone, which are involved in active remodelling, are responsible for the major turnover, while other less dynamic tissues in the full grown individual, such as skin and tendons, may exhibit slow and almost negligible turnover. Although some specific cellular metabolic products have been implicated in stimulating or inhibiting collagen synthesis,84 the mechanisms involved are not understood. Under normal circumstances of growth and activity,
mechanical stress, piezoelectric phenomena, cell density, and cell-cell interactions are likely to affect cell membranes and modulate the synthesis and secretion of collagen. This area of research is difficult to investigate and, therefore, has not received much attention and our knowledge is scanty. Some aspects of these events will be discussed when we review the synthesis of collagen by cultured cells and the effects of specific drugs and inhibitors on such systems. Control could be exerted at both the transcriptional and translational levels within the cells, as well as during the extracellular assembly and crosslinking. In general the activity of cells and tissues in regards to their ability to synthesize collagen are assessed by their ability to synthesize hydroxyproline or by measurement of the activities of specific enzymes, the proline and lysyl hydroxylases being the most commonly assayed. Endogenous
Modulators
Many endogenous factors produced by cells in culture or derived from tissues have been investigated for their ability to regulate collagen accumulation; these have been documented in a recent review.85 Peptides removed from the NH,terminal end of procollagen during conversion to collagen are able to inhibit collagen synthesis in a reticulocyte cell-free system86’87 as well as when added to the media of cultured cells.88 Castor’s group was the first to demonstrate the presence of a series of factors that were capable of inducing a state of hypermetabolic activity in cultured synovial cells.“’ This material, assigned the name connective tissue activating peptide (CTAP), is a peptide of lO,OOO-15,000 daltons that promotes synovial cell proliferation (suggesting a role in pannus formation) and stimulates hyaluronate synthesis, but does not influence collagen synthesis. The search for endogenous factors that stimulate collagen synthesis led to the isolation from homogenates of experimentally (Ccl,) injured liver” of four fractions that, upon elution from Sephadex G-25, increase collagen synthesis up to eightfold in fibroblast cultures. It was subsequently reported that cytosol extracts and the supernatants from cultured, isolated egg granulomas obtained from livers of mice with infection due to Schistosoma mansoni
COLLAGEN STRUCTURE AND METABOLISM
stimulated fibroblasts to incorporate tritiated thymidine and to proliferate in vitro.” Since this fibrotic lesion results from an inflammatory granulomatous response, these investigators examined the nondialyzable fractions derived from antigen-stimulated T-cells for their ability to stimulate fibroblast proliferation and collagen synthesis.92 These preparations stimulated thymidine incorporation and increased both collagen and noncollagen protein synthesis when added to fibroblast medium, thus confirming earlier reports that lymphokine-rich supernatants derived from phytohemagglutinin (PHA)stimulated mononuclear cells and promoted collagen accumulation in cultures of fibroblasts.93 In addition to lymphkines, the presence of macrophage Factors capable of stimulating fibroblast proliferation94 and collagen synthesis by libroblasts has also been reported.q5 In contrast, the extracts from unstimulated mononuclear cells produced no effect on collagen synthesis when added to cultured fibroblasts, while supernatants from PHA-stimulated cells produced a 90% inhibition of collagen synthesis.” On the other hand, media from nonstimulated lymphocytes was shown to inhibit collagen synthesis by chondrocytes while stimulating that by fibroblasts.” Such conflicting findings can probably be explained by the presence in the media of both stimulating and inhibiting factors, and possibly by the concentration dependence of such effects, which could, of course, vary from experiment to experiment. It is therefore possible that circulating metabolites can reach the connective tissue cells and modulate their biosynthetic activity. In addition to these factors, hormones and drugs can influence such a pattern of behavior, but their effects will be discussed separately.
The endocrine control of connective tissue metabolism has received little attention. Of all the hormones known, only the glucocorticoids have generated significant interest because of their ability to inhibit the production of collagen. This topic has been the subject of a recent review.9x The most conspicuous side-effect.of systemic glucocorticoid therapy is somatic growth inhibition. This problem is especially disturbing in
11
children chronically treated with relatively low doses of glucocorticoids. As little as 45 vs 6 mg of cortisone or prednisone (6 mg/m*/day), a dose corresponding to the average endogenous daily secretion of hydrocortisone from the adrenals, suppresses somatic growth.99 Multiple daily injections of cortisone to young rats decreases the newly synthesized collagen content of skin but not the pool of mature collagen”” and can decrease urinary hydroxyproline excretion.“’ In subcutaneous rat granulomas, a single injection of betamethasone decreases hydroxyproline formation to a greater extent than total Procollagen molecules did protein synthesis.“* not accumulate in the granuloma and were fully hydroxylated.“’ Other investigators have confirmed these observations.” The fluorinated glucocorticoids are the most potent in selectively decreasing collagen synthesis. Multiple pharmacologic injections of glucocorticoids were shown to decrease prolyl hydroxylase activity in liver, granuloma, skin, aorta, lung, and human skin.lo4 The growth of most diploid fibroblasts is inhibited by glucocorticoids;“’ however, human lung”’ and skin’“‘~ lo9 fibroblasts represent major exceptions to this observation since proliferation of these cells is enhanced by glucocorticoids. In contrast to rodent fibroblasts, the enhanced proliferation rate of human skin fibroblasts occurs only while the cells are in log-phase growt.t~.“0 In the case of human WI-38 cells, hydrocortisone is also capable of significantly increasing the number of population doublings that the cell strain can achieve prior to senescence.‘“’ When chick embryo leg tendon cells were treated with betamethasone, hydroxyproline formation was decreased to a greater extemt than was total protein synthesis.“’ This fluorinated glucocorticoid also markedly depressed prolyl hydroxylase activity. Glucocorticoid effects on collagen synthesis have also been studied in normal skin and keloid fibroblasts.“L-“4 Hydrocortisone inhibited collagen synthesis in normal fibroblasts by 60% while noncollagen protein synthesis was increased.“’ In keloid fibroblasts, hydrocortisone did not decrease collagen synthesis. However, the synthetic fluorinated glucocorticoid, triamcinolone, markedly decreased collagen synthesis in keloid-derived fibroblasts.“3 Although prolyl
MARCEL E. NIMNI
hydroxylase activity is decreased, the degree of prolyl hydroxylation of procollagen was not affected.“4”‘5 The glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures.“6 Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50%70% inhibition of collagen synthesis and accumulation in polyvinyl spongeinduced granulomas, whereas total protein synthesis was inhibited by only about 25%. Growth rates, total protein synthesis, and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3 x lo-’ M hydrocortisone to the culture medium caused a 30% 50% inhibition of both collagen and noncollagen protein synthesis in fibroblasts from either source. Prolyl hydroxylase activity was reduced in both sponge granulomas from glucocorticoidtreated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as was prolyl hydroxylase activity while there was no effect on peptidyl prolyl hydroxylation. When the synthesis of prolyl hydroxylase by polysomes isolated from the dermis of neonatal rats was investigated, it was found that polysomes derived from control and glucocorticoidtreated rats synthesized similar amounts of enzyme, even though this enzyme is decreased in the tissues of the treated animals.“’ On the other hand, the dermal polysomes from the glucocorticoid-treated rats synthesized significantly less collagen than controls in the wheat-germ lysate assay system. Polysomal m-RNA isolated from the steroid-treated dermal polysomes also synthesized less collagen in a nuclease-treated reticulocyte lysate system. The mechanism of this reported selective reduction of polysomal procollagen m-RNA may relate to a suppression of procollagen gene expression. Glucocorticoids have been reported to decrease ribonucleic acid content and impair ribonucleic acid precursor accumulation in bone also decrease ribonucleic cells.“8 Glucocorticoids acid synthesis in growing mouse fibroblasts conwith a decrease of transcriptional comitantly”’ activity in isolated nuclei.‘*’ The administration of pharmacologic doses of glucocorticoids to neonatal animals suppresses cartilage growth and bone formation, probably
by inhibiting DNA synthesis.‘*’ The marked reduction of thymidine incorporation observed in proliferating cartilage cells is associated with a decline in protein and RNA synthesis and a receptor-mediated event. Developing long bones show an initial retardation or inhibition of calcification, but this is followed by the appearance of more numerous and larger matrix vesicles which, in turn, seem to lead to enhanced calcification. ‘*‘,‘**These events may be relevant to the mode of induction of ectopic calcification processes that can lead to functional and structural abnormalities. It is widely believed that glucocorticoids initiate their action by binding to specific receptor proteins in the cytoplasm of target tissues.‘23-‘26 Locally injected glucocorticoids can improve osteoarthrosis or acute refractory rheumatoid arthritis, but they may also induce progressive joint destruction. Their effects on joints could be mediated through direct hormonal influence on chondrocytes, the constitutive cell of cartilage. Until recently, glucocorticoid receptors had been described only on embryonic chick growth cartilage.‘*’ Evidence exists that glucocorticoid receptors also exist in cultured articular chondrocytes obtained from shoulder and knee joints of twomonth-old rabbits.‘** Vitamin A has been shown to overcome the inhibitory effects of cortisone on tensile strength and collagen deposition in the early stages of wound healing ‘29.‘30and the reduction of inflammatory cell infiltration, fibroplasia, capillary budding, and deposition of collagen fibers in granulomas following glucocorticoid administration is reversed by concurrent vitamin A thera“I It was also shown that vitamin A blocked PY. the inhibitory effect of glucocorticoids on collagen, protein, deoxyribonucleic acid and lipid deposition in granuloma tissue.13* Besides vitamin A, bioflavinoids have also been shown to block the inhibitory effect of glucocorticoids on collagen synthesis and deposition.‘33 Anabolic steroids do not block glucocorticoid inhibition of fibroblast cell proliferation and collagen synthesis in cell cultures,“’ but do diminish glucocorticoid-mediated depression of collagen synthesis. DIFFERENT
TYPES
OF COLLAGEN
Over a decade has passed since we first realized that all collagen fibers within a particular organism are not made up of identical molecules.
13
COLLAGEN STRUCTURE AND METABOLISM
Since 1970 a great deal of experimental work has been devoted to understanding these various collagen types, their molecular structure, biosynthesis, cells of origin, distribution, and turnover. In spite of great advances it is still not clear how the structure of these molecules relates to their function and it is hoped some concepts may emerge from a review of the existing data. The different collagen types have been identified using Roman numerals, which have been assigned as they are purified and characterized (Fig. 14). In addition to these major types (I-V) many lesser, albeit well characterized, collagens have been described. These have been identified using a variety of capital letters and Greek symbols. Type I Collagen Before 1969 this was the only mammalian collagen known. The basic molecule was called “tropocollagen” or “collagen molecule”; the latter term has survived. It is composed of three chains, two identical, termed (~1 chains, and one different from the other two, called a2. Using the new terminology we now call the chains cul type I or al (I) and cr2 type I or (r2(1) or simply cu2, since there is no a2 chain among the other collagen types. This molecule has been characterized both physically and chemically (amino acid sequence, sedimentation, molecular dimensions, carbohydrate attachments, viscosity,
TYPE
I
etc.).13’ Type 1 collagen is most abundant in skin, tendon, ligament, bone, cornea, etc., where it comprises between 80% and 99% of the total collagen. Bone matrix is essentially all type I collagen. The proportion of type I collagen in a particular tissue can vary at different sites, during development, with age and pathology. The most common technique used to isolate this molecule, and distinguish it both qualitatively and quantitatively from the other collagens, involves the use of solvents of different ionic strength and pH followed by differential salting out13’ (Table I). This fractionation is usually followed by identification of the intact chains and CNBr-derived peptides using polyacrylamide gel electrophoresis.‘37 Such separations are shown below (Fig. l5), followed by a schematic diagram that illustrates the distribution of methionines along the (Y chains and the sites where peptide bonds are cleaved by CNBr and responsible for the generation of characteristic patterns (Fig. 16). A more detailed analysis of the CN Br-derived peptides can be accomplished when these are separated by two-dimensional electrophoresis.‘3x The first dimension relies on differences on isoelectric point and the second on molecular weight. A separation of peptides using this procedure is shown below (Fig. 17). The amino acid composition of the better characterized human collagen types are shown in Table 2. The amino acids that are notable by the fact that they differ significantly from that of type I collagen are indicated in bold numbers. On many occasions these differences have been used Table
1.
Collagen
Selective
Fractionation
by
Salt Precipitation Collagen(s1Preclpltatmg
From:
of N&l
Neutral
0.5 M
IM)
Salt Solution
Acetic Ac,d
COllCWltratlOll
Types I, II, Ill
0.7
1.2
Types IV. V 7s
TYPE IU 1.5-1.8
Type III Type IV
1.8 2.0 Fig. 14. Type
Diagram
I is present
cartilage.
and
and es a minor are differences sylation. gen.
type
of the 3 interstitial in
skin,
Ill in blood
component in chain
Disulfide
bone,
vessels.
are only
of collagen.
etc.,
type
developing
in skin and other
composition
crosslinks
types
tendon,
seen
in type
2.2-2.5 3.5
tissues,
tissues.
and degrees
II in There
of glycoIll colla-
4.0 b4.0)
Cys-Rich Aggregate 7s TYPO 1 TYPO I Type I-Trimer Type II Type I-Trimer Tvpe V
MARCEL
Z.
I
_ .__L
II
1411
I.I+IIIR
III
IIIR
I’IIIR
E. NIMNI
BAPN
I YYt= Characteristic
Fig. 15. to the
CNBr
collagen
peptides
chains
alone
has a diagrostic
value
peptide
derived
patterns
from
types
and as mixtures. for identifying
corresponding al(I).
II, and
This peptide collagen
III
pattern
types.‘37
to identify collagen types or their mixtures and to suspect the presence of new or abnormal collagen species.“’ Type II Collagen The major difficulty encountered by earlier investigators interested in isolating collagen from cartilage was associated with its extraction in a native form from fresh tissues. This problem has been overcome in a variety of ways, most of which rely on the prior removal of proteoglycans and of the nonhelical protease sensitive crosslinks to solubilize the collagen molecule, or on the
The distribution
Fig. 16. cross-bars collagen
types
peptides elution tion
along which
from
along
molecular ing from
the
a
I, II, and Ill. Cleavage are
numbered
CM-cellulose the
of methionines
individual
chain).
weight.
Amino
the N-terminal
with
following
columns Their
chains
acid residues end.
by up
make
CNBr
gives
their
sequence
(rather
length
is indicated that
reflect
than
rise to
their
their
are numbered
of
posi-
relative start-
Fig.
17.
2-dimensions first
dimension
tric
focusing)
weight.‘=
The
separation
enhances by virtue and
of
resolution.
in the
of their second
CNBr
derived
Peptides electrical dimension
peptides
separate charge by
in
in the
(isoelecmolecular
15
COLLAGEN STRUCTURE AND METABOLISM
Table 2. Amino Acid Comoosition of the Human Collaaen Chains (Residues11000 Amino Acld
al(l)
a210
nl(lll
al(lll) 0
CYlWl
n2lV)
UlW)
Total Residues) rr3Wl 1
1
2
3
5
108
93
97
125
133
106
110
91
98
93
Aspartic Actd
42
44
43
42
51
50
49
42
46
50
Threonine
16
19
23
13
20
29
21
19
17
25
SeWlt?
34
30
25
39
37
34
23
34
25
28
Glutamlc Acid
73
68
89
71
79
89
100
97
107
98
3-Hydroxyproline 4-Hydroxyproltne
1
7
ProlIne
124
113
120
107
65
107
130
98
109
119
Gl)WllW
333
338
333
350
328
331
332
330
334
327
AlaWla
115
102
103
96
37
64
39
49
54
49
0
0
0
2
O-l
0
0
21
35
18
14
28
27
17
5
10
8
13
11
6
14
9
13
29
15
19
30
26
22
52
37
4
2
3
2
2
12
12
13
8
29
Half-Cystlne Valine Methionlne ls0laWXle Leucine Tyros~ne Phenylalantne Hydroxylystne Lyslne Hlstldlne Argrwva
1
0
0
29
28
18
9
8
10
9
17
20
15
16
36
56
35
39
4
2
2
3
11
12
9
11
11
9
12
20
5
49
23
36
43
38
40
26
18
15
30
9
13
14
15
19
15
3
12
2
6
6
10
6
14
6
11
50
50
50
46
26
48
40
42
45
48
28
34
Gal-Hydroxylysine
1
4
2
3
5
7
Glc-Gal-Hydroxylyslne
2
12
30
5
29
17
administration of BAPN to animals before killdone in this connecing. ‘w’~~’The experiments tion and on the initial characterization of this molecule have been reviewed by Miller.‘4’ Isolation and analysis of collagens from a variety of cartilagenous structures have shown that cartilage collagen is made up primarily of molecules containing three identical LYchains.‘39-‘43 These chains have chromatographic and electrophoretic characteristics similar to the crl chains of type I collagen. Because of intrinsic differences suggesting that their synthesis is directed by a different gene, the cartilage chains are now designated as o(, (II). The most signiticant features of cartilagecollagen are its high hydroxylysine content and its glycosidically bound carbohydrates, since it can contain up to fivefold more of these residues than the homologous chain from skin. Proteolytic enzymes that cleave the protein backbone of the proteoglycans have been of great help in attempting to elucidate the chemistry of the ground substance. In particular, papain has shown a marked specificity for depolymerizing the proteoglycan complexes of cartilage. intravenous injection of crude papain causes a softening and eventual collapse of the normally erect ear of rabbits.lJ4 This is accompanied by a marked decrease in the basophilic and metachromatic
staining and a decrease of the chondromucoprotein content of cartilage.“S.‘46 The collagen matrix does not seem to be affected, and these changes are reversed within a few days. Purified papain will cause a rapid disintegration of bovine articular cartilage at physiologic pH and temperature.‘47 A similar effect of papain had been previously demonstrated using chondroprotein from pig laryngeal cartilage as a substratc.‘4X Due to the temperatures used in most of the experiments (37”-60°C), collagen became rapidly degraded. During the course of some of our experiments it appeared that the difficulties encountered in isolating collagen from cartilage were related to the presence of large amounts of glycosaminoglycans in the tissue. After removal of significant amounts of these substances by papain at 4°C the collagen could be solubilized by increasing the ionic strength of the media. The residual glycosaminoglycans in the crude extract tend to coprecipitate with collagen during high-speed centrifugation, and only after most of these are removed does collagen remain in solution. The purified collagen extracted from bovine articular cartilage exhibits all the characteristics of native soluble collagen, as judged by its high intrinsic viscosity, its optical rotation, and its melting-temperature profile.“’ After denatura-
16
MARCEL E. NlMNl
tion it gives rise to one single component having the same electrophoretic mobility, elution pattern from CM-cellulose, and sedimentation velocity as the (Y, chain from skin collagen. It contains 22 hexose residues per 1,000 amino acid residues, of which eight to nine are glucose and 13 galactose. All galactose residues are bound via 0-glycosidic bonds to hydroxylysine. After periodate oxidation the hydroxylysines with attached sugars were not degraded and could be recovered in the acid hydrolysate. Fig. 18 shows the elution patterns of collagen from bovine articular cartilage and skin collagen. The hexoses, which remain tightly bound to the purified collagen, were separated by paper chromatography and showed a glucose : galactose ratio of 1.6. Only trace amounts of &components were detected in the crude or purified extracts of cartilage collagen, as well as in the collagen extracted in lesser amounts by nondegradative procedures, such as 1M MgCl,, I M NaCl, or water. All of these fractions showed one single component that migrated like CZ~from bovine skin. Limited proteolysis with pepsin has also been used in the isolation of type II collagen from pig LA
0.8-
CARTILAGE al (II)
0.4z no (u 5 go.8 b in d”
200
400
600 SKIN
al
_
0.4
-
0
200 Effluent
400
600
(ml)
Fig. 18. Chromatagraphic patterns of type I from bovine skin (bottom) and Type II from bovine articular cartilage collegens (top) fractionated on CM-cellulose.
laryngeal cartilage’49 and infant articular cartilage.“’ It should be noted that collagen extracted from hyaline cartilage as the result of limited proteolysis has been found to contain little, if any, type I collagen. On the other hand, cartilagenous tissues such as chick sternum and human intervertebral discs contain variable mixtures of types I and II co11agen.‘5’x’52 Limited proteolysis using pepsin or papain has proven to be a valuable method to prepare large quantities of type II collagen. It is of interest in this connection that whereas cartilage collagen from young animals, when solubilized with papain (or pepsin), gives rise primarily to (Y chains upon denaturation, collagen from the skin of the same species but of older age exposed to similar mild proteolytic degradation in the cold, gives rise to large amounts of polymeric material and fl chains upon denaturation.‘39 This reflects the greater susceptibility of the terminal extension of young cartilage collagen to proteolytic cleavage. As the cartilage ages, probably because of the presence of new or more stable crosslinks, it becomes more resistant to solubilization following proteolysis of the tissue. This observation may be worthy of further investigation since it may provide insight into why mature cartilage is more likely to develop degenerative changes. Although type 11 collagen is the major collagen type present in cartilge, it is also present in significant amounts in other connective tissues. The intervertebral disc contains a central gel-like region, the nucleus pulposus, which is surrounded by concentric rings of highly ordered dense collagen fibers known as the annulus fibrosus. The nucleus pulposus is a highly hydrated gel of collagen fibers enveloping glycosaminoglycan molecules similar to those found in hyaline cartilage. The cartilaginous end plate of the intervertebral disc has a composition similar to hyaline cartilage.‘5’-‘54 In contrast, the nucleus pulposus and annulus fibrosus represent a mixed connective tissue matrix of at least two collagen types. The principal collagen type found in the nucleus pulposus is type II collagen. This collagen type represents greater than 85% of the total collagen present, the remainder being type 1 collagen. The structure of the annulus fibrosus is morphologically more complicated than that of the nucleus. Two distinct fiber forms can be seen
COLLAGEN STRUCTURE AND METABOLISM
ultrastructurally in the annulus. Most prominent are the dense parallel fiber bundles forming the concentric ringed pattern radiating from the nucleus pulposus. Surrounding these rings is an amorphous matrix that is rich in proteoglycans. In the human, type I collagen accounts for approximately 50% of the total collagens identified in the annulus at five years of age.ls3 The amount of type I collagen increases with age and represents approximately 66% of the total collagens from year 16 onwards. In addition, the distribution of type I and type II collagens is not uniform. The predominant collagen of the transition zone between the nucleus and the annulus is clearly type II collagen, but the amount of type I collagen increases dramatically as one examines samples radially towards the outer rings of the annulus, where the predominant collagen is type I. It would seem as if the dense fiber bundles are composed of type I collagen, while the amorphous surrounding matrix is richer in type II collagen and proteoglycans. This tissue will be discussed in more detail in connection with degenerative disc disease. Another tissue that contains appreciable amounts of type II collagen is the vitreous.‘55-‘58 Comparisons of collagens extracted from cartilage and vitreous by pepsin treatment show differenccs in amino acid composition, carbohydrate content, and the presence of additional N chains that migrate more slowly than GUI(II). It has recently been reported that rabbit vitreous in addition to cul (II) collagen contains one of the newer collagen species present in hyaline cartilage,3.‘5x to be discussed later. Type II collagen is synthesized during the chondrogenic stages of development of the mesoderm.‘59.‘h” During amphibian limb regeneration the tissues undergo three distinct stages of development during the transition from mesenthyme to cartilage and bone. The synthesis and presence of type II collagen correlate with chondrogenic activity. A similar developmental pattern has been observed in the bone matrix induced endochondral calcification system.‘h’ Type I11 Collagen When the residue of human skin remaining after prolonged extraction with neutral salt and dilute acetic acid solutions is subjected to digestion with CNBr, a series of peptides appear that do not correspond to any of the known peptides
17
derived from types I or II collagens.‘62.‘h3 Because of the similarities of two of these peptides to well characterized regions of the LY’(I) and O(~(II) chains it seemed related to the l-family of chains and was therefore designated o(, (III). When human dermis was digested with pepsin under conditions that allowed the collagen molecules to maintain their helical conformation, it was found that type I molecules could be separated from type III by differential salt precipitation at pH 7.5.‘64 The type III molecules were composed of three identical chains, cy, (I) and 01~. The pepsin-resistant portion of the new chains is similar in size to the previously isolated collagen chains and can migrate with CX,(1) and LY,(III) on conventional sodium dodecyl sulfateepolyacrylamide gels during electrophoresis, although in some systems it can be seen to migrate slightly slower.‘h’ Characteristic of this collagen is the relatively high degree of hydroxylation of proline, its higher glycine content (more than 33% of the residues) and above all the presence of intramolecular disulfide bonds involving two cysteine residues close to the C-terminal region of the triple helix. Because the ratio of type I to type 111 collagen changes with age, type 111 being predominant in fetal skin, this type of collagen is many times referred to as fetal or embryonic collagen. Some interesting conclusions can be derived from observing Epstein’s data on the ratio of types I to III collagen present in the dermis of people of various ages as well as in the insoluble residue remaining after the soluble collagen has been removed. In early fetal life (16 weeks) type III collagen is more abundant than type I collagen. This coincides with the rapid rate of synthesis seen in the developing fetus.lhh At time of birth the ratio of I to III is 2.6 in the total de:rmis but 1.7 in the insoluble dermis. In fetat fife type III collagen seems to enhance significantly strength of the dermis, since insolubility and crosslinks go hand-in-hand. The possibility that type III collagen can become instantly crosslinked through intermolecular disulfide bridge formation is supported by the recent findings of Cheung et al.16’ The ability of this collagen to rapidly crosslink through this mechanism could be of great advantage during early developrnent and wound healing, where collagen is deposited at a rapid rate in an area where no previous connective tissue existed.
18
The distribution of types 1 and II1 collagen in human skin has been assayed by biochemical and immunohistochemical techniques.‘68-‘7’ Whereas biochemical determinations of collagens failed to show relative changes in dermis cut with a dermatome, immunohistochemical studies seemed to indicate that type III collagen is present primarily in the papillary dermis, just beneath the epidermis and around dermal blood vessels and appendages. The reasons for discrepancies are not clear but age-related changes in solubility or preferential masking of the antigenic determinants could be responsible. Type III collagen, when reconstituted in vitro, gives rise to thinner fibers than type I collagen.‘72 The physiologic significance of these findings is not yet clear. Although the denaturation temperatures of type III and type I collagen molecules do not differ, renaturation is more rapid and complete with the type III preparations, due to the presence of interchain disulfide bonds.‘73 This property has been used to further purify type III collagen which still retains type I collagen as a contaminant.‘74 Separation and quantitation of types I and III (Y, chains has most recently been accomplished using HPLC and HEMA 1000 Glc (a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate covalently coated with glucose).‘75 Conversion of type I procollagen to collagen is more efficient than that of type III procollaThese findings are consistent with gen. ‘37*‘76,‘77 the greater yields of type III procollagens extracted by neutral salt solutions from tissues and cell cultures, and with the estimated halflives determined in growing rabbit skin: 26 min for type I procollagen and 3.9 hr for type II1.‘78 The amino acid sequence of the (Y’ (III) chain, consisting of 1028 residues, was determined by Fietzek et a1.‘79 Following the demonstration that type III collagen was a normal constituent of skin (lO%20% of the total collagen), it has been found in many other connective tissues.“’ Normal bone matrix may be the only tissue containing type I collagen that lacks type III. It is present in variable amounts associated with type I collagen in lung, heart muscle, heart valves, uterus, nerves, liver, placenta, unbilical cord, blood vessels, spleen, gingiva, kidney, lymph nodes, sclera, and other eye structures. Blood vessels are partic-
MARCEL E. NIMNI
ularly rich in type III collagen. The actual amounts determined vary with the method of assay, but 20%-30% of the total collagen in human aorta seems to be type III.‘8’,‘82 The media seems to contain the highest proportion of type III collagen and the atherosclerotic plaque the least. Contrary to what had been observed in earlier studies,‘83 where atherosclerotic plaque was found to have more type I than III collagen, more recent work’84 has failed to substantiate such a dramatic reversal even though a small shift in favor of type 1 collagen was observed. In the latter studies, cyanogen bromide treatment solubilized approximately 80% of the total collagen in the intima and plaque and approximately 65% of that in the media. Solubilization with pepsin was much less, generally under 20%. In vitro biosynthetic studies with arterial tissues have also yielded conflicting data since both an increase and a decrease in the relative proportions of types I and III have been reported in diseased tissue.i85 It is conceivable that, as in dermal wound repair, there is in the early stages of plaque formation a preponderance of type III and later a reversion in the III : I ratio towards normal. It is of significant interest with this type of collagen that cells in fibrous joints, such as these present in the sagital sutures in young rabbit calvaria, which normally synthesize only type I collagen can be stimulated to synthesize significant quantities of type III collagen when tensile mechanical stresses are applied to this structure.‘86 Further studies are required to elucidate the sequence of events and the implications of these findings. Type I Trimer Collagen In addition to the genetically distinct types of interstitial collagens described, another molecular form of collagen, al(I)-trimer or type Itrimer has been demonstrated.‘87-‘9’ The LYI(I)trimer consists of three polymers that are genetically identical to the al-chains of type I collagen. It was originally isolated from a virus-induced tumor and its synthesis was observed in cultures of polyoma virus-induced mouse tumors.‘92 Human skin has also been shown to contain al (I)-trimer.“’ Its amino acid composition is, in general, similar to that of al(I) from type I collagen, except that hydroxylysine is increased
19
COLLAGEN STRUCTURE AND METABOLISM
and lysine is decreased. The content of 3-hydroxyproline was also increased, suggesting that both chains are derived from the same gene, but that the cotranslational or posttranslational hydroxylation of lysyl residues is more extensive. This could be a result of a slower recognition and folding of the cul (I) chains in the trimer molecule in the cisternae of the rough RER. Since the synthesis of type I trimer is an early event in the dedifferentiation of chondrocytes in culture,“” and accompanies a variety of unusual circumstances associated with cultured cells, its potential for monitoring abnormalities in the collagen biosynthetic pathway may be of significant interest.“’ IL)’ Type IV Collagen
and Other Basement
Membrane-Associated
Macromolecules
Type IV collagen is the major component of basement membranes and is generally regarded as the most characteristic of a large number of macromolecules that comprise these structures. Basement membranes are specialized connective tissue structures that underlie epithelia and endothelia and perform a multiplicity of structural and functional roles. For instance, they are involved in cell differentiation and orientation, membrane polarization, selective permeability to macromolecules, and are a target for a large number of diseases. The thickening of glomerular basement membrane in diabetics and the deposition of antibodies and complement in bullous pemphigoid, Goodpasture syndrome, and dermatitis herpetiformis are some of the associated pathologic processes. The literature on basement membrane is extensive and the reader is referred to recent reviews on the subject.‘y4~‘y8 In addition to collagen, a large number of other macromolecules, which include glycoproteins such as laminin, fibronectin, proteoglycans, and other less welldefined structures, participate in the formation of basement membranes. This discussion will focus on some of the better documented aspects of basement-membrane research: it will discuss individually the most relevant characteristics of the macromolecules that have been isolated from basement membranes while trying to provide the reader with an overview of the molecular structurc and organization of this vital connective tissue matrix. For a more detailed discussion of
the macromolecules involved, the Timpl and Martin is suggested.ly4
review
by
Type IV Collagen
Kidney glomeruli and lens capsules have been frequently used as a source of basement membranes since they can be readily isolated from other tissue elements.‘99 By treating these tissues with pepsin, Kefalides’“’ was able to solubilize and characterize a collagenous protein, now called type IV. that contains a single type of chain, the cul (IV) chain. It was suggested that basement membranes were composed of type IV collagen linked to noncollagenous glycoproteins by disulfide bonds.‘“x,‘O’ Spiro et al.,‘0’.‘04 using denaturing solvents and reducing agents, were able to extract collagenous proteins ranging in size from 20,000 to over 200,000 daltons from isolated glomeruli. These investigators proposed that glomerular basement membrane was composed of a number of dissimilar peptide subunits with collagenous and noncollagenous sequences of variable length. Type IV collagen differs from interstitial collagens in its amino acid composition (Table 2). In comparison to the interstitial collagens, it contains higher amounts of hydroxylated amino acids (including 3-hydroxyproline) and a lower content of alanine and arginine. The glycine content is less than 3370, indicating the presence of noncollagenous segments. Most of the hydroxylysine residues are substituted by glucosyl-cu( I-2)-galactosyl$ groups linked to thse hydroxyl group. Heteropolysaccharide side chains consisting of glucosamine, mannose, galactose, fucose, and sialic acid have also been identiiied as part of the type IV collagen molecules.20c~2”h Carbohydrate accounts for 10% of the mass of type IV collagen, a higher level than that found in most other collagens. A schematic diagram of type IV is shown in Fig. 19. It consists of a large triple helical domain and noncollagenous extensions that make it resemble procollagen. There are frequent interruptions of the triplet sequence Gly-X-Y within the triple-helical domain with glycine replaced by other amino acids.“‘7 Soluble forms of type IV collagen can be extracted with acidic solvents (usually dilute acetic acid) from the matrix of the EHS tumor20x and from bovine lens capsu1e.20’~“” When electrophoresed the reduced tumor col-
20
MARCEL E. NIMNI
,
-
Fig. lagen
lg.
Basement
is a major
characteristically droxyproline of
contains
and sugar
its nonhelical
ment.
A protease
region insoluble
which
membrane
component
of
significant
residues
extensions sensitive
complicates
collagen: basement
amounts
and seems
in the
problem
IV
col3-hy-
to retain
most
within of
It
of
extracellular
area is located the
type
membrane.
environthe helical
isolation
from
matrices.
lagen migrates as two chains al(lV) and ~u2(lV), with apparent molecular weights of about 160,000 and 140,000.210~2” The chains are not separated by molecular sieve chromatography and their resolution on electrophoresis may be due to compositional differences more than to differences in molecular weight. Evidence exists for a third constituent polypeptide chain. Ultrastructural studies suggested that the pepsin-solubilized collagens contain globular structures at each end of the molecule.2’2~2’3 Pepsin and other proteases are used to dissolve otherwise insoluble collagen. Considerable heterogeneity is observed in the type IV collagen solubilized by pepsin from various tissues such as glomeruli or kidney cortices, lens capsule, placenta, muscle and the EHS tumor, with polypeptides that ranged from M, = 15,000 to 140,000.‘94 Type IV collagen contains nonhelical regions interrupting the helical domain which are sensitive to various proteases, resembling that found in the triple helical domain, of the complement component Clq where it is associated with a bend in the triple helical strands.2’4 These breaks in the helix of type IV collagen may impart a similar flexibility to these molecules. The chains in the triple-stranded molecules of type IV collagen are connected by disulfide bonds. In the al(IV) chain, the disulfides are located at the N-terminal end of the triplehelical domain.2’5 In spite of the sequence peculiarities indicating breaks in the helix, type IV collagen has a thermal stability similar to that of interstitial collagens which is not altered by the presence or absence of disulfide bonds. Ultracentrifugal analysis showed a molecular weight of 450,000 t
50,000 for acid-soluble mouse type IV collagen, consistent with molecules composed of three 150,000-dalton chains. Type IV collagen solubilized by limited proteolysis precipitates when warmed but the precipitate is composed of randomly oriented fibrils lacking periodicity.2’2 Based on such observations, it was proposed that in tissues type IV collagen is arranged as a random meshwork of single molecules connected by covalent bonds.2’3 The size of the polypeptides comprising the triple helix of type IV collagen was estimated by chemical analyses to be in the range of 11 O,OOO145,000, although more recent work on type IV collagen obtained from EHS sarcoma would suggest that anything smaller than 180,000 is a degradation product, and that type IV procollagen (molecular weight around 180,000 daltons per chain) is incorporated as such into basement membrane without processing.2’5 Considering a content of about 10% carbohydrate, these estimates indicated that the triple helix is at least as long as that of interstitial collagens (290-300 nm) but could be as much as 20%-30% longer. ,94.208,216.211 Type V Collagen
Type V collagen was discovered in pepsin digests of placental membranes2’s~2’8~2’9and other tissues. Type V collagen is more soluble than other collagens, particularly at high concentrations of NaCl (333.5 M) and at neutral pH, conditions that readily precipitate the interstitial collagens. Its amino acid composition in large part resembles interstitial collagens except for a high ratio of hydroxylysine to lysine and a low content of alanine. Similar to interstitial collagens, the hydroxylysines are only partially glycosylated with glucosylgalactose or galactosyl groups. Three chains al(V), cu2(V), and &(V) formerly named B, A and C2”) have been obtained to date from type V collagen. They are similar in size, based on their behavior on molecular sieve chromatography, to the 01 chains of interstitial collagens except that the al(V) chains are larger.220.22’The chains of type V collagen show a slightly lower electrophoretic mobility compared to the chains of type I collagen.2’8~222-22sThis may be due to a higher carbohydrate content or anomalous behavior, since their SLS crystallites
21
COLLAGEN STRUCTURE AND METABOLISM
are similar in length to those formed by interstitial collagen and resemble more those of the interstitial collagens than they do basementmembrane collagen.“‘.“’ In addition to the type V chains described, cartilage contains two additional chains that have been labeled E and F.226They are similar to the other chains in their CNBr and limited V8 protease profiles, indicating nonidentity but significant homology. Table 2 summarizes the amino acid composition of the various chains isolated from human hyaline cartilage. The E and F chains together comprise about 10%-l 5% of the total cartilage collagens, and are present in approximately equal amounts, suggesting that they may be part of two different molecules. Recently a series of disulfide-bonded macromolecular aggregates have been isolated from calf skin,“’ placental tissue,228 articular cartilage,“’ chicken hyaline carti1age,230 bovine nasal cartilage vitreous, and human intervertebral discs.“’ In some respects these materials resemble type IV collagen but, on the other hand, they are distributed in many tissues that lack basement membranes. Their association with fractions which extract with type V collagen suggests that they could be extensions of this collagen. Heterotrimeric disulfide-bridged procollagen molecules corresponding to (Y1(V) and c~2(V) (B and A chains) have been described recently.23’ The peptides remaining after collagenase regions are removed with bacterial collagenase are large, and in contrast to the ones present in the interstitial collagens (types I, II, and Ill), may be retained in tissues. The relationship of these procollagen extensions to the disulfide-bonded materials to be described later (7-S collagen) is not known, and further work is required to establish any connections. Type V collagen seems to be particularly abundant in vascular tissues, where it appears to be synthesized by smooth muscle cells, although it is also present in relatively large amounts within the avascular cornea1 stroma. Antibodies specific for type V collagen are closely associated with cell surfaces. In muscle it surrounds the individual myotubules functioning as a boundary, not only between cells but between cells and connective tissue elements of the peri- and epimysium.‘34 In differentiated cartilage, antibodies to type V collagen localize around the
pericellular matrix within the chondrocyte lacunae.235 Electron immunohistochemical localization of type V collagen in rat kidney shows that it is present in the renal interstitium as individual fibers in close apposition to interstitial collagens and vascular basement membranes.23h These observations suggest that type V collagen may be a unique form of collagen that contributes to cell shape by localizing on the surface of the cells and to the formation of an exocytoskeleton, as well as to binding to other connective tissue components. 7-S Collagen This molecule was first identified in a bacterial collagenase digest of the EHS tumor matrix2” and, although collagenase resistant, it has a composition that resembles type IV collagen. Chromatographic and physical studies indicated that it is a relatively homogeneous species. A large form of 7-S collagen (M, = 360,000) can be obtained when the digestion with bacterial collagenase is carried out at 20°C whereas a shorter variant with M, = 225,000 is produced from the long form by digestion with collagenase at 37°C. Electron microscopy shows the short form as compact, rod-like particles, with a length of 29 nm; the long form shows, in addition, four thinner strands (length, 30 nm) extending from both ends of the central core in a symmetric fashion.“” Both forms of 7-S collagen have a highly stable triple-helical domain that melts at 70°C compared to 38”-41°C for other collagens. Reduction of disulfide bonds under nondenaturing conditions produces a single melting profile with T,,, = 48%. This indicates that cysteine (40-45 residues per 1,000) are located within the more stable domain. Both domains also show other differences in chemical and immunologic properties which, in part, may be due to short noncollagenous structural elements. Common to both domains is a high carbohydrate content (20%25’%), including glucosylgalactosyl groups bond to hydroxylysine, and heteropolysaccharide chains containing mainly glucosamine and mannose.237 The reduced protein yields a mixture of peptides ranging from 15,000 to greater than 150,000 daltons, reflecting proteolytic cleavage during the preparation. The data available so far seem to suggest that 7-S collagen is a unique
22
MARCEL
structure that may result from segments of type IV collagen crosslinked by disulfide bonds. Noncollagenous Basement
Proteins Associated
with
Membranes
Basement membranes also contain significant amounts of noncollagenous glycoproteins that presumably account for their positive periodateSchiff reaction.‘38
Laminin Laminin comprises almost 50% of the matrix proteins of the EHS tumor, most of which can be extracted in neutral buffers of moderate ionic strength.239 It has also been isolated from cultured endodermal and teratocarcinoma cells, in some instances in native form after collagenase treatment.‘94 Immunohistochemical localization of laminin indicates that it is abundant in all basement membranes.240 The amino acid composition of laminin distinguishes it from fibronectin.24’ It contains approximately 1295-l 5% carbohydrate.242 While the native protein migrates in electrophoresis as a narrow band, reduction of disulfide bonds produces two broad, faster migrating bands with M, = 200-220,000 and 400-440,000. Ultracentrifugal analyses indicate that laminin has a molecular weight in the range of 800,0001,000,000 daltons both in neutral buffer and under dissociating conditions (6 M guanidine). Laminin interacts with heparin, heparan sulfate,242 and type IV collagen, and links endothelial cells to basement membranes.243 Fibronectin Fibronectin is a major biosynthetic product of cultured fibroblasts.244 It is similar to the coldunsoluble globulin of human plasma described earlier.245,246 It is also produced by a variety of cells, 247including endothelial and smooth muscle cells and some epithelial cells. Immunohistology studies have shown that fibronectin is produced early in development and is associated with most embryonic basement membranes, but is not always detectable in fully developed basement membranes.248v249 It can be found as fibrillar deposits crosslinked via disulfide bonds,250 presumably involving the free sulfhydryls in the molecule, and by yglutamyl crosslinks formed from glutamyl and lysyl residues by transglutaminase. Such cross-
E. NIMNI
links can occur between fibronectin and collagen.25’ Most chemical studies have been carried out with the plasma form of fibronectin. Fibronectin from the matrix or cell surface may be slightly different from the plasma form, particularly with respect to its hydrophobic nature, tendency to aggregate and protease sensitivity.252.253 Fibronectin has a molecular weight of 450,000 daltons and is thought to be composed of two identical polypeptide chains. Each chain of plasma fibronectin contains three to four carbohydrate side chains that are linked N-glycosidically to asparagine and consist of glucosamine, mannose, galactose, and sialic acid.254,255 Fibronectin binds to a variety of macromolecules, including several types of collagens and denatured co11agen.256~258One of the major binding sites for fibronectin on the collagen molecule has a hydrophobic nature and resides in cyanogen bromide peptide al(I)-CB7, near or including the cleavage site for animal collagenase.257~259 Denatured collagen competes with native collagen in bonding fibronectin260 and, since fibronectin inhibits fibrillogenesis, it may play a role in regulating this process as well as fiber size and distribution. The region on fibronectin responsible for its collagen-binding activity is located in the NH,terminal third of each chain,26’ and since there are two such sites, it may enable the molecule to interact with collagen to form extended polymers. The collagen-like region of Clq, a subcomponent of the first complement component, can also bind to this region and coexist as complexes.2h2 Fibroblasts adhere to collagen substrates through attachment to fibronectin.260,2h3 Fibronectin has been visualized by electron microscopy of shadowed specimens as long, thin flexible strands13’ that have recently been shown to bind to actin and that have many features in common with actin-binding protein and laminin.265’266 Since the points of attachment of fibronectin to the cell surface correspond to that of the actin cables in the cytoplasm, one may speculate on its involvement in establishing a continuum between the intracellular and extracellular domains. Fibronectin also binds several glycosaminoglycans, particularly heparin,2673268 an affinity that has facilitated its extraction from tissues.269
COLLAGEN STRUCTURE AND METABOLISM
Proteoglycans
Glycosaminoglycans were first detected in basement membranes of embryonic tissues.270~27’ Polyanionic sites in native basement membranes observed by staining the glomerulus with ruthenium red were shown to disappear after treatment with heparitinase or heparinase*“.*‘* as did the permeability of the kidney and the basement membrane of the glomerulus.272~273 Heparin sulfate and hyaluronic acid were isolated after proteolytic digestion from purified glomerular basement membranes, where they accounted for approximately 1% of the dry weight of the material.“’ A heparin sulfate containing proteoglycan, BM- 1 proteoglycan, (M, = 500,000-l ,OOO,OOO) containing equal amounts of protein and heparan sulfate has been extracted from the EHS tumor matrix.275 Antibodies prepared against these proteoglycans react mainly with the protein core and localize in various basement membranes. In addition to the glycoproteins discussed above, a variety of other macromolecules have been detected in basement membranes; these are discussed in the reviews cited. lmmunochemical Reactivity of Basement-Membrane Components
Considerable interest in this subject arose when it was first discovered that individuals suffering from a variety of autoimmune diseases produced antibodies against basement-membrane components. Bullous pemphigus, chronic glomerulonephritis, and Goodpasture glomerular syndrome have attracted most attention in this connection.27h~277 Patients with the Goodpasture syndrome produce antibodies that react exclusively with alveolar and renal basement membranes, causing lung hemorrhages and renal failure. The antigenic component has been associated with noncollagenous proteins*” and the carbohydrate region of basement membrane collagen.279 In contrast, patients with bullous pemphigoid, a blistering disease of the skin, have circulating antibodies that react primarily with epidermal basement membrane. A variety of animal models have been developed to produce renal damage by injecting antigens such as intact basement membranes or glomerular extracts. These are strongly
23
immunogenic agents capable of eliciting autoimmune responses even when administered in insoluble form.280,‘8’ Rabbit antisera against type IV collagen or laminin injected into mice localizes on the renal and alveolar basement membranes and produces lesions resembling those in Goodpasture syndrome.282 Antigenic determinants to type IV collagen have been identified on the noncollagenous segments of the molecule and on its triple-helical portion.‘94 Rabbit antibodies usually recognize helical antigenic determinants. Laminin and fibronectin both elicit strong immunologic responses.239.283 The major antigenie determinants of laminin are localized in the center of the native molecule. Other antigenic determinants have been identified on a pepsin fragment, and on the reduced and alk:ylated chains. The various basement membrane proteins seem to behave as unique antigens and usually do not share antigenic determinants. In general, comparison of the specific components by absorption or immunochemical assays seems to indicate that type IV collagen, 7-S collagen, or laminin whether obtained from tumors, endodermal cells, or from authentic basement membranes are similar. Cross-reactivity was also found for fibronectins obtained from a variety of sources. Well characterized antibodies to basement-membrane components have been purified by affinity chromatograph:y and used to localize these proteins in tissues and cultured cells. At the iight microscope level, complete codistribution is found with few exceptions for laminin, type IV collagen and the basement-membrane proteoglycan. Type V collagen usually codistributes with the basement membrane proteins and also stained areas that lack basement membranes. Radioimmunoassays and enzyme immunoassays have been developed for the detection of type IV collagen, 7-S collagen, fibronectin, laminin, and laminin fragments in the nanogram range. (For a more extensive discussion the reader is referred to the review by Timpl and Martin.‘94) These immunologic techniques are becoming powerful tools that are certain to add to our understanding of the biochemistry and metabolism of normal basement membranes and their pathogenesis in a number of autoimmune, degenerative, and endocrine disorders.
24
MARCEL E. NIMNI
A CONTINUUM
BETWEEN CYTOPLASM AND
EXTRACELLULAR
MATRIX
The role of the extracellular matrix in determining cell shape, orientation, movement, and metabolic activity has generated considerable interest.284 Cornea1 fibroblasts, which have the capacity to migrate through extracellular matrices, can provide a good model to understand this phenomenon.285 The migrating cornea1 fibroblast is an elongate, bipolar cell, possessing a leading pseudopodium and a trailing cell process that, by successive attachments and retractions, seems able to change shape and location. Using antibodies to various fibrillar proteins, it was observed that fibroblasts grown in collagen gels exhibit significant ultrastructural differences from those attached to glass surfaces.285 The fibroblasts grown on glass exhibit a network of well-defined fibers, “stress fibers” that stain for actin, a-actinin, and myosin. These fibers seem to traverse the cell in all directions radiating from the cell surface along lines of stress. In contrast, the cells grown in collagen gels fail to show such patterns but, rather, show diffuse cytoplasmic staining, and actin and a-actinin but not myosin, and seem to concentrate in the cell cortex and in filipodia. It would seem reasonable to visualize how developing and adult living tissues could possess both of these characteristics at different times, and in different regions of the cell depending on their degree of activity. Even though such stress fibers have not been detected in vivo,286 it is conceivable that such structures could be found in areas of cell attachment. In many ways the phenomena described resembles the organization of microfilaments in smooth muscle cells at the time of contraction. The association of myosin to actin and a-actinin in the stress filaments, and the absence of myosin in the cortical microfilaments of the “nonstressed” cell supports this analogy (Fig. 20). The possible penetration of fibronectin into the cell membrane, its affinity for actin, its general characteristics and distribution, and the fact that the chemotaxis of the fibroblast requires a cytoskeletal organization that seems to be associated with the presence of fibronectin2” suggests that a network must exist which connects the extracellular matrix with the cell cytoplasm (and probably the cell nucleus). This
Fig.
20.
A fibronectin
cell to the surface molecule bonds collagen ment
composed
located and
located
Fibronectin sulfated
may present
cell (stress
link”4 allow
to the
disulfide
via
is attached
period.
a fibrin
binding
fibronectin
to
forms
or tangentially
attachment and
binding
clearly to
is close
a to
is a “trans-
identified actin
of organization oriented
area.’
site* and
site3 which
attach
a site on
cleavage
in this diagram
has not been
to
arrange-
The
collagenase
site. Shown which
linked ends,
site on fibronectin
in addition
in different fibers
nm
a
a dimeric
in a quarter-staggered
glycoseminoglycan
membrane
attaching
Fibronectin,
subunits
by a 68
close
is shown
fiber.
C-terminal
binding
contains
the cell attachment
(AF)
two
the placed
separated
a specific
collagen
which
of
near
molecules
involves
molecule
of a collagen
and
filaments within
the
microfilaments).
network could link the collagen-proteoglycan extracellular matrix via fibronectin, laminin, or chondronectin (or combinations of these molecules with other basement-membrane components discussed earlier) to the actin-binding protein and the microfilamentous network within the cell. The transmembrane mode of communication is not known, but transmethylation reactions involved in chemotaxis of fibroblasts supports membrane involvement.287 Epithelial cells may also be part of such a communication network, but less information is available. Evidence exists for the presence of a receptor for type I collagen on the cell surface of fibroblasts, but not on the apical surface of epithelial cells grown in culture.288 Epithelial cells do not bind directly to collagen but seem to do so through laminin.289 On the other hand, the basal cell surface of cornea1 epithelial cells seems to respond directly to collagen molecules added to the media, without the presence of intermediates, suggesting that this surface may contain a collagen receptor that interacts across the plasmalemma with the actin-rich cortical cytoskeleton.“’ This modality of interaction seems also to hold true at a more macrostructural level. The electron microscopic analysis of the extracted mus-
COLLAGEN STRUCTURE AND METABOLISM
cle-tendon junctions shows that the relationship between the terminal myofilaments and the lamina densa of the basal lamina is retained, despite the extensive extraction of the plasma membrane by nonionic detergentsz9’ Fine filaments (2-7 nm) are seen to connect the lamina densa with an electron-dense intracellular layer into which terminal actin filaments appear to insert. These fine hlaments are considered to represent an important component of the structural linkage between myofilaments and connective tissue and hence to be a significant component of the tension transmitting mechanism. Their precise nature is not known, but some part of the filaments must pass through the hydrophobic compartment of the plasma membrane and thus must be a transmembrane component of considerable tensile strength. These studies suggest that detergentextractable membrane lipids play no significant role in the transmission of tension at the muscleand that fine filaments are tendon junction, responsible for transmitting tension from myofilaments, through the plasma membrane, to the lamina densa of the basal lamina. These studies, among others, strongly support the existence of a well-defined network that links the cytoplasm with the extracellular space, a network that may be able to relay information between the intracellular and extracellular compartments, and probably from cell to cell by means of a series of interactions with components of the extracellular matrix. COLLAGEN
DEGRADATION
The changing patterns of the connective tissue matrix during growth, development, and repair following injury, require a delicate balance between synthesis and degradation of collagen and proteoglycans. Under normal circumstances this balance is maintained, while in many diseased states it is altered, leading to an excessive deposition of collagen or to a loss of functional tissue. The first animal enzyme capable of degrading collagen at neutral pH was isolated from the culture fluid of tadpole tissue.2” This was shown to cleave the native molecule into two fragments in a highly specific fashion at a temperature below that of denaturation of the substrate.“’ These fragments were characterized by electron microscopy and shown to reflect the cleavage of a native collagen molecule at a
25
specific site closer to the C-terminal end of the molecule, yielding segments of 25% and 75% the length of the native collagen molecule; the larger fragment was termed TC, and the smaller fragment TC,. Collagenolytic enzymes have been obtained following cell and organ culture from a wide range of tissues from animal species in Iwhich collagen is present.294m299 In general, these enzymes have a number of fundamental properties in common: they all have a neutral pH optima; they are not stored within the cell but, rather, are secreted either in an inactive form or bound to inhibitors. Figure 21 summarizes schematically its fundamental aspects enzyme and its mode of action. They appear to be zinc metalloenzymes requiring calcium, and are not inhibited by agents that block serine or sulphydryltype proteinases.300 They are inhibited by kelating agents such as EDTA, 1,lO-o-phenanthroline and cysteine, which may inactivate zinc and perhaps other metals required for enzymatic activity, and the zinc in the latent enzyme can be replaced by other divalent cations such as Co. Mn, Mg, and Cu.“’ Nearly all the collagenases studied so far have a molecular mass that rianges from 25,000 to 60,000 daltons. The enzymes are usually present in a latent or inactive form. In some instances they seem to be associated with the presence of a zymogen, but in most cases are bound to an inhibitory protein component that can be removed to form the active enzyme; this step is accompanied by a decrease in molecular weight. Although proteolytic enzymes have been mostly used for activation, some latent collagenases can be activated by nonproteolytic agents, such as cheotropic salts or organic mercurial compounds, suggesting that the collagenase and inhibitors, though forming a tight complex, might not be peptide-linked as in a proenzyme. Chelating agents can inhibit a metalloenzyme either by binding to the enzyme through the metal-ion to form an inactive complex, or by detaching the functional metal-ion to produce an inactive apoenzyme. The collagenase produced by rabbit bone cultures generates an inactive apoenzyme upon removal of zinc.“’ The apocollagenase is unstable and undergoes a slow transition to become irreversibly inactivated if zinc is not readily restored. The latent collagenase with the bound inhibitor is much more resistant to
26
MARCEL
E.
NIMNI
Table 3. Substances Which Have Been Shown to Modulate Collagenase Activity ACtWatOrS
lnhtitors
Tryps18’*
NEUTRAL
EDTA3”
Plasmin
COLLAGENASE
Lisosomal
PROTEAYS
I
Proteinase?
Metallo-Proteinase305 Mast
I-lo-o-Phenanthroline3” a 2-Macroglobulin3’z~3’3
Cell-Proteinase306
Senne
Proteinase”07
Cartilage
Protein”‘4
Nal”’
Platelet
NaSCN”’
Rheumatoid Bone
4-Aminophenyl
Mercuric
Acetatesw
Factor
Culture
Human
43’s
Synovwm3’6 Facto?”
Skin Fibroblasts””
Tumours3’9.320
Chloromercuribenzoate, Mersaly13” Cystww.
Oxidized
Gluta-
Human
Tendor?’
throne”’ Relaxin3’5
Cysteinezg’ Reduced
Glutathionezg7
Fibronectir? Bovine
/==r;‘O& ____.--.._. @ I’
:. -,
Fig.
21.
degradation
Sequence
of
of collagen
,
. . . . . .._..s
I
J
events
fibers
,f
_
- : d .
,
which
by the
,’
I
can
enzyme
lead
to
the
collagenase.
(1 I A variety of factors have been described which stimulate connective tissue cells to synthesize collagenase. glycosydases and neutral proteases. (2) The proteoglycan degrading enzymes remove the mucopolysaccharides which surround collagen fibers and expose it to collagenase. (31 Inactive collagenase is secreted. (4) The enzyme is usually found in the extracellular space bound to an inhibitor. 15) An activating enzyme removes the inhibitor. 16) Glycosidases complete the degradation of the proteoglycans. (7) The active collagenase binds to fibrillar collagen. (8) Collagenase splits the first collagen molecule into two fragments (TC, and TC.) which denature and begins to unfold at body temperature. The enzyme now moves on to an adjacent molecule. (9) The denatured collagen fragments specific
are
now
neutral
susceptible proteases
to
other
proteases.
degrade the
collagen
(10)
Non-
polypep-
tides.
irreversible inactivation than apocollagenase, indicating that the inhibitory portion is a stabilizing factor. There seems to be critical dependence of collagenase activity on the concentration of free zinc (Zn+ +). In excess, zinc can act as an enzyme inhibitor. Table 3 lists some of the agents and conditions that have been shown to modulate
Aorta323
the activity of collagenase by shifting the equilibrium between active and latent forms, by altering the availability of cations essential for enzymatic activity, or by modifying the redox potential around the enzyme inhibitor complex. The cells that synthesize collagenase are influenced to a great extent by the environment in which they live. This includes the permanent resident cells of the connective tissues and adjacent structures and the migratory cells that accumulate as a result of injury, inflammation or immune phenomena, as well as the products secreted by these cells. Epithelial cells and factors secreted by such cells may also play a significant role in the development and remodeling of connective tissues by virtue of their ability to regulate collagenase production by the mesenchymal cells.324x325 Media conditioned by epithelial cells from the adult rabbit cornea is capable of both stimulating and inhibiting the production of latent collagenase by stromal cells from the same source.326 Cytochalasin B is necessary for these effects to be manifested, and optimal stimulation is engendered by low density epithelial cell cultures. A large number of soluble factors have been shown to stimulate collagenase production by mesenchymal cells in culture, among them the prostaglandins (PGEY~),~~~ particularly when combined with endotoxins and proteolytic enzymes’** such as plasmin, trypsin, chymotryp-
27
COLLAGEN STRUCTURE AND METABOLISM
sin, pancreatic elastase, papain, bromelain, thermolysin or cu-protease, but not thrombin. As mentioned earlier, proteases may act not only as stimulators but may activate latent collagenase by acting on the zymogen or specifically degrading the inhibitor bound to the enzyme. Other stimulators of collagenase production are heparin,32y CAMP,“’ collagen,33’.332 type II collagen-derived CNBr peptides,33’ and cytocholasin B.‘-‘4 The stimulatory effects of soluble collagen on collagenase production by cultured fibroblasts is greatly enhanced by a human blood mononuclear cell factor (interleukin 1)332 that has recently been shown to be a potent stimulator of fibroblast proliferation.335 In addition, phorbol ester,j3’ thyroxine,337 colchicine,33x latex beads,33y and hydroxyapatite and calcium pyrophosphate crystals340 have been shown to stimulate collagenase production by cultured cells. Glucocorticoids34’ and progesterone342 inhibit collagenase production, whereas nonsteroidal antiinflammatory drugs inhibit prostaglandin Ez formation but do not significantly affect synthesis of collagenase.343.344 Retinoids that are known to enhance epithelial cell differentiation and decrease proliferation of such cells345.346inhibit collagenase production by rheumatoid synovial cells.‘47 Both the naturally occurring all-transretinoic acid and the synthetic compound 13cis-retinoic acid completely inhibited collagenase production and reduced prostaglandin E, synthesis by monolayer cultures of human rheumatoid synovial cells, at concentrations which were nontoxic to the cells. Enzyme
Specificity
Mammalian collagenases display a great deal of specificity by hydrolizing a single polypeptide bond on each (Y chain of the native triplestranded collagen helix. A significant amount of work has been devoted to understanding the unique characteristics of the cleavage site. The cleavage site of the (Y 1 chain was identified by electron microscopy34x and later by sequence analysis.324.349.35n
Although no conclusive physico-chemical explanation is available to describe the specificity of the cleavage, there are indications that the helix in that region is thermodynamically less stable. The actual bond cleaved in all species studied is a Gly-Leu or Gly-lle link (Fig. 22).
Fig. genase tide
22.
bond
action 775-776
Cleavage
showing
(Gly-lie)
of the
which
enzyme.
is highly
site of chick-skin
the amino The
conserved
acid sequence is selectively sequence from
collagen around
by collathe
hydrolyzed around
species
peptide
pep-
by the bond
to species.
There are slight differences in the amino acid sequence surrounding the scision site: these may account for the differences in the rates at which various collagens are degraded.“’ “’ Type I and III collagens seem to be digested at approximately similar rates, while type II collagen exhibits the slowest rate of hydrolysis. Purified mammalian collagenases do not substantially degrade type LV collagen.3s4 “’ On the other hand, a collagenase derived from a metastatic murine tumor cleaves type IV collagen but not type I, II. 111, or V.‘“’ Proteinase from human polymorphonuclear leukocytes35x,‘59 from mast cells360 and a metalloproteinase from the culture fluid of a metastatic mouse tumor”’ can also cleave type IV collagen. Type V collagen can be degraded by a metalloproteinase secretesd by activated macrophages3h2 as well as enzymes from mast cells35y and tumor cells.‘“” From the culture media of rabbit bone explants, several metalloproteinases have been identified with abilities to degrade proteoglycans. laminin, fibronectin, pepsin-solubilized type IV collagen, and insoluble type IV collagen. A fraction capable of degrading gelatin was also able to solubilize type V and the la, 2n, and 3tr cartilage collagens.3”4 The presence of such a large variety of enzymes in a single preparation support:j the idea that normal connective tissue cells are able to produce a family of metalloproteinases with the combined abilities to digest all the native components of the extracellular matrix. The binding of human skin fibroblast col#agenase to reconstituted collagen has been recently studied in detail.“’ The enzyme interacts tightly with the collagen fiber and appears to remain bound to the macromolecular aggregate during the degradation process. Approximately 10% of the collagen molecules in the reconstituted col-
28
MARCEL E. NIMNI
lagen appears accessible for binding, in close agreement with the theoretical number of molecules estimated to be present on the surface of the fiber. The in vitro data obtained seemed to indicate that digestion proceeds until completion without the enzyme returning to the solution but, rather, hopping from one molecule to another. These observations also would explain why collagenase activity is enhanced when the enzyme is exposed to dilute suspensions of reconstituted polymeric collagen rather than compact fibers of large diameter.366 This mode of action of the enzyme, and its modality of handling the substrate, may explain the low turnover numbers observed (25 molecules of collagen cleaved per enzyme per hour), one of the lowest turnover numbers associated with an enzymatic reaction. It is possible that in vivo the rates could be even slower, due to the presence of enzyme inhibitors and because the active enzyme may have to compete with the latent enzyme for binding sites on the collagen fiber.367 Another factor that seems to slow down the breakdown of collagen is the presence of crosslinks. The introduction of artificial methylenebridges with formaldehyde368 or of native-crosslinks by the use of purified lysyl oxidase369 increases the resistance of collagen to collagenase degradation. Native collagen fibers crosslinked by glutaraldehyde cannot be digested even by bacterial collagenase.370 Collagen from individuals of increasing age becomes more resistant to enzymatic digestion, suggesting that an agerelated accumulation of crosslinks may be responsible.37’ It is therefore possible that crosslinking of collagen plays a role not only in generating mechanical stability to the fibers but also in the regulation of collagen turnover in vivo. Cell to Cell Interactions Collagenase Activity
and the Modulation
of
Tissue culture techniques are of paramount value in understanding the nature of the enzymes involved in connective tissue degradation. The interactions of a variety of cells seem to play a major role in this connection and to be part of a major regulatory mechanism.372.373 Numerous types of cells are present within the connective tissues, including, in inflammatory situations, the invading polymorphonuclear leukocytes and
macrophages. Each of them might participate in the production of the enzymes responsible for collagen and proteoglycan degradation or be the source of soluble factors that may activate the resident cells to produce such catabolic enzymes. Multinuclear giant cells are found frequently in fixed sections of rheumatoid synovium.374 These are usually located in the synovial cell layers near the surface, and not in the deeper layers. Ln addition, when rheumatoid cells are put into monolayer culture, multinucleate cells are observed to form in vitro.375 Many dendritic cells in rheumatoid synovial cell cultures have more than one nucleus. A lymphokine, a soluble factor produced by activated lymphocyte/monocytes, has been characterized that induces multinucleate cell production in cell culture. Because multinucleate cells often are found in areas of connective tissue turnover, Brinckerhoff and Harris376 explored the possibility that these cells could have functional capabilities for increased proteolytic enzyme secretion. Using a one minute exposure to polyethylene glycol (PEG), rabbit synovial fibroblasts growing in monolayer culture could be induced to fuse with one or more neighboring cells, producing a final count of up to 40% polykaryons (cells with two or more nuclei) per culture. After this time, and for up to 28 days, culture in the absence of serum, collagenase in latent form was released from cultures containing giant cells in quantities tenfold that of control cultures. The cooperation between lymphocytes, macrophages and fibroblasts to cause the degradation of connective tissue matrices has been reviewed by Vaes et al.“’ It has also been reported that mononuclear cells isolated from whole human peripheral blood and fractionated by erythrocyte rosetting into T- and B-lymphocytes can secrete collagenase into the media. Removal of the macrophages from the “non-T cells” did not seem to affect collagenase production by the lymphocytes3’* The complexity of these interactions and an excellent chronological review of the development of the different findings that contribute to our understanding of this field is provided by Krane.379 The stimulation of adherent fibroblasts derived from human synovium to produce collagenase can be modulated by a variety of soluble
29
COLLAGEN STRUCTURE AND METABOLISM
cell factors, among which a monocyte-macrophage-derived substance of a molecular weight in the range of 14,000-24,000 daltons termed MCF (mononuclear cell factor) seems to be the most active.“’ Human MCF has many properties in common with murine interleukin 1 (lymphocyte activating factor). Interactions of monocytemacrophages with other cells, such as T-lymphocytes or with immune complexes (or Fc fragments of IgG) or with macromolecules of the extracellular matrix, such as collagen (type II and III), further modulate cell function by stimulation of MCF. This results in changes in PGE, and collagenase production, cell proliferation, and collagen and fibronectin synthesis. Cultured chondrocytes can be stimulated to produce collagenase by a protein fraction (corresponding to a molecular weight of about 14,000 daltons) obtained from endotoxin38’ and lipopolysaccharide-stimulated382 macrophages. Human blood mononuclear cells in short-term culture secrete a factor that induces degradation of matrix proteoglycans and collagen from cartilage explants in culture.383 This matrix degrading activity seems to be associated with monocytes, is stimulated by PHA, and seems to possess characteristics similar to factors described earThe relationships of these factors to lier. ‘7x.38’.384 MCF and interleukin I have not yet been defined. Using another experimental approach, thioglycollate-elicited mouse peritoneal macrophages were cultured in contact with an extracellular matrix produced by rat smooth muscle cells. Both the live macrophages and their conditioned media hydrolyzed glycoproteins, elastin and collagen.3x5 The glycoproteins in the matrix markedly inhibited the rate of digestion of collagen and elastin. When plasminogen was added to the media, activation of plasminogen to plasmim resulted in hydrolysis of the glycoprotein components which then allowed enhanced degradation of the fibrous protein to occur.38h Degradation
of Cartilage
Since degradation of the proteoglycans in the cartilage matrix seems to precede the breakdown of collagen, it is important to understand this process before focusing on the collagenolytic events. A recent symposium devoted to osteoarthritis, the proceedings of which were published
in this journal, devoted a significant effort to this problem.“’ Although earlier work had focused on the participation of lysozomal enzymes or acid hydrolases, 388~390it is now apparent that these enzymes cannot have direct access and be effective in the in situ degradation of the extracellular proteoglycan matrix, particularly in tissues such as cartilage, where the chondrocytes have limited mobility. Currently most studies are focusing on neutral proteases.39’.392 Sapolsky et a1.“j3 extracted from human articular cartilage a metaldependent neutral protease that digested proteoglycans at pH 7.25; similar enzymes are present in cultures of articular chondrocytes394m3q’7 and polymorphonuclear cells.398 Another approach to understanding the degradation of cartilage matrix is illustrated by the observation of Fell and Jubb399 that synovial tissue in organ culture can degrade living cartilage. It was later shown that the synovial tissue was producing an acidic protein of about 17,000 daltons that was stimulating the chondrocytes to resorb the matrix proteoglycans.400,40’ Other connective tissues, such as the sclera, also produce this protein, which has been termed catabolin. It is a heat stable protein (70°C), pI = 4.6, and its synthesis can be suppressed by cortisol. The relationship of catabolin to other factors or to cytokins, proposed to regulate connective tissue catabolism, is not yet clear and there is yet no direct evidence that catabolin causes matrix resorption by stimulating the chondrocytes to secrete proteinases. Cellular
Events in the Degradation
of
Cartilage
Mononuclear phagocytes generated by the in vitro differentiation of rabbit bone marrow cells, when cultivated in a serum-free medium with cartilage, are able to degrade the proteoglycan matrix in a matter of a few days402 This proteoglycan degradation is associated with secretion by the cells of a metal-dependent neutral proteinase. The enzyme is not stored in the macrophages but, rather, is synthesized just before secretion. Under these conditions, no significant degradation of collagen was observed. Rabbit skin or synovial fibroblasts in culture are also able to degrade cartilage proteoglycans under similar circumstances due to the secretion of a rnetaldependent neutral proteinase similar to that produced by the macrophage, but after a few days
30
will degrade the collagen as well, by secretion of collagenase.403.404 Wahl et al.4o5 observed that soluble lymphocyte products (lymphokines) secreted by mitogenor antigen-stimulated spleen cells, enhanced the production of collagenase by macrophages. Since macrophages produce both collagenase and substances that will activate other mesenchymal cells to produce this enzyme, the sequence of events remains uncertain. Hauser and Vaes406 observed a further activation of the macrophage-fibroblast collaboration in the degradation of cartilage; they noted that when rabbit spleen cells are stimulated with either antigens or mitogens they release soluble products that, in turn, stimulate macrophages.4073408These lymphocyte factors have two effects on the macrophages: first, they stimulate the secretion of proteoglycan-degrading neutral proteinases and of collagenase by the macrophages themselves; second, they enhance the production by macrophages of factors that enhance the rate of collagen degradation achieved by fibroblasts. These observations illustrate how macrophages and fibroblasts may successfully collaborate in the degradation of cartilage or other connective tissue matrices made of collagen and proteoglycans and how this activity can be initiated or maintained by properly stimulated lymphocytes. This concept supports the view that macrophages and fibroblasts may be among the main effector cells responsible for an immunologically controlled destruction of chemically altered connective tissues. Chondrocytes do not usually synthesize collagenase in tissue culture, but may be able to do so in the presence of a factor secreted by endotoxin-treated peritoThese observations, couneal macrophages.38’ pled with the fact that human osteoarthritic cartilage in culture may release collagenase,409 indicate that chondrocytes have the capacity to degrade matrix collagen. Nevertheless, the mechanism that causes cartilage destruction in joints is not clearly understood. Removal of proteoglycans seems to be an essential prerequisite for collagen to be actively degraded. While neutral proteases made by the chondrocytes are linked to this initial event, the collagenolytic pathway remains more elusive. The presence of various cell types that can manufacture collagenase, as well as the abundance of inhibitors that can modulate the activity of this
MARCEL E. NIMNI
enzyme, makes this pathway to understand.
URINARY
EXCRETION
DEGRADATION
even more difficult
OF COLLAGEN PRODUCTS
Because of the relatively large amounts of hydroxyproline (12%14%) present in collagen, the assay of this amino acid in body fluids has raised considerable interest. Hydroxyproline is excreted in a peptide-bound form: less than 5% is excreted as free hydroxyproline.4’0,4” Studies using radioactive gelatin and the synthetic dipeptide L-prolyl-L-hydroxyproline showed that the major routes of excretion of collagen metabolites are via lungs and kidneys. Approximately 75% of the peptides released by the degradation of collagen are hydrolyzed to its constituent amino acids and catabolized to carbon dioxide with excretion by the lung; the remaining 25% of the peptides released are excreted in the urine. The prolyl-hydroxyproline peptide appears to be excreted almost quantitatively in the urine, presumably because of its resistance to peptidase activity.4’2 The normal values for urinary hydroxyproline excretion in humans depend on age, sex, body size, and dietary intake of hydroxyproline. When the values are determined on gelatin-free diets and corrected for body surface area, the effect of these variables can be minimized and normal values, reported by a number of laboratories using adequate methods for assays of urinary hydroxyproline, are in agreement413 Altered urinary hydroxyproline values are found in conditions affecting collagen metabolism systemically, such as endocrine disorders, or in conditions accompanied by relatively extensive involvement of bone collagen. Small local changes in connective tissue do not usually cause significant changes in hydroxyproline excretion.4’4 In addition, urinary excretion of hydroxyproline has been reported to be increased in growing children and in many pathologic conditions such as acromegaly, Paget disease of bone, hyperthyroidism, Marfan syndrome, hyperparathyroidism, and primary and metastatic bone tumors. Other conditions that may be accompanied with altered hydroxyproline values include certain skin diseases, thermal burns, some conditions associated with changes in hormonal levels, and administration of certain drugs.4’5
31
COLLAGEN STRUCTURE AND METABOLISM
In most of the above-mentioned conditions, the changes in the excretion of free hydroxyproline and peptide-bound hydroxyproline are of the same magnitude, and the ratio of free hydroxyproline to total urinary hydroxyproline changes only slightly. However, during early infancy, and in hydroxyprolinemia, hyperparathyroidism, severe uremia, and some aminoacidurias the ratio of free hydroxyproline to total urinary hydroxyproline is significantly elevated. A comment should be made regarding the presence of hydroxyproline in Clq, one of the components of the complement system.4’h This compound, a glycorprotein with a molecular weight of approximately 400,000, daltons contains I9 residues of glycine and two to three residues of hydroxyproline per 100 amino acids. The plasma concentration of Clq in normal individuals is around 6.8 mg/kg, and the daily plasma-pool turnover rate is around 4.5 mg/kg. In certain diseases, however, the turnover of Clq can increase threefold to ninefold over normal. In any case, one should be aware of this additional source of hydroxyproline in body fluids. If the hydroxyproline-containing peptides derived from Clq are metabolized in the same fashion as gelatin,“7,4’x one would find each day in urine about 2 mg of OH-proline originating from Clq, which could represent about 15% of the amount of daily hydroxyproline excreted at the lower end of the normal spectrum. Clq would therefore contribute much less to the total output of younger people or those with connective-tissue diseases, whose excretion is elevated. Nevertheless, under circumstances where Clq is turning over rapidly, the implications of such a contribution should be kept in mind. The glycosides, galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine that are characteristic moieties of the collagen molecule, have been identified in urine and used as criteria of collagen degradation.4’“~422 The usefulness of this technique is based on the fact that these hydroxylysine bound glycosides are not metabolized and their total body pools are small relative to their daily renal clearance.42’ Since different collagens contain various proportions of these two glycosides, attempts have been made to quantitate their ratios in the urine in an attempt to identify their tissue of origin. It should be kept in mind that Clq has a high proportion of its hydroxylysine glycosylated4’4 and may contribute up to
50% of the total Hyl-Gal-Glu found in the urine.423 Recently, desmosine, the lysyl-derived crosslink is elastin, and dehydrohydroxylysinonorleutine and 3_hydroxypyridinium, the collagen crosslinks, have been identified in human urine and in patients with Paget disease.4’s The amounts of 3-hydroxypridinium excreted in 24 hr may vary with age; 3-hydroxypyridinium values of leucine equivalents per 24 hr in a 13year-old female and 22-year-old male were 55 1 and 85 nmoles, respectively. The fact that a significant amount of the collagen synthesized by cells may be degraded intracellularly before secretion, and that such degradation products will also appear in the urine,42h further complicates the phenomenon. It has been estimated that between 10%6OQl of the newly synthesized collagen can be degraded intracellularly by a variety of cell types.“‘!’ This pathway can serve as a regulatory role to determine the rates, types, and distribution of the collagen secreted. Abnormalities in the control of such intracellular degradation should be investigated in conditions which affect the quality and quantity of collagen deposited in the extracellular matrix, such as fibrosis, diabetes mellitus, and scurvy. CROSSLINKING Crosslinking renders the collagen fibers stable, and provides them with an adequate degree of tensile strength and visco-elasticity to perform their structural role. The degree of crosslinking, the number and density of the fibers in a particular tissue, as well as their orientation and diameter, combine to provide this function. Crosslinking begins with the conversion to peptide-bound aldehydes of specific lysine and hydroxylysine residues in collagen, a reaction that was illustrated earlier (Fig. 10). It involves the oxidative deamination of the t-carbon of lysine or hydroxylysine to yield the corresponding semialdehydes (allysine or hydroxyallisine) and is mediated by lysyloxidase.42x4” Since this enzyme remains tightly bound to collagen, purified by conventional precipitative methods, incubation of such collagen at 37°C neutral pH and physiologic ionic strength, will cause additional aldehydes to form on the molecule.4”’ This effect can be enhanced by tissue extracts that possess lysyl oxidase activity or by the purified enzyme. This
MARCEL
32
strong affinity of lysyl oxidase for collagen has been used for purifying the enzyme by affinity adsorption.432 Enzymatic activity is inhibited by P-aminopropionitrile, chelating agents such as EDTA and D-penicillamine, and isonicotinic acid hydrazide and other carbonyl reagents. Lysyl oxidase exhibits particular affinity for the lysines and hydroxylysines present in the nonhelical extensions of collagen, but can, at a slower pace, also alter residues located in the helical region of the molecule.433 It has been proposed that it binds initially to the carboxyl-terminal nonhelical end of the fibrillar collagen since it has been observed that the t-NH, groups in this region are the first to be converted to aldehydes.434
marized in Fig. 24. Alkaline hydrolysis of the reduced collagen yielded the reduced form of the aldol-condensation product that generates intramolecular crosslinks (Fig. 25). Although these experiments provided us with basic information on the crosslinking precursors and their early reaction products, they did not allow all of the intricacies of the crosslinking process to unfold. Simple Schiff bases are acid labile, which means that dilute organic acids, such as acetic acid should be able to cleave them. Yet we know that a significant amount of collagen is insoluble in this reagent, particularly when the tissue originates from older individuals. Crosslinking
Intramolecular
and Intermolecular
Crosslinks
Inhibitors of crosslinking, such as BAPN and D-penicillamine, made it clear that aldehydic groups were essential for formation of crosslinks (Fig. 23). Additional progress was made when it was discovered that agents that can add across double bonds or stabilize Schiff bases, such as CN-, and NaBH,, could decrease the solubility of collagen and increase its mechanical the use of tritiated strength.435-‘38 In particular, NaBH, became useful to identify and characterize reducible crosslinks. Analysis of the radioactive compounds showed the presence of aldehydes that have been converted to the parent alcohols (e.g., hydroxynorleucine) and reduced Schiff bases (lysinonorleucine, hydroxylysinonorleucine, and dihydroxylysinonorleucine).439 These fundamental chemical reactions are sum-
Formation
In Vitro
Further insight into the mechanism linking has been provided by studies
of crossin which
R
Y q=o
4=--o - C= 0 H
bH-(CH,),
/I/H
NH,- CH,-(CH,),-
+
k
k
f
I
?
F=O
c=o
L;H-OfzkCH=N-CH,-(CHzk+H
SCMFF
YH R
BASE
YH R
Y
c=o
c=o
~H-/cH~)~-C~
-NH-CH,-(CH,/!!_
tH iH
i/H k
k L Ysbvo/vo~L
Fig. Formation
lar crosslinks occur sation
groups
I collagen.
in the nonhelical reaction
aldehydes links
in type
of intramolecular
on
between
within the
regions
a single
other
of lysine
hand
present
intermolecu-
Intramolecular
and involve
lysine
and
or
molecule. involve
in different
crosslinks
an aldol conden-
hydroxylysine Intermolecular aldehydes molecules.
and
derived crossc-amino
$H NH
F
Fig. 23.
E. NIMNI
24.
Schiff
lysine-derived This interaction
stabilization stable.
The
reaction
and
is primarily
intermolecular hydoxylysine.
base
aldehyde
EUCIM
crosslinks
responsible and
It is acid-labile to
render
reduced
lated derivatives)
the
forms
occurring
an unmodified can
and seems
can be isolated
after
lysine
to require
fiber
(lysinonorleucine
either heat-
of of
further
and
acid-
or its hydroxy-
reduction.
a
group.
for the formation
involve
collagen
between
t-amino
33
COLLAGEN STRUCTURE AND METABOLISM
R
R
&O
;T=o
k-:-CH,I,-C=O
;vH
+
O--C-(CH,I,-hH H
H
k
I
NH R
1
9.
c=o
R
?=a
c-rcH,,,-C=CH-CH,-(~~-~H f?=o H
;vH K
NH k
2
,
4
6
8
Incubation Period (Weeks)
ALDOL Fig. 25.
kx-j¶ UNSATURATED The
aldol
aldehydes
located
molecule.
This product
reaction
involves
in the nonhelical generates
ALDEHYDE) two
extensions
lysine-derived of a collagen
the intramolecular
cross-
link.
Fig.
The
collagen
NaBsH,
to identify
insoluble
reduced
the
bases
amine,
coincides insoluble
crosslinks
bases
obtained
at 37°C.
aldehydes
Schiff
hydroxylysinonorleucine to become
fibers,
is incubated
sors. The precursor the
Schiff
neutral salt-soluble collagen obtained from rat skin was aged in vitro. The collagen fibers become increasingly insoluble as a function of time; within the first two weeks some of the collagen can be redissolved by simply cooling the preparation to 4OC, but later the fibers become insoluble. Following this period, and for another two weeks, some of the collagen can be solubilized by 0.5-M acetic acid or 0.2-M cysteamine, pH 7.0,440 but later these agents are unable to solubilize collagen. In order to interpret these findings, the various collagen fractions were reduced with NaB3H4 and analyzed on an amino acid analyzer for tritium incorporation. After removal of the uncrosslinked collagen by cooling to 4”C, the residue, which remained insoluble, was treated with NaB3H4, various amounts of Schiff bases, in addition to reduced aldehydes and aldol condensation products, could be detected.43’,44’ The Schiff bases identified earlier as lysinonorleucine44’ and hydroxylysinonorleucine?4’ Increase rapidly during the first two weeks of incubation (Fig. 26). In collagens from soft tissues there is a good correlation between the amount of /3 components seen after denaturation and the concentration of cu-P-unsaturated aldol. In bone, this point is less clear, and it is possible that some fl components arise from intermolecular crosslinks of the Schiff base type. Removal of the N-terminal nonhelical peptides by limited
26.
soluble
and
appear
as
(HLNL).
when
The
M acetic
native
be labeled
crosslinking
decrease
with
precur-
as OH-norleucine
lysinonorleucine
with the ability in 0.5
can
ILNL)
and and
in reducible
of the maturing
fibers
acid and 0.2 M cyste-
pH 7.0.
cleavage with CNBr or by enzymes depletes soluble collagen of the nonhelical aldol crosslinks and generates slightly shortened (Ychains, which migrate as do the corresponding a(! and cy2 chains in the parent molecule. The exact significance of intramolecular crosslinking (aldol condensation product) is not known but, as we shall see later, it may proceed to become part of a tetra functional inter-intra-molecular crosslink such as His-OHmerodesmosine. After reaching a maxima, the concentration of reducible crosslinks begins to decline, reaching almost zero after eight weeks of incubation. This decline in reducible crosslinks correlates with increasing insolubility of the collagen fibers, indicating that acid stable and nonreducible crosslinks are formed. In general, lysine-derived crosslinks seem to predominate in soft connective tissues such as skin and tendon, whereas hydroxylysine-derived crosslinks are prevalent in the harder connective tissues such as bone, cartilage, and dentine, which are less prone to yield soluble collagens. In addition to lysinonorleucine (Fig. 24), a compound first identified in elastin, other Schiff bases have been identified (Fig. 27). These are hydroxylysinonorleucine, which results from the association of a lysine-derived
34
A.
B.
MARCEL E. NIMNI
....CH,-
CH,-
r
N- CH-CH, .,..
lminium form
Y
lminium form
..__CH,-$-Ci$-NH-CH,-?i-CH, 0 OH
. ...
Keto - imina
Fig. 27. Two types of crosslinks are illustrated. (A) A crosslink between a lysine from one collagen polypeptide and a lysine derived aldehyde from another. The Schiff base formed is in the iminium configuration and the double bond is susceptible to cleavage by acetic acid and HCI. (B) A crosslink formed between a hydtoxylysine from one molecule and a hydroxylysine derived aldehyde from another molecule. This crosslink is prevalent in bone and in collagens rich in hydtoxylysine. The initial imminium form contains a hydroxyl group conjugated with the N=C bond. This can form the enamine which readily tautomerizes to the ketoimine. The tautomeric form is stable to acid hydrolysis and explains the insolubility of bone collagen.
aldehyde with a hydroxylysine residue, and dihydroxylysinonorleucine, which originates from a hydroxylysine-derived aldehyde and an unmodified hydroxylysine residue. These hydroxylysinecontaining crosslinks seem to be the most prevalent intermolecular crosslinks in native insoluble collagen. The chemical structures of the principal-lysine and hydroxylysine-derived crosslinks identified in collagens are shown in Fig. 28. Recently, several other crosslinks have been identified and their location established. These more complex polyfunctional crosslinks can con-
tain histidine, or can result in the formation of pyridinium ring structures. The earlier work in this connection has been reviewed and many of the possible crosslinking entities discussed in great detail.444 A “post-histidine” peak443 that relates to the aldol-condensation complex in a product: precursor fashion turned out to be the polyfunctional crosslinking compound histidinohydroxy-merodesmosine44s (Fig. 29). This compound seems to result from a Michael addition of histidine N to the 0 carbon of the /3 unsaturated aldehyde (aldol) followed by condensation with the e-amino group of hydroxylysine to form the iminium compound dehydro-His-OHMerDes.446 Reduction with NaBH, converts the Schiff base to the secondary amine, His-OHMerDes, which further stabilizes the crosslink. A recent detailed study of this crosslink has shed significant light on its modality of formation and its location within the collagen fiber.447 Following cleavage of this crosslink (at pH 4.3) into its precursors, its formation was investigated by re-equilibrating collagen fibers at pH 7.4 and 37%. The pepsin digest of the insoluble collagen, previously iodinated to destroy all the histidines except the one involved in formation of the crosslink, can give rise to CNBr peptides containing the crosslink that can then be isolated using chromatographic procedures. The structure of this compound, indicating the relative position of the four amino acids that combine to form it, is shown in Fig. 29. r-----------
;NHy$H-COOH
1
NH,-CH-COW :I 1’ (CH,), : : CHOH ’ , CH; ;mt “1” CH,
,
$H~H
WC), NH;-LH-C&H HYDROXYLYSINONORLEUCINE
MHYDROXYLYSlNONORLEUCCHE
Intermolecular crosslinks involving one lysine Fig. 28. and one hydroxylysine residue (hydtoxylysinonotleucine) and two hydtoxylysine residue (dihydtoxylysinonotleucine). The dotted lines enclose the contributing amino acids and the asterisk denotes the insertion of radioactive ‘H atoms used for the purpose of reducing and stabilizing the double bond as well as for identification.
HISTIDINO-
HYDROXYMERODESMOSINE
A tettafunctional covalent crosslink which Fig. 29. bridges three different molecules. Two of the residues are part of an aldol condensation product (intramolecular crosslink) and therefore associated with one single molecule.
35
COLLAGEN STRUCTURE AND METABOLISM
The peptides that contained the dehydro-HisOHMerDes, retained the histidine and had a composition similar to that of LYlCB5-8 located close to the N-terminus of the 01 chain. A diagram showing the location of the crosslink dehydro-histidinohydroxymerodesmosine in bovine skin insoluble collagen is shown herein (Fig. 30). Two molecules in a quarter-staggered arrangement displaced by 4 D periods show how 2 carboxyl-terminal nonhelical peptides can condense via an aldol reaction to form a Michael adduct with histidine in position 89. The aldol hi&dine, through its unreacted aldehyde group, is free to react with a hydroxylysine residue to complete the crosslink. More recent work emphasizes the need for the amino acids in question to be properly positioned for this tetrafunctional crosslink to form. A genetic variant of type I collagen, which contains a sequence transposition adjacent to histidine, is present in small amounts in calf skin. The collagen fibers that possess this microheterogeneity are not able to complete the formation of this crosslink, but can only form a trifunctional crosslink hydroxyaldolhistidine.448 Because of the pos-
sible significance of the histidine-containing crosslinks, more work is necessary to define its biosynthesis, distribution, and age-dependency. Following the observation in 1977 that achilles tendon collagen contained a naturally fluorescent crosslinking amino acid (subsequentl:y identified as a 3-hydroxypyridinium derivative), considerable interest has arisen in connection with this compound.449~5’ This crosslink is particularly abundant in hyaline cartilage, which may be the richest source of hydroxypyridinium crosslinks, since it contains about one residue per collagen moleculc,4i’ an amount that represents more than 40 times the dehydro-dihidroxylysineonorleucine present, the main reducible Schiff base type crosslink. The structure of this p:yridinium compound is shown in Fig. 3 I. It seems to arise from the condensation of two hydroxylysyl aldehydes and a hydroxylysine residue. Nevertheless, its biosynthesis may proceed. as will be discussed, through the interaction of pre-existing bifunctional crosslinks, explaining the observar-----------l
i HN L_:,\CH
,COOH .-J
1 I------
-l
/’ &j,J,____----+JH,
/’
riiq ,&$H,CH,-CH r-------l I if’;! I II I Hei+ ICH ’ L___+--&__ J INI
1
‘COOH i L_____l
..a, 3 D SHIFT WTERMINh.L Fig.
30.
molecules known
Schematic aligned
intermolecular residues
the
first
C-terminal
sites
are
the
hydroxylysyl
residue a
gN of
an
927 crosslinks
2 chain.
The
an a 1 chain of another
also
form
similar
an
residue
C-terminal
aldol
type of
crosslink
via a Michael
crosslink
between
helical
region
observation
an
that
1
chain
residue 17’
can
gN. The
residue
residue
Histidine
87
with
89 from adds
to
a
the this
an intermolecular 927
and bone
end
220
supporting
in the collagen
PYRIDINOLINE
17’may
crosslink
molecule
or
hydroxylysyl
The residue
Recently,
do form
are The
to 9” of an a 1 chain
a 1 chain.
in dentin
aldehydes
a
to hydroxyllysyl
adjacent
hydroxylsyl
was found
of
molecule.
addition.
of a 1 chains
through
intramolecular
of the other
region
The
hydroxylysine
be recognized.
C-terminal
of the 1 chain crosslinks
from
to 927
CI 1 chains
The
by (-----------I.
region
sites
I collagen
positions.
between
residue
to one or two
to
indicated
N-terminal
crosslinking
of type
staggered
formed
hydroxylysyl
crosslink
residue
and 4-D
crosslink
gN from
among
representation
in 3-D
crosslinking
C-TERMINAL
in the the
helix.“’
Fig. which
31.
This
joins
three
generated sine-derived
newly
described
adjacent
by one hydroxylysine aldehydes
or
trifunctional
collagen by
residue
molecules
hydroxylysine-5-keto-morleucine
from
a hydroxylysine
and a hydroxylysine
can
be
and two hydroxyly-
spontaneous
two
crosslink
interaction
residues
of
formed
aldehyde.4s*
MARCEL E. NIMNI
tion that the reducible, keto-imine crosslinks disappear from skeletal connective tissue with age. Fibrocartilage of the knee meniscus, a collagen that is essentially all type I, in contrast to the type 11 of hyaline cartilage, also has a high ratio of hydroxypyridinium to reducible crosslinks. Adult bone collagen contains fewer hydroxypyridinium residues than does cartilage and more reducible ketoimines,45’ which presumably provide this tissue with relatively stable, yet more readily biodegradable crosslinks necessary for it to withstand continuous remodeling. Quantitative analyses of crosslinking residues in various skeletal connective tissues suggest a precursor-product relationship between dihydroxylysinonorleucine and the hydroxypyridinium compound. Recently, a pyridinium crosslink has been located in dentine collagen. The peptides linked are alCB-5 and (cxICB-6),, reflecting the presence of a collagen microfibril with its molecules packed in a hexagonal array454 (Fig. 32). The study of collagen crosslinking has advanced steadily and, even though hindered by the difficulty in dealing with an insoluble threedimensional matrix composed of quarter staggered molecules, many crosslinking regions, primarily those involving the nonhelical extension peptides, have been identified.455-457 It is interesting in this connection that covalent crosslinks between type I and type III molecules have been recently identified.45R In addition to these aldehyde-derived crosslinks, intermolecular disulfide crosslinks have been recently identified between type III col-
A sequence
Fig. 32. molecules rable
is shown
set of molecules.
between dotted
molecules lines
poly-CB6
are
adjacent
disulfide
crosslinked
side-to-side
solid
lines
molecules.
crosslinks
and
collagen a compacrosslinks
subunits.
subunits
relevant
peptides
with
represent
microfibrillar
between
particularly
crosslinked
intermolecular
The
within
crosslinks
quarter-staggered crosslinks
of end-to-end
crosslinked
and
involving
the non
The
latter
to
the
existence
of
to
the
discovery
of
in type
set
Ill collegen.‘B7
of
lagen molecules, suggesting a rapid and efficient modality of crosslinking within fibers in rapidly proliferating and newly developing connective tissues.‘67 COLLAGEN
AND
AGING
The physical, as well as the chemical properties of collagen change with age. The content of soluble collagen of skin decreases with age, whereas the tensile strength and the insoluble collagen become progressively greater.459.460 The earlier reports are reviewed by Harkness46’ and Nimni.23.462
The age-dependence of the turnover of collagen was studied in growing rats.463 At different time intervals over a 11%day period, skin biopsies were performed in growing rats which had received an initial tracer dose of 14C-glycine. Specific-activity values for the different fractions of skin collagen isolated were corrected for growth, taking into account changes in surface area, skin thickness, and pool size of the particular fraction. The 0.15-M and 0.5-M NaCl fractions in the normally growing rats decayed almost exponentially, with half-lives of 17 and 20 days. The curve for the insoluble collagen did not reflect the presence of a single component, but seemed to indicate a progressive insolubilization. A half-life of 28 days was evident during the period of rapid growth, whereas a value of 300 days could be extrapolated toward the end of the experiment. Corticosteroid treatment (0.11 and 0.36 mg per day) decreased the turnover rate of all fractions and caused a decrease in the amount of collagen extractable by neutral salt. These values are only relative, since absolute determinations would require knowledge of precursor pool sizes as well as isolation of the different collagen types present, which are likely to differ in their individual turnover rates.464466 Transformation of skin collagen with age can, at present, best be described in terms of intramolecular and intermolecular crosslinks, but direct evidence of an increase in the number of crosslinks is scanty. Using stress-strain measurements, an increase in the number of crosslinks in rat skin with age was shown to occur, but the nature of the crosslinks remained unclear.4”7 Increased crosslinking of collagen chains with age has been reported,468.469 an it has been found
37
COLLAGEN STRUCTURE AND METABOLISM
that the isometric tension produced during thermal contraction increases with age.47M73 Changes in the physicochemical properties of collagen with age have been attributed to the formation of both covalent and noncovalent crosslinks. Neutral salt-soluble collagen, which has a low concentration of p components, will generate intramolecular bonds if gelled at 37’C. These intramolecular bonds seem to precede the formation of stable intermolecular crosslinks, since these gels can redissolve when cooled, to yield a soluble collagen with a higher content of /I components of intramolecular origin.474.475 Soluble collagen from rat tail tendons contained increasing amounts of /II and y components, indicating slow formation of crosslinks between collagen components with advancing age. The fact that the slowing down of animal metabolism by cooling prolongs life in poikilothermic species has raised the question of whether similar findings would occur in warmblooded animals, and in what way collagen might be implicated in such a phenomenon. Using the criteria of chemical contraction and relaxation of collagen as an indication of age, the effect of cooling on the speed of aging of collagen in vitro and of hibernation in the fat dormouse was investigated.47h Findings by these investigators suggest that a decrease in temperature slows the maturation of collagen. Although it is attractive, the crosslinking theory of aging awaits further experimental support. Obviously, stabilization of collagen fibers may occur by other means than by formation of new covalent crosslinks. An increase in the number of weaker forces that stabilize macromolecules and their aggregates, such as Van der Waals bonds, ionic interactions, hydrophobic bonds, and combinations of such forces, could account for changes in the physical and chemical properties of the collagen fibers. For instance, a slow-time dependent exclusion of intermolecular water could lead not only to an increase in hydrophobic contacts but could strengthen existing ionic bonds by placing them in an environment of decreased dielectric constant (Fig. 5). Changes in the concentration and composition of proteoglycans surrounding collagen may also play a major role. It is therefore necessary to contemplate many factors in attempting to understand
the age-related changes extracellular matrix. INHIBITION
that
OF COLLAGEN
take place in the
CROSSLINKING
Lathyrism
In connective tissue disorders lathyrism is used to describe primarily a defect in collagen metabolism associated with the ingestion or injection of BAPN (P-amino propionitrile and its chemical analogues) or extracts of the sweet pea or others members of the lathyrus family. The syndrome comprises two entities that have been comprehensively reviewed.477 The terms neurolathyrism and osteolathyrism were coined by Selye4” to describe the two seemingly unrelated metabolic effects. Neurolathyrism, occurs in a variety of animals, including man; thus far three distinct chemical agents have been isolated from the aforementioned plants that seem to be responsible for this condition. Clinical manifestations include spastic paraplegia, and degenerative changes are noted in histopathologic sections of the spinal cord. Epidemics of neurolathyrism have been described over the centuries and a historical review is provided in the reference cited above. Osteolathyrism
Osteolathyrism relates to the more recently described479 connective tissue form of the disease that is observed mainly in rats, turkeys. chicks, and other animals; it has not been described in humans. In this entity the skeletal changes observed differ from species to species, and vary with age, being much more pronounced in younger animals. The epiphyseal plate is a. prime of target.48” Typically one sees a proliferation cartilage cells with an irregular zone of calcified cartilage, retardation of endochondral ossification, loss of orientation of chondrocytes and of cohesion of cartilage, and loosening and detachment of the tendinous and ligamentous insertions. Exostoses occur in the long bones and mandible in the areas of muscle insertion, probably from the mechanical distortion of the weakened matrix. Vascular lesions are common and are frequently manifested in the form of aneurisms (angiolathyrism). In addition to BAPN, several other compounds have been described as having lathyrogenic activity.4x’ These include
38
MARCEL E. NIMNI
organic nitriles and ureides, such as semicarbazide and hydrazines.477 Several compounds have been shown to potentiate the effects of BAPN (inhibitors of monoamineoxidase, copper deficiency) and to inhibit the toxicity (Ca, Zn, Cu, reserpine, propanolol).477 The connective tissue abnormalities appear to be associated with crosslinking defects in collagen and elastin reflected by an increased solubility of these macromolecules in hypertonic neutral salt solutions,482 and to inhibition of lysyly oxidase activity.483 Since Cu deficiency also inhibits this enzymatic activity, the similarities of the defects induced by these two mechanisms are readily explainable. Dermalathyrism
Induced
by Penicillamine
Administration of peniciilamine to animals and humans causes an accumulation of neutral salt-soluble collagen in skin and various soft tissues.48ti87 Two of the more characteristic properties of penicillamine, namely the ability to trap carbonyl compounds and to chelate heavy metals, are of primary significance in impairing collagen crosslinking. The former property manifests itself in all effective dose ranges studied, whereas the latter occurs only at high dosages, far beyond those administered to humans.4s8 The collagen extracted from tissues of animals treated with D-penicillamine is able to form stable fibers in vitro and is not deficient in aldehydes, as is that from BAP-treated animals. In fact, its aldehyde content is even higher than normal, suggesting that the mechanisms of action of BAPN and penicillamine are different (Fig. 33).
Fig. 33. action
Schematic
of penicillamine.
vitro,
will
them
from
interact
intramolecular bility
of the
peptide-bound
with
subsequently and collagen
apy is discontinued thiazolidine
drawing This
complex
summarizing
compound,
aldehydes
on collagen
participating
intermolecular defect
seen
formed
aldehydes.
in the
crosslinks. when
can be explained between
the
mode
in viva as well
of
as in
preventing formation The
penicillamine
by the instability penicillemine
of
reversitherof the and the
Using the method of equilibrium dialysis, it was observed that cysteine, penicillamine, and other analogues bind specifically to free aldehydes on collagen. Both the free amino and sulphydryl groups are necessary for binding to occur. The collagen-mercapto-ethylamine product is in equilibrium with its constituents and can be completely dissociated by exhaustive dialysis.489 The relationship between the dose of penicillamine and the aldehyde content of collagen has been documented.48s+490 Collagen from the skin of young rats contains an average of 0.87 residues of aldehydes per 1,000 amino acid residues. D-peniciiiamine caused the concentration of aldehydes to increase, reaching a maximum of 200 mg/kg of body weight per day. As the dose was increased, however, the aldehyde concentration declined, until at the highest dose it resembled lathyrism induced by BAPN. Although all collagen preparations are able to form native collagen fibers with native 64OA periodicity, only those containing significant amounts of aldehydes form stable crosslinks. Thus collagen from animals treated with high levels of penicillamine, where lysyl oxidase is inhibited by virtue of the Cu being chelated, is incapable of forming stable crosslinks when reconstituted in vitro, and in this connection behaves like lathyritic collagen.49’ From these experiments one can conclude that penicillamine has the ability to block aldehydes and to inhibit lysyl oxidase activity. At low dosage it acts primarily by blocking the aldehyde residues present on the collagen molecule, whereas at a higher dose it also affects the activity of lysyl oxidase.488 This latter observation agrees with the fact that 10m4 M penicillamine partially blocks lysyl oxidase activity in vitro while lo-’ M inhibits it completely.492 Although the mechanism of this activity has not been defined, it may be related to the ability of penicillamine to chelate divalent cations or to interact with other cofactors. Depolymerization of Incompletely Crosslinked insoluble Collagen by Penicillamine
Fractionation of collagen according to its solubility has proven to be of value in disclosing the process of collagen maturation and crosslinking. After rat skin has been extracted exhaustively with 0.45 M NaCl, an additional amount of
39
COLLAGEN STRUCTURE AND METABOLISM
collagen can be dispersed by either dilute organic acids (citric, acetic) or by compounds such as cysteamine or penici11amine.493 In rats less than 60 days old, almost all the skin collagen can be dispersed by amino-thiols to yield a collagen that is soluble in neutral salt. The intrinsic viscosity of this material, 20-25 dl/g (I 3 dl/g), reflects a mixture of collagen molecules with polymeric aggregates. This collagen will withstand centrifugation at forces greater than 100,000 g for several hours without appreciable precipitation.494 In older animals, however, the amount of that is collagen soluble in dilute organic acids and amino-thiols becomes progressively less. In addition to preventing the crosslinking of newly synthesized collagen, peniciilamine seems to enhance in neutral salt solutions the solubility of a collagen fraction that otherwise would remain insoluble, this particular collagen fraction, rendered soluble by administration of penicillamine, is equivalent to that able to be dissolved in vitro by amino-thiols and by 0.5N acetic acid. The fact that both in vivo and in vitro one is able to generate a collagen enriched in aldehydes suggests that penicillamine can also enhance the solubility of collagen by labilizing Schiff base type crosslinks.49’.496 The size of this degradable pool is inversely proportional to the age of the animal; thus penicillamine exhibits maximum effect when given to young animals. Because of the rapid rate of collagen synthesis and turnover, the pool of thiol-soluble material is large at this time.497 By using total skin mass as an index of absolute changes caused by penicillamine, a decline in the amount of total insoluble collagen during the first two weeks of treatment was observed. Continued administration of D-penicillamine failed to affect the remaining insoluble collagen. The soluble collagen that continues to accumulate during treatment may be a part of a rapidly turning over pool or may correspond to the more recently synthesized material that has not been allowed to mature.498.499 Taking into account the variability in rates of collagen synthesis with age, it is possible to estimate that the half-life of this form of collagen-containing labile Schiff bases is about IO days in two-month-old rats.4y7 This period reflects the time required for the Schiff base to be transformed into one of the
more stable forms of covalent crosslinks, discussed previously. Subsequent studies have shown how penicillamine inhibits collagen crosslinking.50” I’enicillamine selectively inhibits the crosslinking of soft-tissue collagen and is much less elective than fi-aminopropionitrile in affecting bone collagen. This collagen, with a high hydroxylysine content, is relatively unaffected in experimental animals fed penicillamine. Contrary to expectations, it was shown that the synthesis of bifunctional crosslinks involving hydroxylysine, :such as N6:6’-dehydro-5.5’-dihydroxylysinonorleucine, increases in those animals treated with penicillamine. The thiazolidine ring interactions may not be sufficiently stable to block the synthesis of these bifunctional crosslinks involving hydroxylysine intermediates highly favored by the geometry of the fibril. On the other hand periarticular collagen from immobilized rabbits treated with penicillamine to decrease contractures reveal an almost complete inhibition of crosslinks formation.50’ These findings explain why the effects of penicillamine are significantly greater on soft tissues than on bone, since the former involve lysine-derived aldehyde intermediates. which will more readily form thiazolidine structures with penicillamine. The stability of the ketoimine form was first demonstrated during the determination of the reduced compound in bone and dentin collagen.“’ The stability of such a crosslink derived from two hydroxylysine residues, explains how it can still form in the presence of physiologic levels of penicillamine. Such a crosslink would be favored over the thiazolidine structure which form when lysine derived aldehydes are present. Figure 27, discussed earlier, illustrates the differences in behavior of these crosslinks; from these experiments it should become apparent why the action of penicillamine varies from tissue to tissue. Its ability to block crosslinking depends on the types of crosslinks that stabilize that particular collagen fiber, skin and soft tissues being more susceptible to the action of this drug, and bone being much less affected. WOUND
HEALING
There is an inherent tendency of tissues to repair after they have been damaged, with the nature of the repair process varying with the site
40
and mode of injury. Connective tissues repair by regeneration of their extracellular matrices. Therefore, a major part of the process involves the deposition of newly synthesized collagen and proteoglycans. Healing of experimentallyinduced skin lesions has been studied extensively in animal models. The cellular population of healing wounds changes from one associated with the initial inflammatory response, (i.e., granulocytes and macrophages) to that associated with a proliferative and anabolic response (fibroblasts). Immediately after wounding, no obvious fibroblasts are present. Before recognizable fibroblasts enter the wound, cells that resemble immature fibroblasts are seen in the perivascular connective tissue. The development of these cells into mature fibroblasts has been followed through the different stages of wound repair.’ 503Collagen synthesis has been reported to increase significantly by 24 hr in open skin wounds of the rat.‘04 A high proportion of type III collagen is produced in response to turpentine injection or to sponge implantationSo and in the initial phase of wound repair in humans.506 More quantitative studies on the nature of the collagen synthesized during the early phase of wound healing5” show an increase in synthesis of type III collagen 10 hr after infliction of the wound; by 24 hr, the percentage of type III collagen synthesized return to a normal value. The early increase in type III collagen observed by these investigators is probably derived from local fibroblasts that are activated by the wounding process, and the early type III collagen deposited may be important in establishing the initial wound structure and in providing a basic lattice for subsequent healing events. The ability of type III collagen to crosslink via disulfide bands may greatly assist in this connection.16’ However, it is unlikely that type III collagen contributes significantly to wound tensile strength as the greatest increase in tensile strength is not observed until later phases of wound repair. In young guinea pigs the rate of collagen synthesis in dermal scars is initially high, but gradually approaches that of the surrounding dermis after a period of about six months.508 Concomitant with the increased rate of collagen synthesis, the extent of hydroxylation of lysine in the early wound is significantly elevated, but declines to approach normal values about three weeks after wounding.
MARCEL
E. NIMNI
Dihidroxylysinonorleucine, derived from two residues of hydroxylysine, is the major reducible crosslink detectable in the early wounded tissue,5o9 but this is subsequently replaced by hydroxylysinonorleucine.5’o In addition to this increase in hydroxylation of lysine and hydroxylysine-derived crosslinks, the amounts of type III collagen is also increased in guinea pig dermal scars compared to uninvolved normal skin. It is noteworthy that the sequence described resembles that seen during early phases of growth and development. Further studies in the quantitative and qualitative distribution of collagen types in a variety of normal and injured tissues might thus provide further insight into the specific role that the different collagen types play in relationship to the normal structure and function of the connective tissues. VITAMIN
C, COLLAGEN WOUND
SYNTHESIS,
AND
HEALING
Well-healed, mature scars in man can disrupt because of asorbic acid deficiency. This phenomenon was recognized in ancient times when sailors on extended sea voyages, living on diets devoid of fresh fruits and vegetables, noted a breakdown in skin scars that had been healed for years. The participation of ascorbic acid in the hydroxylation of peptide-bound proline has been described.23,462 The collagen synthesized in scorbutic guinea pig tissue and in several fibroblast cell lines5”,5’2 in the absence of ascorbic acid contains reduced amounts of hydroxyproline, and the extensively underhydroxylated collagen produced by fibroblasts in culture accumulates within the cell. This raises the question of whether such accumulated collagen may further inhibit collagen synthesis by translational repression. Several earlier observations substantiate this possibility. Electron micrographs of wounds of normal and scorbutic guinea pigs show that vitamin C deficiency caused the cisternae of the endoplasmic reticulum to become dilated and polysomes to become detached.5’3 Using a sucrose gradient it was shown that the heavy collagen-synthesizing polysomes were reduced by vitamin C deficiency, giving rise to 80s monosomes.5’4 Other experiments using scorbutic guinea pigs failed to show a marked accumulation of underhydroxylated collagen, but, rather, decreased collagen synthesis as well as larger amounts of
41
COLLAGEN STRUCTURE AND METABOLISM
diffusible hydroxyproline were noted.5’5 These observations could be due to an increased degree of degradation of partially hydroxylated collagen. It is possible that translational inhibition due to intracellular accumulation as well as increased susceptibility to degradation, are both part of the basic connective tissue defect seen in scurvy. These observations are supported by more recent findings suggesting that the regulatory role of ascorbic acid goes beyond that of acting as a cofactor for hydroxylation.5’6 In addition it stimulates the synthesis of DNA5” and of proteoglycans”” by articular cartilage cells in organ or cell culture. A decrease in the biosynthesis of collagen alone would not be sufficient to account for the dramatic way in which the deficiency manifests itself in old wounds, an event which more likely reflects a combination of anabolic and catabolic events at the wound site.5’9 Metabolic activity in general remains increased, even after the healing process is completed. When the rate of collagen synthesis is reduced because of ascorbate deficiency, the state of equilibrium is altered, and the persisting collagenase activity results in a loss of collagen from the healed wound, and the old scar disrupts as collagen is resorbed. TISSUE
SPECIFIC
FIBROBLASTS
A tendency to investigate the biochemical activities of fibroblasts has been evident without regard to their site of origin, thus assuming that all fibroblasts are alikes. This may lead to errors in interpretation and prevent us from gaining insight into the pathogenesis of various circumscribed diseases. As an example, serum from patients with pretibial myxedema can stimulate fibroblasts to synthesize increased amounts of collagen and hyaluronic acid.52n In this study skin fibroblasts from the shoulder and lower extremities of normal individuals, and from patients with pretibial myxedema (PTM) were grown in culture. When the cells reached the monolayer stage, they were labeled with ‘H-glucosamine and tested for hyaluronic acid synthesis in the presence of either serum from PTM patients or normal human serum. It was of interest that the fibroblasts derived from the pretibial area of normal as well as affected individuals synthesized two to three times more hyaluronic acid when incubated with PTM sera than when incubated with normal
human serum. Fibroblasts cultured from skin of the back or prepuce, on the other hand, did not respond to PTM sera. This heat-stable, proteasesensitive, dialyzable fibroblasts-stimulating factor was not a 7Sy-globulin. The enhanced sensitivity to PTM sera exhibited by fibroblasts from the lower extremities could explain why the lesions in this disease are restricted primarily to a particular area of the lower extremities. Although controversy exists as to whether the serum from patients with scleroderma could specifically stimulate fibroblasts to produce collagen s2’~52sit is clear that serum factors, whether specifically or nonspecifically, stimulate collagen synthesis by fibroblasts originating from patients with this disease. The selective pattern of distribution of many fibrotic diseases may be tied directly to such a selectivity or may result from secondary adaptations of cells at various locations to differences in environmental factors (biochemical or mechanical) which, in turn, sensitizes them to such circulating factors. There is no doubt that fibroblasts from different regions of the dermis exhibit different abilities to synthesize collagen. Cultures from the lower dermis (reticular fibroblasts) accumulate significantly more collagen than do those from the upper dermis (papillary fibrob,asts),“23,524.S26 both in culture and in vivo. Numerous observations support the view that mesenchymal cells, including fibroblasts, from different locations are endowed with unique specificity and characteristics.5’7.5’” For example, estrogens that have been shown to significantly influence the metabolism of connective tissues associated with endocrine control arc also capable of affecting the metabolism of many connective tissues with seemingly non sex-related functions (bones, blood vessels, etc.) Estrogens are able to affect differently the ability of dermaland lung-derived fibroblasts to synthesize collagen,‘28 supporting the view that fibroblasts develop differently from organ to organ. The modality in which this specificity is acquired is not known and could provide an area for fruitful investigation. COLLAGEN
SYNTHESIS
BY CELLS
IN CULTURE
The uniqueness of fibroblasts and their importance in the development of the extracellular matrices of most connective tissues is widely recognized.529 The morphologic and functional
42
characteristics of these cells can vary according to the circumstances under which they are observed. They are widely used as experimental models because they seem to retain their phenotype in culture and express many of the characteristic patterns of behavior seen in vivo, thereby providing a valuable tool to the researcher interested in replicating in vitro abnormalities of the connective tissues seen in a variety of diseases. While normal tissue fibroblasts differ morphologically from fibroblasts grown in culture, many features of the cultured cells resemble those of fibroblasts involved in active metabolic processes (e.g., granulation tissues). Human fibroblasts in culture synthesize both types I and III collagens3 with type I accounting for 70%-90% of the tota1.532 In culture, the rates at which these proteins are synthesized is constant and apparently rigidly controlled.533 However, the proportions differ if cells are cultured with larger amounts of serum (increased type III/I ration).534 Cells obtained from patients with certain diseases such as the Ehlers-Danlos type IV syndrome make little or no type III collagen,535*536 whereas cells from patients with osteogenesis imperfecta exhibit an increased type III/I ratio.537l538 Cell density also alters the ratio of collagen types synthesized by fibroblasts.539 Normal fibroblasts from human and guinea pig skin produce proportionately more type III collagen at high cell density, probably as a result of a reduction in the synthesis of type I collagen. Similar findings are observed with human lung fibroblasts, which synthesize 26%-68% more type III collagen at confluency than at low cell density.540 In vivo, fibroblasts appear as nondividing cells that can, however, be induced to divide by the presence of stimulating factors, such as those associated with tissue damage.54’*542 In culture, conditions can also be altered so as to stimulate their proliferation. Fibroblasts of low and high population doubling potentials grow to confluency when plated on a plastic surface but cease to divide within four days when incorporated into collagen lattices.543 While division is arrested in such a lattice, this state can be reversed if cells are permitted to leave the lattice and again populate a plastic substrate. It thus appears that fibroblasts in tissue-like lattices may be responsive to controls similar to those experienced by connective tissues cells.
MARCEL
E. NIMNI
In order to maximize the information that can be obtained from fibroblasts in culture, many efforts have been made to standardize their growth544 and to assay quantitatively the amounts of procollagen produced.545 Under such optimal conditions, 80% of the procollagen in the media is type 1 and the remaining 20% is type III. CHONDROCYTES, PHENOTIPIC LABILITY, AND OSTEOARTHRITIS
Chondrocytes are highly differentiated mesenchymal cells that synthesize a unique blend of collagen and proteoglycans. These cells are embedded in the metachromatic matrix they produce, a mixture of chondroitin-4 and six sulfates, small amounts of other glycosaminoglycans, and significant amounts of type II collagen. 546~547 The nature of type II collagen has been discussed and earlier work on the lability of the chondrogenic phenotype that causes chondrocytes in culture to “dedifferentiate” and produce other collagen types has been reviewed.548 For their proper function, and for the maintenance of the differentiated phenotype, chondrocytes seem to require a carefully controlled, highly specific microenvironment. Alterations of this environment, either in vivo or in vitro can cause significant changes in cellular metabolism, leading to an alteration of the extracellular matrix. The possibility that such changes could predispose and be ultimately responsible for cartilage degeneration and accelerated wear and tear, have stimulated significant amounts of work to further understand the factors and the mechanisms involved. Early biochemical studies suggested that articular cartilage cells present in areas of degeneration could be switching their synthetic capacity from producing type II collagen to type 1 collagen.549 These findings were supported by immunohistochemical observations that some chondrocytes were surrounded by a fluorescent halo when reacted with type I collagen antibodies.550 Other studies55’,552 have demonstrated a higher solubility of collagen in osteoarthritic cartilage as compared to normal, but no type I collagen could be detected in the soluble fraction. In other instances no a 2 chain was seen in the pepsin solubilized collagen from osteoarthritic collagen,553 while a similar degree of hydroxylation of lysine in control and osteoarthritic-insoluble collagen was observed.554 This
COLLAGEN STRUCTURE AND METABOLISM
was taken as an indication of no major shift in the type of collagen synthesized by osteoarthritic cartilage. Studies using animal models for osteoarthritis have confirmed these observations,iiF.C(h These discrepancies can now be explained as a result of detailed biochemical and histologic analysis of human osteoarthritic cartilage in various stages of degeneration.557 From this study it seems that type I collagen is present in sizable amounts only when histologically identifiable fibrocartilage is formed. In other areas, where hyaline cartilage still persists, only type 11 collagen can be detected biochemically. Nevertheless, in the hypermetabolic areas of degenerating hyaline cartilage, it is possible to clearly see by immunofluorescence small amounts of type I collagen, which is probably present in ins&icicnt amounts (less than 3%) to be detected chemically.‘“” The ultimate significance of this alteration is not known. It would seem as if the small amounts of type I collagen synthesized by the chondrocytes in actively degenerating hyaline cartilage are a reflection of an altered metabolic activity, rather than being directly responsible for the formation of an unsuitable matrix that more readily degenerates. The fibrocartilage, on the other hand, can be interpreted as a healing response by cells that originate most likely from the subchondral bone. The healing response in question may be similar to that observed when full thickness lesions of the articular cartilage, those extending down to the subchondral bone, are produced.558 Such lesions rapidly fill with fibrocartilage,s59 predominantly type 1 collagen in the early stages of formation,“’ but which becomes mostly type II after eight weeks (Fig. 34).‘“’ The induction of cartilage formation at unusual locations has been reported5*’ (vascular lesions, a calcific heart valves, pseudoarthritis, etc.). An example of such a phenomenon is illustrated in Fig. 35. In this case the cruciate ligament of a rabbits knee was cut, and allowed to hang loose in the synovial space. After several months cartilage cells can be detected in the degenerating ligament, probably originating from fibrocytes which have responded to changes in mechanical stresses or to chondrogenic factors present in the joint space. The chondrogenic phenotype is labile and cultured chondrocytes tend to readily “dedifferentiate” when grown in
Fig.
Regeneration
34.
wounding
with
a drill
dral area.
After
eight
nous never hyaline
matrix
ment
was
therefore
This able
the subchondral
the hole filled with of type
fashion
progenitor
the
a poor cells
subchon-
a cartilage-
II collagen,
with
illustrates
area to differentiate
knee after
into the
generating
observation
to cause
in a rabbit
penetrated
primarily
in a continuous
cartilage,
ing surface.
weeks
consisting
blends
of cartilage
which
how
but
preexisting
weight the
bear-
environ-
originating
from
into chondroblasts.=
monolayer cultures,“’ and switch from s:ynthesizing type II to type I collagen. This process can be accelerated, as discussed earlier, by addition to the media of BrdU562 or embryo extracts Viral transformation of chick embryo-derived cultured chondrocytes caused a decrease in the amounts of type II collagen synthesized and increase in the amounts of fibronectin in the media, but did not cause the cells to produce type I collagen.“’ The normal pattern of proteoglycan and collagen synthesis by chondrocytes can also be altered by an excess of vitamin A in the media.“h4 The changes in phenotypic expression occur concomitantly with changes in cell shape, from polygonal to spindle or fibroblastic-like. Immunofluorescence experiments with antibodies to various collagen types5” showed that most fibroblast-like cells among chondrocytes stained positively for type I collagen but not for type II. However, type I collagen synthesis is also found among polygonal chondrocytes, suggesting that
MARCEL E. NIMNI
Fig. 35. Anterior cruciate ligament of a rabbit cut and allowed to dangle freely, attached from one end, in the knee-joint space. As a result of change in mechanical stresses to which the cells were exposed many cells within the ligament differentiated into chondrocytes and produced a characteristic metachromatic matrix. (Courtesy of Dr. E. Gendler.)
no absolute correlation exists between cell shape and collagen phenotype. It is also important to emphasize that the majority of the chondrocytes stain positively for either type I or type II collagen, and less than 1% stain simultaneously for both types. This could mean that the loss of the cartilage phenotype is not a gradual process but occurs rather abruptly5@ for any one particular cell. As discussed earlier attachment of fibroblasts and epithelial cells to collagenous matrices is mediated by the glycoproteins fibronectin and laminin, respectively, while chondrocytes are attached to type II collagen by a distinct factor, known as chondronectin.567 Antibodies to chondronectin, a glycoprotein of approximately 180,000 daltons, localize on the cell surface of the chondrocytes rather than in the matrix, and can inhibit their attachment to type II collagen.567 Although chondrocytes in culture can become metabolically active, in adult cartilage their metabolic activity is relatively low and mitosis has rarely been demonstrated.568 When degenerative changes occur in the joint cartilage, chondrocytes recover their ability to divide.569 Local traumatization of adult rabbit joint cartilage induces mitosis of chondrocytes not only in the traumatized joint but also in the normal cartilage of sham-operated or unoperated control joints.570 All cartilage of the patella groove of the femur of adult rabbits was excised while the other knee underwent arthrotomy or was left intact.
Radioactive thymidine and autoradiographic techniques demonstrated that labeled chondrocytes appeared not only around the excised cartilage but also scattered in the tibia and femoral condyles. Whether this stimulation of chondrocytes is due to a decrease in cell growth-inhibiting factor or to a liberation of stimulating substances is not known, but the mechanisms of this activation represents a significant question.57’ In this connection, much attention is being given to the role of somatomedin, growth factors and other metabolites that may stimulate the metabolic activity of chondrocytes. It has been recently shown that prostaglandin E2 stimulates cyclic AMP levels in cultured chondrocytes. The relative abundance of prostaglandin E2 in these cells suggests that it may have an important physiologic role in controlling cyclic AMP synthesis in chondrocytes.572 Stimulation of prostaglandin E, biosynthesis by a Ca++ ionophore is also accompanied by increased levels of cyclic AMP. Sodium meclofenamate and indomethatin, both potent inhibitors of prostaglandin cyclooxygenase, inhibited the ionophore-stimulated prostaglandin E2 production, which correlated with a reduction in cyclic AMP. How increased intracellular cyclic AMP concentrations affect chondrocyte metabolism are unknown at the present time. The state of differentation of chondrocytes can be monitored in a more quantitative fashion by determining the collagen types they produce. Rabbit articular chondrocytes grown in monolayer culture synthesize collagens other than type II, and the proportion of these collagen species changes during subculture.573 These additional collagens were identified by direct cyanogen bromide peptide analysis as types I, I-trimer, and III; partial characterization of two new collagen chains, X and Y, has been completed.574 Further evidence suggested that during primary chondrocyte culture, the synthesis of two collagen species, types 11 and III, increased at the same time and to the same degree. In addition, X,Y synthesis was stimulated prior to these changes, implying that this collagen may be regulated independently.575 The relative changes in collagen types produced by chondrocytes subcultured through five different passages is shown in Fig. 36. In order to further understand the factors that contribute to the stability (or instability) of the chondrogenic phenotype, cultured cells were
45
COLLAGEN STRUCTURE AND METABOLISM
ItA
4
\
. 3
; : \
I
2
p”
1
WEEKS
CULTURE Fig. 36.
Synthesis
subculture:
radioactive
acrylamide.
The
for
the
of collagen
of total
sample,
the
was
phenotype
41%
trimer.
type
of chains of fifth
I, 25%
13% type
were
sample
X,Y
fractionated peak
analyzed,
was the
during
during
was corrected
on 5%
chains
corrected number
Collagen
nine weeks
of
in
types
IN
synthesized
in culture. collagen
by cartilage
The radioactivity
for cell number
native
CULTURE slices
of each peak
and for the stoichiometry
molecules.
ND
not
of
detect-
able.57*
of al (1) and al (II) chains, in the native
culture (type
Ill and 1% type
Fig. 37.
by chondrocytes
of each
proportion
and the stoichiometry collagen
collagens
radioactivity
fraction
cells per
NO.
collagens.
chondrocyte
V collagen), II. ND=not
The
progeny 20%
type
detectabla.676
compared to chondrocytes in organ culture, which are able to retain their original shape, as well as those in a more physiologic environment (Fig. 37). Similarly to cultured cells during early stages of primary culture, transfer of fresh tissue to organ culture enhanced the metabolic activity of these cells although they did not show significant changes in collagen phenotype. This degree of stimulation has subsequently been shown to correlate with the concentrations of fetal calf serum present in the media. These experiments demonstrate both the sensitivity of the chondrogenic phenotype to environmental manipulation and the presence of metabolic stimulating factors in the tissue culture media. It is apparent from these studies that the differentiated phenotype of rabbit articular
I
chondrocytes, which consists primarily of type 11 collagen and cartilage-specific proteoglycane, is lost during serial monolayer culture and replaced by a complex collagen phenotype consisting predominately of type 1 collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes are able to re-express their differentiated phenotype during suspension culture in firm gels of 0.5% low T, agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains.‘77.“78 Collagen gels are also able to provide a suitable matrix in which embryonic chick chondrocytes proliferate and deposit a territorial matrix that resembles a chondrocytic lacuane.57x~i7” Figure 38 (phase contrast photograph) shows the growth characteristics of dedilferentiated chondrocytes after they have been transferred to
MARCEL E. NIMNI
collagen /:
SYNTHESIS
/
-I
DISH
L 4O
1
2
4
DAYS Fig. cyte
39.
IN
during
collagen,
as counts
per
suspended
in 0.5% f3sS0,)
CTAB-precipitable isolated
Phase
Fig. 38. drocytes ture
contrast
passage)
and subsequent
Exactly ited
(fifth the
same
proliferation
domains matrix indicate
(MD) fusion
culture was
(PI,
limited
around (MF)
both
between
release
in agarose
field
sites where
the culture
micrographs
after
of cultured
from
single
death
Cultures (CD),
and
exhibmatrix
cells and aggregates,
neighboring
examples
cul-
for 1.7, and 13 days.
photographed. cell
chon-
monolayer
of these
and
cells. Arrowheads events
occurred
as
developed.578
agarose. Also shown are the changes in metabolic activity that accompany the transition. Most marked is the increase in synthesis of collagen which are an indication of re-expression of the differentiated chondrocyte phenotype (Fig. 39). A more specific and quantitative way of docu-
12
were
14
CULTURE differentiated
culture.
correction
the
being
number
released
f3H-thymidine)
analyzed
radioactivity.
of synthesis
are expressed
for
(4”) or after DNA
chondro-
Rates
and proteoglycan
after
agarose.
as ‘H-leucine collagen
of the
agarose
cultures
teogycans
10
AGAROSE
protein
minute
cells in monolayer
measured
8
Reexpression
phenotype
for DNA,
6
as Protein
incorporated.
and
of and pro-
pronase-soluble, synthesis
Radioactivity
was in the
was also measured.“’
menting this phenomenon of reversion involves analysis of the collagen phenotypes produced. Figure 40 shows the individual collagen chains synthesized by dedifferentiated chondrocytes (4th subculture) grown as monolayered cells, and of similar cells which were placed in agarose in suspension and maintained in this condition for 14 days. Also shown are a series of dedifferentiated chondrocytes exposed to different number of subcultures (3-6) and transferred to agarose for 10 days. In all instances reversion to normal phenotype can be clearly seen (Fig. 41). These studies, particularly those dealing with the reversibility of the dedifferentiated state, emphasize the usefulness of the tissue culture approach to understand the more subtle changes that occur in the in vivo systems. As we are able to further identify the factors (physical and chemical) that contribute to this reversible pattern, we should be able to apply our findings to the development of new modalities of treatment of diseases where chondrocytes fail to properly remodel or repair damaged cartilage.
47
COLLAGEN STRUCTURE AND METABOLISM
4” 1
(days)
4”
;gar;se
1
1 5’
10
14
I
I
Fig. 40. tiated
Time
collagen
(100,000
(4”).
agarose
the
rose) on
course
chondrocyte
cpm)
a 3.5%-10% for
(l-10 trimer
(I
l(l)
hydroxyproline
The
from
known
type
bands
containing,
from
gel
duration
culture
culture
in SDS
and
of agarose collagen
labeled
CPl
collagenous
interstitial
BIOMECHANICAL
I
laga-
fractionated pro-
culture
for each lane. Also indicated
derived
l(H).
this
(5”) were
gradient
differen-
radioactive
monolayer
from
culture
The
is indicated
and
derived
monolayer acrylamide
of a
of the
Purified
the fourth
derived
fluorography.
days)
positions
phenotype.
from
cultures
and the fifth
cessed
of reexpression
collagen
are the
or type
and
CP2
peptides
I
are not
collagens.“’
PROPERTIES
CONNECTIVE
OF THE
TISSUES
While discussing the biology and chemistry of collagen we should always keep in mind that this macromolecule, by virtue of its becoming organized into fibers and in conjunction with other components of the connective tissue, is providing
3”
I
4*
1
1
5’
6O
MAMAMAMAA
Ill-
lo-day ated.
Effects
differentiated
dioactive
Collagen medium
of subculture chondrocyte
collagen agarose
deficient
o-
-
Fig. 41. the
CF
from culture
from
on the collagen
each monolayer (A)
derived
agarose
(CF) were
phenotype.
culture
from
cultures
reexpression
(M) and the
it were grown
also analyzed.“’
of Ra-
fractionin
calcium
organs with a mechanical function, either of a structural nature or as a selective barrier. So much information is available on this subject that any attempt to discuss it in any detail would be impractical, but some of the fundamental principles which emanate out of the unique physicochemical characteristics of connective tissues shall be reviewed, more to challenge the imagination of the reader than to provide him with the new data in the field. For detail several excellent reviews on the subject are available in journals in biomechanics and medical devices.58”~5xh To understand the elastic, viscous, and frictional properties of the connective tissues, one should look at the physicochemical properties of the individual components and the compartments to which they are restricted. The primary function of collagen, which can be associated with its fibrous conformation, is that of resisting or transmitting forces. Why do we have so many different types of collagens? We cannot answer this question now, but by observing the molecular structure of the various collagen types. their mode of organization at the ultrastructural level, their distribution, the diameter of such fibers, or the lack of hbrillar organization (such as in the basement membranes) one may intuitively begin to relate structure to function. Quantitation of these functions will undoubtedly come. Stress-Strain Behavior of Collagen Fibers Aligned Parallel to Each Other The biomechanical properties of collagen fibers have mostly been studied primarily in tissues where these fibers are oriented in a parallel fashion (i.e., tendons, ligaments). The stressstrain characteristics are well described in the literature, SXZ.iX7.58X A stress-strain curve for tendon, and the kind of information that can be derived from it is illustrated in Fig. 42. Quantitative interpretation of such data is difficult, however, because so many variables (e.g., water content of the specimen, measurement of cross-section, collagen content, presence of various collagen types, heterogeneous population of molecules and crosslinks, presence of other connective tissue components such as elastin, proteoglycans) significantly affect behavior. Which is the most meaningful part of the stressstain curve? The toe region (a) reflects the slack that is associated with elimination of the crimp-
48
MARCEL
E. NIMNI
skin with ageing. This would be difficult to do using stress-strain particularly in a tissue that has fibers oriented in a nonparallel fashion. The thermal shrinkage of collagen and the role of the intermolecular crosslinks in providing for the contractile force is shown in Fig. 43. Interactions STRAIN Fig. 42. nous
Changes
tissues
during
(A) Elimination aligned
fibers.
in the
internal
the stretching
of crimpling. (C) Failure
structure
of a tendon
(B) Resistance
of collegeor ligament.
displayed
by the
point.
ling of the fibers; during this phase the elastic modulus increases steadily. The linear part of the curve is where stress increases rapidly (b). This is the area from which the modulus is usually calculated and reflects the actual contribution of collagen molecules aligned parallel to each other and stretching of the crystalline network. Nevertheless, one still has to distinguish at this stage between intrafibrillar distortion and the slipping of fibers side by side. The increase in modulus reaches a plateau during the linear part of the curve; it is this value that is usually used to determine the “stiffness” of the specimen. When the point of maximum stress is reached, the tissue breaks (c); this is the failure point, and can occur suddenly, in bone, or can be preceded by a leveling off towards the strain axis, as in skin, ligaments, and tendons. Detailed analysis of the various stages of the viscoelastic behavior of such tissues is provided in the reviews previously cited. Thermal
of Collagen with Proteoglycans
(0)
Denaturation
In order to understand the physical properties of connective tissues such as cartilage and intervertebral disks, it is important to have an understanding of the salient features of the proeoglycan molecules59@594 (Fig. 44). There are essentially two types of glycosaminoglycans, those with weak negative changes, such as hyaluranic acid, and those with strong negative charges such as the chondoitin sulfates, heparins, and dermatan sulfate, the latter comprising the largest of proteoglycan species. Their distribution and physicochemical characteristics, which contribute to distinct functions, are also unique. Hyaluronic acid, with weak negative charges associated with the carboxylic acid residues present in glucuronic acid, has a tendency to form hydrated gels, and can therefore contribute signifiA
C
B
HEAT 1
of a Collagen Network
Since the stiffness of the stretched fibers relates to the degree of crosslinking of molecules within a fiber, other more sensitive methods have been used to evaluate this parameter. Denaturation of collagen by heat or chemicals can produce a collapse of the helical structure and a shrinkage in the direction of the longitudinal axis of the fiber. The isometric tensions developed during thermal shrinkage, as well as the hydrothermal swelling that accompanies this event, have been used to evaluate the physical properties of the collagen networks.589 Such an approach, particularly measurements of hydrothermal swelling, may allow us to quantitate differences in the degree of crosslinking of collagen that occur in
Thermal
Fig. 43. direction the
oriented
become those
The native
found
to
heat,
crosslinks
provided. there
are
when
the
If native
normally are
shrinkage
The degree
held
in magnitude
no thermal
are no bridges
between
together
On
the
such a
shrinkage the collapsing
rat
are
in (A).
by
these
If in addition by the
dialdehyde stability
when
it does
other
treated
hand
(C).
if
case
with
because
molecules
to
such as
as is the
occurs
a
such
tissues
by the greater
(B).
of
into
as shown
of contraction,
from
go
are introduced,
whatsoever,
originates
penicillamine,
and
crosslinks
“shrinks.”
is delayed
The
axis
molecules
adult
are generated
and force
no crosslinks tissue
in occurs
new crosslinks
crosslinks
collagen
collapse
and the tissue
is greater
the
of the tissue
crosslinks
artificial
As they
occur
network.
to the longitudinal
(gelatin).
molecules
“collapsing”
glutaraldehyde, occur,
by
contraction
the native when
fibers.
coil conformation
present,
of a collagen
is parallel
collagen
denatured
random as
shrinkage
of shrinkage
D-
there
COLLAGEN
STRUCTURE
AND
METABOLISM
49
Fig. 45.
negative
fluid)
and
positive Fig. 44. are usually
Collagen closely
ground
substance.
cule
cartilage,
in
containing (PGS)
and
fibers
This
to
acid
proteins
(A)
structure.
The
PGS
consists
which
negatively
charged
the
chondroitin
sulfate
(CSj
with
diagram
adjacent
hyaluronic link
do not exist
associated
in a vacuunt.
the proteoglycans depicts
a collagen
a proteoglycan
(HA), which
help
to
and keratan
sulfate
subunits
stabilize
core
glycosaminoglycan
mole-
aggregate
proteoglycan,
of a protein
They of the
(PC)
the from
chains
of
(KS) radiate.
cantly to the viscoelastic fluidity of synovial fluid and to the turgency of the skin of an infant. On the other hand, the negatively charged polysaccharides that contain sulfonic acid residues are able to develop strong ionic bonds with the positively charged amino acids on the surface of the collagen fibers, particularly lysine, hydroxylysine, and arginine. Such tissues are more compact, resilient, less hydrated, and exhibit the viscoelastic behavior typified by hyaline articular cartilage. They are also more collagenous than their fluid counterparts. Synovial fluid has no collagen, the vitreous has only small amounts of type 11 collagen, and the skin of a newborn rabbit has less than 2% collagen; this in contrast to the adult rabbit, which has more than 1.5% collagen.” Figure 45 illustrates these two extremes, the hyaluronic acid gel (A) and compact connective tissue (B) containing collagen and proteoglycans with strong negative charges such as chondroitin sulfate. The physicochemical properties of these two types of connective tissues, their viscoelastic properties, diffusion of macromolecules and of small ions through their midst and exclusion of molecules of various molecular weights (such as immunoglobulins) are, understandably, different Hydrodynamics
and Viscoelasticity
Articular cartilage is characterized by a high degree of hydration and owes its shock-absorbing
(A)
weak
fibers. to
shows
charges
surface. droitin
tendency
on
the
salt
collagen.
cations
other
with
affinity
divalent
cations
compact
structure
hand.
in hyaline
react
with
the
(+ +) or
due
to
by
the
group, the
assocation
the
collagen it can link
to
unstable.
charged
enhanced This
synnovral
of
by the sulfate
with
(e.g..
collagen
positively is
as shown. seen
on
are relatively
generated
linkages This
the
to
surface
such as calcium
groups
bonds
on
charges
gels
no
charged
sulfate,
a glycosaminoglycan
viscous
almost
but these
negative
acid,
forms
present
Via divalent
negatively
tight
Hyaluronic charges
the
cell
(Bj Chonstronger will form
residues presence generates
on of the
cartilage.
capacity to its ability to shift the water molecules from one domain to another. Water is not evenly distributed through the extracellular space,59h rather the upper 25% of articular cartilage contains approximately 85% water, but as the depth increases, the degree of hydration decreases in a linear manner to about 70%. However, these data differ with age, site of biopsy, and patholog:y. It is hypothesized that one of the major forces causing destruction of cartilage in osteoarthritis is hydration, which, through a process of swelling, causes the structure to collapse.597.598 Ultrastructural studies of the cartilage of dogs in which traumatic osteoarthritis is developing showed a disorganization of the perilacunar collagen associated with an increased volume of the tissue.s99 The rupture of the collagenous framework seems to occur as a result of osmotic pressure imbalance within the tissue that is caused by a dilution of the proteoglycan fixed charged density.600 Although most of the water in cartilage is located within the proteoglycan domain, the collagenous network may also play a role in the hemodynamic shift. When cartilagenous tissue is in contact with a saline solution or a physiologic fluid, the proteoglycans within the matrix exert an osmotic pressure r which depends on their concentration,60’ across the interphase. It is this osmotic pressure that is able to counteract an externally applied force and enables cartilage to remain hydrated under load. For cartilage to be in thermodynamic
50
MARCEL E. NIMNI
equilibrium with the synovial fluid, it must be balanced by a combination of tensile stresses exerted by the collagen network of the tissue (PC) and that coming from outside of the network, or applied pressure (Pa): (1) r = Pa + PC, or, expressed in another way, (2) Pa = Ps = ?r - PC, which means that at equilibrium the applied pressure has to be equal to the net swelling pressure of cartilage.600 The osmotic pressure of proteoglycans in solution has been measured at physiologic concentrations and is associated with the excess of ions present as a consequence of the fixed negative charges (FNC) of chondroitin and keratan sulphates. The molecular size or degree of aggregation of proteoglycans does not affect osmotic pressure.60’ Figure 46 shows a network of collagen fibers that contain part of a proteoglycan subunit. It represents resting cartilage where the osmotic pressure of the proteoglycan domain is neutralized by the built-in stresses exerted by the collagen network, which in turn prevents the tissue from swelling. If outside pressure is applied, the collagen pressure will be released as the cartilage begins to decrease in volume. The applied load will then compensate for the decreased contribution by collagen. The graph shows how the swelling pressure of cartilage (Ps) increases as a function of the FCD contributed by the sulfated glycosaminoglycans. The collagen in cartilage consists almost entirely of type II collagen, a collagen with unique characteristics. X-ray diffraction studies of collagen from human intervertebral discs showed differences in the intermolecular spacing of the collagen molecules within the fibers. Whereas the molecules in type I collagen are
Fig. 46.
Collagen
fibers
a proteoglycan
subunit
conditions
collagen
swelling sure
(PSI
is proportional
generated tional
the presure
by the
to their
are shown
within
their
pressure
of cartilage. to
the
fixed
proteoglycans,
concentration
constraining midst.
Under
part
(PC)
is neutralizing
The
net
charged which
the
swelling
pres-
density
(FCD)
in turn
in the tissue.MO
of
resting
is propor-
separated by a distance of 14 A, collagen type II molecules are separated by a distance of 16-17 A. No differences were noted after the specimens were dried.602 The intermolecular volume of fully hydrated collaged fibrils from a number of mineralized and nonmineralized tissues of adult rats has been determined by an exclusion technique and by monitoring specific x-ray diffraction parameters.603 Measurements made on wet rat tail tendon show that 55% of the volume of a fibril is occupied by collagen molecules and 45% by water.604 Assuming a cylindrical geometry for the fibers, one can estimate that the minimum measured increase in the lateral separation between type II molecules compared to type I is approximately 2A, which translates into a 30% intrafibrillar volume increase.602 From these estimates it can be calculated that a wet fibril of type II collagen may contain 50%-100% more water than does a fiber of type I collagen that has the same number of molecules. One can easily envision how additional water in type II fibers can be advantageous to tissues such as cartilage and nucleous pulposus, whose main functions are to absorb or distribute compressive loads, in contrast to type I fibers present in tendon and skin, which transmit tension. Such hydrated fibers can act as hydraulic shock absorbers, and in this way contribute to one of the major functions of the type II collagencontaining tissues. The etiology of this increased hydration is not clear, but could result in part from the higher degree of glycosylation of hydroxylysine, a characteristic of this “sugar coated” molecule. Proteoglycans surround the collagen fibers, but never penetrate into the interfibrillar space (Fig. 47).6o5 During the early stages of osteoarthritis, cartilage exhibits a loss of metachromasia due to a diminution of its glycosaminoglycan content.606 In vitro studies on the wear and tear of articular cartilage showed that removal of divalent cations (primarily calcium and, to a lesser extent, magnesium) by EDTA caused the tissue to exhibit a faster rate of wear.6o7 The cation binding properties of cartilage proteoglycans, particularly to calcium, have been long recognized6’* and it is possible that the loss of glycosaminoglycans seen in osteoarthritis and the decreased resistance to wear of affected cartilage may be associated with the loss of intermolecular stabilizing cations, such as calcium.
COLLAGEN
STRUCTURE
AND
METABOLISM
Figure 47 summarizes in a schematic fashion the mechanism that may further contribute to the changes in hydrostatic pressure within the various cartilage domains, and includes the contribution of intrafibrillar water to the overall picture. Role of Mechanical Factors in Determining the Organization of Collagen and Proteoglycans The normal mechanical stresses that act upon the connective tissues seem to be essential for maintenance of the cellular activities required for the synthesis, turnover, and organization of macromolecules of the extracellular matrix (Fig. 48). Reduction of such stresses by inactivity or immobilization results in loss of muscle mass (atrophy of disuse), bone mass (osteoporosis), and cartilage. The effects of immobility on the periarticular connective tissues is more difficult to recognize but just as significant, as it often leads to contracture, which is the principal complication of fracture treatment using cast immobilization. The largest change found in the composition of the stress-deprived periarticular connective tissues is reduction in the concentration of glycosaminoglycans and water.609 Consistent with such changes is the observation that the immobilized connective tissues show significant changes in the collagen crosslinking profile.6’0 The increase in sodium borohydridereducible intermolecular crosslinks must reflect
Fig. 47.
Water
and small
gen-proteoglycan among
the
various
pressure.
In this
domains
are shown
movement pressure
network
changes.
domains
diagram
of water
ions of
the
as distinct molecules
move
through
cartilage in
and
response
proteoglycan while
to
changes and
the arrows
shifting
the collaredistribute in
collagen
depict
to compensate
the for
Fig. 48.
Healing
ligaments
in rabbits.
(Courtesy
of Drs. W.
of surgically (A)
repaired
Mobilized
Akeson
joint.
and S. Woo.
anterior IB)
cruciate
Immobilized.
and D. Amiel).
the rapid turnover of the immobilized tissue. Hydroxylysinonorleucine and histidinohydroxymerodismosine are the major crosslinks contributing to this increase. This remodeling of the periarticular connective tissues, illustrated by an increased abundance of newly synthesized unorganized collagen, leads to a mechanically less stable ligament that is more susceptible to mechanical damage. On the other hand, the larger number of crosslinks could cause stiffness and are probably responsible for the development, of contractures. Administration of 17 fl-estradiol appears to exert a protective effect against such contractures by minimizing the loss of water and glycosaminoglycans from such tissues.“’ Cartilage tissue responds in a similar way. Immobilization by casting the knee of a normal dog results in rapid degeneration of its articular surface.6’2 This defect is accompanied by decreases in thickness of the cartilage, uranic acid and proteoglycan content, and by a defective aggregation of the newly synthesized proteoglycans. When the period of immobilization is less
52
MARCEL E. NIMNI
than six weeks, the defects are promptly reversed by ad libitum ambulation. On the other hand, vigorous exercise inhibits reversal of the atrophic changes induced by immobilization.6’3 Changes in knee cartilage after amputation of the ipsilateral paw resemble those produced by immobilization.6’4 From the examples cited, involving immobilization of ligaments and cartilage, the potential for additional research to lead to a greater understanding of the way in which mechanical factors affect the metabolism and structure of connective tissues becomes obvious.
FIBROSIS
Accumulation of collagen in excessive amounts is a major pathologic event that underlies several clinical conditions, including pulmonary fibrosis, liver cirrhosis, retrocorneal fibrous membrane formation, as well as various forms of dermal fibrosis, such as scleroderma, keloids, and familial cutaneous hypertrophic scars, collagenoma. Although in many of these diseases the terminal fibrotic lesion is considered to be the sequela of cellular injury, the cell populations injured and the endogenous mediators responsible for the postinjury fibrotic response vary from organ to organ. In many instances we seem to be dealing with an uncontrolled repair mechanism, where less organized and less specific connective tissue replaces a previously functional and carefully constructed matrix. In other instances we see an imbalance in the homeostasis of the extracellular matrix, where synthesis of macromolecules exceeds breakdown, the end result being an excessive accumulation of collagen. In order to understand some of the general features of fibrosis, we shall look at a few of the better investigated fibrotic diseases and experimental animal models.
Fibrosis and Cirrhosis
of the Liver
Although hepatic fibrosis is only one distinctive feature of liver cirrhosis, understanding of its pathophysiology has progressed rapidly and current efforts are directed at unveiling the mechanisms that connect cell injury with collagen deposition and at developing methods for early detection and designing new forms of thera615.616
PY.
In human liver disease and in animal models with liver fibrosis, proline has been found to increase in the liver. Although normal liver is capable of producing proline from glutamic acid, the amount synthesized is small. In Ccl,induced liver fibrosi?” or after ethanol administration6’* the formation of proline from glutamic acid is increased. In rats treated with Ccl,, inhibition of proline oxidase, the mitochondrial enzyme involved in proline degradation, contributes to the elevation of free proline.6’9 Proline concentration is critical for collagen biosynthesis and its incorporation into liver collagen in vitro is directly proportional to its concentration in the incubation medium.6’5 Normal human liver contains 5.5 mg of collagen per gram of fresh tissue6*‘; in rat liver the amount is much smaller (0.91 -t 0.15 mg/g).6’9 However, both human and rat liver contains the same types of collagen. In human liver, 33% is type I collagen, 33% is type III collagen, and the remainder is a mixture of type IV and type V collagens.h20 In a rat model of reversible Ccl,-induced liver fibrosis in the ratio of type I/III collagens remained constant throughout all stages of the disease.“2’ In human cirrhotic liver containing less than 20 mg of collagen per gram of fresh tissue, the ratio of type I/III collagen was also normal.620 However, in another study, higher concentrations of type III collagen were found in normal human liver (47%) and the ratio of type I/III was elevated in cirrhotic liver.622 A similar increase in type I collagen was observed in advanced cirrhotic liver containing more than 20 mg of collagen per gram of tissue.620 The distribution of liver collagens has been examined with the use of monospecific fluorescent-labeled collagen antibodies.623m625 The heavy collagen bundles of the liver correspond to type I collagen, the fibrous septa, the projections of the septa and the perivascular areas stain strongly with anti-type 1 collagen antibodies. Type III collagen corresponds to some but not all of the reticulin fibers. Antibodies directed against the procollagen terminal peptides also localize on liver type III collagen. Type IV collagen has been localized in sites containing basement membranes623 and type V collagen in the portal triads and the terminal venule areas. A marked increase of type 111 collagen within the parenchyma is observed in
53
COLLAGEN STRUCTURE AND METABOLISM
iver fibrosis. Septum formation is due to collapse of necrotizing hepatocytes as well as collagen The final cirrhotic stage is charleosynthesis. ,Icterized by thick collagen fiber bundles derived Yom type I collagen molecules. A distinct pattern of collagen neosynthesis is seen in liver fibrosis that is characterized first by ‘3asement membrane collagen deposition, second ‘3~ type 111 collagen deposition, and third by type I collagen deposition.
LIVER
CONTRACTION
One of the striking features of terminal cases of liver cirrhosis is the decrease in liver size. Since collagen per se has no contractile capacity, cellular elements that possess a contractile system may play a role in liver shrinkage. In this connection it has been shown that human626 and animalh” livers with fibrosis contain myofibroblasts that can be grown from explants of fibrotic tissue.h2s The presence of contractile myofibroblasts, and their positive response to drugs, such as herotonin and epinephrine,627 suggest that these cells are responsible for scar contraction and liver shrinkage. The increase within cirrhotic liver of all collagen types, with predominance of type I collagen, further supports the notion that the process of scar formation in liver is similar to that in skin and other tissues.
DIAGNOSIS
AND
TREATMENT
OF LIVER
FIBROSIS
Some novel approaches that may aid in the early diagnosis of liver fibrogenesis have been recently reviewed.h’5.628,629These include assay of the specific enzymes involved in posttranslational modification of collagen, and metabolites used in the synthesis of collagen and degradation products, including the procollagen peptides specific for the various collagen types. The problem with these assays is that they are not tissuespecific and the values obtained usually reflect changes in the overall metabolism of tissue collagen. Regarding treatment, two compounds with .mtifibrogenic activity, but which act by difrerent mechanisms, are proving to be of value. These are penicillamine for the treatment of 3rimary biliary cirrhosis6i0 and of colchicine in
other types of cirrhosis.6’5 The results obtained seem to warrant trolled use of these compounds. PULMONARY
encouraging further con-
FIBROSIS
Collagen is a major component of lung tissue and its concentration and distribution is affected by a number of adverse systemic and environmental factors. Pulmonary fibrosis is frequently the end result of such events. Regardless of the etiology, these diseases are usually characterized by a progressive loss of lung volume and functioning alveolar-capillary units. In order to understand the factors that may modulate the bisynthesis of collagen and lead to fibrosis of the lung, we shall discuss two situations that have received particular attention. These are idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis. 1PF is grouped with the fibrotic lung disorders because patients with this disease have characteristic restrictive physiologic findings as well as increased fibrous tissue within the alveolar interstitium.63’ By electron microscopy, one can visualize at least three morphologic forms of collagen in the normal alveolar interstitium632: (1) most prominent are the parallel arrays of 500~1,000 Athick, crossbanded fibers; (2) the randomly arrayed 160-350 A-thick fibrils, which differ from the 1OO- 150 A-thick microfibrillar component of elastic fibers (the former are larger, randomly arrayed, and show crossbanding, whereas the latter are smaller, usually found on the periphery of amorphous elastin, and are not crossbanded); (3) basement-membrane collagen, which usually appears amorphous or finely lilamentous. In the normal adult lung, more than 90% of the collagen is type I and IIIh3* normally found in a ratio of approximately 1.5-2.5 to 1.“’ Morphologic evaluation of the connective tissue in IPF has demonstrated thickening of alveolar interstitium with fibrous tissue, as well as basement membrane disruption and thickening. On the basis of collagen type fluorescence. two distinctive patterns of fibrosis were recognized while observing a variety of lung diseases.h34 Areas of mature collagen surrounding vessels and bronchi and in established scar tissue, for example in asbestotic pleural plaques, are ,virtually exclusively type I collagen. By contrast,
54
areas of early active fibrosis like sarcoid nodules and organizing pneumonia, which usually contained variable numbers of fibroblasts and chronic inflammatory cells, are characterized by an increased proportion of type III collagen and a greater intensity of both types I and III collagen fluorescence.634 It has been suggested that IPF is a disease in which there is a shift in ratio of interstitial collagens toward more type I collagen; in IPF, type I collagen represents approximately 80% of the lung collagen.633 Such a shift characterized by an increase in the thick, cross-banded fibers is seen on light and electron microscopy. Because type I collagen fibers are less elastic than those of other collagen types, the parenchyma would be less compliant compared with that of normal lung. Thus, a shift in collagen types without a change in total collagen content would be consistent with the morphologic and physiologic alterations found in IPF. In agreement with this, there was no significant differences in the collagen content between patients with IPF compared with that of control subjects.635 In addition, there was no correlation between collagen content and the morphologic assessment of the degree of fibrosis. Furthermore, the rates of collagen and noncollagen protein synthesis in explants of lung from patients with IPF demonstrated no significant difference compared with those of the control subjects. The results of this study are consistent with the concept that IPF is a disease of an alteration in quality, form, and location of collagen rather than simply a disease of increased interstitial collagen. Another study636 suggests that patients with IPF have active collagenase activity in their lower respiratory tract, thus providing a mechanism by which the initial disordering of interstitial collagen might occur. Thus, even though the rate of collagen production by lung cells may be normal in IPF, if cellular order is disrupted, as is the case in this disease, one would expect deposition of collagen in abnormal areas, and the morphologic finding of focal accumulations of collagen seen in IPF. An interesting experimental model that may also help us to understand the development of fibrosis relies on the use of bleomycin. This drug causes pulmonary fibrosis as an undesirable side effect,637 and has been used to produce fibrosis
MARCEL E. NIMNI
experimentally in mice,638 rats,639*640 hamsters 64’3642 and baboons.643 The baboons display all the physiologic features of interstitial lung disease, including edema and infiltration of the alveolar septums by inflammatory cells. The lobar content of collagen in the biopsy specimens increased 48% over that of the controls, and content of elastin nearly doubled.643 Following cessation of treatment the excess connective tissue proteins persist despite a resolution of inflammation, and their accumulation due to increased rates of synthesis also continues. The net rate of collagen synthesis in shortterm lung explant cultures from rats examined after administration of a single intratracheal dose of bleomycin showed that after a delay of three to four days, the net rate of collagen synthesis was significantly increased over that in untreated control animals.640 This increase was sustained during approximately 10 days and subsequently returned to normal or below normal values. The increased rate of collagen synthesis could not be explained by increased free radioactive proline pools or by age differences among the animals. The period of increased rate of collagen synthesis preceded the detectable increases in lung collagen content. In hamsters, collagen accumulation is also increased as a result of the enhanced rates of collagen synthesis. When lung fibroblasts are cultured in the presence of conditioned media obtained from explants of normal and bleomytin-exposed hamsters, it was observed that the conditioned medium decreased fibroblast proliferation and collagen production in a dose-dependent manner.642 This observation may help to explain why, after a period of enhanced collagen production, the synthesis of this protein is restored to normal in the experimental model. Exogenous PGE, and c-AMP also suppressed fibroblast proliferation and collagen production. The suppressive activity factor present in lungconditioned medium has an apparent molecular weight of 15,000-20,000 daltons, is heat stable, and may represent a means for limiting collagen accumulation following injury. To evaluate how tissue fibroblasts control collagen production, HFL-1, a diploid human lung cell strain, was studied during periods of rapid cell growth and relatively slow growth over 25 population doublings.644 Although the specific activity of intracellular free proline and the per-
55
COLLAGEN STRUCTURE AND METABOLISM
centage hydroxylation of proline in collagen varied considerably depending on the growth rate of the cells, collagen production by HFL-1 was constant, even during periods of rapid cell growth. Thus, under conditions of a stable environment, populations of soft tissue fibroblasts rigidly control collagen production, probably by internally modulating their levels of procollagen mRNA.645 This intrinsic homeostasis is probably disturbed by locally produced or systemic factors that arise as a result of the various forms of injury that are able to induce lung fibrosis. Further understanding of the control mechanisms involved should increase our ability to prevent, arrest, and reverse this damage. KELOIDS
AND
HYPERTROPHIC
SCARS
Keloids can be defined as abnormal scars that extend beyond the confines of the original wound and rarely regress, while hypertrophic scars are raised sears within the confines of the original wound and frequently regress spontaneously.646 Whereas hypertropic scars develop soon after keloids may not appear for many surgery, months. Their distribution, relationship to the degree of injury, and response to motion and to surgery also differ significantly.647,648 Both these entities are characterized by excessive deposition of collagen in healed skin wounds,649 and underlying abnormalities in both the synthesis and degradation of collagen have been suggested in their pathogenesis. The rates iof collagen synthesis, the level of proline hydroxylase6” and several other enzymes, as well as the number of fibroblastP’ are all increased in keioids. The growth kinetics for fibroblasts derived from normal skin, normal scar and keloid are similar; however, the rate of collagen synthesis per fibroblast is greater for the keloid derived cells6” The reason for this difference is not known although several hypotheses have been advanced.h52 The levels of collagenase in keloids have been reported to be increased,653 decreased,654 and unchanged655 when compared to normal skin.655 It has long been known that scars maintain a higher metabolic activity and rate of collagen turnover than the adjacent normal tissue6j6 and, in spite of variable levels of collagenase, collagen may not be removed because of the presence of inhibitors to this enzyme, such as cY-globulins, which are deposited more heavily in keloids and
hypertrophic scars than in normal scars or skin.6s7 There seem to be no significant differences in the ratio of types I and 111 collagen produced by fresh biopsies and fibroblasts from keloids compared to normal human skin controls.658 This is surprising since both keloids and hypertrophic scars seem to contain larger amounts of type III collagen than does normal skin6s9 and also exhibit changes in lysine and hydroxylysine-derived crosslinks resembling those seen in experimentally induced wounds. The fact that under culture conditions the fibroblasts derived from keloids do not differ from normal in the types of collagen produced may reflect the presence in vivo of local factors of a biochemical or mechanical nature that could be responsible for the abnormal cell behavior and induction of the defect. The recently described trifunctional crosslink, pyridinoline, has been reported to be present in hypertrophic scars.66” Since hydroxylysine is a precursor of such a crosslink, its presence is not surprising, and it is possible that such a stable crosslink may contribute to the unusual accumulation of collagen in such tissues. Because of the inhibitory effects of corticosteroids on protein synthesis and of collagen in particular, these agents have been used to treat these conditions. Injection of corticosteroids, particularly triamcinolone, into the lesion will cause keloids to soften and regress in size.h6’ When the effects of triamcinolone acetonide were tested on fibroblasts isolated from normal dermis and keloids, 662 it was observed that the DNA content and cell division of normal fibroblasts was inhibited to a greater extent than that of keloid fibroblasts. On the other hand, the drug enhanced the production of TCA-soluble 14Chydroxyproline in both cell types, suggesting that collagen degradation could be increased by this corticosteroid. FIBROTIC
RESPONSE FOREIGN
TO IMPLANTATION
OF
MATERIALS
Most biomaterials cause a host tissue inflammatory response resulting in collagen synthesis and deposition around the implanted material. In some instances, marked inflammation and collagen synthesis, is desired in order to produce a firm fibrous capsule of collagen, as around the sewing ring of a valve implant to secure it in place. Similarly, the repair of a mandibular
56
defect is best corrected by a biomaterial which not only fills the defect, but also stimulates collagen production to allow a strong fibrous connection between bone and the biomaterial. In other instances, however, minimal collagen deposition is desirable. For example, breast augmentation requires a material with minimal collagen response, thereby producing a soft, natural breast. The granulomatous response that results from the implantation of clay correlates well with the significantly increased relative rates of collagen synthesis. Sand, on the other hand, which causes minimal histologic reactivity, does not increase the relative rate of collagen synthesis compared to control wounds.663 Implantation of soft silicone rubber prostheses is followed by growth of an enclosing connective tissue capsule that may undergo a process of contracture that results in pain, tissue deformity, and extrusion of the prosthesis.664,665 Capsules developing in animals are histologically and ultrastructurally similar to those seen in humans, and also show contracture.666’667 The total collagen content of experimental capsules implanted in rats reaches a plateau with little change after 60 days.668 When fibrous tissue capsules around siliconegel and saline-filled breast implants were examined by light, transmission, and scanning elecregularly arranged dense tron microscopy, connective tissue capsules were seen.667 This tissue contains bundles of collagenous fibers that are densely packed and lie parallel to each other, forming structures of great tensile strength. The outer surface of the connective tissue capsule contains reticulum fibers, small in diameter, that branch to form a net-like framework which supports the collagenous material. At the inner, surface fibrocytes and histiocytes are present in single layers and form an epithelial-like structure. The connective tissue of the capsules also contains capillaries and fibrocytes, which are usually deployed along bundles of collagen fibers and appear as fusiform elements with long processes. Contractile fibroblasts (myofibroblasts) are also found in these fibrous capsules. However, deformation of the augmented breast does not seem to be caused by the action of the myofibroblasts, because their number is too small. Rather it is more likely caused by the inelastic arrangement of large amounts of collag-
MARCEL E. NIMNI
enous material layered around the capsule. Contracting capsules seem to present elevated levels of total glycosaminoglycans and chondroitin-4sulphate compared to noncontracted capsules.668 There is evidence that the ultrastructure of contracted capsules is similar to that of hypertrophic scars.665,667-669The kinetics of collagen deposition and of proteoglycan accumulation in fibrous capsules surrounding implants are similar to those of normal wound healing. DEGENERATION INTERVERTEBRAL
OF THE DISC
The intervertebral disc consists of three parts: the annulus fibrosus, the cartilage plates above and below, and the nucleus pulposus in the center. During adult life the intervertebral disc undergoes a series of progressive morphologic and physicochemical changes, that begin in the teens and become significant in the fourth and fifth decades of life.67o,67’ The two major components responsible for these changes are the annulus fibrosus and the nucleus pulposus. The annulus in the adult lumbar spine is formed by a series of concentric encircling lamellae, which are thicker anteriorly than posteriorly. The peripheral lamellae attach themselves to the bony edge of the vertebral body in the way of Sharpey fibers, while the remaining lamellae continue into the cartilagenous plates. The nucleus pulposus, centrally located, consists of a three-dimensional network of collagen fibrils enmeshed in proteoglycan gel. It occupies about 30%-50% of the disc cross-sectional area of the disc. It contains few cells resembling chondrocytes. The water content of the disc is highest at birth, being around 90% in the nucleous and 78% in the annulus; with age, these values decrease to about 70% in both areas. This change in hydration is coupled to changes in the chemical composition and mechanical properties. After childhood the nucleus is no longer sharply demarcated from the annulus, and the collagen fibers become gradually coarser and merge.672 The proportion of mucopolysaccharides decreases and with age changes its nature, the ratio of keratan sulphate to chondroitin sulfate becoming increased.673 In mature rabbits the administration of androgens, estrogens, and growth hormone is able to reverse these changes.674 The proteoglycans become smaller in molecular size and form fewer aggregates with
57
COLLAGEN STRUCTURE AND METABOLISM
hyaluronic acid.675 These changes seem to impair the ability of the nucleus to resist and redistribute loads, and the greater stresses imposed on the annulus fibrosus can lead to its eventual mechanical failure. Early studies showed that the collagen in the intervertebral disc had a higher content of hydroxylysine than was expected from the total collagen content.676 The collagen is not distributed homogenously in this tissue, as the outer regions of the annulus contain the largest amount of collagen.677 The collagen is heterogeneous and consists of a mixture of type I and II collagen (Fig. 49).678 Serial section from the outside towards the center of the nucleus pulposus reveals variable ratios of type I to type II collagen. The outer fibers in the pig are exclusively type I collagen and the inner mostly (95%) type II collagen. In the human these numbers are somewhat different since the annulus contains consistently larger proportions of type II collagen.679 As in all other connective tissues, the mechanical strength of this structure is related to the degree of crosslinking between the individual
TYPE
Fig.
49.
Distribution
disc. The outer collagen with
while
varying
(adapted
from
part
of collagen
of the annulus
the inner proportions
nucleus in
the data of Eyre
in the
fibrosus pulposus
the et al.).
I
intervertebral is mostly
is mostly
intermediate
type type zones
I II
collagen molecules, and the diameter and distribution of the fibers. Marked differences are observed in the reducible crosslinks of discs progressing down in young lumbar spines. 680The content of reducible crosslinks declines between Ll-L2 and L5-Sl in both nucleus and annulus collagens. Also, when spines with a grossly degenerated disc were studied, the disc above the degenerated one, but not the degenerated one itself, was rich in reducible crosslinks, normally absent from discs at that age. This was interpreted as a sign of recent synthesis of new collagen, suggesting that the normal disc had compensated in some way for the defective disc below. The new collagen was predominantly type I in both annulus and nucleus, based on an enrichment of hydroxylysinonorleucine and histidino-hydroxymerodesmosine, and an analysis of LYchains labeled radioisotopically during incubation of disc tissue in vitro.680 The relative contributions of types I and II collagens of the normal annulus to the crosslinking profiles are difficult to assess, though the profiles loosely fit a combination of those for type 1 collagen of insoluble tendon and type II of hyaline cartilage. The identification of elastic fibers within, the disc6*’ raises the possibility that such tissue makes a contribution to disc elastic function, however, the proportion present is not high. Immature elastic fibers, similar to those seen in other developing tissues, have been observe:d in the human fetal disc.6”2 The presence of elastic fibers within the annulus fibrosus could be of importance in supporting collagen fiber recovery after deformation, as has been found in other tissues. There is some evidence to suggest that both the collagen and glycosaminoglycans of some connective tissues are abnormal in scoliosis.6x’ The stability of the polymeric collagen of the skin from adolescents with idiopathic scoliosis was found to be significantly less than that of control subjects, except for two patients with mature skeletons, in whom the polymeric collagen was normal. One of three patients with congenital scoliosis also had polymeric collagen of low stability. In an attempt to further resolve these questions, the collagen extractability and distribution across normal and scoliotic discs was investigated.684v685 The collagen content of the scoliotic nucleus pulposus was found to be higher
58
MARCEL E. NIMNI
than normal, particularly at the apex of the curve, but no consistent correlation was found with the spinal mobility or degree of curvature.685 The collagen content and type of the annulus fibrosus from scoliotic discs was shown to be abnormally distributed, again only in those discs encompassed by the curve.6843685These results contrast with the pepsin extractability of collagen in the annulus fibrosus, which was abnormal in all the scoliotic discs examined, independent of location. While the precise interpretation of these latter findings is complex, it would seem that a subtle defect in collagen does exist within the scoliotic disc which, coupled with extraspinal influences, may play an important role in progression of the scoliotic curve. Attempts have been made to alter the properties of these discs by infiltration with proteolytic enzymes that will remove collagen and proteoglycans, but results are speculative.686 Degenerating discs, particularly in the elderly, tend to calcify687 as mineral is deposited in the annulus and in the perilacunar regions of the end plates. Other conditions that can lead to degeneration and calcification have been discussed.688 Because of its physiologic significance, the clearly age-related chemical changes observed, the unusual distribution of collagen types relative to the shape and function of this tissue, and the clinical implications associated with the high degree of dissability associated with the degeneration of the intervertebral disc, this structure deserves to be further studied.
SCLERODERMA (PROGRESSIVE SYSTEMIC SCLEROSIS)
Progressive systemic sclerosis is considered to be one of the most classical collagen diseases. It is characterized by extensive fibrotic infiltration of many organs with a concomitant accumulation of collagen. Most commonly involved is the skin, but other internal organs such as the lung, heart, kidney, and gastrointestinal tract are frequently affected. Two stages are evident in the development of this disease: a cellular stage characterized by perivascular or diffuse cellular infiltration of the dermis and subcutaneous tissues and a fibrotic stage that is associated with deposition of collagen. Most determinations of collagen in scleroderma have been done on skin, where it was observed that the most characteris-
tic feature consisted of replacement of subcutaneous tissue by dense collagen fibers.689*69a The etiology of the disease is not known although several recent reviews and symposia have addressed themselves to this question.69’*692 Here we shall briefly address the question of collagen deposition in an attempt to relate it to other forms of fibrosis. In general, fibroblasts from the skin of patients with scleroderma seem to produce more collagen in tissue culture than do their normal counterparts, although again this is not a universal finding.69’ The serum from scleroderma patients may enhance this effect, particularly if the fibroblasts are derived from the reticular dermis.693 As discussed earlier, normal skin contains primarily type I collagen (approximately 80%) and type III collagen (approximately 20%). Deviations from this ratio in skin of patients with scleroderma have been found by many investigators who studied this disease,69’ and such findings seem to correlate with the state of activity of the tissue. In general, the findings seem to parallel those of wound healing and other situations where there is a sudden burst of collagen synthesis. It appears that in the early stages of the disease there is an increased synthesis of type III collagen over that of type I, but this ratio reverts to normal when the disease activity subsides and the ultimately formed fibrotic tissue contains normal proportions of type I and III collagens. By immunofluorescent microscopy using specific antibodies against types I and III collagen, 694it was observed that in the cellular stage the dermis and adipose tissue contained perivascular or diffuse cellular infiltrates accompanied by enhanced deposition of type III collagen. The lower dermis also showed an increase in type III collagen. In the fibrotic stage the papillary layer shows a reduction and clumping of type III collagen and the lower dermis show compact collagen, mostly of the type I type. Although these observations are not quantitative, they reflect the general pattern of fibrosis that begins with enhanced production of type III collagen and leads to an ultimately normal proportion of type III (approximately 20%). These observations are confirmed by chemical analysis of scleroderma lungs, which show types 1 and III collagens to be present in the same proportions as in immunormal 1ung.695 Using the enzyme-linked nosorbent assay method antibodies to types I and
59
COLLAGEN STRUCTURE AND METABOLISM
IV collagens were detected in the sera of patients with scleroderma.696 Antibodies of type IV CO]Iagen are present in significant amounts and correlated with the presence of abnormal pulmonary diffusion capacity, so did antibodies to type 1 collagen, suggesting that autoantibodies to basement membrane and interstitial collagens may participate in the pathogenesis of this disease. Further support for the involvement of an immunologic component in this disease is provided by the fact that lymphocytes from scleroderma patients, when compared to those from normal donors were found to produce soluble factors that induce fibroblasts to increase synthesis of collagen without enhancing cell proliferation.697 The role of other cell types (platelets, endotheha1 cells) and their metabolic products,69’ collagen or procollagen degradation products,698 as well as fibronectin699 and other connective tissue macromolecules on the control of collagen synthesis and deposition remains a subject of interest. DlJPUYl-REN
CONTRACTURE
AND
MYOFIBROBLASTS
This is a disease that results in flexion contracture of one or more fingers. The process is not limited to the fingers, however, since it can also involve the soles of the feet and penis. The changes are usually progressive and irreversible and are associated with characteristic changes in the longitudinal fasciculi of the palmar aponeurosis. Histologic observation reveals a proliferation of connective tissue cells leading to the formation of characteristic nodules and contracted longitudinal bands of collagen. The nodules appear in the early phases of the disease and the collagen deposition reflects the more advanced stages.“’ For many years it was assumed that the contractile effect was associated with the intrinsic ability of the collagen fibers to shrink when (denatured, but this concept cannot be substantiated. A more feasible hypothesis arose when it was observed that many of the cells present in the involved area had characteristics similar to smooth muscle cells and exhibited a contractile apparatus: such cells have been termed myofii~rob1asts.70’~702The fibrillar system that develops
in these cells consists of parallel bundles of fibers, averaging 60 A in diameter, usually oriented along the long axis of the cell. Strips of granulation tissue containing such cells respond to a variety of pharmacologic agents in a manner similar to smooth muscle cells.“* It recently has been shown that fibroblasts exert forces larger than those needed for locomotion and that this excess traction may function in the rearrangement of extracellular matrices.‘03 Fibroblasts in vitro are capable of contracting hydrated threedimensional collagen gels into tissue-like structures, the extent of contraction varying directly with the cell concentration and inversely with the collagen concentration.704 Recent observations have been made which support the view that the fibroblast can modulate between the contractile and “normal” phenotype. Periodontal ligament fibroblasts contracting collagen gels can change their phenotype in accordance with the stages of gel compaction.705 In the early and active phase of gel compaction, the fibroblasts exhibited numerous cell processes containing microfilamentous material and few organelles, little RER and few Golgi membranes, but many free ribosomes, numerous structures resembling gap junctions, and a rich cell coat. Fibroblasts changed to a more rounded, relaxed state after 24 hr when the gel was compacted. At this stage, extensive RER and Golgi membranes, small peripheral microfilament bundles, little cell coat, and few, if any, gap junctions were observed. On the other hand, fibroblasts plated on plastic exhibited prominent RER and Golgi membranes, peripheral microfilament bundles, and junctions of the adherent type. The results suggest that the myofibroblasts could represent a reversible phenotypic modulation of fibroblasttype cells to a contractile state. When compared to age-matched control aponeurosis, lesions of Dupuytren disease contain larger amounts of water, collagen, and chondroitin-sulphate, as well as increased proportions of soluble collagen and reducible cross links.7”h The lesions show also increased amounts of type III collagen and an increased hydroxylation and glycosylation of the reducible crosslinks. All these parameters are characteristic of granulation and scar tissues. Type 111 collagen was located by immunofluorescence on thin argyrophilic fibers and within large fiber bundles that appeared to be disrupted into microbundles. The
60
MARCEL E. NlMNl
increase in type III collagen and the presence of myofibroblasts in the apparently unaffected aponeurosis suggest that the disease could be initiated within the aponeurosis and propagated by cells migrating along the collagen bundles. In general, the chemical changes observed reflect a situation of rapid synthesis and turnover of collagen since, compared to normal tissues, the involved fascia shows elevated levels of hydroxylysine, reducible aldimine intermolecular crosslinks and the presence of hydroxylysinohydroxynorleucine, virtually absent from normal adult palmar fascia.“’ The nature of the disturbance that leads to the proliferation of myofibroblasts and to the accumulation of abundant connective tissue of an immature nature is not known. The disease process can progress only as long as there are nodules present which have not yet undergone complete resolution. The nodules completely disappear, marking an end to the involutional (contraction) stage and the establishment of the residual stage (final stage) in which no further progression occurs. But the contractures previously incurred during the involutional stage, if not treated surgically persist without alterpostoperation ation.“’ Frequency of recurrence depends upon the stage of the disease at the time the operation is performed. Operations performed during the residual stages rarely recur. In a late involutional stage, complete excision of the nodules is followed by recurrence in 20% or less. In the proliferative early formative stage of the nodules, the recurrence rate is higher and especially so if parts of the nodules are not resected. CORNEAL
ical and thermal burns, and cornea1 graft failure. Diverse terms are used to describe cornea1 fibrosis, the most common of which are retrocorneal fibrous membrane (RCFM),“* or posterior collagenous layer (PCL).‘09 This abnormal tissue appears between Descemet membrane and the cornea1 endothelium. Histopathologic studies show a multifibrillar layer posterior to the normal Descemet membrane, the thickness varying in different regions of the cornea. Ultrastructural studies show that the RCFM is composed of fibrils, ground substances and cells (Fig. 50). The cells have a characteristic fibroblastic morphology in contrast to the polygonal shape of cornea1 endothelial cells. The origin of the fibroblast-like cells in RFCM remains controversial. While they could be derived from stromal fibroblasts7” or from transformed cornea1 endothelial cells, 7’1m7’3the endothelium is the most likely source, as substantiated in numerous experimental models. In culture, fibroblastic cells isolated from
FIBROSIS
Disease and damage of the cornea1 endothebum and its basement membrane (Descemet membrane) frequently cause loss of function of the cornea1 endothelium, cornea1 edema, and progressive cornea1 opacity and scarring (cornea1 fibrosis). Such fibrosis is a common final outcome for a number of cornea1 diseases and is a leading cause of blindness. The various ocular and cornea1 disorders that have been associated with such cornea1 fibrosis include Peter anomaly, congenital hereditary cornea1 dystrophy, hereditary cornea1 edema, macular dystrophy, posterior polymorphous dystrophy, Fuch dystrophy, interstitial keratitis, herpes simplex keratouveitis, postoperative retrocorneal membrane, chem-
Electron micrograph showing the changes Fig. 60. associated with the development of a retrocorneal fibrous membrane (RCFM). Cornea1 stroma (St) Descemets’ men brane (Dm) fibrocyte (f) and endothelial cell (Endo).
61
COLLAGEN STRUCTURE AND METABOLISM
RCFM synthesize predominantly type I procollagen and secrete it into the medium. The CO!lagen molecules extracted from the cellular fraction and RCFM in organ culture demonstrate a-size chains, the final product of processing. These findings suggest that procollagen peptidase activities are associated with cell surfaces and/or the extracellular matrix associated cell surface. The stoichiometry of type I collagen shows two CY,and one LY*chains, indicating that type 1 trimer is not present on this tissue. On the other hand, the major collagenous peptide synthesized by rabbit cornea! endothelial cells is basement membrane collagen.7’437’5 However, type ITT collagen is synthesized by bovine709 but not by rabbit cornea! endothelial cells. Other components of the RCFM such as proteoglycans and glycoproteins, have not yet been studied. Little is known about how transformation of one cell type to another occurs. It has been suggested that endothelial cells are pluripotent and become either epithelial-like7” or fibroblastextracts from leukocytes like; 7”x~7”9lysosomal have been reported to induce endothelium to acquire an epithelial-like pattern.7’” However, little is known about what causes such transformation. When injury is caused to the cornea! endothelium, either directly or indirectly, polymorphonuclear leukocytes migrate to the wound (Fig. 5 I ). Under such conditions, collagenase released by the leukocytes is activated and damages the exposed Descemet membrane. Leukocytic lysosomal enzymes can also cause cell damage.
~nwation wn byr d DM,
GWMOI ibrolb (RCFM)
TmrfmnDd ndomllum
Fig. 51. Schematic diagram showing the sequence of events following the application of a frozen probe to the cornea of a rabbit which leads to retrocarneal fibrosis.
Therefore, the cells participating in the repair process are faced with an abnormal extracellular matrix (which may negatively modulate their behavior) and may suffer damage. Consequently, the endothelial cells could redifferentiate into fibroblast-like cells which then deposit an abnormal extracellular matrix and generate a barrier to light transmittance.7’7 The scar remains permanently in situ and the regenerating endothelium may resume production of a physiologic collagen type. For more detailed information on RCFM, the review by Waring et al. 709is recommended. NERVE REGENERATION AND SCAR: FORMATION
The factors that modulate growth and regeneration of nervous tissue are part of a most interesting biologic and clinical problem. Studies done both in vivo and in vitro have shed significant light on the active role played by collagen in enhancing or inhibiting this process. The classic observations by Key and Retzius7” ancl Ranvier719 have laid the foundation for subsequent studies on the microscopic anatomy of the peripheral nerves. The former suggested the presence of an epineurium, perineuriurn, and endoneurium, which correspond to the perifascicular tissue, the lamellated sheath and the intrafascicular tissue described by Ranvier.‘19 Electron microscopists have examined the nature of the connective tissue that surrounds peripheral nerve fibers.720,72’ The Schwann cells of myelinated nerve fibers are ensheathed by a basement membrane that is continuous across the nodes of Ranvier from one internodal segment to the next. These, in turn, are surrounded by a condensation of endoneural collagen fibers. The structural organization of peripheral nerves includes within the perineurium two tissue constituents: the population of nerve fibers and the endoneural connective tissue. The fine structure of cross-section of a peripheral nerve is consistent with this concept since it shows a background of fairly evenly distributed collagen fibrils, running para.llel to the axis of the nerve fibers, the latter appearing to be suspended between them. An entirely different pattern of organization of collagen fibrils becomes apparent when isolated nerve fibers are studied, particularly with the scanning electron microscope.722
62
MARCEL E. NIMNI
Observation of the isolated nerve fibers of rat sciatic nerve show that the majority of the collagen fibers form a tightly woven cuff around individual nerve fibers. A relatively small fraction of the fibrils form an extremely loose, cobweb-like reticulum spun between the individual nerve fibers that holds them within a wide-mesh net. This continuum manifests itself along the entire nerve fiber, running parallel to the axis and often taking a slightly sinusoid curse (Fig. 52). These findings clearly support the preelectron microscope concept of Key and Retzius, generated more than 100 years ago, proposing a distinct membranous sheath around each individual nerve fiber, and are further supported by the work of Thomas, who observed (using the electron microscope) a condensation of collagen around nerve fibers. Following surgical transection of a peripheral nerve, regeneration proceeds from the stump by axonal sprouting and cell proliferation, causing the connection between the proximal and distal stumps to become reestablished. These cells proliferate and migrate to form a union scar.723 Regenerating axons invade the scar and cytologic differentiation towards a Schwann cell
Fig.
52.
Peripheral
old child during fibers
aligned
magnification
nerve
autopsy.
obtained
Showing
from
a three-year-
cross sections
parallel
to the direction
12,500
x; courtesy
of collagen
of the nerve.
of Dr. Mei
Liu.)
(Original
seems to occur from undifferentiated mesenchyma1 cells. The direction and course of the regenerating axon seems to be determined by the primitive mesenchymal cells of the granulation tissue, which exhibit a high degree of affinity and provide mechanical support for the regenerating axon. It has often been postulated that the central nervous system is capable of regeneration, but that its efforts are defeated by other processes, such as mesenchymal scar formation, which by depositing collagen creates a barrier that physically blocks the advance of regenerating axons into and across the wound. Chemical analysis showed that little collagen is present in unwounded spinal cord compared with surrounding mesenchymal membranes.724 Nevertheless, significant collagen synthetic potential was found within the spinal cord, a tissue of neurectodermal origin. The rate of collagen synthesis per unit of collagen content in the unwounded spinal cord is high and is equivalent to that of wounds at their maximum collagen synthetic rates. Wounding the spinal cord, pia mater, and dura mater caused substantial elevations in rates of collagen synthesis and deposition in these tissues. These synthetic rates remained at maximum levels throughout the eight-week study, a prolonged period when compared with other wounded tissues. Prolyl hydroxylase activity is extremely high in unwounded spinal cord, and after wounding a marked accumulation of collagen is seen to occur.724 The cell type responsible for this collagen synthetic activity has not been identified. In order to further understand the interactions between granulation tissue and nerve growth several experimental approaches have been used, one of which involves study of the migration of peripheral nerves through a preformed tissue space.725 For this purpose a tubular channel was created by implanting a silastic rod. When the rod was removed, a pseudosynovial tube was created that was then used to guide the growth of a resected sciatic nerve. Although growth of the nerve occurred within the tubular space it did not seem to be guided by the walls of the tube. In the process of regeneration, a perineural connective tissue structure was formed which was similar to that which surrounds the normal peripheral nerve. This experiment strongly indicates that the regenerating nerve fibers are responsible for
COLLAGEN STRUCTURE AND METABOLISM
the development and maintenance of the structural organization of the perineurium, mediated, at least in part, by the production of neurotropic substances that may play a key role in guiding axonal growth during normal development and regeneration of the peripheral nerves.726,727 The process by which axons regenerate following freeze injury to the optical nerve of the newt has been investigated.728 It seems as if the basal lamina which persists inside the optic sheath provides continuity across the site of injury and orients the axon sprouts in an orderly process of axon regeneration without neuroma formation. Types I and III collagen have been detected in human femoral nerve.729 Observation with the electron microscope shows two different populations of collagen fibers, based on their diameters:“” the collagen fibers in the endo- and perineurium have distinctly smaller diameters than those of the epineurium. These investigators have developed a method that relies on changes in birefringency observed in tissue sections stained with Picrosirius and that allows type I collagen to appear as thick, bright (strongly birefringent) red or yellow fibers, whereas type III collagen shows up as thin, pale (weakly birefringent) greenish fibers. When applied to nerve sections, this method localized collagen type III in the endoneurium and type 1 in the epineurium. Leprous lesions, which are known to induce proliferation of collagen in nerves, do so by enhancing the deposition of type III collagen in the peri- and endoneurium and deposition of type I in the epinerium.‘j’ This polymorphism of collagen, coupled with the different modalities of organization that it can acquire, are probably important in dictating the growth of neurons. The proper orientation and type of collagen present seem to be a must, while massive amounts of dissorganized collagen pose a strong hindrance to regeneration. Collagen, which has frequently been used as a substrate in tissue culture because of its ability to provide favorable conditions for cell adhesion, growth and differentiation, is required for the ensheathment and myelination of nerve fibers by Schwann cells.7’2 The capacity of collagen substrates to bind and grow neurons differs markedly with the method of preparation and the amount of collagen plated per unit area.733 It has been shown recently that human plasma fibronectin, when absorbed to a variety of conven-
63
tional culture substrata, promotes rapid neurite outgrowth from aggregates of embryonic chick neural retina cells.734 A clonal cell line (RN-2) obtained from a chemically-induced rat Schwann cell tumor has been described.735 This cell line produces unusual collagenous polypeptides736 and has been examined as a model for the demyelinization induced by viruses.737 This same cell line synthesizes procollagen of an unknown type and fibronectin.73” In peripheral nerves, fibronectin has been detected in the basement membrane around the Schwann cells, and is especially abundant in the nodes of Ranvier, suggesting that the tibronectin in this area could be produced by Schwann cells. Another interesting approach that has been used in the last few years to culture cells in general and neurons in particular involves the use of hydrogels, a class of polymeric materials that are swollen extensively in water but that do not dissolve in it. Collagen-hydroxyethylmethacrylate hydrogels can be prepared by polymerizing monomeric hydroxyethylmethacrylate in the presence of various concentrations of soluble native collagen.739 The resulting transparent hydrogels have been evaluated as substrata for growth of IMR-90 human embryonic lung fibroblasts. Without collagen, no significant growth occurred, whereas a dose-response curve expressing maximal cell growth against collagen concentration could be constructed quite readily by the use of appropriate hydrogels. Because of the potential of this method to probe into the mechanisms of cell adhesion and differentiation, it has been used to investigate nerve fiber growth.740 Cultured neurons become attached to hydrogels substrates prepared from 2-hydroxyethylmethacrylate but grow few nerve fibers unless fibronectin, collagen, or nerve growth factor is incorporated into the hydrogel. Antibodies to fibronectin inhibit nerve fiber growth on hydrogels containing fibronectin, which suggests that growing neurons interact directly with proteins trapped in the hydrogel. The adhesive requirements for attachment of neurons appear distinct and possibly less specific than those for fiber growth. It therefore seems as if defined hydrogel substrates offer a controlled method for analyzing complex substrates that support nerve fiber growth and neuronal differentiation. Further studies along these lines should lead
64
MARCEL E. NIMNI
us to further understanding of the role the connective tissue macromolecules play in modulating the process of nerve growth under normal and pathologic conditions. PHARMACOLOGY
OF THE FIBROTIC REACTION
A variety of chemicals have been investigated for the purpose of inhibiting fibrosis and accelerating the removal of collagen. The inhibitors of crosslinking, the lathyrogens and penicillamine, have been discussed earlier. Since collagen with lesser crosslinks is more readily degraded by collagenase, it was expected that these agents would facilitate its removal. The potential of /3-aminopropionitrile (BAPN) in the prevention and treatment of wound scar contractures, peritendineous and perineural adhesions, and other pathologic defects resulting from the polymerization of collagenous structures, were recognized several years ago.74’*742Although human trials showed promise, wide clinical use of this drug is lacking, due to toxic side-effects,743*744 the mechanism of which is not clearly understood. In animals this could be associated with the decreased food consumption induced by the drug, either via monoamineoxidase inhibition leading to alterations in the serotonin content of the brain, or via depletion of Zn associated with regulation of acuity of the taste buds. The latter hypothesis of serum Zn seem unlikely,745 since reduction levels seems secondary to an inhibition of protein synthesis by the liver. Zn levels can be restored by administering Zn while reducing the lathyrogenic effects of BAPN without correcting the body weight loss. Because of these toxic effects, the clinical use of BAPN has been limited. In view of this systemic toxicity, the topical route of administration has been explored.746 It was shown that 14C-BAPN can be absorbed readily across the skin of rats, the free base more so than the fumarate salt. The lysyl oxidase activity of a subcutaneously induced granuloma was significantly inhibited by a single small dose of BAPN applied to the intact skin over the implant, and this was accompanied by increased local extractability of collagen. This route of administration may thus prove of value in reducing scar contracture and in decreasing tendon adhesions and joint stiffness while minimizing systemic toxic effects.
Penicillamine, on the other hand, in addition to displaying considerable side effects at the dose required to inhibit collagen crosslinking, generates a reversible effect. Once the drug is discontinued or the dose lowered the aldehydes on collagen blocked by the presence of the drug will become free and normal crosslinks will form. (Fig. 33). So this compound has to be used with this limitation in mind, and may only prove useful in limited situations, such as early or acute stages of scleroderma and transient fibrotic episodes. Its apparent effectiveness in the treatment of alcoholic liver cirrhosis may be associated with this mode of action. As a corollary to the studies on the synthesis and structure of collagen a variety of chemical agents have emerged that have the ability to interfere with many of the biosynthetic steps involved. Many of these have a significant potential for the treatment of fibrosis, many are toxic, and still others are nonspecific for collagen and would interfere with other biosynthetic process. Rojkind, as a result of his interest in the treatment of liver fibrosis, has studied these compounds in a systematic fashion and grouped them into six categories.747.748 A brief description of their mode of action and possible toxic effects follows. Proline Analogues (L-azetidine-2-carboxylic acid, cis-hydroxyproline, cis-fluoro proline, 3,4-Dehydro-DL-proline, cis-Bromoproline) This group includes several compounds that are similar but not identical to proline and that compete with this imino acid for its functions. L-azetidine-2-carboxylic acid competes with proline for transport749 and acylation of tRNA.“O It is incorporated into collagen and is not hydroxylated by collagen prolylhydroxylase. Tendon cells incubated with cis-hydroxylproline can incorporate it into collagen to the extent that it can occupy up to 19% of the imino acid positions. With cis-fluoroproline the degree of replacement colcan reach 27%.“’ The under-hydroxylated lagen chains are unable to form triple helical structures and are not secreted but they are digested inside the cell by nonspecific proteolytic enzymes.749 Even though proline analogues are incorporated efficiently into collagen molecules, they are also incorporated into other proteins and could
COLLAGEN STRUCTURE AND METABOLISM
alter their function. Although these compounds are highly effective in vitro and in experimental studies are animals,748 detailed pharmacologic still lacking, and thus they can not be used for the treatment of human disease. Iron Chelators [cY,a-dipyridyl, 9,9-dihydroxy-7-methylbenzo bromide
quinolizinium
(GPA 1734)/
These have been used in many systems in vitro but are not suitable for use in vivo because of their toxicity. They remove ferrous ion required by the two hydroxylating enzymes, thus inhibiting hydroxylation of prolyl and lysyl residues.752 The nonhydroxylated collagen remains inside the cell where it is degraded. These compounds are used in the preparation of under-hydroxylated collagen, as a substrate for the hydroxylating enzymes. Lathyrogens acetonitrile.
(P-amino propionitrile,
amino
etc.)
These compounds, which inhibit at relatively low concentration the activity of lysyl oxidase and the subsequent formation of intramolecular and intermolecular crosslinks, have been discussed in detail. Aldehyde
Trapping
Cysteamine,
Agents (Penicillamine,
etc.)
At low doses, penicillamine combines with free aidehyde groups in collagen, thus preventing the formation of intra- and intermolecular crosslinks.753 The bond formed, a thiazolidine ring, is unstable and dissociation occurs when the concentration of penicillamine in a given tissues decreases. Upon discontinuing treatment, many aldehyde groups are re-exposed and formation of crosslinks quickly occurs.753 At high doses (toxic for the animals) penicillamine inhibits the activity of Iysyloxidase.488,754 Penicillamine combines very readily with pyridoxal phosphate, thus producing a deficiency of this vitamin: this toxic effect can be avoided by daily administration of vitamin B,. Antimicrotubular Related
Agents
(Colchicine
and
Compounds)
Colchicine inhibits microtubule assembly and the transcellular movement of collagen.755~757 It
induces the production of collagenase in synorelease vium75x and enhances protease-activated decreases colof procollagenase.759 Colchicine lagen deposition and breaking strength of wounds in rats but only at a dose that produces systemic side effects. 760Colchicine has been used in the treatment of human liver fibrosis in a group of severely ill patients. After several weeks of treatment with a dose of 1 mg per day, these patients showed a striking clinical improvement. Long-term treated patients showed normalization of biochemical parameters (serum albumin and proline levels)76’ and side effects were minimal. Nonspecijc
Compounds
Corticosteroids can be included in this group and their effects on collagen metabolism have been described in detail earlier. Another compound that has attracted attention of individuals working in cell culture is the monovalent ionophore monensin, that interferes with a variety of eukaryotic cell functions. Monensin inhibits secretion of macromolecules,762~763 its major effect on secretion appears to be in the Golgi complex, which becomes markedly distended in cells treated by this drug.762-763Secretion of types I and I1 collagen, fibronectin, and cartilage proteoglycan are also inhibited in cultured cells treated with monensin.765m766 These macromolecules also accumulate within the treated Ce,,s.765-767
The macromolecules secreted by monensintreated cells have abnormalities in some of their posttranslational modifications. Chicken embryo chondrocytes treated with monensin secrete undersulfated cartilage proteoglycans.76x Normal intracellular processing of the N-asparagine-linked oligosaccharides on fibronectin”” and other macromolecules76x is also disrupted by monensin. Although obviously nonspecific for collagen, such a drug may help us understand factors involved in the intracellular translocation of collagen and its secretion into the extracellular space. It is obvious that there is a great need to further investigate drugs that will inhibit excessive deposition of collagen, alter the phenotypes expressed by cells at the site of injury, and modulate its synthesis and turnover. Combinations of drug therapy, surgery and physical
66
MARCEL E. NIMNI
manipulation should make it possible to regulate cell behavior in such a way that the biosynthesis of the connective tissue macromolecules proceeds in a normal and controlled fashion. Maintenance of a normal extracellular matrix is essential for the overall homeostasis of the organism.
ACKNOWLEDGMENT
I thank Drs. David Cheung, Paul Benya, E. P. Kay, Ken Nishimoto, and J. Vernon Luck, Sr. for their helpful suggestions and discussions, as well as the many colleagues who provided me with preprints of their unpublished work.
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