Gerontology: Collagen disease

2 Gerontology: Collagen Disease D A V I D A. H A L L

Collagen constitutes approximately one-third of the total mass of the body, and hence it may be deduced that any variations in either the content or nature of this component occurring as the result of the ageing process may well have an appreciable effect on the continuing function of the organism as a whole. This assessment of the role of collagen in age-mediated changes in bodily function has been accepted only for the past 20 or 30 years. Before that, collagen, in common with the other main fibrous constituent of connective tissue, elastin, was believed to be an essentially inert protein with little function other than supportive in the body as a whole. The often quoted observations by Neuberger, Perrone and Slack (1951) did little to rectify this long-held belief. These workers reported that collagen was relatively inert from a metabolic point of view and they inferred that, once it was laid down in the body it remained a permanent constituent of the tissue into which it was incorporated. In fact they claimed that the half-life of collagen was roughly equivalent to the half-life o f the animal of which it formed a part. Unfortunately they chose rat tail tendon as the tissue of choice for their experiments, mainly because of the ease with which collagen from this source could be prepared in a relatively pure state, devoid of noncoUagenous proteins into which and from which the radioactive amino acids which they administered would more easily pass. As will be demonstrated later, tendon is among the more stable of the collagenous tissues. Had Neuberger and his colleagues examined collagen from any one of a number of other sites in the body they might have reported that in many tissues this protein, although less metabolically active than many others, is in a state of continual degradation and resynthesis. In view of the fact that this relegation of collagen into a class of nonmetabolically active proteins arose as the result of relatively sophisticated biochemical studies, it is of considerable interest that the first suggestion, that collagen might after all alter both in structure and in function as age progresses, resulted from what were, initially at least, relatively simple studies of the physical properties of the same tissue. Verzar (1963) inferred from the results of his studies of the thermal contraction of rat tail tendon fibres that the increased force required, as the animal aged, to withstand Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - - Vol. 10, No. 1, March 1981.

0300-595X/81/10.01/23

$03.00 © 1981 W. B. Saunders Company Ltd.

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24

DAVID A. HALL

such contraction must be due to the formation of an increasing number of cross-linkages between individual collagen molecules. These observations stimulated a massive upsurge in the study of collagen and its role in the ageing process and although, as will become apparent later, Verzar's views probably do not represent an accurate assessment of the situation any more closely than did those of Neuberger and his colleagues, the resultant mass o f research has done much to explain some of the problems of ageing, and has incidentally contributed largely to present-day knowledge of the structure and metabolism of collagen. T H E S T R U C T U R E OF C O L L A G E N Collagenous tissues perform a variety of functions in the body, ranging from the protective and supportive to the transmissive, and variations in the specialized architecture of the collagen fibre fit it for all these various roles. The physical strength and relative inextensibility of the fibre are direct consequences of the basic structure of the molecular unit - - tropocollagen - - and of the way in which adjacent tropocollagen molecules are packed together and ultimately linked by covalent bonds with one another and with glycosaminoglycans to form the microscopically identifiable fibrils. Basic Structure The primary structure of each of the three individual a-chains, the basic protein molecules, which together make up the tropocollagen molecules, differs from that of most other proteins in that one-third of the 1052 amino acid residues consists of glycine and another third of one or other of the imino acids proline or hydroxyproline. The latter imino acid occurs only in collagen, elastin and the C l q component of complement, and only in the first and last of these in amounts in excess of I or 2 per cent. On account of their particular stereochemical configuration, proline and hydroxyproline, when present in such high concentrations, constrain the polypeptide chain to assume a coiled conformation which is a modification of the poly-1proline II helix (Figure 1), and the hydroxyl group on the latter imino acid provides repeating sites down the length of the molecule at which hydrogen bonds can be formed to form linkages with other a-chains lying adjacent to it. This is of considerable importance in the maintenance of the integrity of the tropocollagen molecule immediately after it has been synthesized and before more stable covalent cross-links have been formed. Another amino acid which is exclusively confined to the collagen molecule is hydroxylysine which provides the site for glycosylation and for linkage with noncollagenous proteoglycans. The major portion of the polypeptide chain, with the exception of 17 amino acid residues at the N-terminal end and 25 at the C-terminal end, consists of a series of repeating triads in which the first residue is glycine, the second either proline or hydroxyproline, and the third may be any one of the 20 different species of amino acid present in the molecule. The two terminal portions - - the so-called telopeptides - - which are not constructed

G E R O N T O L O G Y : C O L L A G E N DISEASE

25

© © © o

A Figure 1. The helical structure of collagen based on the polyproline II model. The three separate a-chains are labelled A, B and C. The structure is such that the m a x i m u m number of hydrogen bonds are formed between adjacent chains.

26

DAVID A. HALL

in this repetitive fashion do not assume the helical configuration. They provide sites for cross-linkage between adjacent a-chains (Figure 2), specifically through the lysine residues at positions 9 and 1047 which, after oxidative deamination to convert them into aldehydes, react with the terminal amino groups of unchanged lysine residues elsewhere in other molecules, to form cross-linkages. The individual a-chains are synthesized on membrane-bound ribosomes in the cytoplasm of the fibroblasts. From the size of the ribosome clusters involved in this synthesis it appears that each a-chain requires a separate mono-cistronic messenger RNA molecule (Lazarides and Lukens, 1971).

~.1 - ---e-

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O 12~4 ~ ,

o41

8

~

3

~

o41

7

~

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6

.....

I j

6

O! 1

I

t

Figure 2. Diagrammatic representation of the cross-linking of a poly-a 1 collagen. The numbers along the length of tee molecule indicate the individual pepfides which can be separated by chromatography following the fission of pepfide linkages adjacent to methionine groups by cyanogen bromide.

There are no specific codons for either hydroxyproline or hydroxylysine and both these residues have to be synthesized by the hydroxylation of proline and lysine residues already incorporated into the polypeptide chains and before they have been released from the ribosomes (Lazarides, Lukens and Infante, 1971). Hydroxylation of either residue is under enzymic control and requires in addition the presence of molecular oxygen, ferric ions and ascorbic acid. In the absence of any one of these cofactors non- or underhydroxylated protein accumulates (Hurych and Chvapil, 1965) and since this 'protocollagen' is incapable of becoming involved in the formation of hydroxyproline or hydroxylysine-based linkages with other molecules, tissues containing high concentrations of this aberrant form of collagen are less stable in a physical sense than those containing normal collagen. This may explain the failure of wounds in scorbutic, ascorbic acid deficient tissues to heal adequately and, in view of the relatively low ascorbic acid levels in the tissues of an appreciable proportion of elderly subjects, may also provide a reason for the retardation of healing sometimes observed in wounds in older individuals.

Tropocollagen Individual a-chains, synthesized on separate ribosomes, are grouped together in threes to form the fundamental collagen molecule - - tropocollagen. The three separate helices are coiled around one another in a

GERONTOLOGY: COLLAGENDISEASE

27

master helix of longer pitch. Tropocollagen immediately after synthesis but after its appearance in the extracellular space consists of three chains joined together by hydrogen bonds. The spatial relationship of the three separate chains is such as to permit the maximum number of hydrogen bonds to be formed. This alignment and registration is accomplished within the cell by the formation of -S-S- linkages formed by the oxidation of thiol groups present in peptides which are terminal to the telopeptides (Dehm et al, 1972). These segments of polypeptide for which Speakman (1971) suggested the name 'registration peptide', after fulfilling their role of locating and linking a-chains so that the necessary optimal number of hydrogen bonds are formed, are removed by enzymes (Bellamy and Bornstein, 1971) either at the cellular membrane or immediately external to it. Once formed, tropocollagen spontaneously aggregates into fibrils, the nature and size of which may be determined by the presence of differently charged macromolecules (Wood, 1960).

Types of Collagen and the Effect of Age Until relatively recently it was believed that although collagens of different fundamental composition could be isolated from different phyla, all mammalian collagens could be characterized by a common amino acid analysis and a specific physical structure as defined by electron microscopy and x-ray diffraction studies (Ramachandran, 1963). It became increasingly apparent, however, that collagens from different sites in the body showed a different degree of solubility to solutions of neutral salts and acid -- a solubility which was not solely dependent on the presence or absence of covalent cross-linkages. It had long been appreciated that the most easily obtainable forms of pure collagen from skin, tendon and artery wall consisted of two different types of a-chain which were present in the ratio of two a;-chains to one a2-chain. Miller and Matukas (1969) observed that the collagen extractable from cartilage contained a genetically distinct a-chain with a different amino acid analysis. As the result of these and subsequent studies it became apparent that there are at least four and probably other forms of collagen (Table 1) and that these are distributed unevenly throughout the tissues of the body. Type III collagen was shown to be present alongside type I in fetal dermis (Epstein, 1974), and a study of these two forms of collagen has led to the evolution of appropriate theories concerning certain age-related changes in collagen. Muir, Bornstein and Ross (1976) reported varying ratios of types I to III collagen in aortic smooth muscle cell cultures. In the dermis (Table 2) the type III collagen which is present to the extent of 30 per cent of the total collagen present in the fetal tissue is replaced in adult dermis by type I whereas the opposite appears to be the case in aortic smooth muscle cell cultures. Layman and Titus (1975), Layman et al (1977) and Reuterberg et al (1977) have shown that there is a reduction in the ratio of type I and type III with increasing age of the subject from whom the smooth muscle cells were originally obtained. Reuterberg and his co-workers, for instance, observed a rise in type III collagen from 15 to 20 per cent in three- to four-

28

DAVID A. H A L L

Table 1. The various type o f collagen, their structures and locations Type I II III IV A+B

Composition

Location

a, ([)_~a2 a, (II)3 a, (lII)3

Skin, bone, tendon, arteries Cartilage Skin, arteries Basement membranes

AB2

month-old fetuses to more than 30 per cent by nine years of age. No further variation, however, was observed between 9 and 67 years of age. Any changes which occur would appear to be associated with the processes of maturation rather than with those of ageing. Type I and type III collagen differ significantly in amino acid analysis, with threonine, alanine, valine, methionine, phenylalanine and hydroxylysine present in between 20 and 50 per cent lower concentrations in type III than in type I and the less prevalent form of hydroxyproline, with the hydroxyl group on carbon atom 3 rather than 4, virtually absent from type III. Probably the most significant difference between the amino acid compositions of the two forms of collagen, however, is the reduction in the number of hydroxylysine residues from ten to five per thousand residues. This residue not only provides one of the centres at which cross-linkage occurs, but also provides sites for glycosylation which are of considerable importance in promoting the aggregation of platelets on the collagen. The dermis therefore becomes more stable but more likely to initiate clotting with increasing age, whereas the protein synthesized by the smooth muscle cells of the arterial wall are not only less stable but also less likely to promote platelet aggregation. The overall differences in amino acid content between the two forms of collagen are so great as to preclude the possibility that they are interconvertible forms of one protein species. They must therefore have been synthesized as the result of the activity of two distinct forms of messenger RNA and hence must be controlled by two separate genes. Orgel (1963, 1970) has suggested that some alterations in protein structure may result from random faults in protein synthesis. The differences between type I and type III collagen are too great for such a theory to be able to provide an adequate explanation. The age-related changes in the relative amounts of Table 2. The effect o f age on the distribution o f types I and Ili collagen in different tissues

Tissue

Age

Type I (070)

Type III (%)

Dermis

Fetal Adult

70 100

30 --

2-4 mo. (fetal) 9 years 67 years

80-85

15-20

70 70

30 30

Artery (smooth muscle cell)

GERONTOLOGY: COLLAGEN DISEASE

29

the two protein species must therefore indicate that the gene responsible for the synthesis of type III collagen in the skin is switched off as age progresses, whereas that for type I is switched o f f in the arterial smooth muscle. Eyre and Muir (1974) have shown that type III also accumulates (to between 25 and 50 per cent) in rheumatoid synovium, indicating that the genetic control of collagen synthesis may be affected by disease conditions as well as by age, although the age relationship of the incidence of rheumatoid arthritis may indicate that, in this instance at least, pathological and physiological phenomena are quite closely related. A G E C H A N G E S IN T H E C O L L A G E N C O N T E N T OF VARIOUS TISSUES The mean content of collagen in various tissues (Table 3) is a value which is closely related to the function of the tissue concerned. However, superimposed on these gross differences are age-mediated variations which m a y have important effects on the functioning of the individual tissues. Table 3. Collagen content o f various tissues (g/lO0 g dry weight) Mineral-free cortical bone Achilles tendon Cornea Cartilage Ligamentum nuchae Aortic media Lung Liver

88 86 68 46-64 17 12-24 10 4

Dermis The collagen content of h u m a n dermis falls steadily with age. The absolute decline in collagen concentration is accompanied by a reduction in thickness of the skin, resulting in a dramatic lowering of the overall amount of skin collagen in a column of full skin thickness beneath unit surface area (Figure 3). These individual values are for female subjects, the male distribution being roughly parallel but about 100 p g / m m 2 higher at all ages (Hall et al, 1974). Hall (1977) has also demonstrated that steroid-induced loss of skin collagen is additional to the loss due to ageing (Figure 4), all the values for subjects receiving prednisolone being below the regression line for collagen content against age for non-steroid treated controls over the whole age range. The additive nature of these two effects may well indicate that the reduction of total skin collagen may be induced by a similar metabolic disturbance in both cases. Hall (1977) and Hall et al (1974) have demonstrated that the excessive reductions in skin collagen which are associated both with steroid treatment and with ideopathic osteoporosis are directly related to the degree of osteoporosis. Steroid osteoporosis is considered to be due, in part at least, to an augmentation of bone marrow degradation to a stage at which resynthesis is incapable of maintaining the status quo, and it would

30

DAVID A. HALL

therefore appear likely that both the physiological age-related loss of collagen and the steroid-induced pathological loss may be due to the same phenomenon--namely, an augmentation of the release of collagenase from endogenous tissue cells or of cathepsins from the lysosomes of wandering cells.

Vascular Tissue Harkness, Harkness and McDonald (1957) examined the collagen content of the vascular tissues of dogs and showed that in the intrathoracic aorta this protein does not exceed 35 per cent at any age whereas in other vessels the concentration may rise to 70 per cent or more. In rats the collagen

mature

collagen pg/sq,

mrn

" 300

.. ; ; 200

.-!

.: g"

"

100

20

40

age

60

years

80

Figure 3. The amount of mature (insoluble) collagenpresent in a column of full skin thickness beneath 1 mm2 of skin surface. Normal subjects. content of the fat free aorta rises from 28 per cent in the tenth week of fetal growth to between 37 and 40 per cent at birth, but thereafter does not appear to rise significantly (Berry and Looker, 1973). However, if the collagen content is expressed as a percentage of the total amount of scleroprotein - - collagen and elastin combined - - the collagen content appears to continue to rise with age since the elastin level falls slightly. Banga (1969), studying both absolute values for collagen concentration and

GERONTOLOGY: COLLAGEN DISEASE

31

its ratio to the total scleroproteins in h u m a n aorta, recorded values ranging from 16 to 90 per cent. She employed various methods for the measurement of the collagen content and obtained values ranging f r o m 28 to 81 per cent over the age range 17 to 76 years if the elastic tissue was removed by elastolysis, but between 17 and 32 per cent over the same age range if the collagen content was calculated from the hydroxyproline content of the entire tissue. Since she did not give any details of the accuracy and effectiveness of her analytical methods, especially the degree of recovery of hydroxyproline from the tissue hydrolysates, it m a y well be that the latter set of values are erroneously low.

total

collagen ~gJsq

mrn

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410

age

bO I

years

80 1

Figure 4. The total amount of skin collagen measured as in Figure 3. Filled circles represent normal subjects, open circles subjects who have been administered steroid (prednisolone) for periods in excess of six months.

Various other workers have either been unable to observe any significant alteration in collagen content in the heart with increasing age (Lev and McMillan, 1961; Montfort and Perez-Tamayo, 1962) or have observed slight decreases (Oken and Boucek, 1957; Wegelius and von Knorring, 1964) or steady increases (Clausen, 1962). Some of these observations, which have been made by the visual assessment of stained sections, have a built-in h u m a n error. Lenkiewiez, Davies and Rosen (1972), employing a

32

DAVID A. HALL

flying spot densitometer to eliminate such error, confirmed Clausen's observations. The volume percentage of the interventricular septum, which stains as collagen, rises from around 22 per cent in early adolescence to between 27 and 31 per cent at the age of 90 years. The value obtained depends on the orientation of the muscle fibres among which the bundles of collagen fibres are distributed. When the muscle fibres, and hence the collagen fibres, were aligned predominantly in a longitudinal direction to the section under study, the values for collagen content appeared lower. A similar 'artefact of technique' affecting the observed value for the concentration of a stained component has also been observed (Hall, 1971) in comparable studies of age changes in the amount of elastica staining material in the dermis. The Skeleton The loss of collagen from bone is associated directly with the loss of calcium phosphate and hence m a n y assessments of collagen changes in this tissue have been deduced from figures for loss of total bone tissue. Bone loss in osteoporosis can be determined directly, post mortem, by measurements of the ash weight of a unit volume of bone, by the densitometric assessment of bone x-rays or by the calculation of an 'index' such as the metacarpal index which relies solely on the linear measurement of ordinary x-ray pictures without recourse to standardized densitometry (Nordin et al, 1970; Dequeker et al, 1971; Exton-Smith, 1978). This method of assessment of bone loss can be correlated directly with absolute values for bone loss as measured by ash-weight determinations. Morgan and Newton-John (1969), using this latter approach, were able to show that between the ages of 40 and 80 years the amount of bone loss could approach 40 per cent. The individual variation is considerable, however, the standard deviation being about 12 per cent of the mean value. Hence at least 1 per cent of the female population at 35 years will have a bone mass and hence bone collagen content which is comparable with the mean value for the whole population at 75 years. Males, who at maturity have a greater mean bone mass than females, start to lose bone at a later age and lose it more slowly than women. The two rates of bone loss remain roughly constant at approximately 5 per cent per decade for men and 10 per cent for women until the women reach their menopause when there is evidence for an increase in the rate of bone loss. Jowsey (1960) has suggested that the pattern of bone turnover alters with age and that this accounts for the increasing loss of tissue. As with all other tissues the status quo is maintained by a balance between synthesis and degradation. Degradation and consequent resorption of bone, occurring mainly at the bone surface, increase with age whereas the formation of bone remains at a relatively constant level. Having assessed the overall changes in the skeleton, it is possible to relate this to alterations in the bone matrix. The degradation of bone collagen is brought about by the stimulation of an endogenous collagenase, first identified in bone explants by Fullmer and Lazarus (1967, 1969). The enzyme exists in an inactive form which is activated by a factor which can

GERONTOLOGY: COLLAGEN DISEASE

33

also be isolated from bone cultures (Eeckhout and Vaes, 1977). The latter is also present in an inactive form and must itself undergo activation before it is capable of affecting the procollagenase (Vaes et al, 1978). Intact bone is not susceptible to this chain of reactions; it is necessary for decalcification to precede the degradation of the protein. Since both processes occur in the vacuole which lies between the osteoclast and its associated bone surface (Vaes, 1980) it is apparent that bone resorption and hence collagen degradation by the activated collagenase is dependent on the stimulation of the osteoclast. There is as yet little evidence as to the way in which this may be age related, but it would appear that changing concentrations of parathormone a n d / o r calcitonin may be responsible.

Articular Cartilage Once again much of the evidence for age-related changes in the collagenous component of cartilage is dependent on related observations on the alterations of cartilage as a whole. Thus although excessive and localized overloading of a joint may result in the type of tissue degradation associated with arthrosis or rheumatoid arthritis it would appear likely that some underlying change in the structure of the tissue predisposes the joint to mechanical degeneration (Hall, 1978). The outer layer of the cartilage, 1 to 7 mm thick, contains a densely packed three-dimensional network of collagen fibres which lie roughly parallel to the surface. With increasing age the thickness of this oriented layer increases and later the outer portions of the network become degenerate. It is of interest that the type of collagen present in cartilage - - type II - - differs from type I which is present in those tissues which have been discussed earlier, in that it is only one-sixth as susceptible to attack by collagenase (Woolley et al, 1978). There is no evidence as yet that collagenases are fully type-specific, and hence it must be assumed that it is the identity of the particular form of collagen present in the cartilage which protects this tissue from degradation in youth. It has previously been assumed that the progressive erosion of the joint surface is related to the development of the arthritic lesion, whether the phenomenon be identified as inflammatory, infective or autoimmune. It is, however, apparent that surface irregularities are as much related to age as they are to any specific disease process (Figure 5) and hence the collagen degradation associated with these surface changes may itself be assumed to be age related. Collagenase can be identified in appreciable amounts by immunofluorescent techniques at the junction of the hypertrophic synovium with the cartilage matrix, but it must be assumed that it is also present throughout the cartilage. Hence the development of surface irregularities may be due to the additive effect of a 'physiological' degree of collagen degradation which is directly associated with age and controlled by the progressive stimulation of an endogenous collagenase secreted by the chondrocytes, and with a 'pathological' response due to a collagenase secreted by the pannus cells, and stimulated by some factor associated with the inflammatory process.

34

DAVID A. HALL

Intervertebral Disc The annulus fibrosus of the intervertebral disc contains an appreciable amount of highly oriented collagen whereas the nucleus pulposus, although penetrated by a loose three-dimensional mat of collagen fibres, does not demonstrate a similar degree of orientation. This difference between the two regions is directly related to their separate functions. The radial transmission by the nucleus of forces applied vertically through the spinal column is controlled by circumferentially aligned fibres in the

A

Figure 5. Tracings of the irregularities of the surface of human femoral cartilage. (A) Condyle from a normal subject aged 26 years. (B) Osteoarthritic condyle from subject aged 63 years. (C) Femoral head: subject with no clinical signs of osteoarthrosis aged 67 years. The vertical axes are exaggerated ten-fold with respect to the horizontal axis which coincides with the surface of the cartilage. It is apparent that the appreciable irregularities occur as a result of age alone but that the effect of arthrosis is two to three times greater.

annulus. The angle of alignment of different layers of collagen fibres in the annulus is modified as pressure is applied, until all the fibres assume a parallel alignment at right angles to the radial forces transmitted through the nucleus (Happey et al, 1974). This prevents the excessive distension of the entire disc. X-ray diffraction studies (Happey et al, 1969) have shown that, as age advances, soluble proteins are deposited in a/J-configuration in both nucleus and annulus. In the nucleus this material is essentially noncollagenous; in fact, Hall and Reed (1974) have demonstrated that the collagen content of the nucleus does not rise by more than 0.075 per cent per year throughout life. In the annulus, however, above the age of 65 years, non-oriented collagen accumulates between the aligned collagen fibres at a rate of about 1 per cent per year.

The Female Reproductive System Cretius (1959) reported an appreciable increase in the collagen content of samples of muscle from human myometrium with increasing age. For nonpregnant mature females the collagen content represents 2.2 per cent of the

GERONTOLOGY: COLLAGEN DISEASE

35

fresh weight whereas for the post-menopausal female it is 4.3 per cent. On the other hand'in myometrium from a pregnant woman it may be as low as 1.4 per cent. On a dry-weight basis these values may be multiplied by 5, indicating that collagen is a major component of this tissue, especially in the elderly. The fall during pregnancy is due to the fact that the eight-fold increase in total uterine weight is not mirrored completely by a similar increase in collagen. Harkness and Harkness (1954) demonstrate, however, that the increase in collagen in the gravid horn of a rat uterus continues in amount up to term whereas the total weight of the uterus peaks five or six days before parturition. Woessner (1965) has suggested that the massive loss of uterine collagen which occurs within four days of parturition is in fact an instance of accelerated ageing, the degradation of collagen by uterine collagenases and cathepsins mirroring to a considerable degree the changes which take place in other tissues over a protracted period of time. The question of collagen degradation is considered in more detail at a later stage in this chapter. CROSS-LINKAGES AND THEIR CHANGES WITH AGE Tropocollagen molecules, immediately following their release into the extracellular space, are not only soluble in 0.2 mol/1 sodium chloride but can be denatured at temperatures between 40 ° and 45°C or by a variety of reagents capable of breaking hydrogen bonds to provide individual achains. When the collagen is derived from skin, tendon, artery wall or bone these a-chains are present in the ratio of two al-chains to one a2-chain. They can be separated from one another by various forms of electrophoresis or in exchange column chromatography, either at sufficiently high temperature or in the presence of reagents which similarly prevent the hydrogen bonds from being reformed. At increasing periods after the synthesis of the tropocollagen molecules, they become increasingly difficult to extract from the tissue with neutral salt solutions, increasing concentrations being required. Moreover, following thermal or chemical denaturation, other species of molecule of higher molecular weight can be identified. Mature type I collagen provides, on denaturation, two forms of dimeric molecule in which either two al-chains or an a~- and an al-chain are firmly bound together. As the collagen becomes increasingly resistant to dissolution trimeric and other species of higher molecular weight can also be identified. The nature of some of these trimers, containing as they do three a~-chains, indicates that stable linkages have been formed not only between the three chains within each tropocollagen molecule, but also between tropocollagen molecules. The solubility of collagenous tissues decreases steadily with increasing age as an increasing number of stable cross-linkages are formed (Figure 6). This change in solubility indicates a failure to synthesize new tropocollagen molecules at a rate which can keep pace with the stabilization and insolubilization of those molecules already present. Verzar (1963) observed that age changes in the solubility of rat tail tendon were inversely proportional to alterations in the forces of thermal contraction and it was on the basis of this correlation that his concept of cross-linkage was formed.

36

DAVID A. HALL

Thermal Stability of Collagen The cross-linkages which are now known to exist in mature collagen fibres were first postulated not as the result of their chemical identification, but following studies of the thermal stability of collagenous tissues of increasing age (Verzar, 1963). If a collagenous tissue is placed in an aqueous solution at temperatures above 40°C it contracts to one-half its length. As it contracts a force is exerted, and if one end of the fibre is fixed and

solub|e collagen

16

~g/ sq.mm

•

Q

• .12

0

•

0

•

•

.: 0 0

~0

.

•

-8 •

• 0

0 0 Q

•

•

•

-4 0 o •

20

40

age

60

yuars

O

80

Figure 6. The fraction of the total skin collagen which is soluble in 2 mol/1 sodium chloride solution.

increasing weights are added to the other it can be shown that the weight required to prevent contraction and hence the force of the contraction increases with increasing age. A typical curve for the thermal contraction of mouse tail tendon against a small weight (Blackett, 1980) is shown in Figure 7. On the basis of similar observations Verzar suggested that the ageing of collagen was accompanied by the formation of an increasing number of cross-linkages. Banga, Balo and Szabo (1956) demonstrated that similar contraction could be accomplished by chemical means (Figure 8) and confirmed Verzar's observations regarding relaxation subsequent to the contraction. Rat tail tendon immersed in 40 per cent potassium iodide contracts increasingly with increasing age. The percentage contraction

37

GERONTOLOGY: COLLAGEN DISEASE

increases from 34 per cent at two months to 66 per cent at 24 to 30 months. Contraction is complete within 15 minutes and the original length of the fibre is regained in a further 10 minutes in the case of the youngest animal, and after 4 hours in the case of a 7-month-old animal, whereas the tendons from a 24 to 30-month-old animal fail to relax at all. Such thermal or chemical denaturation results in the conversion of part at least of the collagen molecule into a state in which it is no longer soluble in collagenase but has assumed a degree of susceptibility to elastase. In this respect the susceptibility to elastase does not signify any conversion of the collagen to elastin but merely reflects the destruction of the tertiary structure of the collagen molecule, thereby rendering certain specific centres near to alanine residues, which are not accessible in the uncontracted collagen molecule, available to the elastase - - which in this instance is not acting as an enzyme specific for elastin but as an endopeptidase with a general mode of attack.

65 SHRINKAGE As o~ INITIAL 60 LEN

%

55 50

45 40. 3~. ,,.,,. ,'*

AGE IN MONTHS 2"5

6

10

14

18

23

28

Figure 7. The effect of age on the degree of thermal contraction of mouse tail tendon heated to 50°C.

Nature of the Cross-Links Evidence for the types of chemical group involved in cross-linkage was first provided by the studies of Levene and Gross (1959) on experimental lathyrism. They noted that un-cross-linkcd rathyritic collagen contained a markedly reduced content of aldehyde groups. Later, Thomas, Elsden and Partridge (1.963) showed that the cross-linkages in the other m a j o r connective tissue protein, elastin, were also dependent on the presence of an aldehyde and that it was derived from lysine. The first stage in the formation of these aldehyde-based cross-links consists in the oxidative deamination of the e-amino group of lysine residues incorporated in the telopeptidc region of one of the a-chains of

38

DAVID A. HALL

collagen, with the consequent conversion of the methylene group to which that amino group is attached to an aldehyde. Two of these lysine-derived aldehydes in the telopeptide regions of adjacent molecules condense with one another to form allysine aldol. a-chain a-chain

>CH-CH2-CH2-CH2-CHO + CHO-CH2-CH2-CH2 -(a-chain > CH-CH2-CH2-CH2-CH = C-CH2-CH2-CH2 (a-chain

I CHO This intramolecular cross-linkage stabilizes the tropocollagen molecule, but in this form has no effect on the stability of the tissue as a whole.

A

0)

Ill

O xz

c q~

I

.

4,0

80 m l n s

12,0

160

200

240

Figure 8. The effect of age on the contraction and subsequent relaxation of rat tail tendon resulting from immersion in a 40 per cent solution of potassium iodide. (A) Two months old. (B) Seven months old. (C) 24 to 30 months old. After Banga (1969).

However, Franzblau, Kang and Faris (1970) have demonstrated that as tissues mature this intramolecular cross-linkage disappears, to be replaced by a more complex structure linking three a-chains together (fraction C). One at least of these three chains may be present in another tropocollagen molecule indicating that the intramolecular cross-linkage merely acts as a precursor of intermolecular linkages which may well be of considerably greater importance in determining the stability of the collagen fibre and hence of the collagenous tissue of which it forms a part.

39

GERONTOLOGY: COLLAGENDISEASE

The other forms of intermolecular cross-links, although also dependent on the primary production of a lysine-derived aldehyde, owe their structure to another typical reaction of aldehydes, the formation of so called Schiff's bases. -CHO + NH2--~-CH = N - - + H20 To perform this reaction the allysine formed in one a-chain interacts with an unoxidized lysine in another, linking the two through the aldimine linkage, -CH=N-. Isolation of such cross-linkages following the hydrolysis of the polypeptide which they link is not possible in view of the fact that the aldimine group itself is not stable. However, if the collagen is reduced before hydrolysis the saturated compounds derived from various Schiff's bases can be separated by column chromatography after hydrolysis of the protein. If radioactive (3H)-borohydride is used to reduce the collagen the individual components of the mixture of cross-linkages can be identified in quite low concentrations from the radioactivity incorporated in the reduced cross-linkage. In this fashion it is possible to identify five different possible cross-links: Dihydroxylysinonorleucine Hydroxylysine hydroxynorleucine Hydroxylysinonorleucine Lysinonorleucine and Histidinohydroxymerodesmosine

DHLNL HLHNL HLNL LNL

the last of these being the so-called fraction C which replaces the allysine aldol in mature tissues. The presence of hydroxyl groups in one or other end of these doubleended molecules arises from the interaction of deaminated hydroxylysine with lysine or deaminated lysine with hydroxylysine or, as in the case of DHLNL, from the condensation of hydroxyallysine and hydroxylysine (Davis and Bailey, 1971). This particular cross-link can undergo a spontaneous Amadori rearrangement from its aldimine form to a structure containing a ketone which is exceptionally stable. Ruiz-Torres (1978) has shown that the amount of DHLNL increases steadily with increasing age in rat tail tendon, and that it does so at the expense of HLNL. However, this effect is nowhere near as clear cut in the skin of this animal. In human tissues, however (Fujii, Kuboki and Sasaki, 1976), the variations in these two cross-linking elements are not nearly as large. In bovine dermis (Figure 9) HLN and the complex histidinohydroxymerodesmosine pass through peaks of concentration after between one and two years. DHLNL, which is present in highest concentration in fetal dermis, falls rapidly during the latter period of pregnancy and after birth whilst hexitol lysines, in which a single hydroxylysine is combined with a galactose molecule or a galactose--glucose disaccharide (and hence do not contribute to the cross-link pattern), increase progressively with age (Robins, Shimokomaki and Bailey, 1973).

40

DAVID A. HALL

Surprisingly enough, embryonic skin is not as soluble as that from young animals. This may be due to the fact that the glycosaminoglycan content of embryonic skin is relatively high (Sobel and Marmorsten, 1956) and the linkages between collagen and glycosaminoglycan are not easily severed by neutral salt solution. m

i®

ro j~

r

I

age years

o

;

Figure 9. Changing concentrations of cross-links in bovine skin with increasing age. (A) Dihydroxylysinonorleucine. (B) Hydroxylysinonorleucine. (C) Histidinohydroxymerodesmosine. Adapted from Robins, Shimokomaki and Bailey (1973).

CHANGES IN THE PHYSICAL PROPERTIES OF COLLAGENOUS TISSUE W I T H INCREASING AGE Loading Studies The tensile strength of tendons, the purest tissue as far as collagen content is concerned, has been calculated to lie within the range 4.7 k p / m m 2 (Stucke, 1950) to 12.7 k p / m m 2 (Cronkite, 1936), the main reason for this degree of variation lying in the difficulty of measuring the cross-sectional areas of the tested samples. The breaking strain of individual fibres has been calculated by Harkness (1961) to lie between 10 and 50 k p / m m L The stress-bearing structures are the individual collagen molecules the breaking load of which, at its weakest point, the -C-N bond, has been calculated by Gustavson (1956) as being 300 k g / m m 2. The difference between these extreme values for fibre and constituent molecule is due to the possibility of slip of one molecule or fibril over another before scission occurs. Vogel (1979) has

GERONTOLOGY: COLLAGEN DISEASE

41

shown that the tensile strength of rat back skin rises from 0.5 k p / m m 2 at one month of age to 1.25 k p / m m 2 at four months and then falls steadily over the next 24 months by about 7 × 10-3 k p / m m 2 / m o n t h . The tensile strength of rat tail tendon, on the other hand, continues to rise over the first year of life from 250 k p / m m 2 to 1000 k p / m m 2 and the ultimate load (recorded in preference to tensile strength on account of difficulties in determining cross-sectional areas) from 0.12 to 0.23 kp. The rates at which these parameters decrease with increasing age above the 12-month point are greater than those at which the skin strength regresses (20.8 k p / m m V m o n t h and 0.0036 k p / m o n t h respectively). Vogel was able to correlate these changes with comparable alterations in the collagen content of the tissues which peaked at the same points in the life span and decreased comparably thereafter. Stress/Deformation Studies More information is obtained if not only the ultimate tensile strength is recorded but also the l o a d / d e f o r m a t i o n relationship plotted. This is not possible for individual molecules or fibrils and proves most easy to study when a sample of intact tissue is employed. Although some studies have been carried out on tendon (Viidik, 1966) the most widely studied tissues are not those consisting exclusively of collagen but mixed tissues such as artery wall and skin where some of the deformation observed when a load is applied is due to the elastic tissue which is also present. These tissues are subject to a variety of stresses throughout life with resulting changes in their physical properties. Skin The load extension curve of skin is sigmoid in shape (Figure 9) and it is the toe of this curve which is associated with the elasticity of the tissue and its weave. The difference between the shapes of the toes of the two curves in Figure 10, which refer to a young and an old human subject (Ridge and Wright), indicates that the angular relationship of adjacent bundles of collagen fibres decreases during ageing due no doubt to a failure on the part of the elastic fibres intertwined round the collagen fibre bundles to return the network to its relaxed state. The differences between the points at which the two curves begin to turn downwards indicate the differences between the ultimate tensile strengths of the tissue at these two ages. The slopes of the central portions of the two curves represent the relationships between load and extension, and had they been truly linear would have represented two values for Young's Modulus. However, they are not linear and empirical formulae have to be derived to explain their shape. Ridge and Wright (1965, 1966) calculated that an equation of the form E = C + KL b

where E equals the extension, L the load and C K and b are constants, fitted the slopes of curves of this type at all ages. The 'constants' C and K are dependent on the dimensions of the sample of skin under test and are also

42

D A V I D A. H A L L

characteristic of the instrument used to extend the sample, b, on the other hand, is fully independent of all these factors and appears to be related solely to the properties of the individual collagen fibres. Therefore, any changes in b associated with some continuously varying parameter such as age will indicate that some particular property of the collagen fibres changes with age. Figure 11 shows the relationship of b to age for male and female populations, indicating that it passes through a peak when the individuals from whom the skin samples are taken reach 40 to 45 years of age.

J

koed

k9

0.5

3.3

:).1

E x t e n s ; o n °/o 10

20

30

Figure 10. Load/extension curves for human skin. (A) 10 years of age. (B) 70 years of age. The central most nearly linear portions of the curves are the regions shown by Ridge and Wright to conform to the equation. E = C + K L b (see Figure 11). From the rising limb of the curve it can be inferred that the stiffness of the collagen fibres of the dermis increases up to the age of 40 to 45. This may be correlated with an increasing number of cross-linkages. It is well known, however (Bailey and Robins, 1973), that these cross-links, once formed, are fully stable, and hence it is unlikely that the subsequent falling limb of the curve represents a disruption of cross-links. The only other possibility is a fission of the main chains themselves. Disruption of the polypeptide structure of the a-chains themselves, even allowing for the continued presence of the cross-links, will permit extension of the collagen fibres to occur (Figure 12). Increasing degrees of degradation will be associated with the activity of tissue collagenases.

GERONTOLOGY: COLLAGEN DISEASE

43

Vascular tissue The load/extension curves for rings cut f r o m the wall of one of the m a j o r arteries (Figure 13) alter shape with increasing age (Roach and Burton, 1959). The percentage increase in the circumference of the ring resulting f r o m a given applied load decreases dramatically with increasing age, falling f r o m 90 to 15 per cent when a load of 15 g / c m width of ring (measured along the axis of the vessel) is applied to tissue f r o m 5- and 80- to 100-yearsold subjects respectively.

'50 b

,30

.lo

Age 10

30

(years~ 50

70

90

Figure 11. Changing values for the power b to which the load has to be raised to expressits time relationship to extension. The upper curve refers to female subjects, the lower one to males.

These physical changes in the artery wall with increasing age are in the main associated with alterations in the orientation of the collagen fibres. In early life the collagen which lies in amongst the smooth muscle cells in the interlamellar spaces of the arterial media is aligned at an angle to the longitudinal axis of the vessel. Fibres in adjacent interlamellar spaces are aligned alternately to the right and the left of this axis. Each successive pulse distends the lumen of the vessel and this exerts a radial pressure on the medial tissue. The collagen fibres rotate relative to the longitudinal axis until fibres in adjacent spaces approach a parallel alignment. During this period the load extension curve o f a ring of aortic tissue will show a flattish curve (Figure 13) in which appreciable extension is accomplished without the application of undue load. As the fibres become increasing!y parallel they come under tension but their relatwe inextensibility provides increasing resistance to the extension of the tissue as a whole and finally a check to the over-distension of the vessel wall. Burton (1967) has shown by mathematical

44

DAVID A. HALL

analysis of l o a d / e x t e n s i o n curves that it is possible to d e m o n s t r a t e that the p r o p o r t i o n o f the collagen fibres which are actually u n d e r t e n s i o n at any p o i n t o n the l o a d / e x t e n s i o n curve for the tissue as a whole increases with increasing age. H e a c c o m p l i s h e d this by differentiating the curves twice, thus p r o v i d i n g values for the ' a c c e l e r a t i o n ' in the t e n s i o n in the vessel wall with respect to extension. Not only do m a x i m u m n u m b e r s of fibres come u n d e r t e n s i o n at lower levels o f extension in older tissues b u t the area u n d e r

>

ii' I

i

I

i

I

I !

!

,,,,~ ....

>l i...

~,, %

/

/

I c

I

rl i

I

vat..,

"-4"-t

4i

It-

l]

I

h

Figure 12. A schematic representation of the degeneration of collagen as affected by age. (A) Young tissue with minimal numbers of cross-links -- complete degradation. (B) 'Middleaged' tissue where the presence of a proportion of cross-linkages results in the collapse of the structure into an amorphous enzyme-resistant form following the fission of main chain bonds. (C) Fibres from fully mature tissue in which the full number of cross-links stabilize the structure and permit the penetration of the enzyme, resulting in complete degradation, even though the cross-links remain intact.

45

GERONTOLOGY: COLLAGEN DISEASE

the second derivative curve, which is proportional to the actual number of fibres under tension, is higher. Hence it is possible to deduce that as a subject ages the collagen fibres in the artery wall increasingly fail to return to their relaxed state in which alternate layers lie at an angle to one another. This explains the increasing cross-sectional area of the adult vessel at ages greatly in excess of the point at which growth has ceased. It also provides evidence for an increasing loss of resilience in these permanently distended blood vessels with increasing age.

Tension

160

C 120 B

80

A 40

Extension •

,

100

Figure 13. The effect of age on the extensibility of rings of aortic tissue subjected to increasing load. The tension is measured in grams/cm width of tissue (measured along the length of the vessel); extension as the percentage increase in the circumference of the ring. (A) Eight years.

(B) 25 years. (C) 82 years. Since part at least of the youthful resilience of the vessel wall is associated with the elastic fibres which lie between the lamellae and which anastamose with one another to f o r m an elastic network surrounding the smooth muscle cells and the collagen fibres, it can be deduced that the failure on the part of the collagen fibres to return to their relaxed state is due to a failure in the properties of this network. This m a y be due either to the degradation of the elastic fibres, under the action of the enzyme elastase, or to the accumulation of inelastic material around these structures. The latter of these two possibilities will be dealt with in the next section.

D E G R A D A T I O N OF COLLAGEN It was noted earlier from studies of load/extension curves that a degradative phase follows maturation in the development of the collagen fibre. For a

46

DAVID A. HALL

long period it was not appreciated that these processes represented two separate but consecutive phases in the life history of the collagen molecule. Hence some of the early studies on collagen degradation were not accepted by many workers in this field in view of the massive support which was extended to the cross-link theory of ageing at that time by gerontologists from a variety of different backgrounds. Now, however, it is possible to disentangle these two theories and to place them in their proper perspective one to the other. Dermis

The first evidence for a specific degradation of collagen which was related to increasing age and occurred in sites within the body was recorded by Unna as early as 1896. Using purely histochemical techniques he observed that with increasing age the clear-cut identification of collagen and elastin became more difficult. Based on both the morphological appearance and the staining properties of skin tissue, he identified three species of fibre which he regarded as being intermediate between collagen and elastin and to which he gave the names collacin, collastin and elacin. This work was further developed by Gillman (Gillman et al, 1955) who, discarding many of the more usual elastica stains, showed that a number of specific methods could enable collagen on the one hand and elastin on the other to be differentiated from other material which he suggested might be called 'elastotically degenerate collagen'. The use of this title, and also of the name 'pseudoelastin' (Burton et al, 1955; Keech, Reed and Wood, 1957; Hall, 1968) has tended to cloud the issue, since it was erroneously assumed by numerous other workers that this implied that collagen was actually converted into elastin during the ageing process. Admittedly, degraded collagen assumes many of the properties of elastin, being more elastic, losing its typical electron microscopic appearance and becoming amorphous like elastin; moreover it loses its typical x-ray diffraction pattern and becomes stainable with elastica stains. The differences between the amino acid analyses of collagen and elastin, however, make a direct conversion of one to the other virtually impossible (Table 4). The apparent elastosis observed in sections cut from the dermis of the outer surfaces of the arms of elderly subjects (Tunbridge et al, 1952) was shown to be located in areas in which there was appreciable electron microscopic evidence for the degradation of the collagen fibres originally present. It was also shown that similar changes in staining properties and electron microscopic appearance could be induced in young dermis in vitro by partial degradation with Clostridium collagenase. One difficulty associated with the interpretation of these findings was the contention on the parts of some workers that these alterations in the properties of dermal collagen were attributable to the effects of exposure to ultra-violet light rather than to age (Smith et al, 1962a, b). Observations by Hall and Slater (1973), however, demonstrated that the effects of age and exposure were similar and were additive and that dermal collagen is affected in this fashion even in fully covered regions such as the abdomen.

47

G E R O N T O L O G Y : C O L L A G E N DISEASE Table 4. Amino acid composition of collagen and elastin (residues/lO00 residues) A m i n o acid Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Leucine Isoleucine Tyrosine Phenylalanine Isodesmosine Desmosine Lysinonorleucine Ornithine Lysine Histidine Arginine Hydroxylysine

a~-Collagen

a2-Collagen

102 43 17 39 74 139 352 121 21 7 21 7 2 13 ---

87 46 19 36 69 123 350 114 34 5 33 15 4 12 ---

-31 2 51 5

-23 11 53 9

Elastin 11 8 7 9 14 97 325 232 145 trace 26 64 13 30 4 6 1.3 1.4 7 trace 5 --

Keech (1954) observed that the electron microscopic appearance of collagenase-treated dermis varied considerably with increasing age. The collagen remaining after young human skin is treated with collagenase for periods which are insufficient to result in complete dissolution is easily converted into gelatin at elevated temperatures. The same is also true of collagen from the skins of very old subjects. At intermediate ages, however, the residue after collagenase treatment cannot be converted into gelatin. This results in a loss of structure when viewed in the electron microscope. An amorphous, electron-dense, thermostable residue remains and this bears some resemblance to elastin. It is interesting to note that Keech's studies were carried out at a stage in the development of the electron microscopic art, when it was necessary for preparations to be teased out before they were deposited on the electron microscope grid. Had the thin section technique, which was developed shortly afterwards, been in use at that time it is likely that such observations would never have been made, since both the material which results from the activity of collagenase and the degraded collagen present in senile elastotic dermis only assume the fragmented appearance observed by Keech, Hall and their colleagues after they have been subjected to mechanical disturbance such as that sustained when they are teased out for application to the electron microscope grid. Few signs of the degradative process can be observed in undisturbed thin sections. Keech certainly observed an artefact, but it was an artefact which assisted her in the explanation of the facts in question. The fact that degenerate collagen is susceptible to attack by elastase, although adding to the confusion which is attributable to the use of the

48

DAVID A. HALL

name pseudoelastin, has helped in the provision of a suitable method for its estimation. Collagen is not susceptible to elastase, hence the effect of this enzyme on a tissue which contains collagen, elastin and collagen degradation products results in the latter two being taken into solution. Extraction with boiling alkali on the other hand dissolves only collagen and its degradation products, leaving elastin untouched. The difference between the values obtained for the content of elastin and partially degraded collagen on the one hand and of elastin alone on the other, therefore, provides a measure of the amount of partially degraded collagen present in a tissue (Table 5). Degenerate collagen fibres have been observed in the dermis of subjects with a variety of pathological conditions, some of which are age related. The degree of degradation as observed in the electron microscope ranges from minor bends and breaks in the microfibrils through the appearance of short lengths of fibres with tapered ends to complete conversion to a fully amorphous state as mentioned above (Hall, 1976). Table 5. Method for the estimation of the pseudoelastin (partially degraded collagen) content

of a tissue 1. Treatment with elastase - - dissolves elastin and pseudoelastin. 2. Treatment with hot alkali - - dissolves collagen and pseudoelastin. Value for 1 minus residue from 2 gives a value for the pseudoelastin content.

These differences in appearance are associated with a marked variability in amino acid composition which can be explained by the occurrence of different degrees of degradation. It has been suggested that cross-links between a-chains once formed and converted in the body into a stable form are unlikely to be degraded. Hence any age changes which do occur must involve degradation of peptide linkages within individual a-chains between existing cross-links. This is confirmed by the conversion of collagen to the electron-dense, amorphous, elastica-staining product by partial degradation with collagenase and pepsin, both of which enzymes will be more likely to break specific peptide linkages than the -C-C or reduced -C-N linkages formed from allysine and lysine residues. Keech's observation that collagen from both young and old subjects degrades completely whereas that from 'middle-aged' subjects is converted into 'pseudoelastin' could indicate that the enzymic degradation of collagen from subjects of this age group is controlled by the degree of cross-linking. Young fibres with few crosslinkages will be fully susceptible to collagenolytic enzymes whereas, in middle age, a limited hydrolysis of the main chains could be followed by a collapse of the structure due to the effect of the intermediate number of cross-links present in fibres from subjects of this age. A higher concentration of cross-links in collagens from older age groups might be expected to hold the individual a-chains apart, thus permitting collagenolytic enzymes to penetrate to the susceptible peptide linkages. This hypothesis does not, however, only implicate the covalent lysine-based linkages referred to earlier. These cross-linkages, although increasing throughout

GERONTOLOGY: COLLAGEN DISEASE

49

fetal life and for a short period of infancy and adolescence, do not continue to increase throughout adult life. The solubility of adult tissues, however, does continue to decrease, and hence it must be assumed that interfibrillar links are formed. In fact Jackson suggested as long ago as 1958 that the fibrils might react with glycosaminoglycans to form high molecular weight complexes. Hall and Reed (1973) and Hall and Slater (1973) have suggested that interactions of this type are especially likely in aged tissues which have been subjected to continued cyclic extension and contraction throughout life. Evidence of a purely chemical nature for this type of interaction arose as the result of the studies by Labella, Vivian and Thornhill (1966) on the amino acid analysis of elastin. Since pseudoelastin and elastin are segregated in the same fraction during purification, apparent age changes in the amino acid analysis of elastin m a y indicate that collagen degradation products become linked to elastin - - possibly through the proteoglycan and glycoprotein contenf of the tissue. Labella reported an age pattern for this phenomenon similar to that observed by Keech; namely, it was only in those of middle age that this amino acid analysis of elastin differed from the classical one. Vascular Tissue

The values which have been reported for the age variations in the elastin content of vascular tissue have been distorted, in part at least, by the simultaneous presence of pseudoelastin in tissues from elderly subjects (Table 6). Only when methods of purification are employed which are capable of separating collagen, elastin and pseudoelastin is it possible to assess the true value for elastin levels. Using the method outlined earlier it has proved possible to measure the amount of pseudoelastin in the vascular tissue, and to demonstrate how it changes with increasing age. Hall and Slater (1973) reported that aortic media from a two-month-old infant contained 125 mg pseudoelastin/g tissue, the value rising through 158 m g / g at 21 years to 241 m g / g at 75 years. It was also possible to demonstrate that Table 6. The apparent changes with age o f elastin content of human aortic intima (I) and media (M) as reported by eight different authors

(% Dry weight) Age in years

Tissue 0-20 M I,M I,M I,M I,M 1,M I,M I,M

48.3

20-50

50-80

44.1-41.4

43.8-41.4

36.5-32.1 -14-33 I 48-40 --

41.1 41.7-30.8

45 28.6

80 +

-20.5-25.9 34 58.5-54.5

43 22.9 -20.9 33 44.6

37 16.8 -26.2 39-18

50

DAVID A. HALL

the polysaccharide content of the elastase-soluble fraction of the aorta rose from 29.1 ~g/mg protein at 21 years of age to 46.4/xg/mg at 75. Fractionation of the extracted material on G50 Sephadex demonstrated that each of the five different molecular weight species contained both protein and polysaccharide, indicating that at all levels of particle size the two are covalently linked to one another. Hall and Slater suggest that firmer links between degraded collagen and polysaccharide are formed with increasing age in tissues such as aortic wall which are subjected to cyclical extension and contraction throughout life. This effect is similar to the one observed by Hall and Reed (1973) in the intervertebral disc. In vitro studies (Hall, 1970), in which a preparation of soluble collagen was allowed to precipitate within a nylon mesh which was continuously stretched and relaxed, demonstrated that mechanical alignment of collagen fibrils during precipitation resulted in the production of a modified form of collagen. This material was less soluble in neutral salt solution or dilute acid, less susceptible to collagenase and more susceptible to elastase. It also retained a greater proportion of the polysaccharide originally present in the soluble collagen preparation. In all these respects it resembled quite closely the degraded collagen or pseudoelastin present in ageing vascular tissue and it may therefore be deduced that relatively stable bonds between degraded collagen and polysaccharide from the surrounding tissue are formed in vascular tissue and probably also in other tissues which are continuously subjected to varying degrees of tension throughout life.

Hormonal Control of Collagen Metabolism It has long been appreciated that changes occur in the connective tissues of subjects who are suffering from hormonal imbalance of natural, pathological or experimental origin. Thus osteoporotic bone loss not only occurs naturally with increasing age but also results from the hormonal imbalance which is associated with the menopause and from the administration of naturally occurring corticosteroids such as cortisone or synthetic ones such as prednisolone. In view of this broad spread of aetiological factors a search has been proceeding for some time to ascertain whether there is a direct relationship, either positive or negative, between the ageing process in connective tissues and hormonal activity. The two major collagenous tissues which have come under study are the skeleton and the dermis, and various workers have demonstrated related changes in both resulting from steroid therapy (Sheppard and Meema, i967). Hall et al (1974) and Hall (1976) have not only studied the overall content of collagen in skin in association with steroid-induced osteoporosis, but have assessed the nature of the collagen' which remains following the steroid-induced loss of part of this protein. They observed that whereas the mean value for the soluble collagen content of the skin of normal subjects aged 57 to 78 was 20 per cent lower than that for the skin of a comparable group aged 20 to 56, there was a 38 per cent increase with age in two age-matched groups of prednisolonetreated subjects. The residue after the extraction of this soluble fraction

GERONTOLOGY: COLLAGEN DISEASE

51

was significantly less susceptible to collagenase attack in the elderly prednisolone-treated group whereas there was no significant difference in this respect between the two normal age groups. It can be deduced from these observations that although the effect of the steroid on collagenase activity is one of inhibition (Dayer et al, 1980) it also prevents the formation of increasing numbers of cross-links between tropocollagen molecules as age increases. This raises the level of soluble un-cross-linked collagen and permits a higher proportion of the partially cross-linked collagen in the residue to degrade under the action of collagenase. These results do not appear at first sight to confirm the observations previously made on wound healing in experimental animals. S0rensen (1966), for instance, reports that the administration of cortisone prevents the accumulation of hydroxyproline in healing wounds. She does not, however, comment on the age of the animals she employed, and it may well be that her experimental stock was more comparable to the younger of the two groups of human subjects studied by Hall and his colleagues. They were also able to examine serial biopsy specimens of skin from a limited number of individuals who had received prednisolone for one year. The effect of the steroid on the collagen content of the skin was directly related to the age of the subject. Loss of collagen during the year increased six-fold between 40 and 80 years of age. Although these studies are by no means completed it is possible to deduce from them that there is in fact a relationship between age-related alterations in the nature and amount of collagen in at least one tissue - - the skin - - and the effects of steroids.

CONCLUSIONS The present state of our knowledge regarding the ageing of collagen enables us to state categorically that the changes which can be observed take place in various sequential phases. The protein is synthesized on a variety of ribosomal complexes in tissue fibroblasts and subsequently suffers numerous post-translational changes throughout life. Some of these, restricted as they are to the early years of life of the organism of which the collagen forms a part, must be regarded as maturative rather than senescent (Hall, 1968; Jackson, 1973), In certain tissues maturation alters the physical properties of the collagen fibres due to the presence of increasing numbers of cross-linkages of various types, to improve the efficiency of the tissue of which the collagen forms a part (Table 7A). Thereafter the degradation of the main chains detracts from their effectiveness in the tissue concerned. In other tissues, increasing numbers of cross-links detract from the function of the collagen molecules (Table 7B) and hence an 'ageing' process sets in far earlier. Any main chain degradation in these sites might result in a degree of rejuvenation. Such changes have not yet been observed, thus indicating that the control of the ageing process is probably tissue specific. If, however, selective main chain degradation could be induced some of these changes might be controlled.

52

DAVID A. HALL

Table 7. The effect o f increasing numbers o f cross-links in collagen on the functioning o f

collagenous tissues A. Deleterious effects Aorta Bone Cornea Organ matrix

-----

decreased elasticity and compliance reduced turnover rate and remodelling reduced transparency reduced permeability to products of cellular activity B. Advantageous effects

Tendon -Skin -Intervertebraldisc - -

increased tensile strength better tone greater resistance to compression

REFERENCES

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