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Elastofibroma: Disturbed elastic fibrillogenesis by periosteal-derived cells? An immunoelectron microscopic and in situ hybridization study
Elastofibroma: Disturbed Elastic Fibrillogenesis by Periosteal-Derived Cells? An Immunoelectron Microscopic and In Situ Hybridization Study J. S. KUMARATILAKE, PHD, R. KRISHNAN, PHD, J. LOMAX-SMITH, PHD, AND E. G. CLEARY, MD Monospecific
antibodies
to elastic
used for immunoelcctron broma.
The
exhibited
elastic-staining
a variety
amorphous fibers.
and microfibrillar
for ionic
phologies. elastin typical
reactike
occurring
of the elastic
cells, and the structure It is plroposed
as a result
periosteal-derived (
I991
by W.B.
cells. Saunders
Elastof~I)I-0rri;i
associated
fibers,
elastin
fibers
in common
and were spe-
biosynthesis
HL~M
was
and staining
the morphology
of the
in these lesions
were
with those of normal
peri-
arise from the periosteum
and that the different
disturbances
with
that were
to microfibril-asso-
The appearances
tissue elements,
irritation
represent
were
mor-
with anti-
studies with a cRNA probe
that active
that elastofibromas
of chronic
morphologies
had variable
microfibrils
elastic
of the collagen
shown to have many features osteum.
fibers
of the
in elastic
substructural
antibodies
within the lesions.
seen
specifically
diameter
with normal
confirmed
patchily
characteristics
several
been
lesions
ratios
in these fibers
In situ hybridization
e&tin
have
of elastofi-
in these
usually
reacted
with monospecific
ciated glycoprotein. for human
form
of 12.nm
of those associated
cifically
material
Most of the elastic
numbers
seen
with differing
and exhibited
this, each
antibodies. large
typically
components
elastic
stains
Despite
relatively
fibers
components
of two examples
of morphologies
The amorphous
affinity
tissue
microscopy
of elastic
elastic
22:1017-1029.
P.THOI.
fiber
fibrillogenesis
by
Copyright
Compaq
is ;I
raw
tunior
coirinionl~itt the lower end of the
that subscapular
occurs
Inost space
of
is usually attached to the underlying ribs arltl sometimes to the adjacent scapula ’ The olccranon pt~ocess is another commonly aff&red site.’ These t11oderatel~ cellular, notiencapsulated masses consist oL:t ~x~llaget~ous stroma containitlg charrefractile fibers with the actcaristic solid. eosinophilic. staining properties of elastic tissue. The elastic-staining fibers exhibit se\,eral fort11s, iticluditig unifortiily thickened. bt-anclied, or rml~ranched fibers; long, irregularly thic kcncd (bcxded or segmented) fibers; and globular prolilrs. Significatit quantities of the protein elastin ha\e been isolated frotii these tun1ors,1 and he elasticstainitig fibet- have Bern sltowt1 to react histochcmically with polvclonal ;inti-(hut1iat1 elastin) antibodies.‘.” peuon\
ove1
50
!;wrs
of
age.
It
_-___
1017
A variety 01 elastic fiber morphologit~s ha\,e beet1 observed in elastofihromas in the electron microscope (EM). Some fibers have a central core of typical atnorphous elastin. but most of the elastic-staining fibers have abnormal ttlorpholoCq and staining properties, c.onsisting either of electron-dense, granular, or fihrillat? masses or less electron-dense amorphous rlastic tissue, sin-rounded hy a variable layer of irregularly arranged niicrofibrils.‘~i Many investigators have reported an increase in the relative amounts of microfibrils and some have seett tr1icrotrtl~ular structures withit the amorphous cottiponcnt .” It1 the single inimunoelectrori microscopic study repot-ted. the reactivity of rabbit anti-,(hut11an aorta cu-elastin) antibody with two tumors was examined using a pre-embedding labeling technique.’ The antibody was sl1owt1 to localize specifically in the outer granular zot1e of the elastic fibers. hut penetration to their interiors was t1ot achieved. Microfibril-rich peripheral /ones were also labeled. the cells of clastofilbromas were Histologically, originally considered to be fihroblasts. and the EM appearances wert’ consistent with that view.’ flowever. the presence of tt1~ofilatiients led some researc,hers to suggest that the c,ells were tt1yofil,rohlasti~. although they lacked a basement men~l~rane.‘i~S Several investigators have noted that these cells have a thickened fihrous lamit1a adjacent to the prominent inner nuclear metlthratle surt-ounding tl1e peripherally dispersed nuclear chromatin.“.” Othet-s, noting the presence of nuclear pores, an asremarked on tt1e resemblance to chottdrocvtes,” sociation reinforced by finding that the stromal collagen was reactive with anti-(type II c.ollagen) attt ibodv.” We have applied modern techniques, inc-luding postembedding inltnunoelectron microscopic studies, to two typical elastofihromas obtained fron1 the subscapular spaces of an elderly woman. The components of the atypical elastic fibers have heen defined immunologically using affinity-purified monospecific., polvclonal atltibodtes against tropoelastin (TE) and the monoclonal antibody (lL44) raised against a hexapeptide unique lo the elastin molecule. Atltibodies specific, for the elastic. tissue tt1ic t-ofibt-il-associated glvcoprotein (3IAGI’) were used to characterize the micro~hrillar compot1ents. The cellular biosynthesis of elastiri was studiecl by in situ hybridizatiotl with a cKNA prohe specific lin- human elastin. Exattlinatic m of periosteum from the rscxpula. rihs, and some long hones has shown that the elastic tissue of this strurrure is relatively electron dense and is surrounded by a thickened glycoproteitt-cotit aininp zone.
HUMAN PATHOLOGY
Volume 22, No. 10 (October
rich in microfibrils that are immunoreactive with the anti-MAGI’ antibodies. The cells of the periosteum were shown to have many features in common with those of the elastofibromas. In view of these findings and taking account of the regular association of elastofibromas with periosteum, it is postulated that they have their origin from the periosteum, in response to chronic physical irritation, and that they represent a complex disturbance of elastic fibrillogenesis. MATERIALS
AND METHODS
subscapular
pH was adjusted to 7.2. Tissues fixed in 1.25% glutaraldehydecontaining fixative were postfixed in 1% osmium tetroxide for 1 hour, dehydrated in a graded series of ethanols, and embedded in Spurr’s resin (polymerized at 70°C for 72 hours). Tissues fixed in 0.25% glutaraldehyde-containing fixative were treated with 1% sodium borohydride before dehydration and were embedded in LR White resin (polymerized at 50°C for 72 hours). Ultrathin sections (silver-gold), cut on Reichert Ultracut E and Om U2 ultramicrotomes using diamond knives. were mounted on naked (Spurr’s sections) or Celloidon-coated (LR White sections) nickel grids.
Antibodies
Tissues Two elastofihromas
1991)
were
obtained from the right and left spaces of a female patient at two surgical proce-
dures 16 months apart. The age of the patient at the time of removal of the first tumor (tumor I) was 78 years. The clinical
history was not remarkable. The tumors had the typical lobular grey fibrofatty appearance of elastofibromas. Parts of each tumor were processed for light microscopy, EM, and in situ hybridization studies. Normal periosteum was obtained at autopsy from the scapula, ribs, and pelvis of two female patients, aged 75 and 82 years, who died acutely from nonosseous diseases. Periosteunl was also obtained at surgery 2 and 6 days after fracture from three patients with fractures of long bones requiring open reduction. The ages of these three patients range from 25 to 78 years. Samples were taken immediately adjacent to the fracture site and 3 to 4 cm proximal to it.
Light Microscopy Each tissue was fixed in 10% buffered neutral formaldehyde and embedded in paraffin wax. Five-micrometer sections were stained with hematoxylin-eosin (H&E), periodic acidSchiff (PAS), and Miller’s elastic tissue stains.
In Situ Hybridization The procedure for in situ hybridization (including cDNA probe preparation, hybridization conditions. stringency washes, and autoradiography) was as detailed previously.’ ’ Briefly, 5~111 parafIin sections were mounted on slides pretreated with Elmer’s glue, deparaffinized, and hydrated in decreasing ethanol solutions before processing. The probe was derived from a 1.1 -Kb fragment, containing the 5’ end of the human elastin cDNA.‘” subcloned into pGEM 2 vector. The “‘S-labeled cRNA probe was then generated by in vitro transcription with T7 polymerase, and sized by mild alkaline digestion to a mass average of 150 bases. The probe-specific activity was approximately 10’ dpm/Fg. It was applied at a concentration of 10” dpm/lO pL/section (2 mm X 2 mm). After autoradiographic development for 4 to 8 days, sections were countersrained with H&E and examined. Sense RNA was labeled with “3 uridine-5’-triphosphate by tl-anscription of the DNA template by SP6 RNA polymerase, and step sections were hybridized with this probe as negative controls. Nonspecifically bound probe was removed from the hybridized sections by digestion with RNAse prior to autoradiography.”
Electron Microscopy Approximately 1 -mm cubes of each tissue were fixed in chilled (4°C) I .25% glutaraldehyde plus 4Yo paraformaldehyde for l? to 18 hours or in 0.25% glutaraldehyde plus 4% paraformaldehyde for 4 hours. The fixatives were made in 6 mmol/ L phosphate-buffered saline (PBS) containing 4% sucrose and 5% polyvinylpyrrolidone (molecular weight, 10,000) and the
1018
Affinity-purified polyclonal anti-TE antibody was prepared in rabbits against porcine aortic TE.‘” The monoclonal antibody, BA4, had been prepared against a synthesized form of the elastin peptide, Val-Gly-Val-Ala-Pro-Gly.‘” Affinity-purified antibody to bovine MAGP, prepared in rabbits, was used for the identification of microfibrillar components of elastic tissue.” Affinity-purified types III and IV collagen antibodies were raised against bovine placental type III and human placental type IV collagen in rabbits. Details of the purification and specificity of anti-TE and antiMAGP antibodies have been published,‘“,“‘,‘h The specificity of the types III and IV collagen antibodies was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.
lmmunolabeling Immunolabeling was performed as described by White et al.‘” Prior to the ovalbumin/PBS (ova/PBS) step, all the sections were incubated with 0.02 mol/L glycine in PBS (for 10 minutes each on five drops). Spurr’s sections were used for studying the distribution of anti-TE, anti-MAGP, and BA4 antibodies. With the anti-MAGP antibody, prior to the glycine/ PBS step, the sections were treated with 50 mmol/L dithlothreitol in 6 mol/L guanidine hydrochloride (GdnHCl) for 5 minutes, after which the excess was drained off on a filter paper. The sections were then treated with I10 mmol/L io-
doacetamide in 6 mol/L GdnHCl for 5 minutes and washed vigorously in Tris-buffered saline, pH 8.0.‘7 LR White sections were used for the immunoelectron microscopic studies of anticollagen antibody disrribution. They were etched with a saturated solution of sodium metaperiodate for 10 minutes and washed vigorously in water prior to the glycine/PBS step. Sections reacted with BA4 monoclonal antibody were incubated with affinity-purified rabbit anti-mouse immunoglobulin G (IgG) for 1 hour at room temperature and washed for 5 minutes each on six drops of ova/PBS prior to incubation with protein A-dextran-gold (12-nm diameter gold particles). Ovalbumin/PBS containing Tween 20 (final concentration, 0.05%) was used for antibody dilution and for all the washing steps with the BA4 monoclonal antibody. Optimal antibody dilutions for maximal labeling with minimal background staining were determined for each antibody separately. Controls were performed on adjacent sections using ova/PBS, affinity-purified nonimmune rabbit IgG in place of the antibody, and antibody adsorbed with excess of the respective antigens. With the BA4 monoclonal antibody, ova/PBS was used in place of the primary antibody as an additional control. Bovine fetal nuchal ligament served as a positive control.
Transmission
Electron Microscopy
Spurr’s sections were stained with alcoholic uranyl acetate and lead citrate for 20 minutes each. LR White sections were
stained with aqueous uranyl acetate and lead citrate for 1 minute and 15 seconds, respectively. Sections were stained for glycoproteins using the periodic acid-bismuth (PA-bis)
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
method. ” The specificity of this reaction for glycoproteins was established hy specific blockin? with sodiunl borohydride. All sections were carbon coated prwr to examination in a Philips EM 300 at 80 kV.
RESULTS Light Microscopy The two elastofibromas were similar in appearance, being composed mainly of fibrous connective tissue, within which were elongated, but rather plump fibroblastic-type cells and inactive fibrocytes. The connective tissue consisted of dense collections of collagen fibers. among which were scattered solid eosinophilic refractile rods of varying thicknesses, many of which were seen in cross-section as solid, roughly circular masses of varying diameter (Fig 1A). These refractile components reacted with elastic-tissue and PAS stains, exhibited several different forms, and occurred in collections of like fibers. Some were solid and had no clear evidence of internal architecture. Other solid-looking fibers had a paler-staining peripheral portion surrounding a more darkly staining central zone, so that in longitudinal section they resembled flattened disks strung along a thread, or a bottle brush within a test tube. In crosssection, the elastic-staining material was arranged radially in these fibers. A thin but more dense, rod-like, elastic-staining core was visible within some of the thicker beaded fibers (Fig 1 B) and occasionally in thinner elastic fibers. Core-containing fibers tended to occur in collections and were more prominent in tumor I than in tumor Il. A third fiber- form occurred as fused irregular masses of electron-dense elastic material in fibers that had serrated borders, in both longitudinal and transverse sections (Fig 1B). The perlosteum consisted mainly of a dense collagenous stroma, within which small masses of elastic-staining, PAS-positive material were visible in a narrow zone near the bone surface, from which they were separated b!, a layer of cells. Adult rib periosteurn was relatively acellular, but that from healing fracture sites contained a proliferation of’ cells resembling fibroblasts (data not shown). In Situ Hybridization The elastofibromas contained collections of fibroblastic-appearing cells, which had high grain counts for elastin mRNX, well above background (Fig 1C). These cells were ofi:en seen in close apposition to elastic elements in the tumor. Background staining was low, as confirmed by the step section exposed to the sense mRNA probe (Fig 11)). These findings point to the presence within the reactive cells of relatively high concentrations of elastin mRNA, indicating that they were actively engaged in etastinogenesis. In elastin-poor regions of the tumors, a lesser proportion of cells reacted with the cRNA probe. indicating that some populations of cells were not engaged in elastin biosynthesis. Electron Mic:roscopy The ultrastructural morphology of the two tumors was similar. Borh consisted mainly of collections of
(Kumarotilake
et al)
tightly packed bundles of collagen fibers which, at low magnification, exhibited normal organization. and both tumors contained collections of what appeared to be abnormal elastic fibers of variable size, shape, and staining. Fibroblastic cells were commonly observed with many thin irregular processes, which extended between the collagen bundles and were often closely apposed to the abnormal elastic fibers (Fig 2A). None of the elastic fibers had ultrastructural appearances typical of normal elastic tissue. They appeared as roughly circular or irregular masses of am amorphous material, which exhibited a variety of morphologies and staining patterns. These included electron-dense granular fibers, some showing a radial pattern in cross section (Fig 2B); more solid-looking fibers containing amorphous material of lesser electron density, often with a narrow electron-dense rim (Fig 2D): and uniformly amorphous, relatively electron-lucent solid fibers with occasional holes, some containing what appeared to be a cell process (Fig 3C). The beaded fibers in light microscopy commonly exhibited the electron-dense granular form in the EM, while the solid fibers contained both electron-dense granular material and more electron-lucent homogeneous material. Fibers with each staining pattern were seen in cross-section. The granular electron-dense material was seen to be arranged predominantly in the radiating spokes pattern and generally had an irregular, serrated edge (Fig 2B and C). ‘There was a tendency for fibers of a particular staining pattern to occur in collections within the tumors. and each type of fiber was seen in a full range of diameters, from the smallest to the largest. Hybrid forms exhibiting characteristics of more than one type of fiber were virtually never seen. The different fiber types are shown, at higher magnifications, in Fig 3. The amorphous portion of each type of fiber was specifically reactive with both the antiTE and the BA4 antibodies throughout (Fig 3). Both antibodies also localized specifically to the amorphous component of elastic fibers in bovine fetal lmchal ligament (data not shown). Figure 3A and B shows examples of the granular- electron-dense fibers and the less electron-dense homogeneous fibers, respective&, each with an investment of typical elastin-associated microfibrils. In both types of fiber, the granularity was more obvious in the central region of the fiber and appeared to be related to the presence there of collections of collagenlike fibrils, similar in diameter (35 to 50 mn) IU the collagen fibrils closely apposed to the surface of the elastic fiber (Fig YB). Occasional collagen fibrils. sectioned longitudinally so that typical periodic striation was visible, were seen in such fibers (Fig 3B). Figure 3C shows a less electron-dense type of elastic fiber in which the amorphous component, which reacted with both of the anti-elastin antibodies, had a fibrillary texture. In some immunoreactive elastic tissue, the amorphous component was homogeneous and had little affinitv for the electron-dense stain. These types of fiber also &mtained collagen fibrils. recognizable by their pattern of striations when sectioned longitudinally. Figure 3D shows another abnormal elastic fiber morphology in which a central core of normal-looking amorphous elastin is enclosed within abnormal electron-dense elastin. Both the
1019
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FIGURE 1. (A and 6) Photomicrographs showing predominantly collagenous stroma containing variable amounts of abnormal elastic-staining fibers. These elastic fibers are seen as thick solid curved rods with circular cross-sections of varying diameters. Some have a beaded look (short arrow) and some have a darker staining core (long arrow). In cross-section, the darker-staining material occupies the central portion of the fiber and is arranged radially. The stromol cells are fibroblastic. (A: H&E stain; magnification x500. 8. Miller’s elastic tissue stain; magnification x.500.) (C and D) Photomicrographs of in situ hybridization of elastin mRNA in elastofibroma. Many stromal ftbroblastic cells exhibit positive signal. The signal persisted after RNase treatment of the hybridized section, Confirming that it was specific for elastin mRNA. (C: H&E stain, labeling with cRNA probe; magnification x425. D: H&E stain, control with mRNA probe; magnification X425.)
1020
FIGURE 2. Electron mlcrographs showing elastic tissue morphology within the predominantly collagenous stroma of an elastofibroma. Uranyl acetate/lead citrate stain. (A) Moderately electron-dense solid elastic fibers (e) are cross-sectloned. .A fibroblastic stromal cell IS seen with many elongated processes surrounding an elastic fiber and bundles of collagen fibrils (c). (Magnification * 5,500.) (B) Transverse section of radially arrayed electron-dense, granular elastic fibers seen among collagen fiber bundles. An occasional cell and an electron-lucent elastic fiber, sectioned longitudinally (arrow), are present. (Magnification ~5.500.) (CJ Longitudinal sectlon of an electrondense granular elastic fiber with an irregular edge, seen among the dense collagenous stroma. (Magnification I 5,500.) [D) Longitudinal section of more solid, less electron-dense elastic fiber with an electron-dense rim. (Magnification * 5,500.)
1021
FIGURE 3. Electron micrographs showing immunolocalization of anti-elastin antibodies in elastic fibers of a variety of morphologic forms. Uranyl acetate/lead citrate stain; A and B labeled with anti-tropoelastin antibody, and C and D labeled with BA4 antibody. (A) Electron-dense granular fiber with a peripheral rim of typical microfibrils (arrowhead). (Magnification X37500.) (6) Less electrondense elastic fiber with a thin rind of peripheral microftbrils (arrowhead). Fibrils 35 to 50 nm in diameter and collagen ftbrils of similar diameter and with faint cross-striations (arrows) are seen within the fiber. (Magnification X38.750.) (C) Cross-section of a less electron-dense elastic fiber in which the amorphous component has a ftbrillary texture. The fiber lacks peripheral microltbrils. A membrane-bound process and some holes are seen within the tiber. (Magnitication X39.500.) (D) Oblique section of a small electron-dense elastic fiber containing a core of apparently normal-looking, electron-lucent amorphous elastic tissue (a). A ftbroblastic cell (arrow) is closely applied to the surface of the fiber. (Magnification ~6,600.) 1-a_
FIGURE 4. E:lectron micrographs showing immunostaining of 12-nm diameter microfibrils in a range of relationships with elastin. Uranyl acetate/lead citrate stain; A and B labeled with anti-MAGP antibody, and C and D labeled with BA4 antibody. (A) Section of elastic fiber with a relatively electron-lucent amorphous component. The peripheral rim of microfibrils (arrowhead) shows good avidity for the elastin-associated microftbrillar antibody. Less intense specific antibody localization is seen over the amorphous elastic material. (Magnifkation X42.000.) (B) A collection of typical elastin-associated microfibrils seen within the extracellular matrix between two ftbroblastic cell processes (arrows). The collection of microfibrils (arrowhead) is adjacent to an elastic fiber with few microfibrils and is intermingled with collagen ftbrils. (Magnification x38.000.) (C) High-magnification view of an microftbrillar collection (arrowhead), immunoreactive with anti-BA4 antibody, among collagen ftbrils. (Magnification \t85,500.) (D) A tangle of nonimmunor’eactive microftbrils (arrowhead) lying within the extracellular matrix, adjacent to a cell process (arrow), an electrondense immunoreactivc elastic fiber, and intermingled with collagen ftbrils. (Magnification x38.5000.)
1023
HUMAN PATHOLOGY
Volume 22, No. 10 (October
1024
1991)
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
c.elitral con: and the abnormal amorphous elastic material were reac.tive with each of the anti-elastin antibodies. Similar typical amorphous cores with normal immunoreac-tivity for the two elastin antibodies were seen within occasional electron-lucent and granular, electron-dense abnormal elastic fibers. A majority of the elastic fibers, of both staining types, had a rind of microfibrils, similar in appearance to the 12-mn diameter microfibrils associated with normal elastic fibers (Fig 3A and B). The thickness of the rind of microfibrils varied from fiber to fiber, as well as around their perimeters. In a number of fibers of each staining type, small collections of similar microfibrils lvere seen within the amorphous elastic material. All the microfibrils seen in association with the different types of abnormal elastic fibers were specifically reactive with the anti-MAGP antibody (Fig 4A). A few immunoreactive elastic fibers of each staining pattern were completely free of peripheral microfibrils (Figs 2B, 2D, and 4B). In control nuchal ligament sections, anti-MAGP antibody di.stribution was restricted specifically to microfibrils. The surrounding matrix and amorphous elastic material were negative (data not shown). In the elastofibromas, anti-MAGP antibody bound not only to the peripheral elastic fiber microfibrils, but also to adjacent although at a lesser labeling amorphous component, density (Fig 4A). The latter was significantly greater than that of the nearby matrix. Collections of 12-nm microfibrils, with or without foci of amorphous elastin, were also seen between collagen bundles and in the vicinity of stromal cells (Figs 3B to D). Such microfibrillar collections were seen more frequently in tumor I than in tumor II. They reacted specifically with the anti-MAGP antibody (Fig 4B) and, not infrequentl>r, also reacted with the anti-elastin antibodies. even when no amorphous elastic tissue was apparent in that region (Fig 4C). Large tangled masses of typical elastin-associated microfibrils, which did not exhibit elastin antibody binding, were also seen within the matrix (Fig 1D). Sections of elastofibroma. stained for glycoproteins with PA-bis stain, showed specific staining of elastic fiherb of all i.hrer morphologies. The staining was not apparently more intense in the electron-dense type of amorphous elastic tissue. In all types of elastic fiber the stain localized not only to the periphery of the fibers, but also deep within the fiber, as seen in Fig 5A. Collagen fibrils and microfibril collections separate from the elastic fibers were also stained. The majority of the collagen fibrils in the stroma were normal in appearance, with uniform fibril diameters.
(Kumaratilake
et al)
There were, however, a number of‘ focal c,olleclions of thickened collagen fibrils. which appeared as typical collagen “flowers” in cross-section, suggesting that they were in loose twists (Fig 5B). Attempts to stain the stromal collagen fibers with antibodies to structural collagens were unsuccessful, even after tissue etching, protease digestion, and extraction with acetic acid, presumably because the tissues had been subjected to standard histologic fixation, which inhibits collagen antibody binding. The stromal cells were elongated and had many relatively thin, cytoplasmic processes (Figs 2A and 5C). These extended between collagen bundles and were sometimes seen in clear spaces within elasl:ic fibers. The cytoplasm of these cells had abundant rough endoplasmic reticulum, dilated cisternae. and vesicles of varying diameter. Many unbanded filaments, similar in size to myofilaments, were apparent within the cytoplasm. These were loosely organized and did not exhibit electron-dense zones; these cells also did not have a basement membrane structure. Their nuclei were prominent and often indented, and the chromatin material was arranged around the periphery of the nuclear membrane. There was a heavy condensation of electrondense material adjacent to the inner nuclear membrane, which was interrupted at intervals by pores (Fig 5C). Occasional stromal cells were surrouncled by a thickened, loosely organized zone of electron-dense staining, superficially resembling a basement membrane (data not shown). However. its organization differed from normal basement membrane, and anti-type TV collagen antibody did not bind to these structures, although it Iocalized specifically, in the section, to the basement mt mbrane of the blood vessels. With all antibodies, backgn)und staining was minimal and no significant antibody binding was observed in negative control tissues.
Periosteum The periosteum consisted predominantly of bundles of tightly packed collagen fibers, among which were scattered some densely-staining elastic fibers and a relatively low number of fibroblastic cells (Fig GA). These cells contained loose collections of 1 O-rim diameter intermediate filaments, which did not show electron-dense zones, and did not have a basement membrane. Their nuclei exhibited an electron-dense fibrous lamina along the inner nuclear membrane (Fig 5D), similar to that seen in the cells of the elastofibroma. The periosteal cells had a number of elongated cell processes. The amorphous component of the elastic fibers was more elec,tron-dense than that of normal (elastic fibers,
FIGURE 5. Electron micrographs of elastofibroma and periosteum. (A) Periodic acid-bismuth stain for glycoproteins. An elastic fiber cut in cross-section with adjacent collagen fibrils. The stain is distributed specifically around the periphery of the elastic fiber, but also penetrates into deeper parts of the amorphous elastic material. (Magnification X34,000.)(B) Cross-banded ‘collagen fibrils, of varying diameters, irregularly twisted along their long axis. In cross-section, the fibrils exhibit considerable variation in diameter, and there are many relatively fine fibrils. The twisted fibrils are large and have a star- or flower-like appearance in cross-section. A cell process (arrow) is seen among the collagen fibrils. (Uranyl acetate/lead citrate stain; magnification k 37,000:) (C) A typical fibroblastic cell in an elastofibroma showing the many finger-like processes (black arrow), loosely arranged intracellular filaments (short white arrow), peripheral nuclear heterochromatin and electron-dense fibrous nuclear lamina (white arrowhead). and a nuclear pore (long white arrow). (Uranyl acetate/lead citrate stain; magnification ;\37,000.) (D) Fibroblastic cell in the periosteum of adult rib showing a more orderly array of intracellular filaments (small white arrow), peripheral nuclear heterochromatin and electron-dense fibrous nuclear lamina (white arrowhead), and a nuclear pore (long white arrow). (Uranyl acetate/lead citrate stain; magnification * 50.000.)
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HUMAN PATHOLOGY
Volume 22, No. 10 (October
1026
1991)
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
but was reactive with the anti-elastin antibodies (Fig 6B). All the elastic fibers were surrounded by a thick electrondense coat, and similar material was seen as inclusions within the amorphous component (Fig 6A and B). In mature periosteum, this electron-dense material obscured both the microfibrils and most of the reactivity with anti-MAGP antibody (data not shown). In healing fractures and in fetal bovine periosteum, relatively large numbers of microfibrils were seen around the periphery of the elastic fibers (data not shown), so that the ratio of microfibrils to amorphous component was high compared with that seen in normal human skin or aortic elastic tissue. Both the electron-dense peripheral zones and the elesctron-dense inclusions within the amorphous elastic tissue reacted specifically with the PA-his stain, indicating the presence of glycoprotein in this distribution (Fig 6C). Large, densely-packed collections of 1?-nm microfibrils were observed in the periosteal extracellular matrix, some clearly separate from amorphous elastic tissue and others containing scattered small masses of amorphous elastic tissue. These microfibrils also reacted specifically with anti-MAGP antibody (Fig 61)). Sometimes they also reacted positively with anti-TE antibodies, despite the lack of recognizable amorphous elastic tissue in those regions (data not shown). DlSCUSSlOlN Almost 30 years have elapsed since the first reported cases of elastofibroma. Although more than 270 cases have now been reported, neither the nature of the tumors nor their origin has been established. The subscapular region is by far the most common location, but other bony sites are aff‘ected, including the olecranon,‘l the greater femoral trochanter,‘!’ the ischium,‘,’ the upper humerus,“’ and the lateral wall of the chest. It is notablle that in each location the tumors were attached to nearby periosteum. The typical eosinophilic refractile fibers that react with elastic tissue stains and that give the lesion its name have been shown in two immunohistochemical studies”,” to react with anti-elastin antibodies, at least one of which was of proven specificity. However, in the one immunoelectron microscopic study reported in the literature,’ not all the elastic-staining fibers reacted with the anti-e&in antibody. The antibody was observed to bind to small masses of granular amorphous elastic tissue, to the periphery of the larger elastic-staining fibers, and to disorganized collections of microfibrils at the periphery of the fibers. However, the tannic acid-positive central areas of larger elastic fibers
(Kumaratilake
et al)
did not react with the antibody, noI. did the bottle brushlike elastic structures with the radial-staining pattern. Fukuda et al suggested that their antibody distribution was restricted by the pre-embedding labeling method used, and by poor antibody penetration into the amorphous components of the mature elastic fibers. on actount of their compactness.’ Using postembedding immunolabeling, we have shown conclusively that both anti-elastin antibodies localized specifically, not only to the periphery, but also to the central regions of all the amorphous components of each of the morphologic types of elastic fibers, including the bottle brush-like elastic fibers with radial masses of electron-dense amorphous elastic tissue. Reactivity with the monoclonal antibody IO the elastin hexapeptide provides independent verific,ation of the immunohistochemical identification of elastic tissue. Both antibodies have been shown to he nlonospecific for elastin’ ‘,“’ dnd, with each, adsorption with purified elastin removed all capacity for binding to elastic tissue in sections. The absence of background >Ntainiqg shows that there was no nonspecific staining. II is concluded that in elastofibromas, immnrloreactive t.lastin is distributed throughout the amorphous c.omponents of each of the abnormal elastic-staining fibers, regardless of their different ultrastructural appearances. It is also present in relation to ~nany of the microfibrillar components in these tumors. The microtibrils in elastofihromas have received relatively little attention in published reports. Rlost investigators appear to have assumed them to be typical elastin-associated microfibrils; indred, tile published niicroCvaphs give no reason to suspect that microfibrilla1 morphology is abnormal. A number of researchers’.‘.” have observed that the elastic fibers contain a high ratio of microfibrils to amorphous elastin. mos1.1~ in a mantle around I he periphery of the fiber. Akhtar and Millex appear to he inferring that microfibrils were increased in number relative to amorphous elastic tissue when the) suggested there was maturation arrest of elastic fibrillogenesis.” In our study, many of the abnormal elastic fibers were seen to have a peripheral coating of 1t’-nrrl diameter microfibrils. which were not recognizably different from those associated with normal elastic fibers and which reacted specifically with the microfibrillar anti-MA<;P antibody. The number of such microfibrils vanled from fiber to fiber and around the perimeter of a single fiber. Some fibers of each of the morphologic types had no peripheral microfibrils. Occasionally, microfibrils were included within the electron-dense, granular amorphous elastic tis~~___
--
FIGURE 6. Electron micrographs of elastic tissues in periosteum of adult rib. (A) Low-magnification view of the elastic tissuecontaining layer of the periosteum showing electron-dense elastic fibers (arrows) and fibroblastic stromal cells interspersed among collagen fibrils. (Uranyl acetate/lead citrate stain; magnification ~6,600.) (B) High-magnification view of periosteal elastic fiber showing the electron-dense staining of the peripheral region (arrow) of the fiber, masking the microfibrils visible in younger individuals. The section h’as been reacted with anWBA4 antibody and the immunogold is seen specifically labeling the amorphous component of the elastic: fiber. (Uranyl acetate/lead citrate stain; magnification ~69.000.) (C) High-magnification view of ‘an elastic fiber showing bismuth deposition in the distribution of glycoproteins in the peripheral and internal regions of the elastic fiber. Collagen fibrils are also seen to stain, but less intensely, with the glycoprotein stain. (Periodic acid-bismuth stain for glycoprotein; magnification i 77,000.) (D>l A tangled mass of microfibrils (arrowheads), reacting specifically with the anti-MAGP antibody, intermingled with collagen fibrils and lying adjacent to a typical periosteal elastic fiber. The appearances are very like those seen in the elastofibroma, as shown in Fig 4B and D. (Uranyl acetate/lead citrate stain; magnification X46.000.) 1027
HUMAN PATHOLOGY
Volume 22, No. IO (October
sue, and their presence was confirmed by sparse immunostaining of this amorphous elastic material by the antiMAGP antibody (Fig 4A). Overall, the relative number of microfibrils was greater than that seen in normal developing elastic tissues.“.?2 In some instances, collections of such microfibrils, not associated with amorphous elastic component, were seen scattered within the collagenous matrix. Many of these collections were shown to bind antielastin antibodies. These observations are interpreted as indicative of new elastic tissue formation, and this view is confirmed by both the in situ hybridization data, showing collections of cells rich in elastin mRNA, and by the morphologic evidence of actively synthesizing fibroblastic-type cells within the elastofibromas. The relative excess of ~crofib~~ls over amorphous material is compatible with the notion of maturation arrest or delayed maturation of the elastic fibers,!‘,‘” but does not establish either hypothesis. The several different morphologies of the elastic fibers observed in this study have all been reported previously. They have been interpreted as indicating disturbed elastic fibrillogenesis,” admixture of an electron-dense material with the elastin to affect elastic fiber formation,~ degeneration of elastin,” and even elastotic degeneration of collagen.” Our data provide evidence for disturbed elastic fibrillogenesis, as well as for the possible presence of admixed glycoproteins, but we found no evidence of elastic degeneration. The concept of elastotic degenerati~~rl of collagen is inco~~patible with modern biochemical and immunologic understanding of collagen and elastin composition. The electron-dense elastic fibers with the granular appearance were seen at high resolution to contain many hbrils similar in size (35 to 50 mn diameter) and appearance to the surrounding collagen fibrils. In many sections, collagen fibrils with a typical cross-banding pattern could be recognized deep within these fibers. Such fihrils were not seen within elastic fibers in normal developing elastic tissue, indicating that their presence here is not the result of chance oblique sections of irregular elastic fibers.” These findings suggest that the granular elastic material represents deposition of elastin, in a more electron-dense form, on a framework of collagen fibers. ~nfortuxlately, both the tumors had been fixed in formalin before being processed for EM, and this fixation is known to inhibit reactivity of structural collagens with anticollagen antibodies. Thus, we were unable to confirm iminunolo~cally that these 35 to 50nm fibrils were collagenous. We did not observe the “short flexible t,ubulofibrillar structures” that Govoni et al described in some fibers.” These were admixed with microfibrils and, on the basis of enzyme digestions, were claimed to be complexes of elastin and galactosaminoglycarl-containing proteoglycans. These t.ubulofibrillar structures may have been confused with the collagenous fibrils, but this seems unlikely as it was noted that the former were resistant to collagenase digestion. However, no comment was made as to the presence of collagen-like fibrils within the granular amorphous elastic fibers.” As both our tumors were fixed routinely, sections were not processed specifically for studies of proteoglycans. 1028
1991)
The occasional observation of cores of- normallooking amorphous elastic tissue within abnormal elastic fibers of a variety of sizes, morphologies, and staining patterns was interpreted as indicating that, in these fibers, disordered elastic c~)tllponents were being deposited on the surface of preformed, normal elastic fibers (Fig 3D) rather than representing a progression to normalappearing elastic tissue within abnormal elastic fibers, as proposed by Akhtar and Miller.” Fibers with such cores occurred relatively infrequently, much less frequently than they are represented in the published electron micrographs, and it seems likely that they are overrepresented in the literature because such fibers were indisputably elastic, whereas the other types of elastic fiber r~~[~rpholo~es were not clearly elastin-containing in the absence of immunoelectron microscopic identification. The increased electron density of much of the amorphous elastic tissue points to the likelihood that another negatively charged matrix ct~rnponent is involved in fibrillogenesis in these tumors. The PA-bis staining studies indicated the presence of glycoproteins within many of the abnormal elastic fibers; this could account, in part, for their increased electron-dense staining. Whether the presence of such glyc~~proteins, even in increased amounts, could affect fibrillogenesis is not known. The presence of additional electron-dense materials within these newly forming elastic fibers camiot be excluded. We note that newly formed elastic fibers in normal fetal tissues are more electron dense than mature elastic fibers (unpublished data). The nature of the cells responsible for the production of the abnormal matrix components of elastofibromas has been a subject of dispute: many investigators have claimed that they are typical fibroblasts,’ while others have claimed that they have the morphology of myofibroblasts.“x In this study, the cells had many of the characteristics of fibroblasts and none were typical I~~y~)fibroblasts.They did contain abundant intracellular filaments, but focal densities were not observed along their course. Many cells exhibited a “fibrous lamina” adjacent to the inner nuclear membrane, marginal chromatin distribution, and the obvious nuclear pores that others have reported, but basement men~brane-like structures were not demonstrated in relation to these cells in this study. Such cells have been noted to be similar in appearance to an actively synthesizing chondrocyte.!’ The demonstration that anti-type-II collagen antibody binds to collagen fibers in an elastofibro~~a”’ is consistent with such an origin. Abnormal collagen fibers, many of which demonstrated appearances typical of unravelling collagen fibrils, were seen in areas of both elastofibromas. Waisman and Smith illustrated collagen fibers with very similar appearances in some portions of their elastofibroma’ and Govoni et al commented on the presence of “infrequent.. larger star-shaped” collagen bundles.” Similar appearances of collagen fibers have been reported in several genetic diseases of the skin,“’ and they are also occasionally seen in sun-exposed skin.“” The significance of their presence is obscure, but the possibility of interference with collagen fibrillogenesis by the presence of some other component cannot be excluded.
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
Most investigators appear to have accepted that elastofihromas are derived from cells of the exrracellular matris, usually of the subscapular space.‘!’ Despite the apparent consensus on this point, examination of the litrrature reveals that in the vast majority of cases, the lesions were reported to be attached to the periosteum of‘ the ad_jacenr scapula. ribs, or other bones. These observations 1~1 us to examine the possibility that, rathe t hm “invading” the periosteum, elastofibromas actually arise from it. Normal periosteurn of the scapula, ribs, ancl long hones ~vas found tc: contain significant amounts of elastic tissue in small masses in its deeper regions, csvrn in elderly patients. Periosteal elastic tissue was found to have many features in l:xmln~on with those observed in elastofibromas. These include the electron-dense staining of both the amorphous elastic tissue and of the generous peripheral rind of’ microfibrils associated with them. the increased rvactit’ity with PA-his stain in hot h amorphous rlastic tissuch and the microfihrils, and the increased ninnhers of‘ rnic7-ofibrils immunoreactive with antiMAGP antibody seen in the developing periosleum (unpublished data). These microfibrils occurred in associat it m with arnorpliolls elastic tissue, in collections within the estracrllular matrix (including between collagen fibers), alrd in the apparent absence of amorphous elastic tissue or of immunoreactive elastin. Periosteal cells also exhibited many features in common with those of the &~stofibroma. Thev were elongated, plump fihroblastic c.ells with a large nucleus, typically with a marginal chromatin distribution. a fib&s nuclear larnina, nuclear pores, and collec~tions of ordered cytoplasmic filaments. Thus, it tin be concluded on morphologic grounds that periostcum mav well be the source of the cells of elastofibromas. Furthermore, these tumors have almost all occurred in regions that are subjected to repetitive mechanical stimulation. most commonly between the scapula and the ribs and in the infraolecranon region. ‘I‘hree cases have been reported in which the lesions were attach4 to the ischium; it is pertinent that one of these Iyatient.\ wx an amateur cyclist” while another had rec‘(xnt 1~ had surgical treatment to an adjacent ischial The frequent occurrence of elastofibroma fra0ure.’ among fiunilies in Okinawa, Japan’ suggests that a predisposition to these lesions may be inherited in some (‘astas. However, even in these families, few cases were obser& before the age of40 years, and the distribution 01‘the lesions correlated with the likelihood of repetitive nlilror trauma. These observations lead us to propose that clastofibromas arise from the periosteum. in susceptible individuals. in response to repeated physical irritation. The prrdominant occurrence in females is an interesting a1spPc.t to be examined further. Elastofibro~na thus appears to be a good model in wl1it.h it ma? be possible to analyze further the processes The recent availability of a of elastic hbrillogenesis.
(Kumarotiloke
et al)
antibodies
to elastic
used for immunoelcctron broma.
The
exhibited
elastic-staining
a variety
amorphous fibers.
and microfibrillar
for ionic
phologies. elastin typical
reactike
occurring
of the elastic
cells, and the structure It is plroposed
as a result
periosteal-derived (
I991
by W.B.
cells. Saunders
Elastof~I)I-0rri;i
associated
fibers,
elastin
fibers
in common
and were spe-
biosynthesis
HL~M
was
and staining
the morphology
of the
in these lesions
were
with those of normal
peri-
arise from the periosteum
and that the different
disturbances
with
that were
to microfibril-asso-
The appearances
tissue elements,
irritation
represent
were
mor-
with anti-
studies with a cRNA probe
that active
that elastofibromas
of chronic
morphologies
had variable
microfibrils
elastic
of the collagen
shown to have many features osteum.
fibers
of the
in elastic
substructural
antibodies
within the lesions.
seen
specifically
diameter
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confirmed
patchily
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several
been
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ratios
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In situ hybridization
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Most of the elastic
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Despite
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The amorphous
affinity
tissue
microscopy
of elastic
elastic
22:1017-1029.
P.THOI.
fiber
fibrillogenesis
by
Copyright
Compaq
is ;I
raw
tunior
coirinionl~itt the lower end of the
that subscapular
occurs
Inost space
of
is usually attached to the underlying ribs arltl sometimes to the adjacent scapula ’ The olccranon pt~ocess is another commonly aff&red site.’ These t11oderatel~ cellular, notiencapsulated masses consist oL:t ~x~llaget~ous stroma containitlg charrefractile fibers with the actcaristic solid. eosinophilic. staining properties of elastic tissue. The elastic-staining fibers exhibit se\,eral fort11s, iticluditig unifortiily thickened. bt-anclied, or rml~ranched fibers; long, irregularly thic kcncd (bcxded or segmented) fibers; and globular prolilrs. Significatit quantities of the protein elastin ha\e been isolated frotii these tun1ors,1 and he elasticstainitig fibet- have Bern sltowt1 to react histochcmically with polvclonal ;inti-(hut1iat1 elastin) antibodies.‘.” peuon\
ove1
50
!;wrs
of
age.
It
_-___
1017
A variety 01 elastic fiber morphologit~s ha\,e beet1 observed in elastofihromas in the electron microscope (EM). Some fibers have a central core of typical atnorphous elastin. but most of the elastic-staining fibers have abnormal ttlorpholoCq and staining properties, c.onsisting either of electron-dense, granular, or fihrillat? masses or less electron-dense amorphous rlastic tissue, sin-rounded hy a variable layer of irregularly arranged niicrofibrils.‘~i Many investigators have reported an increase in the relative amounts of microfibrils and some have seett tr1icrotrtl~ular structures withit the amorphous cottiponcnt .” It1 the single inimunoelectrori microscopic study repot-ted. the reactivity of rabbit anti-,(hut11an aorta cu-elastin) antibody with two tumors was examined using a pre-embedding labeling technique.’ The antibody was sl1owt1 to localize specifically in the outer granular zot1e of the elastic fibers. hut penetration to their interiors was t1ot achieved. Microfibril-rich peripheral /ones were also labeled. the cells of clastofilbromas were Histologically, originally considered to be fihroblasts. and the EM appearances wert’ consistent with that view.’ flowever. the presence of tt1~ofilatiients led some researc,hers to suggest that the c,ells were tt1yofil,rohlasti~. although they lacked a basement men~l~rane.‘i~S Several investigators have noted that these cells have a thickened fihrous lamit1a adjacent to the prominent inner nuclear metlthratle surt-ounding tl1e peripherally dispersed nuclear chromatin.“.” Othet-s, noting the presence of nuclear pores, an asremarked on tt1e resemblance to chottdrocvtes,” sociation reinforced by finding that the stromal collagen was reactive with anti-(type II c.ollagen) attt ibodv.” We have applied modern techniques, inc-luding postembedding inltnunoelectron microscopic studies, to two typical elastofihromas obtained fron1 the subscapular spaces of an elderly woman. The components of the atypical elastic fibers have heen defined immunologically using affinity-purified monospecific., polvclonal atltibodtes against tropoelastin (TE) and the monoclonal antibody (lL44) raised against a hexapeptide unique lo the elastin molecule. Atltibodies specific, for the elastic. tissue tt1ic t-ofibt-il-associated glvcoprotein (3IAGI’) were used to characterize the micro~hrillar compot1ents. The cellular biosynthesis of elastiri was studiecl by in situ hybridizatiotl with a cKNA prohe specific lin- human elastin. Exattlinatic m of periosteum from the rscxpula. rihs, and some long hones has shown that the elastic tissue of this strurrure is relatively electron dense and is surrounded by a thickened glycoproteitt-cotit aininp zone.
HUMAN PATHOLOGY
Volume 22, No. 10 (October
rich in microfibrils that are immunoreactive with the anti-MAGI’ antibodies. The cells of the periosteum were shown to have many features in common with those of the elastofibromas. In view of these findings and taking account of the regular association of elastofibromas with periosteum, it is postulated that they have their origin from the periosteum, in response to chronic physical irritation, and that they represent a complex disturbance of elastic fibrillogenesis. MATERIALS
AND METHODS
subscapular
pH was adjusted to 7.2. Tissues fixed in 1.25% glutaraldehydecontaining fixative were postfixed in 1% osmium tetroxide for 1 hour, dehydrated in a graded series of ethanols, and embedded in Spurr’s resin (polymerized at 70°C for 72 hours). Tissues fixed in 0.25% glutaraldehyde-containing fixative were treated with 1% sodium borohydride before dehydration and were embedded in LR White resin (polymerized at 50°C for 72 hours). Ultrathin sections (silver-gold), cut on Reichert Ultracut E and Om U2 ultramicrotomes using diamond knives. were mounted on naked (Spurr’s sections) or Celloidon-coated (LR White sections) nickel grids.
Antibodies
Tissues Two elastofihromas
1991)
were
obtained from the right and left spaces of a female patient at two surgical proce-
dures 16 months apart. The age of the patient at the time of removal of the first tumor (tumor I) was 78 years. The clinical
history was not remarkable. The tumors had the typical lobular grey fibrofatty appearance of elastofibromas. Parts of each tumor were processed for light microscopy, EM, and in situ hybridization studies. Normal periosteum was obtained at autopsy from the scapula, ribs, and pelvis of two female patients, aged 75 and 82 years, who died acutely from nonosseous diseases. Periosteunl was also obtained at surgery 2 and 6 days after fracture from three patients with fractures of long bones requiring open reduction. The ages of these three patients range from 25 to 78 years. Samples were taken immediately adjacent to the fracture site and 3 to 4 cm proximal to it.
Light Microscopy Each tissue was fixed in 10% buffered neutral formaldehyde and embedded in paraffin wax. Five-micrometer sections were stained with hematoxylin-eosin (H&E), periodic acidSchiff (PAS), and Miller’s elastic tissue stains.
In Situ Hybridization The procedure for in situ hybridization (including cDNA probe preparation, hybridization conditions. stringency washes, and autoradiography) was as detailed previously.’ ’ Briefly, 5~111 parafIin sections were mounted on slides pretreated with Elmer’s glue, deparaffinized, and hydrated in decreasing ethanol solutions before processing. The probe was derived from a 1.1 -Kb fragment, containing the 5’ end of the human elastin cDNA.‘” subcloned into pGEM 2 vector. The “‘S-labeled cRNA probe was then generated by in vitro transcription with T7 polymerase, and sized by mild alkaline digestion to a mass average of 150 bases. The probe-specific activity was approximately 10’ dpm/Fg. It was applied at a concentration of 10” dpm/lO pL/section (2 mm X 2 mm). After autoradiographic development for 4 to 8 days, sections were countersrained with H&E and examined. Sense RNA was labeled with “3 uridine-5’-triphosphate by tl-anscription of the DNA template by SP6 RNA polymerase, and step sections were hybridized with this probe as negative controls. Nonspecifically bound probe was removed from the hybridized sections by digestion with RNAse prior to autoradiography.”
Electron Microscopy Approximately 1 -mm cubes of each tissue were fixed in chilled (4°C) I .25% glutaraldehyde plus 4Yo paraformaldehyde for l? to 18 hours or in 0.25% glutaraldehyde plus 4% paraformaldehyde for 4 hours. The fixatives were made in 6 mmol/ L phosphate-buffered saline (PBS) containing 4% sucrose and 5% polyvinylpyrrolidone (molecular weight, 10,000) and the
1018
Affinity-purified polyclonal anti-TE antibody was prepared in rabbits against porcine aortic TE.‘” The monoclonal antibody, BA4, had been prepared against a synthesized form of the elastin peptide, Val-Gly-Val-Ala-Pro-Gly.‘” Affinity-purified antibody to bovine MAGP, prepared in rabbits, was used for the identification of microfibrillar components of elastic tissue.” Affinity-purified types III and IV collagen antibodies were raised against bovine placental type III and human placental type IV collagen in rabbits. Details of the purification and specificity of anti-TE and antiMAGP antibodies have been published,‘“,“‘,‘h The specificity of the types III and IV collagen antibodies was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.
lmmunolabeling Immunolabeling was performed as described by White et al.‘” Prior to the ovalbumin/PBS (ova/PBS) step, all the sections were incubated with 0.02 mol/L glycine in PBS (for 10 minutes each on five drops). Spurr’s sections were used for studying the distribution of anti-TE, anti-MAGP, and BA4 antibodies. With the anti-MAGP antibody, prior to the glycine/ PBS step, the sections were treated with 50 mmol/L dithlothreitol in 6 mol/L guanidine hydrochloride (GdnHCl) for 5 minutes, after which the excess was drained off on a filter paper. The sections were then treated with I10 mmol/L io-
doacetamide in 6 mol/L GdnHCl for 5 minutes and washed vigorously in Tris-buffered saline, pH 8.0.‘7 LR White sections were used for the immunoelectron microscopic studies of anticollagen antibody disrribution. They were etched with a saturated solution of sodium metaperiodate for 10 minutes and washed vigorously in water prior to the glycine/PBS step. Sections reacted with BA4 monoclonal antibody were incubated with affinity-purified rabbit anti-mouse immunoglobulin G (IgG) for 1 hour at room temperature and washed for 5 minutes each on six drops of ova/PBS prior to incubation with protein A-dextran-gold (12-nm diameter gold particles). Ovalbumin/PBS containing Tween 20 (final concentration, 0.05%) was used for antibody dilution and for all the washing steps with the BA4 monoclonal antibody. Optimal antibody dilutions for maximal labeling with minimal background staining were determined for each antibody separately. Controls were performed on adjacent sections using ova/PBS, affinity-purified nonimmune rabbit IgG in place of the antibody, and antibody adsorbed with excess of the respective antigens. With the BA4 monoclonal antibody, ova/PBS was used in place of the primary antibody as an additional control. Bovine fetal nuchal ligament served as a positive control.
Transmission
Electron Microscopy
Spurr’s sections were stained with alcoholic uranyl acetate and lead citrate for 20 minutes each. LR White sections were
stained with aqueous uranyl acetate and lead citrate for 1 minute and 15 seconds, respectively. Sections were stained for glycoproteins using the periodic acid-bismuth (PA-bis)
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
method. ” The specificity of this reaction for glycoproteins was established hy specific blockin? with sodiunl borohydride. All sections were carbon coated prwr to examination in a Philips EM 300 at 80 kV.
RESULTS Light Microscopy The two elastofibromas were similar in appearance, being composed mainly of fibrous connective tissue, within which were elongated, but rather plump fibroblastic-type cells and inactive fibrocytes. The connective tissue consisted of dense collections of collagen fibers. among which were scattered solid eosinophilic refractile rods of varying thicknesses, many of which were seen in cross-section as solid, roughly circular masses of varying diameter (Fig 1A). These refractile components reacted with elastic-tissue and PAS stains, exhibited several different forms, and occurred in collections of like fibers. Some were solid and had no clear evidence of internal architecture. Other solid-looking fibers had a paler-staining peripheral portion surrounding a more darkly staining central zone, so that in longitudinal section they resembled flattened disks strung along a thread, or a bottle brush within a test tube. In crosssection, the elastic-staining material was arranged radially in these fibers. A thin but more dense, rod-like, elastic-staining core was visible within some of the thicker beaded fibers (Fig 1 B) and occasionally in thinner elastic fibers. Core-containing fibers tended to occur in collections and were more prominent in tumor I than in tumor Il. A third fiber- form occurred as fused irregular masses of electron-dense elastic material in fibers that had serrated borders, in both longitudinal and transverse sections (Fig 1B). The perlosteum consisted mainly of a dense collagenous stroma, within which small masses of elastic-staining, PAS-positive material were visible in a narrow zone near the bone surface, from which they were separated b!, a layer of cells. Adult rib periosteurn was relatively acellular, but that from healing fracture sites contained a proliferation of’ cells resembling fibroblasts (data not shown). In Situ Hybridization The elastofibromas contained collections of fibroblastic-appearing cells, which had high grain counts for elastin mRNX, well above background (Fig 1C). These cells were ofi:en seen in close apposition to elastic elements in the tumor. Background staining was low, as confirmed by the step section exposed to the sense mRNA probe (Fig 11)). These findings point to the presence within the reactive cells of relatively high concentrations of elastin mRNA, indicating that they were actively engaged in etastinogenesis. In elastin-poor regions of the tumors, a lesser proportion of cells reacted with the cRNA probe. indicating that some populations of cells were not engaged in elastin biosynthesis. Electron Mic:roscopy The ultrastructural morphology of the two tumors was similar. Borh consisted mainly of collections of
(Kumarotilake
et al)
tightly packed bundles of collagen fibers which, at low magnification, exhibited normal organization. and both tumors contained collections of what appeared to be abnormal elastic fibers of variable size, shape, and staining. Fibroblastic cells were commonly observed with many thin irregular processes, which extended between the collagen bundles and were often closely apposed to the abnormal elastic fibers (Fig 2A). None of the elastic fibers had ultrastructural appearances typical of normal elastic tissue. They appeared as roughly circular or irregular masses of am amorphous material, which exhibited a variety of morphologies and staining patterns. These included electron-dense granular fibers, some showing a radial pattern in cross section (Fig 2B); more solid-looking fibers containing amorphous material of lesser electron density, often with a narrow electron-dense rim (Fig 2D): and uniformly amorphous, relatively electron-lucent solid fibers with occasional holes, some containing what appeared to be a cell process (Fig 3C). The beaded fibers in light microscopy commonly exhibited the electron-dense granular form in the EM, while the solid fibers contained both electron-dense granular material and more electron-lucent homogeneous material. Fibers with each staining pattern were seen in cross-section. The granular electron-dense material was seen to be arranged predominantly in the radiating spokes pattern and generally had an irregular, serrated edge (Fig 2B and C). ‘There was a tendency for fibers of a particular staining pattern to occur in collections within the tumors. and each type of fiber was seen in a full range of diameters, from the smallest to the largest. Hybrid forms exhibiting characteristics of more than one type of fiber were virtually never seen. The different fiber types are shown, at higher magnifications, in Fig 3. The amorphous portion of each type of fiber was specifically reactive with both the antiTE and the BA4 antibodies throughout (Fig 3). Both antibodies also localized specifically to the amorphous component of elastic fibers in bovine fetal lmchal ligament (data not shown). Figure 3A and B shows examples of the granular- electron-dense fibers and the less electron-dense homogeneous fibers, respective&, each with an investment of typical elastin-associated microfibrils. In both types of fiber, the granularity was more obvious in the central region of the fiber and appeared to be related to the presence there of collections of collagenlike fibrils, similar in diameter (35 to 50 mn) IU the collagen fibrils closely apposed to the surface of the elastic fiber (Fig YB). Occasional collagen fibrils. sectioned longitudinally so that typical periodic striation was visible, were seen in such fibers (Fig 3B). Figure 3C shows a less electron-dense type of elastic fiber in which the amorphous component, which reacted with both of the anti-elastin antibodies, had a fibrillary texture. In some immunoreactive elastic tissue, the amorphous component was homogeneous and had little affinitv for the electron-dense stain. These types of fiber also &mtained collagen fibrils. recognizable by their pattern of striations when sectioned longitudinally. Figure 3D shows another abnormal elastic fiber morphology in which a central core of normal-looking amorphous elastin is enclosed within abnormal electron-dense elastin. Both the
1019
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FIGURE 1. (A and 6) Photomicrographs showing predominantly collagenous stroma containing variable amounts of abnormal elastic-staining fibers. These elastic fibers are seen as thick solid curved rods with circular cross-sections of varying diameters. Some have a beaded look (short arrow) and some have a darker staining core (long arrow). In cross-section, the darker-staining material occupies the central portion of the fiber and is arranged radially. The stromol cells are fibroblastic. (A: H&E stain; magnification x500. 8. Miller’s elastic tissue stain; magnification x.500.) (C and D) Photomicrographs of in situ hybridization of elastin mRNA in elastofibroma. Many stromal ftbroblastic cells exhibit positive signal. The signal persisted after RNase treatment of the hybridized section, Confirming that it was specific for elastin mRNA. (C: H&E stain, labeling with cRNA probe; magnification x425. D: H&E stain, control with mRNA probe; magnification X425.)
1020
FIGURE 2. Electron mlcrographs showing elastic tissue morphology within the predominantly collagenous stroma of an elastofibroma. Uranyl acetate/lead citrate stain. (A) Moderately electron-dense solid elastic fibers (e) are cross-sectloned. .A fibroblastic stromal cell IS seen with many elongated processes surrounding an elastic fiber and bundles of collagen fibrils (c). (Magnification * 5,500.) (B) Transverse section of radially arrayed electron-dense, granular elastic fibers seen among collagen fiber bundles. An occasional cell and an electron-lucent elastic fiber, sectioned longitudinally (arrow), are present. (Magnification ~5.500.) (CJ Longitudinal sectlon of an electrondense granular elastic fiber with an irregular edge, seen among the dense collagenous stroma. (Magnification I 5,500.) [D) Longitudinal section of more solid, less electron-dense elastic fiber with an electron-dense rim. (Magnification * 5,500.)
1021
FIGURE 3. Electron micrographs showing immunolocalization of anti-elastin antibodies in elastic fibers of a variety of morphologic forms. Uranyl acetate/lead citrate stain; A and B labeled with anti-tropoelastin antibody, and C and D labeled with BA4 antibody. (A) Electron-dense granular fiber with a peripheral rim of typical microfibrils (arrowhead). (Magnification X37500.) (6) Less electrondense elastic fiber with a thin rind of peripheral microftbrils (arrowhead). Fibrils 35 to 50 nm in diameter and collagen ftbrils of similar diameter and with faint cross-striations (arrows) are seen within the fiber. (Magnification X38.750.) (C) Cross-section of a less electron-dense elastic fiber in which the amorphous component has a ftbrillary texture. The fiber lacks peripheral microltbrils. A membrane-bound process and some holes are seen within the tiber. (Magnitication X39.500.) (D) Oblique section of a small electron-dense elastic fiber containing a core of apparently normal-looking, electron-lucent amorphous elastic tissue (a). A ftbroblastic cell (arrow) is closely applied to the surface of the fiber. (Magnification ~6,600.) 1-a_
FIGURE 4. E:lectron micrographs showing immunostaining of 12-nm diameter microfibrils in a range of relationships with elastin. Uranyl acetate/lead citrate stain; A and B labeled with anti-MAGP antibody, and C and D labeled with BA4 antibody. (A) Section of elastic fiber with a relatively electron-lucent amorphous component. The peripheral rim of microfibrils (arrowhead) shows good avidity for the elastin-associated microftbrillar antibody. Less intense specific antibody localization is seen over the amorphous elastic material. (Magnifkation X42.000.) (B) A collection of typical elastin-associated microfibrils seen within the extracellular matrix between two ftbroblastic cell processes (arrows). The collection of microfibrils (arrowhead) is adjacent to an elastic fiber with few microfibrils and is intermingled with collagen ftbrils. (Magnification x38.000.) (C) High-magnification view of an microftbrillar collection (arrowhead), immunoreactive with anti-BA4 antibody, among collagen ftbrils. (Magnification \t85,500.) (D) A tangle of nonimmunor’eactive microftbrils (arrowhead) lying within the extracellular matrix, adjacent to a cell process (arrow), an electrondense immunoreactivc elastic fiber, and intermingled with collagen ftbrils. (Magnification x38.5000.)
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HUMAN PATHOLOGY
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1024
1991)
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IN ELASTOFIBROMA
c.elitral con: and the abnormal amorphous elastic material were reac.tive with each of the anti-elastin antibodies. Similar typical amorphous cores with normal immunoreac-tivity for the two elastin antibodies were seen within occasional electron-lucent and granular, electron-dense abnormal elastic fibers. A majority of the elastic fibers, of both staining types, had a rind of microfibrils, similar in appearance to the 12-mn diameter microfibrils associated with normal elastic fibers (Fig 3A and B). The thickness of the rind of microfibrils varied from fiber to fiber, as well as around their perimeters. In a number of fibers of each staining type, small collections of similar microfibrils lvere seen within the amorphous elastic material. All the microfibrils seen in association with the different types of abnormal elastic fibers were specifically reactive with the anti-MAGP antibody (Fig 4A). A few immunoreactive elastic fibers of each staining pattern were completely free of peripheral microfibrils (Figs 2B, 2D, and 4B). In control nuchal ligament sections, anti-MAGP antibody di.stribution was restricted specifically to microfibrils. The surrounding matrix and amorphous elastic material were negative (data not shown). In the elastofibromas, anti-MAGP antibody bound not only to the peripheral elastic fiber microfibrils, but also to adjacent although at a lesser labeling amorphous component, density (Fig 4A). The latter was significantly greater than that of the nearby matrix. Collections of 12-nm microfibrils, with or without foci of amorphous elastin, were also seen between collagen bundles and in the vicinity of stromal cells (Figs 3B to D). Such microfibrillar collections were seen more frequently in tumor I than in tumor II. They reacted specifically with the anti-MAGP antibody (Fig 4B) and, not infrequentl>r, also reacted with the anti-elastin antibodies. even when no amorphous elastic tissue was apparent in that region (Fig 4C). Large tangled masses of typical elastin-associated microfibrils, which did not exhibit elastin antibody binding, were also seen within the matrix (Fig 1D). Sections of elastofibroma. stained for glycoproteins with PA-bis stain, showed specific staining of elastic fiherb of all i.hrer morphologies. The staining was not apparently more intense in the electron-dense type of amorphous elastic tissue. In all types of elastic fiber the stain localized not only to the periphery of the fibers, but also deep within the fiber, as seen in Fig 5A. Collagen fibrils and microfibril collections separate from the elastic fibers were also stained. The majority of the collagen fibrils in the stroma were normal in appearance, with uniform fibril diameters.
(Kumaratilake
et al)
There were, however, a number of‘ focal c,olleclions of thickened collagen fibrils. which appeared as typical collagen “flowers” in cross-section, suggesting that they were in loose twists (Fig 5B). Attempts to stain the stromal collagen fibers with antibodies to structural collagens were unsuccessful, even after tissue etching, protease digestion, and extraction with acetic acid, presumably because the tissues had been subjected to standard histologic fixation, which inhibits collagen antibody binding. The stromal cells were elongated and had many relatively thin, cytoplasmic processes (Figs 2A and 5C). These extended between collagen bundles and were sometimes seen in clear spaces within elasl:ic fibers. The cytoplasm of these cells had abundant rough endoplasmic reticulum, dilated cisternae. and vesicles of varying diameter. Many unbanded filaments, similar in size to myofilaments, were apparent within the cytoplasm. These were loosely organized and did not exhibit electron-dense zones; these cells also did not have a basement membrane structure. Their nuclei were prominent and often indented, and the chromatin material was arranged around the periphery of the nuclear membrane. There was a heavy condensation of electrondense material adjacent to the inner nuclear membrane, which was interrupted at intervals by pores (Fig 5C). Occasional stromal cells were surrouncled by a thickened, loosely organized zone of electron-dense staining, superficially resembling a basement membrane (data not shown). However. its organization differed from normal basement membrane, and anti-type TV collagen antibody did not bind to these structures, although it Iocalized specifically, in the section, to the basement mt mbrane of the blood vessels. With all antibodies, backgn)und staining was minimal and no significant antibody binding was observed in negative control tissues.
Periosteum The periosteum consisted predominantly of bundles of tightly packed collagen fibers, among which were scattered some densely-staining elastic fibers and a relatively low number of fibroblastic cells (Fig GA). These cells contained loose collections of 1 O-rim diameter intermediate filaments, which did not show electron-dense zones, and did not have a basement membrane. Their nuclei exhibited an electron-dense fibrous lamina along the inner nuclear membrane (Fig 5D), similar to that seen in the cells of the elastofibroma. The periosteal cells had a number of elongated cell processes. The amorphous component of the elastic fibers was more elec,tron-dense than that of normal (elastic fibers,
FIGURE 5. Electron micrographs of elastofibroma and periosteum. (A) Periodic acid-bismuth stain for glycoproteins. An elastic fiber cut in cross-section with adjacent collagen fibrils. The stain is distributed specifically around the periphery of the elastic fiber, but also penetrates into deeper parts of the amorphous elastic material. (Magnification X34,000.)(B) Cross-banded ‘collagen fibrils, of varying diameters, irregularly twisted along their long axis. In cross-section, the fibrils exhibit considerable variation in diameter, and there are many relatively fine fibrils. The twisted fibrils are large and have a star- or flower-like appearance in cross-section. A cell process (arrow) is seen among the collagen fibrils. (Uranyl acetate/lead citrate stain; magnification k 37,000:) (C) A typical fibroblastic cell in an elastofibroma showing the many finger-like processes (black arrow), loosely arranged intracellular filaments (short white arrow), peripheral nuclear heterochromatin and electron-dense fibrous nuclear lamina (white arrowhead). and a nuclear pore (long white arrow). (Uranyl acetate/lead citrate stain; magnification ;\37,000.) (D) Fibroblastic cell in the periosteum of adult rib showing a more orderly array of intracellular filaments (small white arrow), peripheral nuclear heterochromatin and electron-dense fibrous nuclear lamina (white arrowhead), and a nuclear pore (long white arrow). (Uranyl acetate/lead citrate stain; magnification * 50.000.)
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1991)
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but was reactive with the anti-elastin antibodies (Fig 6B). All the elastic fibers were surrounded by a thick electrondense coat, and similar material was seen as inclusions within the amorphous component (Fig 6A and B). In mature periosteum, this electron-dense material obscured both the microfibrils and most of the reactivity with anti-MAGP antibody (data not shown). In healing fractures and in fetal bovine periosteum, relatively large numbers of microfibrils were seen around the periphery of the elastic fibers (data not shown), so that the ratio of microfibrils to amorphous component was high compared with that seen in normal human skin or aortic elastic tissue. Both the electron-dense peripheral zones and the elesctron-dense inclusions within the amorphous elastic tissue reacted specifically with the PA-his stain, indicating the presence of glycoprotein in this distribution (Fig 6C). Large, densely-packed collections of 1?-nm microfibrils were observed in the periosteal extracellular matrix, some clearly separate from amorphous elastic tissue and others containing scattered small masses of amorphous elastic tissue. These microfibrils also reacted specifically with anti-MAGP antibody (Fig 61)). Sometimes they also reacted positively with anti-TE antibodies, despite the lack of recognizable amorphous elastic tissue in those regions (data not shown). DlSCUSSlOlN Almost 30 years have elapsed since the first reported cases of elastofibroma. Although more than 270 cases have now been reported, neither the nature of the tumors nor their origin has been established. The subscapular region is by far the most common location, but other bony sites are aff‘ected, including the olecranon,‘l the greater femoral trochanter,‘!’ the ischium,‘,’ the upper humerus,“’ and the lateral wall of the chest. It is notablle that in each location the tumors were attached to nearby periosteum. The typical eosinophilic refractile fibers that react with elastic tissue stains and that give the lesion its name have been shown in two immunohistochemical studies”,” to react with anti-elastin antibodies, at least one of which was of proven specificity. However, in the one immunoelectron microscopic study reported in the literature,’ not all the elastic-staining fibers reacted with the anti-e&in antibody. The antibody was observed to bind to small masses of granular amorphous elastic tissue, to the periphery of the larger elastic-staining fibers, and to disorganized collections of microfibrils at the periphery of the fibers. However, the tannic acid-positive central areas of larger elastic fibers
(Kumaratilake
et al)
did not react with the antibody, noI. did the bottle brushlike elastic structures with the radial-staining pattern. Fukuda et al suggested that their antibody distribution was restricted by the pre-embedding labeling method used, and by poor antibody penetration into the amorphous components of the mature elastic fibers. on actount of their compactness.’ Using postembedding immunolabeling, we have shown conclusively that both anti-elastin antibodies localized specifically, not only to the periphery, but also to the central regions of all the amorphous components of each of the morphologic types of elastic fibers, including the bottle brush-like elastic fibers with radial masses of electron-dense amorphous elastic tissue. Reactivity with the monoclonal antibody IO the elastin hexapeptide provides independent verific,ation of the immunohistochemical identification of elastic tissue. Both antibodies have been shown to he nlonospecific for elastin’ ‘,“’ dnd, with each, adsorption with purified elastin removed all capacity for binding to elastic tissue in sections. The absence of background >Ntainiqg shows that there was no nonspecific staining. II is concluded that in elastofibromas, immnrloreactive t.lastin is distributed throughout the amorphous c.omponents of each of the abnormal elastic-staining fibers, regardless of their different ultrastructural appearances. It is also present in relation to ~nany of the microfibrillar components in these tumors. The microtibrils in elastofihromas have received relatively little attention in published reports. Rlost investigators appear to have assumed them to be typical elastin-associated microfibrils; indred, tile published niicroCvaphs give no reason to suspect that microfibrilla1 morphology is abnormal. A number of researchers’.‘.” have observed that the elastic fibers contain a high ratio of microfibrils to amorphous elastin. mos1.1~ in a mantle around I he periphery of the fiber. Akhtar and Millex appear to he inferring that microfibrils were increased in number relative to amorphous elastic tissue when the) suggested there was maturation arrest of elastic fibrillogenesis.” In our study, many of the abnormal elastic fibers were seen to have a peripheral coating of 1t’-nrrl diameter microfibrils. which were not recognizably different from those associated with normal elastic fibers and which reacted specifically with the microfibrillar anti-MA<;P antibody. The number of such microfibrils vanled from fiber to fiber and around the perimeter of a single fiber. Some fibers of each of the morphologic types had no peripheral microfibrils. Occasionally, microfibrils were included within the electron-dense, granular amorphous elastic tis~~___
--
FIGURE 6. Electron micrographs of elastic tissues in periosteum of adult rib. (A) Low-magnification view of the elastic tissuecontaining layer of the periosteum showing electron-dense elastic fibers (arrows) and fibroblastic stromal cells interspersed among collagen fibrils. (Uranyl acetate/lead citrate stain; magnification ~6,600.) (B) High-magnification view of periosteal elastic fiber showing the electron-dense staining of the peripheral region (arrow) of the fiber, masking the microfibrils visible in younger individuals. The section h’as been reacted with anWBA4 antibody and the immunogold is seen specifically labeling the amorphous component of the elastic: fiber. (Uranyl acetate/lead citrate stain; magnification ~69.000.) (C) High-magnification view of ‘an elastic fiber showing bismuth deposition in the distribution of glycoproteins in the peripheral and internal regions of the elastic fiber. Collagen fibrils are also seen to stain, but less intensely, with the glycoprotein stain. (Periodic acid-bismuth stain for glycoprotein; magnification i 77,000.) (D>l A tangled mass of microfibrils (arrowheads), reacting specifically with the anti-MAGP antibody, intermingled with collagen fibrils and lying adjacent to a typical periosteal elastic fiber. The appearances are very like those seen in the elastofibroma, as shown in Fig 4B and D. (Uranyl acetate/lead citrate stain; magnification X46.000.) 1027
HUMAN PATHOLOGY
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sue, and their presence was confirmed by sparse immunostaining of this amorphous elastic material by the antiMAGP antibody (Fig 4A). Overall, the relative number of microfibrils was greater than that seen in normal developing elastic tissues.“.?2 In some instances, collections of such microfibrils, not associated with amorphous elastic component, were seen scattered within the collagenous matrix. Many of these collections were shown to bind antielastin antibodies. These observations are interpreted as indicative of new elastic tissue formation, and this view is confirmed by both the in situ hybridization data, showing collections of cells rich in elastin mRNA, and by the morphologic evidence of actively synthesizing fibroblastic-type cells within the elastofibromas. The relative excess of ~crofib~~ls over amorphous material is compatible with the notion of maturation arrest or delayed maturation of the elastic fibers,!‘,‘” but does not establish either hypothesis. The several different morphologies of the elastic fibers observed in this study have all been reported previously. They have been interpreted as indicating disturbed elastic fibrillogenesis,” admixture of an electron-dense material with the elastin to affect elastic fiber formation,~ degeneration of elastin,” and even elastotic degeneration of collagen.” Our data provide evidence for disturbed elastic fibrillogenesis, as well as for the possible presence of admixed glycoproteins, but we found no evidence of elastic degeneration. The concept of elastotic degenerati~~rl of collagen is inco~~patible with modern biochemical and immunologic understanding of collagen and elastin composition. The electron-dense elastic fibers with the granular appearance were seen at high resolution to contain many hbrils similar in size (35 to 50 mn diameter) and appearance to the surrounding collagen fibrils. In many sections, collagen fibrils with a typical cross-banding pattern could be recognized deep within these fibers. Such fihrils were not seen within elastic fibers in normal developing elastic tissue, indicating that their presence here is not the result of chance oblique sections of irregular elastic fibers.” These findings suggest that the granular elastic material represents deposition of elastin, in a more electron-dense form, on a framework of collagen fibers. ~nfortuxlately, both the tumors had been fixed in formalin before being processed for EM, and this fixation is known to inhibit reactivity of structural collagens with anticollagen antibodies. Thus, we were unable to confirm iminunolo~cally that these 35 to 50nm fibrils were collagenous. We did not observe the “short flexible t,ubulofibrillar structures” that Govoni et al described in some fibers.” These were admixed with microfibrils and, on the basis of enzyme digestions, were claimed to be complexes of elastin and galactosaminoglycarl-containing proteoglycans. These t.ubulofibrillar structures may have been confused with the collagenous fibrils, but this seems unlikely as it was noted that the former were resistant to collagenase digestion. However, no comment was made as to the presence of collagen-like fibrils within the granular amorphous elastic fibers.” As both our tumors were fixed routinely, sections were not processed specifically for studies of proteoglycans. 1028
1991)
The occasional observation of cores of- normallooking amorphous elastic tissue within abnormal elastic fibers of a variety of sizes, morphologies, and staining patterns was interpreted as indicating that, in these fibers, disordered elastic c~)tllponents were being deposited on the surface of preformed, normal elastic fibers (Fig 3D) rather than representing a progression to normalappearing elastic tissue within abnormal elastic fibers, as proposed by Akhtar and Miller.” Fibers with such cores occurred relatively infrequently, much less frequently than they are represented in the published electron micrographs, and it seems likely that they are overrepresented in the literature because such fibers were indisputably elastic, whereas the other types of elastic fiber r~~[~rpholo~es were not clearly elastin-containing in the absence of immunoelectron microscopic identification. The increased electron density of much of the amorphous elastic tissue points to the likelihood that another negatively charged matrix ct~rnponent is involved in fibrillogenesis in these tumors. The PA-bis staining studies indicated the presence of glycoproteins within many of the abnormal elastic fibers; this could account, in part, for their increased electron-dense staining. Whether the presence of such glyc~~proteins, even in increased amounts, could affect fibrillogenesis is not known. The presence of additional electron-dense materials within these newly forming elastic fibers camiot be excluded. We note that newly formed elastic fibers in normal fetal tissues are more electron dense than mature elastic fibers (unpublished data). The nature of the cells responsible for the production of the abnormal matrix components of elastofibromas has been a subject of dispute: many investigators have claimed that they are typical fibroblasts,’ while others have claimed that they have the morphology of myofibroblasts.“x In this study, the cells had many of the characteristics of fibroblasts and none were typical I~~y~)fibroblasts.They did contain abundant intracellular filaments, but focal densities were not observed along their course. Many cells exhibited a “fibrous lamina” adjacent to the inner nuclear membrane, marginal chromatin distribution, and the obvious nuclear pores that others have reported, but basement men~brane-like structures were not demonstrated in relation to these cells in this study. Such cells have been noted to be similar in appearance to an actively synthesizing chondrocyte.!’ The demonstration that anti-type-II collagen antibody binds to collagen fibers in an elastofibro~~a”’ is consistent with such an origin. Abnormal collagen fibers, many of which demonstrated appearances typical of unravelling collagen fibrils, were seen in areas of both elastofibromas. Waisman and Smith illustrated collagen fibers with very similar appearances in some portions of their elastofibroma’ and Govoni et al commented on the presence of “infrequent.. larger star-shaped” collagen bundles.” Similar appearances of collagen fibers have been reported in several genetic diseases of the skin,“’ and they are also occasionally seen in sun-exposed skin.“” The significance of their presence is obscure, but the possibility of interference with collagen fibrillogenesis by the presence of some other component cannot be excluded.
ELASTIC
FIBRILLOGENESIS
IN ELASTOFIBROMA
Most investigators appear to have accepted that elastofihromas are derived from cells of the exrracellular matris, usually of the subscapular space.‘!’ Despite the apparent consensus on this point, examination of the litrrature reveals that in the vast majority of cases, the lesions were reported to be attached to the periosteum of‘ the ad_jacenr scapula. ribs, or other bones. These observations 1~1 us to examine the possibility that, rathe t hm “invading” the periosteum, elastofibromas actually arise from it. Normal periosteurn of the scapula, ribs, ancl long hones ~vas found tc: contain significant amounts of elastic tissue in small masses in its deeper regions, csvrn in elderly patients. Periosteal elastic tissue was found to have many features in l:xmln~on with those observed in elastofibromas. These include the electron-dense staining of both the amorphous elastic tissue and of the generous peripheral rind of’ microfibrils associated with them. the increased rvactit’ity with PA-his stain in hot h amorphous rlastic tissuch and the microfihrils, and the increased ninnhers of‘ rnic7-ofibrils immunoreactive with antiMAGP antibody seen in the developing periosleum (unpublished data). These microfibrils occurred in associat it m with arnorpliolls elastic tissue, in collections within the estracrllular matrix (including between collagen fibers), alrd in the apparent absence of amorphous elastic tissue or of immunoreactive elastin. Periosteal cells also exhibited many features in common with those of the &~stofibroma. Thev were elongated, plump fihroblastic c.ells with a large nucleus, typically with a marginal chromatin distribution. a fib&s nuclear larnina, nuclear pores, and collec~tions of ordered cytoplasmic filaments. Thus, it tin be concluded on morphologic grounds that periostcum mav well be the source of the cells of elastofibromas. Furthermore, these tumors have almost all occurred in regions that are subjected to repetitive mechanical stimulation. most commonly between the scapula and the ribs and in the infraolecranon region. ‘I‘hree cases have been reported in which the lesions were attach4 to the ischium; it is pertinent that one of these Iyatient.\ wx an amateur cyclist” while another had rec‘(xnt 1~ had surgical treatment to an adjacent ischial The frequent occurrence of elastofibroma fra0ure.’ among fiunilies in Okinawa, Japan’ suggests that a predisposition to these lesions may be inherited in some (‘astas. However, even in these families, few cases were obser& before the age of40 years, and the distribution 01‘the lesions correlated with the likelihood of repetitive nlilror trauma. These observations lead us to propose that clastofibromas arise from the periosteum. in susceptible individuals. in response to repeated physical irritation. The prrdominant occurrence in females is an interesting a1spPc.t to be examined further. Elastofibro~na thus appears to be a good model in wl1it.h it ma? be possible to analyze further the processes The recent availability of a of elastic hbrillogenesis.
(Kumarotiloke
et al)