Differences in the Distribution of Versican, Decorin, and Biglycan in Atherosclerotic Human Coronary Arteries Paulo Gutierrez, MD,* Kevin D. O’Brien, MD,‡ Marina Ferguson,† Seppo T. Nikkari, MD, PhD,† Charles E. Alpers, MD,† and Thomas N. Wight, PhD† Departments of †Pathology and ‡Medicine (Cardiology), University of Washington, Seattle, Washington; *Coracao Institute, Hospital das Clinicas, São Paulo, Brazil
11 The distributions of versican, biglycan, and decorin have been examined in segments of normal and atherosclerotic human coronary arteries using antibodies directed against the core proteins of these macromolecules. Versican immunostaining was prominent throughout the extracellular matrix (ECM) in regions of the vessels that contained abundant smooth-muscle cells, such as in diffuse intimal thickenings, fibrous caps, and in zones of loose, myxoid connective tissue. Versican also was present in smooth-muscle–rich thrombi and at borders of the lipid-rich cores of advanced atherosclerotic lesions. Biglycan immunostaining was observed in diffuse intimal thickenings, fibrous caps, and myxoid areas, but, unlike versican, it was abundant in the lipid-rich core of advanced plaques. However, biglycan immunostaining was absent in smooth-muscle cell–enriched thrombi. Decorin immunostaining paralleled biglycan immunostaining except that it was conspicuously absent in the myxoid areas of the plaque and markedly reduced in diffuse intimal thickenings. Both biglycan and decorin immunostaining were consistently associated with some of the microvessels in the thrombi and in advanced atherosclerotic plaques. Taken together, these results indicate that specific proteoglycans distribute to topographically defined regions of normal and atherosclerotic human coronary arteries and that these different distributions may indicate a diversity of functions in normal and pathologic processes of the arterial wall. Cardiovasc Pathol 1997;6:271–278 © 1997 by Elsevier Science Inc.
Atherosclerosis is characterized by an increase in both the cellular and acellular components of the intimal layer of medium and large arteries (1,2). The acellular compartment comprises a large percentage of the atherosclerotic lesion mass and consists of a mixture of collagens and elastic fibers embedded in a viscoelastic gel containing proteoglycans, hyaluronan, glycoproteins, and water (3,4). Considerable interest has been devoted recently to the proteoglycan component of atherosclerotic lesions as these molecules interact with molecules involved in lipid retention, calcification, and thrombosis and are thought to participate in the regulation of these atherosclerotic events (5–8). Furthermore, proteoglycans interact with vascular cells and with Manuscript received October 7, 1996; revised January 13, 1997; accepted January 31, 1997. Address for reprints:Thomas N. Wight, PhD, Department of Pathology, Box 357470, University of Washington, School of Medicine, Seattle, WA 98195-7470.
growth factors to modify vascular cell adhesion, migration, and proliferation (9,10), which are cellular processes fundamental to the development of vascular disease (1,2). Although the involvement of proteoglycans in the atherosclerotic process is well documented (see reviews 3–8 and 11– 18), the identities of the specific types of proteoglycans and their location and distribution in human vascular lesions are poorly understood. Recent studies have characterized specific classes of proteoglycans that are defined by both the core protein amino acid sequence and the type of glycosaminoglycan (GAG) associated with the core protein (19). Three types of proteoglycans that have been identified in blood vessels and that are synthesized by vascular endothelial and smoothmuscle cells are versican, decorin, and biglycan (20–30). Versican belongs to a family of large, aggregating proteoglycans that share both similarities of structure and the ability to bind to hyaluronan (19,31–33). Versican is a large
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chondroitin sulfate (CS)–containing proteoglycan present in blood vessels that fills the extracellular space not occupied by fibrous components. It contains 15 to 17 negatively charged chondroitin sulfate chains that interact to provide the tissue with resistance to compression (4,34–36). Decorin and biglycan are members of the small leucine-rich family of proteoglycans characterized by conserved cysteines that form disulfide-bonded loops near both termini of the protein core and by highly homologous internal leucine-rich repeats that make up approximately 80% of the core protein (19,37,38). Decorin and biglycan bear, respectively, one or two dermatan sulfate (DS) chains. Both of these proteoglycans are present in the vascular tissue (22,27,28,39) and are synthesized by vascular cells (21,23–26,30). Decorin binds to type I collagen fibrils (see review 37) and regulates collagen fibrillogenesis. Similarly, it recently has been shown that biglycan also can associate with collagen in vitro (40), but the role of biglycan in blood vessels is not known. Despite the fact that proteoglycans and glycosaminoglycans are known to accumulate in various forms of vascular disease, few studies have identified the location and distribution of specific types of proteoglycans in human atherosclerosis. In this study, monospecific antisera to the core proteins of versican, biglycan, and decorin were used to identify their distribution in normal and atherosclerotic human coronary arteries obtained from the explanted hearts of patients undergoing cardiac transplantation. The results demonstrate that versican is prominent in regions of the plaque that are enriched in smooth-muscle cells, whereas biglycan and decorin are localized both to areas where lipid is present and to plaque neovasculature. These differences in distribution suggest a diversity of functions for these three proteoglycans in the development of atherosclerosis.
Material and Methods Human Coronary Arterial Tissue A total of 36 coronary segments were obtained from 15 hearts explanted at the time of cardiac transplantation and were placed in 10% neutral buffered formalin within 2 hours of organ excision, fixed for at least 12 hours, and then processed and paraffin-embedded according to conventional histologic techniques. All patients had medically refractory, symptomatically moderate to severe (New York Heart Association Classes III or IV) congestive heart failure and most accepted criteria for listing cardiac transplantation. Ten patients (all men) had cardiomyopathy due to atherosclerotic coronary artery disease (“ischemic cardiomyopathy”). Five patients (4 men and 1 woman) had nonischemic cardiomyopathies, including idiopathic dilated cardiomyopathy in 1, hypertrophic cardiomyopathy in 1, and congenital heart disease (corrected transposition of the great vessels) in 1. Patients with ischemic cardiomyopathy were slightly older (age range 40–66 years; mean age 51.1 years; median
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age 47.5 years) than patients with nonischemic cardiomyopathies (age range 31–56 years; mean age 45.2 years; median age 51 years). Atherosclerosis, defined by the presence of typical features of luminal narrowing due to regional accumulation of cholesterol, foam cell, and non-foam cell macrophages, and the presence of fibrous caps on histologic examination, resulted in severe luminal narrowing (70% diameter stenosis) of all three major coronary arteries (left anterior descending, circumflex, and right coronary arteries) in all 10 patients with ischemic cardiomyopathy. Of the patients with nonischemic cardiomyopathies, histologic examination revealed the presence of mild atherosclerosis (,50% diameter stenosis) involving all three major coronary arteries in one patient and involving a single coronary artery in an additional patient, whereas the remaining three patients had no histologic evidence of atherosclerosis. Adjacent 6-mm sections from each coronary segment were deparaffinized with xylene, rehydrated with graded alcohols, and washed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with 3% H2O2. To digest GAG chains and expose core protein epitopes, the sections were incubated for 30 minutes at 378C with a 0.2U/ml solution of chondroitin ABC lyase (ICN Biomedicals, Lisle, IL) in 0.8% bovine serum albumin (BSA), 0.6% TRIS, 0.2% NaAc, and 0.3% NaCl in distilled water. The pH was adjusted to 8 with HCl (34). The slides then were incubated at room temperature in a moist chamber with rabbit polyclonal antisera against peptides corresponding to: (a) the carboxyl terminus of human decorin (29) (Chemicon International Inc., Temecula, CA); (b) an internal domain of human versican (32) (kindly provided by Dr. Richard LeBaron, University of Texas at San Antonio, and Erkki Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA); (c) the amino terminus of human biglycan (39) (LF-51, kindly provided by Dr. Larry Fisher, National Institute of Dental Research, Bethesda, MD); and (d) to the core protein of human perlecan (41). The decorin, versican, biglycan, and antisera were used, respectively, at dilutions of 1:500, 1:800, 1:600, and 1:50. The slides then were washed with PBS, after which a biotinylated anti-rabbit secondary antibody was added, followed by an avidin-biotin-peroxidase conjugate. The slides then were immersed in a waterbath at 378C with 3,39-diaminobenzidine, NiCl2, and 3% hydrogen peroxide to yield a black reaction product. The slides were counterstained with methyl green. Negative controls included omission of the primary antisera as well as inclusion of normal rabbit serum at the same dilutions. To further establish specificity of the polyclonal antisera to decorin and biglycan, antigen absorption tests were performed: 1.0 nmole of the peptide used to raise the antisera (gift of Dr. L. Fisher) was added to 100 ml of primary antibody at a dilution of 1:200 in PBS, 1% BSA. After incubation overnight at 48C, the antigen–antibody solution was centrifuged at 14,000 rpm in a microcentrifuge and the supernatant was taken for immunostaining.
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Figure 1. Adjacent sections from a nonatherosclerotic coronary artery. (a) Hematoxylin and eosin stain. Abbreviations: A 5 adventitia; M 5 media; I 5 intima. Immunostaining for versican (b), biglycan (c), and decorin (d). (Magnification: 320, bar 5 200 mm.)
Results Proteoglycan Distributions in Normal Human Coronary Arteries In coronary arteries free of atherosclerosis (Figure 1a), there was patchy intimal staining for versican (Figure 1b) and biglycan (Figure 1c) and (Table 1). Similar patterns of
Table 1. Summary of Immunostaining for Each Proteoglycan in the Different Regions of Human Coronary Arteries
Adventitia Media Nonatherosclerotic diffuse intimal thickening Fibrous cap Lipid-enriched areas Myxoid areas Thrombus in recanalization
Versican
Biglycan
Decorin
2* 1†
1* 1†
1 2
1 Irregular 2 1
1 Irregular 1 Irregular
2 2 1 2
1
1‡
1‡
1, positive; 2, negative. *Only in the inner region. † In laminar patches. ‡ Mostly associated with microvessels.
immunostaining for these two proteoglycans were present in the media as well. Faint immunostaining for decorin was present in the lower third of the intima (Figure 1d). Immunostaining for both versican and biglycan was prominent in the portion of the adventitia adjacent to the external elastic lamina (Figures 1b and 1c), and immunostaining for decorin was present throughout the adventitia (Figure 1d).
Proteoglycan Distributions in Advanced Human Atherosclerotic Plaques Most of the atherosclerotic plaques examined in this study were characterized by a dense connective tissue fibrous cap and a lipid-rich/necrotic core, sometimes with calcification (Figure 2a). These plaques contained regional variations in the distribution of versican, biglycan, and decorin (Figures 2b–d and Table 1). Versican immunostaining was irregularly positive throughout the smooth-muscle–rich fibrous cap and in areas deeper within the plaque beneath the plaque core (Figure 2b). Usually, versican immunostaining was not present in the plaque center that contained cholesterol clefts and necrotic debris, but was prominent at the borders of these areas (Figure 2b). Versican immunostaining was absent from the plaque core, but biglycan and decorin frequently were present in the plaque core, sometimes with
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an overlapping distribution, However, there were other areas within the plaque core that immunostained positive for biglycan and negative for decorin (Figures 2c and 2d). Versican immunostaining was always prominent in the loose myxoid connective tissue regions of the plaque that contained numerous smooth-muscle cells (Figures 3a and 3b; Table 1). Biglycan also was present in regions of the lesion that contained abundant smooth-muscle cells (Figure 3c). Decorin immunostaining was either absent or very weak in these regions (Figure 3d).
Proteoglycan Distributions in Human Atherosclerotic Plaques with Organized Thrombi and in Plaque Neovasculature In plaques that contained organized thrombi, versican immunostaining was intense throughout in regions containing abundant smooth-muscle cells (Figure 4a; Table 1). These regions stained weakly for decorin and biglycan (Figure 4b). In contrast, immunostaining for biglycan and decorin (not shown) usually was associated with the microvessels within the thrombus (Figure 4c). Similarly, some of the atheroscle-
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rotic lesion microvessels, identified by positive staining for the basement membrane HSPG, perlecan (Figure 5a), also had positive staining for biglycan (Figure 5b; Table 1).
Discussion This study has demonstrated that particular types of proteoglycans accumulate in specific regions of human atherosclerotic arteries and suggests that these molecules may have different functional roles in the development of vascular disease. Versican, a large interstitial chondroitin sulfate proteoglycan (CSPG), accumulates in regions of the plaque that are enriched in smooth-muscle cells. There are two regions within advanced atherosclerotic lesions that are enriched in smooth-muscle cells. One region is the fibrous cap, which contains mostly spindle-shaped smooth-muscle cells. The second region consists of foci of stellate-shaped smooth-muscle cells embedded in a loose connective matrix. These latter areas often have been referred to as myxoid and are present in both primary and restenotic human arterial lesions (42). A number of studies have localized CSPG to the extracellular matrix of normal and atherosclerotic arteries (see reviews 3–8 and 12,17,37,43,44), but the
Figure 2. Adjacent sections of an advanced coronary atherosclerotic plaque. (a) Hematoxylin and eosin. Immunostaining for versican (b), biglycan (c), and decorin (d). Note area of plaque that immunostained positive for biglycan and negative for decorin. Note that the immunostaining for versican is confined principally to the fibrous cap and the borders of the lipid core whereas biglycan and decorin appear concentrated in the lipid core. (Magnification: 320, bar 5 200 mm.)
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identity of the proteoglycans that carries the CS chains and their exact location have not been established. The results of the present study indicate that at least a portion of the CS in human atherosclerotic arteries is present as versican and associated with smooth-muscle cell foci. The finding of large accumulations of versican in areas surrounding smooth-muscle cells in different regions of the plaque is consistent with the results of previous studies demonstrating that versican is a major extracellular matrix (ECM) macromolecule synthesized by arterial smooth-muscle cells in vitro (21,23,24,29). For example, the synthesis of versican by arterial smooth-muscle cells is regulated by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-b1 (TGF-b1) (21, 23). Thus, it may be that plaque-associated cytokines such as PDGF and TGF-b1 (45,46) modulate versican expression by arterial smooth muscle cell (ASMC) (31,32) and cause this matrix molecule to accumulate in the ECM. It is interesting to note that antibodies to TGF-b1 block versican accumulation in injury-induced neointimas and reduce intimal thickenings in experimental animals (47). Versican also is present at the border of plaques containing macrophages, and recent studies indicate that versican is a substrate for the macrophage-derived metalloproteinase matrilysin (48). The
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degradation of versican by matrilysin in particular topographic regions of the atherosclerotic lesion may predispose these regions of the plaque to fissure and rupture by exposing structural components of the ECM, such as collagens and elastic fibers, to subsequent attack by other matrix metalloproteinases. Versican is prominent in areas of the plaque that are critical to plaque integrity, such as the fibrous caps and plaque shoulders (48). Differences in the location of versican, biglycan, and decorin relative to plaque lipids suggest differences in the association of these proteoglycans with various lipoprotein classes. Versican contains chondroitin 6-sulfate as its predominant glycosmainoglycan, and this GAG binds to apoB–containing lipoproteins (7,49) and is present in complexes of lipoproteins isolated from human atherosclerotic lesions (37). In fact, Galis et al. (44), have shown co-location of these two macromolecules in atherosclerotic plaques. In addition, we have found that versican co-localizes with some apo-B–containing regions of human atherosclerotic plaques, but is absent from other apo-B–containing regions (50). Such results indicate that there may be differences in the nature of the versican molecule that predisposes it to lipid entrapment. Interestingly, CSPGs isolated from growing arterial smooth-muscle cells bind more avidly to lipo-
Figure 3. Myxoid area of an atherosclerotic plaque. (a) Hematoxylin and eosin (320); inset, higher magnification of the same area (340). Immunostaining for versican (b), biglycan (c), and decorin (d). Note that versican is strongly positive, whereas biglycan stains weakly and decorin is absent in this area. (Magnification: 320, bar 5 200 mm.)
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proteins than CSPGs isolated from nongrowing cultures (51). Modifications in the CS chains have been shown to influence lipoprotein binding (52–57). The presence of decorin and biglycan in the lipid-core regions of the plaque suggests that these proteoglycans preferentially bind lipid within the atherosclerotic lesion. Both decorin and biglycan contain DS chains, and a number of studies show that DS increased in both primary and restenotic human lesions (13–17,29). Furthermore, DS binds avidly to lipoproteins (53,55). Recent studies show remark-
Figure 4. Adjacent sections of an organized smooth-muscle cell– rich thrombus immunostained for versican (a), biglycan (b), and decorin (c). Note that the bulk of the thrombus stains intensely for versican and not biglycan, whereas the small vessels within the thrombus are positive for decorin. (Magnification: (a and b) 320, bar 5 200 mm; (c) 390, bar 5 50 mm.)
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able co-localization of biglycan with apo-E in human coronary lesions (10), suggesting specificity in the association of these two molecules. The finding of large amounts of versican in smooth-muscle–rich thrombi, versus decorin and biglycan’s tendency to localize to neovessels in the thrombi, further illustrates potentially different roles for these two sets of ECM macromolecules. ECMs enriched in molecules such as versican and associated hyaluranon are believed to promote cellular migration by creating a loose and hydrated ECM (10). Such an ECM may be essential for the cellular movement and re-
Figure 5. Adjacent sections of an advanced coronary atherosclerotic plaque. (a) Immunostained for perlecan, a basement membrane HSPG, to demonstrate microvessels. (b) Immunostained for biglycan. Note some of the microvessels in this region also stain for biglycan. (Magnification 332; bar 5 30 mm.)
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modeling that occurs during thrombus formation. Although smooth-muscle cells represent a rich source of versican, endothelial cells synthesize little versican, but large amounts of biglycan (24,56). In fact, biglycan expression is upregulated when endothelial cells are stimulated to migrate (56,57) and decorin expression is characteristic of the angiogenic phenotype (58). Studies demonstrating that neovessels may serve as a route for plasma lipoproteins (59) and leukocyte (59,60) into plaques have highlighted the importance of the neovasculature in plaque growth. Thus, finding of prominent biglycan and decorin immunostaining of some of the neovessels in human coronary plaques raises the possibility that either one or both of these proteoglycans may be required for neovascularization of human atherosclerotic arteries. In summary, specific proteoglycans accumulate in topographically defined regions of human coronary atherosclerotic plaques. The finding that versican and biglycan are enriched in areas of smooth-muscle cell involvement, whereas biglycan and decorin localize to areas enriched in lipid and necrotic debris and neovasculature suggests different roles for these PGs in the atherosclerotic process. This work is supported in part by grants NIH HL4751 and DK47659 (CA), NIH JL02788 (KO), and HL18645 (TNW). Dr. Gutierrez was supported by the National Council of Scientific and Technologic Development CNPq, Brazil. The authors thank Dr. Larry Fisher, from the National Institute for Dental Research, Bethesda, MD, and Drs. Richard Le Baron and Erkki Ruoslahti, from the La Jolla Cancer Foundation, La Jolla, CA, for providing the antibodies to versican, decorin, and biglycan; Dr. Susan Potter-Perigo for critical reading of the manuscript; and Thomas McDonald and the University of Washington Cardiac Transplantation Program.
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48. Halpert I, Sires U, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC. Matrilysin is expressed by lipid laden macrophages at sites of potential rupture in atherosclerotic lesions and localized to areas of versican deposits—a proteoglycan substrate for the enzyme. 1996; Proc Nat Acad Sci USA 1996;93:9748–9753. 49. Olsson U, Camejo G, Olofsson SO, Bondjers G. Molecular parameters that control the association of low density lipoprotein apo b-100 with chondroitin sulfate. Biochem Biophys Acta 1991;1097:37–44. 50. O’Brien KD, Alpers CE, Chiu W, Hudkins R, Wight TN, Chait A. Colocalization of apolipoprotein E and biglycan in human coronary atherosclerotic plaques: a possible pathogenic role for coronary atherosclerosis. Submitted. 51. Camejo G, Fager B, Rosengren E, Hurt-Camejo E, Bondjers G. Binding of low density lipoproteins by proteoglycans synthesized by proliferation and quiescent human arterial smooth muscle cells. J Biol Chem 1993;268:14131–14137. 52. Alves CS, Mourão OAS. Interaction of high molecular weight chondroitin sulfate from human aorta with plasma low density lipoproteins. Atherosclerosis 1998;73:113–124. 53. Sambandam T, Baker JR, Christner JE, Ekborg SL. Specificity of low density lipoprotein–glycosaminoglycan interaction. Arterioscler Thromb 1991;11:561–568. 54. Cardoso L, Mourão PAS. Glycosaminoglycans from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb 1994;14:115–124. 55. Iverius PH. The interaction between human plasma lipoproteins and connective tissue glycosaminoglycans. J Biol Chem 1972;247:2607– 2613. 56. Kinsella MG, Tsoi CK, Järveläinen HT, Wight TN. Selective expression and processing of biglycan during migration of bovine aortic endothelial cells: the role of endogenous basic fibroblast growth factor. J Biol Chem 1997;272:318–325. 57. Järveläinen HT, Iruela-Arispe ML, Kinsella MG, Sandell LJ, Sage EH, Wight TN. Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro. Exp Cell Res 1992;203:395– 491. 58. Srinivasan SE, Dolan P, Rhadakrishnamurthy B, Pargaonkar P, Berenson GS. Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions. Biochem Biophys Acta 175;388:5870. 59. O’Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neovascular expression of E-selectin intercellular adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation 1996;93:672–682. 60. O’Brien KD, Allen MD, McDonald TO, Chait A, Harlan JM, Fishbein D, McCarty J, Ferguson M, Hudkins K, Benjamin CD, Lobb R, Alpers CE. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerosis. J Clin Invest 1993;92:945–951.
11 The distributions of versican, biglycan, and decorin have been examined in segments of normal and atherosclerotic human coronary arteries using antibodies directed against the core proteins of these macromolecules. Versican immunostaining was prominent throughout the extracellular matrix (ECM) in regions of the vessels that contained abundant smooth-muscle cells, such as in diffuse intimal thickenings, fibrous caps, and in zones of loose, myxoid connective tissue. Versican also was present in smooth-muscle–rich thrombi and at borders of the lipid-rich cores of advanced atherosclerotic lesions. Biglycan immunostaining was observed in diffuse intimal thickenings, fibrous caps, and myxoid areas, but, unlike versican, it was abundant in the lipid-rich core of advanced plaques. However, biglycan immunostaining was absent in smooth-muscle cell–enriched thrombi. Decorin immunostaining paralleled biglycan immunostaining except that it was conspicuously absent in the myxoid areas of the plaque and markedly reduced in diffuse intimal thickenings. Both biglycan and decorin immunostaining were consistently associated with some of the microvessels in the thrombi and in advanced atherosclerotic plaques. Taken together, these results indicate that specific proteoglycans distribute to topographically defined regions of normal and atherosclerotic human coronary arteries and that these different distributions may indicate a diversity of functions in normal and pathologic processes of the arterial wall. Cardiovasc Pathol 1997;6:271–278 © 1997 by Elsevier Science Inc.
Atherosclerosis is characterized by an increase in both the cellular and acellular components of the intimal layer of medium and large arteries (1,2). The acellular compartment comprises a large percentage of the atherosclerotic lesion mass and consists of a mixture of collagens and elastic fibers embedded in a viscoelastic gel containing proteoglycans, hyaluronan, glycoproteins, and water (3,4). Considerable interest has been devoted recently to the proteoglycan component of atherosclerotic lesions as these molecules interact with molecules involved in lipid retention, calcification, and thrombosis and are thought to participate in the regulation of these atherosclerotic events (5–8). Furthermore, proteoglycans interact with vascular cells and with Manuscript received October 7, 1996; revised January 13, 1997; accepted January 31, 1997. Address for reprints:Thomas N. Wight, PhD, Department of Pathology, Box 357470, University of Washington, School of Medicine, Seattle, WA 98195-7470.
growth factors to modify vascular cell adhesion, migration, and proliferation (9,10), which are cellular processes fundamental to the development of vascular disease (1,2). Although the involvement of proteoglycans in the atherosclerotic process is well documented (see reviews 3–8 and 11– 18), the identities of the specific types of proteoglycans and their location and distribution in human vascular lesions are poorly understood. Recent studies have characterized specific classes of proteoglycans that are defined by both the core protein amino acid sequence and the type of glycosaminoglycan (GAG) associated with the core protein (19). Three types of proteoglycans that have been identified in blood vessels and that are synthesized by vascular endothelial and smoothmuscle cells are versican, decorin, and biglycan (20–30). Versican belongs to a family of large, aggregating proteoglycans that share both similarities of structure and the ability to bind to hyaluronan (19,31–33). Versican is a large
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chondroitin sulfate (CS)–containing proteoglycan present in blood vessels that fills the extracellular space not occupied by fibrous components. It contains 15 to 17 negatively charged chondroitin sulfate chains that interact to provide the tissue with resistance to compression (4,34–36). Decorin and biglycan are members of the small leucine-rich family of proteoglycans characterized by conserved cysteines that form disulfide-bonded loops near both termini of the protein core and by highly homologous internal leucine-rich repeats that make up approximately 80% of the core protein (19,37,38). Decorin and biglycan bear, respectively, one or two dermatan sulfate (DS) chains. Both of these proteoglycans are present in the vascular tissue (22,27,28,39) and are synthesized by vascular cells (21,23–26,30). Decorin binds to type I collagen fibrils (see review 37) and regulates collagen fibrillogenesis. Similarly, it recently has been shown that biglycan also can associate with collagen in vitro (40), but the role of biglycan in blood vessels is not known. Despite the fact that proteoglycans and glycosaminoglycans are known to accumulate in various forms of vascular disease, few studies have identified the location and distribution of specific types of proteoglycans in human atherosclerosis. In this study, monospecific antisera to the core proteins of versican, biglycan, and decorin were used to identify their distribution in normal and atherosclerotic human coronary arteries obtained from the explanted hearts of patients undergoing cardiac transplantation. The results demonstrate that versican is prominent in regions of the plaque that are enriched in smooth-muscle cells, whereas biglycan and decorin are localized both to areas where lipid is present and to plaque neovasculature. These differences in distribution suggest a diversity of functions for these three proteoglycans in the development of atherosclerosis.
Material and Methods Human Coronary Arterial Tissue A total of 36 coronary segments were obtained from 15 hearts explanted at the time of cardiac transplantation and were placed in 10% neutral buffered formalin within 2 hours of organ excision, fixed for at least 12 hours, and then processed and paraffin-embedded according to conventional histologic techniques. All patients had medically refractory, symptomatically moderate to severe (New York Heart Association Classes III or IV) congestive heart failure and most accepted criteria for listing cardiac transplantation. Ten patients (all men) had cardiomyopathy due to atherosclerotic coronary artery disease (“ischemic cardiomyopathy”). Five patients (4 men and 1 woman) had nonischemic cardiomyopathies, including idiopathic dilated cardiomyopathy in 1, hypertrophic cardiomyopathy in 1, and congenital heart disease (corrected transposition of the great vessels) in 1. Patients with ischemic cardiomyopathy were slightly older (age range 40–66 years; mean age 51.1 years; median
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age 47.5 years) than patients with nonischemic cardiomyopathies (age range 31–56 years; mean age 45.2 years; median age 51 years). Atherosclerosis, defined by the presence of typical features of luminal narrowing due to regional accumulation of cholesterol, foam cell, and non-foam cell macrophages, and the presence of fibrous caps on histologic examination, resulted in severe luminal narrowing (70% diameter stenosis) of all three major coronary arteries (left anterior descending, circumflex, and right coronary arteries) in all 10 patients with ischemic cardiomyopathy. Of the patients with nonischemic cardiomyopathies, histologic examination revealed the presence of mild atherosclerosis (,50% diameter stenosis) involving all three major coronary arteries in one patient and involving a single coronary artery in an additional patient, whereas the remaining three patients had no histologic evidence of atherosclerosis. Adjacent 6-mm sections from each coronary segment were deparaffinized with xylene, rehydrated with graded alcohols, and washed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with 3% H2O2. To digest GAG chains and expose core protein epitopes, the sections were incubated for 30 minutes at 378C with a 0.2U/ml solution of chondroitin ABC lyase (ICN Biomedicals, Lisle, IL) in 0.8% bovine serum albumin (BSA), 0.6% TRIS, 0.2% NaAc, and 0.3% NaCl in distilled water. The pH was adjusted to 8 with HCl (34). The slides then were incubated at room temperature in a moist chamber with rabbit polyclonal antisera against peptides corresponding to: (a) the carboxyl terminus of human decorin (29) (Chemicon International Inc., Temecula, CA); (b) an internal domain of human versican (32) (kindly provided by Dr. Richard LeBaron, University of Texas at San Antonio, and Erkki Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA); (c) the amino terminus of human biglycan (39) (LF-51, kindly provided by Dr. Larry Fisher, National Institute of Dental Research, Bethesda, MD); and (d) to the core protein of human perlecan (41). The decorin, versican, biglycan, and antisera were used, respectively, at dilutions of 1:500, 1:800, 1:600, and 1:50. The slides then were washed with PBS, after which a biotinylated anti-rabbit secondary antibody was added, followed by an avidin-biotin-peroxidase conjugate. The slides then were immersed in a waterbath at 378C with 3,39-diaminobenzidine, NiCl2, and 3% hydrogen peroxide to yield a black reaction product. The slides were counterstained with methyl green. Negative controls included omission of the primary antisera as well as inclusion of normal rabbit serum at the same dilutions. To further establish specificity of the polyclonal antisera to decorin and biglycan, antigen absorption tests were performed: 1.0 nmole of the peptide used to raise the antisera (gift of Dr. L. Fisher) was added to 100 ml of primary antibody at a dilution of 1:200 in PBS, 1% BSA. After incubation overnight at 48C, the antigen–antibody solution was centrifuged at 14,000 rpm in a microcentrifuge and the supernatant was taken for immunostaining.
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Figure 1. Adjacent sections from a nonatherosclerotic coronary artery. (a) Hematoxylin and eosin stain. Abbreviations: A 5 adventitia; M 5 media; I 5 intima. Immunostaining for versican (b), biglycan (c), and decorin (d). (Magnification: 320, bar 5 200 mm.)
Results Proteoglycan Distributions in Normal Human Coronary Arteries In coronary arteries free of atherosclerosis (Figure 1a), there was patchy intimal staining for versican (Figure 1b) and biglycan (Figure 1c) and (Table 1). Similar patterns of
Table 1. Summary of Immunostaining for Each Proteoglycan in the Different Regions of Human Coronary Arteries
Adventitia Media Nonatherosclerotic diffuse intimal thickening Fibrous cap Lipid-enriched areas Myxoid areas Thrombus in recanalization
Versican
Biglycan
Decorin
2* 1†
1* 1†
1 2
1 Irregular 2 1
1 Irregular 1 Irregular
2 2 1 2
1
1‡
1‡
1, positive; 2, negative. *Only in the inner region. † In laminar patches. ‡ Mostly associated with microvessels.
immunostaining for these two proteoglycans were present in the media as well. Faint immunostaining for decorin was present in the lower third of the intima (Figure 1d). Immunostaining for both versican and biglycan was prominent in the portion of the adventitia adjacent to the external elastic lamina (Figures 1b and 1c), and immunostaining for decorin was present throughout the adventitia (Figure 1d).
Proteoglycan Distributions in Advanced Human Atherosclerotic Plaques Most of the atherosclerotic plaques examined in this study were characterized by a dense connective tissue fibrous cap and a lipid-rich/necrotic core, sometimes with calcification (Figure 2a). These plaques contained regional variations in the distribution of versican, biglycan, and decorin (Figures 2b–d and Table 1). Versican immunostaining was irregularly positive throughout the smooth-muscle–rich fibrous cap and in areas deeper within the plaque beneath the plaque core (Figure 2b). Usually, versican immunostaining was not present in the plaque center that contained cholesterol clefts and necrotic debris, but was prominent at the borders of these areas (Figure 2b). Versican immunostaining was absent from the plaque core, but biglycan and decorin frequently were present in the plaque core, sometimes with
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an overlapping distribution, However, there were other areas within the plaque core that immunostained positive for biglycan and negative for decorin (Figures 2c and 2d). Versican immunostaining was always prominent in the loose myxoid connective tissue regions of the plaque that contained numerous smooth-muscle cells (Figures 3a and 3b; Table 1). Biglycan also was present in regions of the lesion that contained abundant smooth-muscle cells (Figure 3c). Decorin immunostaining was either absent or very weak in these regions (Figure 3d).
Proteoglycan Distributions in Human Atherosclerotic Plaques with Organized Thrombi and in Plaque Neovasculature In plaques that contained organized thrombi, versican immunostaining was intense throughout in regions containing abundant smooth-muscle cells (Figure 4a; Table 1). These regions stained weakly for decorin and biglycan (Figure 4b). In contrast, immunostaining for biglycan and decorin (not shown) usually was associated with the microvessels within the thrombus (Figure 4c). Similarly, some of the atheroscle-
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rotic lesion microvessels, identified by positive staining for the basement membrane HSPG, perlecan (Figure 5a), also had positive staining for biglycan (Figure 5b; Table 1).
Discussion This study has demonstrated that particular types of proteoglycans accumulate in specific regions of human atherosclerotic arteries and suggests that these molecules may have different functional roles in the development of vascular disease. Versican, a large interstitial chondroitin sulfate proteoglycan (CSPG), accumulates in regions of the plaque that are enriched in smooth-muscle cells. There are two regions within advanced atherosclerotic lesions that are enriched in smooth-muscle cells. One region is the fibrous cap, which contains mostly spindle-shaped smooth-muscle cells. The second region consists of foci of stellate-shaped smooth-muscle cells embedded in a loose connective matrix. These latter areas often have been referred to as myxoid and are present in both primary and restenotic human arterial lesions (42). A number of studies have localized CSPG to the extracellular matrix of normal and atherosclerotic arteries (see reviews 3–8 and 12,17,37,43,44), but the
Figure 2. Adjacent sections of an advanced coronary atherosclerotic plaque. (a) Hematoxylin and eosin. Immunostaining for versican (b), biglycan (c), and decorin (d). Note area of plaque that immunostained positive for biglycan and negative for decorin. Note that the immunostaining for versican is confined principally to the fibrous cap and the borders of the lipid core whereas biglycan and decorin appear concentrated in the lipid core. (Magnification: 320, bar 5 200 mm.)
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identity of the proteoglycans that carries the CS chains and their exact location have not been established. The results of the present study indicate that at least a portion of the CS in human atherosclerotic arteries is present as versican and associated with smooth-muscle cell foci. The finding of large accumulations of versican in areas surrounding smooth-muscle cells in different regions of the plaque is consistent with the results of previous studies demonstrating that versican is a major extracellular matrix (ECM) macromolecule synthesized by arterial smooth-muscle cells in vitro (21,23,24,29). For example, the synthesis of versican by arterial smooth-muscle cells is regulated by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-b1 (TGF-b1) (21, 23). Thus, it may be that plaque-associated cytokines such as PDGF and TGF-b1 (45,46) modulate versican expression by arterial smooth muscle cell (ASMC) (31,32) and cause this matrix molecule to accumulate in the ECM. It is interesting to note that antibodies to TGF-b1 block versican accumulation in injury-induced neointimas and reduce intimal thickenings in experimental animals (47). Versican also is present at the border of plaques containing macrophages, and recent studies indicate that versican is a substrate for the macrophage-derived metalloproteinase matrilysin (48). The
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degradation of versican by matrilysin in particular topographic regions of the atherosclerotic lesion may predispose these regions of the plaque to fissure and rupture by exposing structural components of the ECM, such as collagens and elastic fibers, to subsequent attack by other matrix metalloproteinases. Versican is prominent in areas of the plaque that are critical to plaque integrity, such as the fibrous caps and plaque shoulders (48). Differences in the location of versican, biglycan, and decorin relative to plaque lipids suggest differences in the association of these proteoglycans with various lipoprotein classes. Versican contains chondroitin 6-sulfate as its predominant glycosmainoglycan, and this GAG binds to apoB–containing lipoproteins (7,49) and is present in complexes of lipoproteins isolated from human atherosclerotic lesions (37). In fact, Galis et al. (44), have shown co-location of these two macromolecules in atherosclerotic plaques. In addition, we have found that versican co-localizes with some apo-B–containing regions of human atherosclerotic plaques, but is absent from other apo-B–containing regions (50). Such results indicate that there may be differences in the nature of the versican molecule that predisposes it to lipid entrapment. Interestingly, CSPGs isolated from growing arterial smooth-muscle cells bind more avidly to lipo-
Figure 3. Myxoid area of an atherosclerotic plaque. (a) Hematoxylin and eosin (320); inset, higher magnification of the same area (340). Immunostaining for versican (b), biglycan (c), and decorin (d). Note that versican is strongly positive, whereas biglycan stains weakly and decorin is absent in this area. (Magnification: 320, bar 5 200 mm.)
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proteins than CSPGs isolated from nongrowing cultures (51). Modifications in the CS chains have been shown to influence lipoprotein binding (52–57). The presence of decorin and biglycan in the lipid-core regions of the plaque suggests that these proteoglycans preferentially bind lipid within the atherosclerotic lesion. Both decorin and biglycan contain DS chains, and a number of studies show that DS increased in both primary and restenotic human lesions (13–17,29). Furthermore, DS binds avidly to lipoproteins (53,55). Recent studies show remark-
Figure 4. Adjacent sections of an organized smooth-muscle cell– rich thrombus immunostained for versican (a), biglycan (b), and decorin (c). Note that the bulk of the thrombus stains intensely for versican and not biglycan, whereas the small vessels within the thrombus are positive for decorin. (Magnification: (a and b) 320, bar 5 200 mm; (c) 390, bar 5 50 mm.)
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able co-localization of biglycan with apo-E in human coronary lesions (10), suggesting specificity in the association of these two molecules. The finding of large amounts of versican in smooth-muscle–rich thrombi, versus decorin and biglycan’s tendency to localize to neovessels in the thrombi, further illustrates potentially different roles for these two sets of ECM macromolecules. ECMs enriched in molecules such as versican and associated hyaluranon are believed to promote cellular migration by creating a loose and hydrated ECM (10). Such an ECM may be essential for the cellular movement and re-
Figure 5. Adjacent sections of an advanced coronary atherosclerotic plaque. (a) Immunostained for perlecan, a basement membrane HSPG, to demonstrate microvessels. (b) Immunostained for biglycan. Note some of the microvessels in this region also stain for biglycan. (Magnification 332; bar 5 30 mm.)
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modeling that occurs during thrombus formation. Although smooth-muscle cells represent a rich source of versican, endothelial cells synthesize little versican, but large amounts of biglycan (24,56). In fact, biglycan expression is upregulated when endothelial cells are stimulated to migrate (56,57) and decorin expression is characteristic of the angiogenic phenotype (58). Studies demonstrating that neovessels may serve as a route for plasma lipoproteins (59) and leukocyte (59,60) into plaques have highlighted the importance of the neovasculature in plaque growth. Thus, finding of prominent biglycan and decorin immunostaining of some of the neovessels in human coronary plaques raises the possibility that either one or both of these proteoglycans may be required for neovascularization of human atherosclerotic arteries. In summary, specific proteoglycans accumulate in topographically defined regions of human coronary atherosclerotic plaques. The finding that versican and biglycan are enriched in areas of smooth-muscle cell involvement, whereas biglycan and decorin localize to areas enriched in lipid and necrotic debris and neovasculature suggests different roles for these PGs in the atherosclerotic process. This work is supported in part by grants NIH HL4751 and DK47659 (CA), NIH JL02788 (KO), and HL18645 (TNW). Dr. Gutierrez was supported by the National Council of Scientific and Technologic Development CNPq, Brazil. The authors thank Dr. Larry Fisher, from the National Institute for Dental Research, Bethesda, MD, and Drs. Richard Le Baron and Erkki Ruoslahti, from the La Jolla Cancer Foundation, La Jolla, CA, for providing the antibodies to versican, decorin, and biglycan; Dr. Susan Potter-Perigo for critical reading of the manuscript; and Thomas McDonald and the University of Washington Cardiac Transplantation Program.
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16. 17.
18.
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20.
21.
22.
23.
24.
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