Versican, Biglycan, and Decorin Protein Expression Patterns in Coronary Arteries: Analysis of Primary and Restenotic Lesions Hong Lin, PhD,* Tsugiyasu Kanda, MD, PhD,† Yoichi Hoshino, MD,‡ Shin-ichi Takase, MD,§ Isao Kobayashi, MD,* Ryozo Nagai, MD,† and Bruce M. McManus, MD, PhD* *Department of Pathology and Laboratory Medicine, The University of British Columbia, Vancouver, BC, Canada; †Department of Laboratory Medicine and ‡Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan; and §Gunma Cardiovascular Hospital, Takasaki, Gunma, Japan
11 The pathobiology of rapid intimal thickening following balloon angioplasty remains unsettled. Proteoglycans (PGs) expressed by smooth-muscle cells (SMCs) are known to participate in vascular responses to injury. In this analysis, patients ranging from age 48 to 79 years (mean 5 58), underwent atherectomy for 36 restenotic tissues (taken 64 to 345 days postangioplasty; mean 5 108) and for 10 primary atherosclerotic plaques. Tissues were formaldehyde-fixed, paraffin-embedded, and histochemically and immunohistochemically stained to determine the temporal and semi-quantitative contribution of major vessel wall PGs, versican, biglycan, and decorin. Versican was the most striking PG in the neointima of restenotic vessels, including a prominent pericellular pattern corresponding to proliferative SMCs, as well as a large extracellular accumulation. Biglycan was limited to the most loose and proliferative neointima and stained less than in primary plaques. Decorin staining was virtually absent in the most proliferative neointimal tissue, whereas it was quite striking in established primary lesions. Thus the earliest response to balloon injury of a coronary artery includes striking expression of versican protein, but the limited expression of biglycan differs from the prominence of the PG in primary atherosclerosis. Versican expression in restenotic lesions is similar to that seen previously in transplant arteriopathy, but the lack of biglycan in atherectomy specimens from restenosis sites is distinctively different from that seen in rapidly progressive transplant vascular disease. Cardiovasc Pathol 1998;7:31–37. © 1998 by Elsevier Science Inc.
Despite the effectiveness and safety of percutaneous transluminal coronary angioplasty (PTCA) for treating ischemic coronary disorders (1–6), restenosis occurs in 30% to 50% of treated patients (7–9). There are no medical regimens or variations of angioplasty that have yet consistently lowered the rate of restenosis after PTCA (10–13). Vessel wall remodeling after PTCA injury is incompletely understood, and new directions are warranted for investigation based on a concept of rapidly reparative events. In particular, the role
Manuscript received June 3, 1996; revised October 21, 1996; accepted May 30, 1997. Address for reprints: Bruce M. McManus, MD, PhD, Department of Pathology and Laboratory Medicine, St Paul’s Hospital, 1081 Burrard Street, Vancouver, Canada V6Z 1Y6; telephone: (604) 631-5200; fax: (604) 631-5208. Cardiovascular Pathology Vol. 7, No. 1, January/February 1998:31–37 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
of extracellular matrix in restenotic lesions deserves more attention. Previous studies have shown that primary atherosclerotic lesions, when excised by atherectomy or as observed at autopsy, have the classical characteristics of fibrous plaques accompanied by foam cells and extracellular lipid. Restenotic lesions following balloon angioplasty are characterized by smooth-muscle cell proliferation and extracellular matrix synthesis, which together form a neointima, histopathologically similar to other rapidly progressive lesions, such as transplant arteriopathy (14,15). Previous immunohistochemical studies by our laboratory (16) have revealed abundant proteoglycan (PG) deposits, especially versican and biglycan, in the rapidly expanding intima of allograft coronary arteries in a pattern distinct from that of native coronary artery disease. We have dem-
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onstrated that certain apolipoproteins, especially apo(a) and apoE, are much more prominent in the intima of transplant arteriopathy than in native atherosclerotic disease. These apolipoproteins and associated PGs, especially versican, constitute the major lipid accumulation and extracellular matrix volume, respectively, in that rapidly evolving disease process (17). It is known that PGs play a fundamental role in self-surface anchorage for lipoprotein lipase on endothelium, in sequestration of lipoproteins in the subendothelial matrix, and in presentation of lipoproteins to phagocytic intimal cells in the setting of native atherosclerosis. Much less is known about restenosis. In the present study, we characterize the amount and distribution of major PGs in restenotic neointima using immunohistochemical techniques and a comparative sample of primary atherosclerotic plaques, also taken by atherectomy.
Materials and Methods Patients Thirty-one unselected, human atherectomy procedures were undertaken from 24 male and 7 female patients with coronary artery disease. The patients were hospitalized in Saiseikai Maebashi Hospital of Japan. Their ages ranged from 48 to 70 years (mean 5 58). The duration postangioplasty was from 64 to 345 days (mean 5 108). Ten primary atherosclerotic lesions and 36 restenotic tissues were included in the analysis.
Tissue Processing Human atherectomy tissues were collected as soon as possible after coronary atherectomy using the Simpson AtheroCath (Devices for Vascular Intervention, Redwood City, CA). The specimens were formaldehyde-fixed and paraffin-embedded, 4- to 5-m sections were cut, placed on SuperfrostTM slides, and baked for 30 minutes at 458C for histochemical and immunohistochemical staining.
Histochemical Staining Serial sections of primary and restenotic coronary artery tissues were stained with a modified Movat’s pentachrome stain. Smooth-muscle cellular components were corroborated by staining with the muscle-actin–specific antibody HHF35 (18).
Immunohistochemical Study Rabbit polyclonal antibodies against biglycan (LF15) and decorin (LF30) were generous gifts from Dr. L. Fisher (National Institute of Dental Research, Bethesda, MD). The antisera were raised against specific synthetic peptides and shown to be monospecific (19–21). A rabbit polyclonal an-
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tibody against versican was kindly provided by Dr. E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA). A fusion protein of versican was used as the antigen to raise the polyclonal antiserum, which was subsequently affinity-purified on a versican synthetic 26-mer peptide column (22). After deparaffinization, the sections were treated with 1 U/ml of chondroitinase ABC in 0.1 M Tris-HCl/50mM calcium acetate/0.1% bovine serum albumin (BSA) pH at 7.2 for 30 minutes at 378C (18), to detach glycosaminoglycan side chains from the core protein. After chondroitinase treatment, the sections were washed thoroughly in Tris-2% BSA for 2 hours at room temperature. The sections were then washed in TBS, incubated in 1:100 dilution of goat anti-rabbit IgG-alkaline phosphatase in the presence of levamisole. Following color development, sections were counterstained with Mayer’s hematoxylin and mounted. The experiment included a negative control using nonimmune normal rabbit serum in place of the biglycan antibody and a positive control using coronary tissue with large amounts of PG present. Negative controls and internal positive controls were included in each run to evaluate consistency of staining from run to run and to allow comparison of results between runs. Slides were stained for decorin and versican by an automated method using the Ventana immunostainer (Ventana Medical Systems, Inc., Tucson, AZ). The Ventana Alkaline Phosphatase Fast Red Detection Kit includes prediluted biotinylated secondary antibody in PBS, alkaline phosphatasestreptavidin conjugate in PBS, fast red A and fast red B in acetate buffer, naphthol in Tris buffer, enhancer (MgCl2) in aqueous solution, and a negative control reagent. The automated procedure is similar to the manual procedure established in the laboratory. Decorin and versican antibodies were diluted to 1:100 in an antibody-diluting buffer (Dimension Laboratories, Inc., Mississauga, Ontario, Canada) that included the primary antibodies for 32 minutes and then a secondary antibody for 8 minutes at 378C.
Grading of Immunohistochemical Stains Slides were scored in a blinded fashion by one investigator for intensity of PG staining against an intensity scale developed for this purpose (0–61) and for PG localization in the specimens including intima, neointima, media and adventitia. The scale of scoring was defined as follows: 0, no staining; 11, trace amount; 21, small amount; 31, small to moderate amount; 41, moderate amount; 51, considerable amount; 61, maximum amount. When staining intensity was not uniform in a particular tissue layer, a focal peak score and an overall score were rendered, and the mean of the two scores was used for final analysis. The repeatability of scoring was randomly tested by the same investigator without his knowledge. This reproducibility is comparable to that obtained with other semi-quantitative grading schemata in histopathologic studies (21).
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Statistical Analysis Staining intensity was compared between the primary and restenotic groups by two-way analysis of variance (ANOVA). The probability of an alpha error was set at p , 0.05 for statistical significance.
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graphical histochemical staining pattern of glycosaminoglycans by Movat’s pentachrome and immunohistochemical staining for versican (16,17). Specimens from primary atherectomy tissue included intima in 10/10 (100%), media in 8/10 (80%), but no adventitia; whereas, restenotic tissue at times included all three layers, intima in 38/38 (100%), media in 16/38 (42%) and adventitia in 6/38 (16%).
Results Histologic Staining
Immunohistochemical Staining
As noted, atherectomy tissue samples taken from human coronary arteries were first characterized with Movat’s pentachrome stain. These stains revealed features typical of primary and restenotic lesions. Movat’s stain distinguished lesions as follows: older atheromatous plaques were distinguished as yellow-cream, with superimposed young restenotic tissue as sea-green (glycosaminoglycans), and a small portion of adventitia with yellow-staining collagenous tissue outward from the external elastic lamina. Musculoelastic laminae with prominent elastin stained black (Figure 1). In previous work we have observed a concordance in the geo-
Versican, biglycan, and decorin were expressed in all three vessel wall layers of primary and restenotic atherectomized tissues. However, the distribution patterns of these three PGs were very different from one PG to another and between study groups. Versican staining was very prominent in the intima and media of restenotic lesions, more so than in primary ones (Figures 2D and 3D). The staining pattern for versican in restenotic neointima was diffuse, typically moderate throughout the thickened intima, but focally more intense in association with deep atheromata and around foam cells. Smooth
Figure 1. Photomicrographs of atherectomy fragments taken from a restenotic left anterior descending artery at 72 days following angiography. The restenotic tissue is sea-green and rich in glycosaminoglycans. An older cream-colored atheromatous plaque is superimposed on young “plaque” of restenotic tissue. A portion of adventitial tissue, including external elastic lamina, is evident. (Movat pentachrome stain. 340.)
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Figure 2. Photomicrographs of atherectomy fragments taken from a restenotic left anterior descending coronary artery at 119 days following angioplasty. (A) Movat’s stain shows young restenotic tissues with intimal thickening (especially leftward) and smooth-muscle cell proliferation. (B) The same field has many muscle-specific actin positive cells, presumptively smooth-muscle cells. (C–E) The proteoglycans (C, biglycan; D, versican; E, decorin) are distinctively in a mirror-image pattern relative to the smooth-muscle cells. The versican positivity is strikingly prominent. (All samples 3220.)
Figure 3. Higher power photomicrographs of material depicted in Figure 2. (A) Movat’s stain. (B) Muscle-specific actin. (C) Biglycan. (D) Versican, (E) Decorin. (All samples 3500.)
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muscle cells (SMCs), except foam cells in both intima and media, had intense staining for versican. Biglycan was less prominent in the intima of restenotic neointima than in the intima of primary coronary lesions, and SMCs were positive for biglycan in neointima of both restenosis and primary lesions. Decorin was almost not identifiable in the neointima of restenotic tissues, although decorin was detected at high levels in the intima of primary atherosclerotic coronary lesions. Strong intracellular expression of decorin was noted in intimal SMCs, and adventitial layers were generally very positive for decorin. In comparison with Movat’s pentachrome staining, versican was geographically correspondent. Thus, alcian-blue staining of acid mucopolysaccharides was mirrored by versican antibody reactivity, as noted in other conditions (16), whereas biglycan and decorin stains were less clearly represented by the Movat’s stain.
Immunohistochemical Grading Comparative results of immunohistochemical grading of versican, biglycan, and decorin in the intima and media were shown in Figure 4. Versican deposition in restenotic intima was apparently higher than in primary lesions (5.1 6 0.6 vs. 2.3 6 1.1, p , 0.05). Biglycan did not stain differently primary and restenotic tissues. However, decorin expression in restenotic specimens was significantly (p , 0.05) lower either in the intima or media than in primary stenotic tissues. In the adventitia of restenotic specimens (n 5 6), decorin (4.7 6 1.2) was prominent and versican (3.0 6 1.5) and biglycan (3.3 6 0.8) also were present. There was no adventitia in primary atherectomy tissues.
Figure 4. Comparison of staining intensity in atherectomy tissues between primary stenosis (solid bars) and restenosis (striped bars). *p , 0.05 versus primary stenosis. Predominant expression of versican in restenotic neointima was compared with primary stenosis, whereas decorin deposition in both of intima and media was significantly (p , 0.05) lower in restenotic tissue than in primary lesional tissue.
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Discussion The present immunohistochemical analysis was focused on the qualitative and semi-quantitative nature of PGs in primary atherosclerotic and restenotic coronary arteries. Each of three PGs evaluated has its own unique pattern of protein expression. The earliest response to balloon injury of a coronary artery includes striking upregulation of versican expression, whereas the limited expression of biglycan differs from the prominence of PG in the primary atherosclerosis. Decorin staining was virtually absent in the most proliferative neointimal tissue, although it was quite striking in established primary lesions. Therefore, versican may be involved in the earliest pathogenetic steps of neointimal restenosis. Previous studies in native atherosclerosis have concentrated on glycosaminoglycans (GAGs). Alavi and Moore (23) have shown in the balloon-injury rabbit model a marked increase in the content of chondroitin sulfate (CS) and dermatan sulfate (DS) in the lesions, whereas the content of heparin sulfate (HS) remained unaffected. This injury-induced increment in GAGs was aggravated by cholesterol feeding. A significant increase in the contents of CS/DS and cholesterol, but loss of HS, also was observed in human atherosclerotic tissues (24). In vitro studies employing rabbit aortic explants have demonstrated that in diet-induced atherosclerosis, aortic tissue synthesized twice the amount of PG as controls, predominantly CSPG (25). Biglycan and decorin are the major small DSPG, and versican is a major large CSPG in blood vessels. Whether differences in molecular weight contribute to more readily demonstrated expression of versican in the distinction between restenotic intima and primary atherosclerosis is uncertain. For certain, virtual absence of decorin and limited prescence of biglycan in restenotic lesions were confirmed, and primary intimal le-
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sions had intense antibody reactivity for both decorin and biglycan. Therefore, CSPG including versican, instead of DSPG, may be responsible for the initiation of restenotic neointimal proliferation. Microscopic studies have shown that DSPG is mainly associated with collagen fibrils, whereas CSPG is preferentially localized in nonfibrous areas of the extracellular matrix (26). CSPG in primary atherosclerotic tissues was prominent in close proximity to SMCs. Riessen et al. (27) illustrated PG composition of primary atherosclerosis as well as restenotic tissues by the analysis with biglycan and decorin antibodies. Biglycan, which colocalized with intense staining for collagen type I and III in their analysis, was most evident in the connective tissue of primary atherosclerosis and in proliferative neointima. Strong decorin expression was found in primary atherosclerosis, whereas restenotic neointima did not exhibit significant staining for decorin. These results are consistent with our observations of biglycan and decorin. Thus, restenotic arteriopathy, characterized by rapid disease progression, exhibits a PG deposition pattern that is distinct from that seen in primary atherosclerosis. In molecular studies, messenger RNA levels of versican and biglycan were increased at the intimo-medial junction in a carotid artery balloon injury model (28). Such observations are consistent with our results in restenotic arteries in human coronaries, wherein there are notable accumulations of versican and biglycan in the neointima. Expression of versican, biglycan, and decorin is known to be regulated by different mechanisms. In rat SMC cultures, decorin, but not biglycan, mRNA levels were significantly increased (29). Versican and biglycan have been found to be synthesized by both aortic SMCs and endothelial cells, whereas decorin was synthesized by SMCs only (20,30). In monkey arterial SMC cultures, platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF- b1) increase versican synthesis (31). If more TGF-b1 or PDGF were synthesized and more were available for binding and release within restenotic blood vessels than in primary atherosclerosis, the apparently distinctive versican and biglycan patterns in restenotic neointima, as opposed to decorin predominance in primary atherosclerosis, may partially explain the basis of our observations. Actually, vascular SMCs in balloon-injured rat aorta do not express more TGF-b1 than normal cells, but respond to TGF-b1 by producing more PGs (32). Multiple other factors contribute to PG patterns, such as the nature of injury, immune response, lipid metabolism, and modification of apoproteins. Further studies will be needed to explore the basis of differential regulation of PG expression. Versican is a CSPG originally isolated from fibroblasts (33). The carboxy-terminal protein includes two epidermal growth factor-like receptors, a lectin-like sequence, and a complement regulatory protein-like domain. Binding elements are identified as cell adhesion molecules (33). The domain at the NH2 terminus of versican is a member of the
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immunoglobulin superfamily, sequences involved in cell recognition, cell adhesion, or immune function (34). Therefore, in neointimal hyperplasia, versican may regulate cell recognition, cell adhesion events between injured endothelium and surrounding matrix, and remodeling of extracellular matrix. Both in chronic vascular rejection (35) and with the occlusion of saphenous vein grafts (36), intimal SMC proliferation occurs in a fashion similar to that seen in coronary artery restenosis. Histopathologic examination of lesions in chronic vascular rejection (37) revealed an extracellular matrix rich in proteoglycans. Although there are no published reports on proteoglycans in occlusion of saphenous vein grafts, histopathologic analysis shows an extracellular matrix rich in glycosaminoglycans, similar to the prominent “ground substance” of restenotic coronary arteries and of transplant vascular disease (chronic vascular rejection). We would speculate that proteoglycans, particularly versican, are more numerous in the extracellular matrix of intimal hyperplasia associated with the most rapidly changing vessels exposed to injurious stimuli, including restenosis, chronic vascular rejection, and occlusion of saphenous vein graft. Thus, although the temporal events and pathways leading to intimal thickening, luminal narrowing, and remodeling may certainly be distinct, the accumulation of matrix that includes substantial proteoglycans probably is an incoming pathway for such responses to injury. In summary, PG deposition in coronary arteries is a prominent feature in both primary atherosclerosis and restenotic arteriopathy. In particular, versican deposition contributes to the extent of early neointima after balloon angioplasty. Differing patterns of PG accumulation in restenotic arteriopathy and in primary lesions support a distinctive pathogenesis for the two diseases despite the eventual similarity of lipid and PG overload (1).
The authors wish to thank Mr. Michael Iagallo and Mrs. Yoshiko Nonaka for excellent histochemical assistance and Dr. Akihiko Nakano for providing atherectomized tissues. We gratefully acknowledge Dr. Larry Fisher, Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD, for providing the biglycan and decorin antibodies and Dr. Erkki Ruoslahti, Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA, for providing the versican antibody. This study was supported by a grant-in-aid (#5-53973) from the Heart and Stroke Foundation of British Columbia and Yukon to B. M. McManus, and a postdoctoral fellowship from the Heart and Stroke Foundation of Canada to H. Lin.
References 1. Vlietstra RE, Holmes DR Jr, Smith HC, et al. Percutaneous transluminal coronary angioplasty: initial Mayo Clinic experience. Mayo Clin Proc 1981;56:287–293. 2. Holt GW, Gersh BJ, Holmes DR Jr, et al. Results of percutaneous transluminal coronary angioplasty for angina pectoris early after myocardial infarction. Am J Cardiol 1988;61:1238–1242. 3. Meyer J, Merx W, Schmitz H, et al. Percutaneous transluminal coro-
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nary angioplasty after intracoronary streptolysis of transmural myocardial infarction. Circulation 1985;71:754–760. Mabin TA, Holmes DR Jr, Smith HC. Follow-up clinical results in patients undergoing percutaneous transluminal coronary angioplasty. Circulation 1985;71:754–760. Cowley MJ, Vetrovec GW, Wolfgang TC. Efficacy of percutaneous transluminal coronary angioplasty: technique, patient selection, salutary results, limitations, and complications. Am Heart J 1981;101:272–280. Kent KM, Bentivoglio LG, Block PC, et al. Long-term efficacy of percutaneous transluminal coronary angioplasty (PTCA): report from the National Heart, Lung and Blood Institute PTCA Registry. Am J Cardiol 1984;53:27C–81C. Meier B, King SB III, Gruentzig AR, et al. Repeat angioplasty. J Am Coll Cardiol 1984;4:463–466. Holmes DR Jr, Vlietstra RE, Smith HC, et al. Restenosis after percutaneous transluminal coronary angioplasty (PTCA): a report from PTCA Registry of the NHLBI. Am J Cardiol 1984;53:77C–81C. Meier B, King SB III, Gruentzig AR, et al. Repeat coronary angioplasty: review of the literature. Eur Heart J 1988;9(suppl C):1–6. Corcos T, David PR, Val PG, et al. Failure of diltiazem to prevent stenosis after percutaneous transluminal coronary angioplasty. Am Heart J 1985;109:926–931. Faxon DP, Sanborn TA, Haudenschild CC. Mechanism of angioplasty and its relation to restenosis. Am J Cardiol 1987;60:5B–9B. Thornton MA, Gruentzig AR, Hollman J, et al. Coumadin and aspirin in prevention of recurrence after transluminal coronary angioplasty: a randomized study. Circulation 1984;69:721–727. Grigg LE, Kay TWH, Valentine PA, et al. Determinants of restenosis and lack of effect of dietary supplementation with eicosapentaenoic acid on the incidence of coronary artery restenosis after angioplasty. J Am Coll Cardiol 1989;13:665–672. Garratt KN, Edwards WD, Kauffmann UP, et al. Differential histopathology of primary atherosclerotic and restenotic lesions in coronary arteries and saphenous vein bypass grafts: analysis of tissue obtained from 73 patients by directional atherectomy. J Am Coll Cardiol 1991;17:442–448. Block PC, Myler RH, Stertzer S, Fallon JT. Morphology after transluminal angioplasty in human beings. N Engl J Med 1981;305:382–385. Lin H, Wilson JE, Roberts CR, et al. Biglycan, decorin and versican in epicardial coronary arteries of human heart allografts: immunohistochemical evidence of particular patterns of deposition, distinctive from native atherosclerosis. J Heart Lung Transplant 1996;15:1233– 1247. Lin H, Ignatescu M, Wilson JE, et al. Prominence of apolipoproteins B, (a) and E in the intimae of coronary arteries in transplanted human hearts: geographical relationship to vessel wall proteoglycans. J Heart Lung Transplant 1996;15:1223-1232. Bianco P, Fischer LW, Young MF, et al. Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissue. J Biol Chem 1990;38:1549–1563. Fisher LW, Termine JD, Young MF, et al. Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of spaces. J Biol Chem 1989;264:4571–4576. Jarvelainen HT, Kinsella MG, Wight TN, Sandell LJ. Differential expression of small chondroitin/dermatan sulfate proteoglycans, PG-I/
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biglycan and PG-II/decorin, by vascular smooth muscle and endothelial cells in culture. J Biol Chem 1991;266:23274–23281. 21. Fisher LW, Hawkins GR, Tuross N, Termine JD. Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone. J Biol Chem 1987;262:9702–9708. 22. LeBaron RG, Zimmermann DR, Ruoslahti E. Hyaluronate binding properties of versican. J Biol Chem 1992;267:10003–10010. 23. Alavi MZ, Moore S. Proteoglycan composition of rabbit arterial wall under conditions of experimentally induced atherosclerosis. Atherosclerosis 1987;63:65–74. 24. Volker W, Schmit A, Oortmann W, Broszey T, Faber V, Buddecke E. Mapping of proteoglycans in atherosclerotic lesions. Eur Heart J 1990; II(Suppl):29–40. 25. Radhakrishnamurthy B, Srinivasan S, Eberle K, Ruiz H, Dalferes ER Jr, Sharma C, Berenson GS. Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis. Biochem Biophys Acta 1988;964:231–243. 26. Volker W, Schmit A, Buddecke E. Cytochemical changes in a human arterial proteoglycan related to atherosclerosis. Atherosclerosis 1989; 77:117–130. 27. Riessen R, Isner JM, Blessing E, Loushin C, Nikol S, Wight TN. Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries. Am J Pathol 1994;144:962–974. 28. Nikkai ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol 1994;144:1348–1356. 29. Asundi VK, Dreher KL. Molecular characterization of vascular smooth muscle decorin: deduced core protein structure and regulation of gene expression. Eur J Cell Biol 1992;59:314–321. 30. Yao LY, Moody C, Schonherr E, Wight TN, Sandell LJ. Identification of proteoglycan versican in aorta and smooth muscle cells by DNA sequence analysis, in situ hybridization and immunohistochemistry. Matrix Biol 1994;14:213–225. 31. Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN. Effects of platelet-derived growth factor and transforming growth factor b1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem 1991;266:17640–17647. 32. Rasmussen LM, Wolf YG, Ruoslahti E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-b-activity. Am J Pathol 1995;147: 1041–1048. 33. Zimmerman DR, Rouslahti E. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 1989;8:2975–2981. 34. Handingham TE. Proteoglycans: many forms and many functions. FASEB J 1992;6:861–870. 35. Hammond EH, Hansen JK, Spencer LS, et al. Vascular rejection in cardiac transplantation: histological, immunopathologic and ultrastructural features. Cardiovasc Pathol 1993;2:21–34. 36. Angelini GD, Newby AC. The future of saphenous vein as a coronary artery bypass conduit. Eur Heart J 1989;10:273–280. 37. Chow LH, Huh S, Jiang J, Zong R, Pickering JG. Intimal thickening develops without humoral immunity in a mouse aortic allograft model of chronic vascular rejection. Circulation 1996;94:3079–3082.
11 The pathobiology of rapid intimal thickening following balloon angioplasty remains unsettled. Proteoglycans (PGs) expressed by smooth-muscle cells (SMCs) are known to participate in vascular responses to injury. In this analysis, patients ranging from age 48 to 79 years (mean 5 58), underwent atherectomy for 36 restenotic tissues (taken 64 to 345 days postangioplasty; mean 5 108) and for 10 primary atherosclerotic plaques. Tissues were formaldehyde-fixed, paraffin-embedded, and histochemically and immunohistochemically stained to determine the temporal and semi-quantitative contribution of major vessel wall PGs, versican, biglycan, and decorin. Versican was the most striking PG in the neointima of restenotic vessels, including a prominent pericellular pattern corresponding to proliferative SMCs, as well as a large extracellular accumulation. Biglycan was limited to the most loose and proliferative neointima and stained less than in primary plaques. Decorin staining was virtually absent in the most proliferative neointimal tissue, whereas it was quite striking in established primary lesions. Thus the earliest response to balloon injury of a coronary artery includes striking expression of versican protein, but the limited expression of biglycan differs from the prominence of the PG in primary atherosclerosis. Versican expression in restenotic lesions is similar to that seen previously in transplant arteriopathy, but the lack of biglycan in atherectomy specimens from restenosis sites is distinctively different from that seen in rapidly progressive transplant vascular disease. Cardiovasc Pathol 1998;7:31–37. © 1998 by Elsevier Science Inc.
Despite the effectiveness and safety of percutaneous transluminal coronary angioplasty (PTCA) for treating ischemic coronary disorders (1–6), restenosis occurs in 30% to 50% of treated patients (7–9). There are no medical regimens or variations of angioplasty that have yet consistently lowered the rate of restenosis after PTCA (10–13). Vessel wall remodeling after PTCA injury is incompletely understood, and new directions are warranted for investigation based on a concept of rapidly reparative events. In particular, the role
Manuscript received June 3, 1996; revised October 21, 1996; accepted May 30, 1997. Address for reprints: Bruce M. McManus, MD, PhD, Department of Pathology and Laboratory Medicine, St Paul’s Hospital, 1081 Burrard Street, Vancouver, Canada V6Z 1Y6; telephone: (604) 631-5200; fax: (604) 631-5208. Cardiovascular Pathology Vol. 7, No. 1, January/February 1998:31–37 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
of extracellular matrix in restenotic lesions deserves more attention. Previous studies have shown that primary atherosclerotic lesions, when excised by atherectomy or as observed at autopsy, have the classical characteristics of fibrous plaques accompanied by foam cells and extracellular lipid. Restenotic lesions following balloon angioplasty are characterized by smooth-muscle cell proliferation and extracellular matrix synthesis, which together form a neointima, histopathologically similar to other rapidly progressive lesions, such as transplant arteriopathy (14,15). Previous immunohistochemical studies by our laboratory (16) have revealed abundant proteoglycan (PG) deposits, especially versican and biglycan, in the rapidly expanding intima of allograft coronary arteries in a pattern distinct from that of native coronary artery disease. We have dem-
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onstrated that certain apolipoproteins, especially apo(a) and apoE, are much more prominent in the intima of transplant arteriopathy than in native atherosclerotic disease. These apolipoproteins and associated PGs, especially versican, constitute the major lipid accumulation and extracellular matrix volume, respectively, in that rapidly evolving disease process (17). It is known that PGs play a fundamental role in self-surface anchorage for lipoprotein lipase on endothelium, in sequestration of lipoproteins in the subendothelial matrix, and in presentation of lipoproteins to phagocytic intimal cells in the setting of native atherosclerosis. Much less is known about restenosis. In the present study, we characterize the amount and distribution of major PGs in restenotic neointima using immunohistochemical techniques and a comparative sample of primary atherosclerotic plaques, also taken by atherectomy.
Materials and Methods Patients Thirty-one unselected, human atherectomy procedures were undertaken from 24 male and 7 female patients with coronary artery disease. The patients were hospitalized in Saiseikai Maebashi Hospital of Japan. Their ages ranged from 48 to 70 years (mean 5 58). The duration postangioplasty was from 64 to 345 days (mean 5 108). Ten primary atherosclerotic lesions and 36 restenotic tissues were included in the analysis.
Tissue Processing Human atherectomy tissues were collected as soon as possible after coronary atherectomy using the Simpson AtheroCath (Devices for Vascular Intervention, Redwood City, CA). The specimens were formaldehyde-fixed and paraffin-embedded, 4- to 5-m sections were cut, placed on SuperfrostTM slides, and baked for 30 minutes at 458C for histochemical and immunohistochemical staining.
Histochemical Staining Serial sections of primary and restenotic coronary artery tissues were stained with a modified Movat’s pentachrome stain. Smooth-muscle cellular components were corroborated by staining with the muscle-actin–specific antibody HHF35 (18).
Immunohistochemical Study Rabbit polyclonal antibodies against biglycan (LF15) and decorin (LF30) were generous gifts from Dr. L. Fisher (National Institute of Dental Research, Bethesda, MD). The antisera were raised against specific synthetic peptides and shown to be monospecific (19–21). A rabbit polyclonal an-
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tibody against versican was kindly provided by Dr. E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA). A fusion protein of versican was used as the antigen to raise the polyclonal antiserum, which was subsequently affinity-purified on a versican synthetic 26-mer peptide column (22). After deparaffinization, the sections were treated with 1 U/ml of chondroitinase ABC in 0.1 M Tris-HCl/50mM calcium acetate/0.1% bovine serum albumin (BSA) pH at 7.2 for 30 minutes at 378C (18), to detach glycosaminoglycan side chains from the core protein. After chondroitinase treatment, the sections were washed thoroughly in Tris-2% BSA for 2 hours at room temperature. The sections were then washed in TBS, incubated in 1:100 dilution of goat anti-rabbit IgG-alkaline phosphatase in the presence of levamisole. Following color development, sections were counterstained with Mayer’s hematoxylin and mounted. The experiment included a negative control using nonimmune normal rabbit serum in place of the biglycan antibody and a positive control using coronary tissue with large amounts of PG present. Negative controls and internal positive controls were included in each run to evaluate consistency of staining from run to run and to allow comparison of results between runs. Slides were stained for decorin and versican by an automated method using the Ventana immunostainer (Ventana Medical Systems, Inc., Tucson, AZ). The Ventana Alkaline Phosphatase Fast Red Detection Kit includes prediluted biotinylated secondary antibody in PBS, alkaline phosphatasestreptavidin conjugate in PBS, fast red A and fast red B in acetate buffer, naphthol in Tris buffer, enhancer (MgCl2) in aqueous solution, and a negative control reagent. The automated procedure is similar to the manual procedure established in the laboratory. Decorin and versican antibodies were diluted to 1:100 in an antibody-diluting buffer (Dimension Laboratories, Inc., Mississauga, Ontario, Canada) that included the primary antibodies for 32 minutes and then a secondary antibody for 8 minutes at 378C.
Grading of Immunohistochemical Stains Slides were scored in a blinded fashion by one investigator for intensity of PG staining against an intensity scale developed for this purpose (0–61) and for PG localization in the specimens including intima, neointima, media and adventitia. The scale of scoring was defined as follows: 0, no staining; 11, trace amount; 21, small amount; 31, small to moderate amount; 41, moderate amount; 51, considerable amount; 61, maximum amount. When staining intensity was not uniform in a particular tissue layer, a focal peak score and an overall score were rendered, and the mean of the two scores was used for final analysis. The repeatability of scoring was randomly tested by the same investigator without his knowledge. This reproducibility is comparable to that obtained with other semi-quantitative grading schemata in histopathologic studies (21).
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Statistical Analysis Staining intensity was compared between the primary and restenotic groups by two-way analysis of variance (ANOVA). The probability of an alpha error was set at p , 0.05 for statistical significance.
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graphical histochemical staining pattern of glycosaminoglycans by Movat’s pentachrome and immunohistochemical staining for versican (16,17). Specimens from primary atherectomy tissue included intima in 10/10 (100%), media in 8/10 (80%), but no adventitia; whereas, restenotic tissue at times included all three layers, intima in 38/38 (100%), media in 16/38 (42%) and adventitia in 6/38 (16%).
Results Histologic Staining
Immunohistochemical Staining
As noted, atherectomy tissue samples taken from human coronary arteries were first characterized with Movat’s pentachrome stain. These stains revealed features typical of primary and restenotic lesions. Movat’s stain distinguished lesions as follows: older atheromatous plaques were distinguished as yellow-cream, with superimposed young restenotic tissue as sea-green (glycosaminoglycans), and a small portion of adventitia with yellow-staining collagenous tissue outward from the external elastic lamina. Musculoelastic laminae with prominent elastin stained black (Figure 1). In previous work we have observed a concordance in the geo-
Versican, biglycan, and decorin were expressed in all three vessel wall layers of primary and restenotic atherectomized tissues. However, the distribution patterns of these three PGs were very different from one PG to another and between study groups. Versican staining was very prominent in the intima and media of restenotic lesions, more so than in primary ones (Figures 2D and 3D). The staining pattern for versican in restenotic neointima was diffuse, typically moderate throughout the thickened intima, but focally more intense in association with deep atheromata and around foam cells. Smooth
Figure 1. Photomicrographs of atherectomy fragments taken from a restenotic left anterior descending artery at 72 days following angiography. The restenotic tissue is sea-green and rich in glycosaminoglycans. An older cream-colored atheromatous plaque is superimposed on young “plaque” of restenotic tissue. A portion of adventitial tissue, including external elastic lamina, is evident. (Movat pentachrome stain. 340.)
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Figure 2. Photomicrographs of atherectomy fragments taken from a restenotic left anterior descending coronary artery at 119 days following angioplasty. (A) Movat’s stain shows young restenotic tissues with intimal thickening (especially leftward) and smooth-muscle cell proliferation. (B) The same field has many muscle-specific actin positive cells, presumptively smooth-muscle cells. (C–E) The proteoglycans (C, biglycan; D, versican; E, decorin) are distinctively in a mirror-image pattern relative to the smooth-muscle cells. The versican positivity is strikingly prominent. (All samples 3220.)
Figure 3. Higher power photomicrographs of material depicted in Figure 2. (A) Movat’s stain. (B) Muscle-specific actin. (C) Biglycan. (D) Versican, (E) Decorin. (All samples 3500.)
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muscle cells (SMCs), except foam cells in both intima and media, had intense staining for versican. Biglycan was less prominent in the intima of restenotic neointima than in the intima of primary coronary lesions, and SMCs were positive for biglycan in neointima of both restenosis and primary lesions. Decorin was almost not identifiable in the neointima of restenotic tissues, although decorin was detected at high levels in the intima of primary atherosclerotic coronary lesions. Strong intracellular expression of decorin was noted in intimal SMCs, and adventitial layers were generally very positive for decorin. In comparison with Movat’s pentachrome staining, versican was geographically correspondent. Thus, alcian-blue staining of acid mucopolysaccharides was mirrored by versican antibody reactivity, as noted in other conditions (16), whereas biglycan and decorin stains were less clearly represented by the Movat’s stain.
Immunohistochemical Grading Comparative results of immunohistochemical grading of versican, biglycan, and decorin in the intima and media were shown in Figure 4. Versican deposition in restenotic intima was apparently higher than in primary lesions (5.1 6 0.6 vs. 2.3 6 1.1, p , 0.05). Biglycan did not stain differently primary and restenotic tissues. However, decorin expression in restenotic specimens was significantly (p , 0.05) lower either in the intima or media than in primary stenotic tissues. In the adventitia of restenotic specimens (n 5 6), decorin (4.7 6 1.2) was prominent and versican (3.0 6 1.5) and biglycan (3.3 6 0.8) also were present. There was no adventitia in primary atherectomy tissues.
Figure 4. Comparison of staining intensity in atherectomy tissues between primary stenosis (solid bars) and restenosis (striped bars). *p , 0.05 versus primary stenosis. Predominant expression of versican in restenotic neointima was compared with primary stenosis, whereas decorin deposition in both of intima and media was significantly (p , 0.05) lower in restenotic tissue than in primary lesional tissue.
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Discussion The present immunohistochemical analysis was focused on the qualitative and semi-quantitative nature of PGs in primary atherosclerotic and restenotic coronary arteries. Each of three PGs evaluated has its own unique pattern of protein expression. The earliest response to balloon injury of a coronary artery includes striking upregulation of versican expression, whereas the limited expression of biglycan differs from the prominence of PG in the primary atherosclerosis. Decorin staining was virtually absent in the most proliferative neointimal tissue, although it was quite striking in established primary lesions. Therefore, versican may be involved in the earliest pathogenetic steps of neointimal restenosis. Previous studies in native atherosclerosis have concentrated on glycosaminoglycans (GAGs). Alavi and Moore (23) have shown in the balloon-injury rabbit model a marked increase in the content of chondroitin sulfate (CS) and dermatan sulfate (DS) in the lesions, whereas the content of heparin sulfate (HS) remained unaffected. This injury-induced increment in GAGs was aggravated by cholesterol feeding. A significant increase in the contents of CS/DS and cholesterol, but loss of HS, also was observed in human atherosclerotic tissues (24). In vitro studies employing rabbit aortic explants have demonstrated that in diet-induced atherosclerosis, aortic tissue synthesized twice the amount of PG as controls, predominantly CSPG (25). Biglycan and decorin are the major small DSPG, and versican is a major large CSPG in blood vessels. Whether differences in molecular weight contribute to more readily demonstrated expression of versican in the distinction between restenotic intima and primary atherosclerosis is uncertain. For certain, virtual absence of decorin and limited prescence of biglycan in restenotic lesions were confirmed, and primary intimal le-
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sions had intense antibody reactivity for both decorin and biglycan. Therefore, CSPG including versican, instead of DSPG, may be responsible for the initiation of restenotic neointimal proliferation. Microscopic studies have shown that DSPG is mainly associated with collagen fibrils, whereas CSPG is preferentially localized in nonfibrous areas of the extracellular matrix (26). CSPG in primary atherosclerotic tissues was prominent in close proximity to SMCs. Riessen et al. (27) illustrated PG composition of primary atherosclerosis as well as restenotic tissues by the analysis with biglycan and decorin antibodies. Biglycan, which colocalized with intense staining for collagen type I and III in their analysis, was most evident in the connective tissue of primary atherosclerosis and in proliferative neointima. Strong decorin expression was found in primary atherosclerosis, whereas restenotic neointima did not exhibit significant staining for decorin. These results are consistent with our observations of biglycan and decorin. Thus, restenotic arteriopathy, characterized by rapid disease progression, exhibits a PG deposition pattern that is distinct from that seen in primary atherosclerosis. In molecular studies, messenger RNA levels of versican and biglycan were increased at the intimo-medial junction in a carotid artery balloon injury model (28). Such observations are consistent with our results in restenotic arteries in human coronaries, wherein there are notable accumulations of versican and biglycan in the neointima. Expression of versican, biglycan, and decorin is known to be regulated by different mechanisms. In rat SMC cultures, decorin, but not biglycan, mRNA levels were significantly increased (29). Versican and biglycan have been found to be synthesized by both aortic SMCs and endothelial cells, whereas decorin was synthesized by SMCs only (20,30). In monkey arterial SMC cultures, platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF- b1) increase versican synthesis (31). If more TGF-b1 or PDGF were synthesized and more were available for binding and release within restenotic blood vessels than in primary atherosclerosis, the apparently distinctive versican and biglycan patterns in restenotic neointima, as opposed to decorin predominance in primary atherosclerosis, may partially explain the basis of our observations. Actually, vascular SMCs in balloon-injured rat aorta do not express more TGF-b1 than normal cells, but respond to TGF-b1 by producing more PGs (32). Multiple other factors contribute to PG patterns, such as the nature of injury, immune response, lipid metabolism, and modification of apoproteins. Further studies will be needed to explore the basis of differential regulation of PG expression. Versican is a CSPG originally isolated from fibroblasts (33). The carboxy-terminal protein includes two epidermal growth factor-like receptors, a lectin-like sequence, and a complement regulatory protein-like domain. Binding elements are identified as cell adhesion molecules (33). The domain at the NH2 terminus of versican is a member of the
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immunoglobulin superfamily, sequences involved in cell recognition, cell adhesion, or immune function (34). Therefore, in neointimal hyperplasia, versican may regulate cell recognition, cell adhesion events between injured endothelium and surrounding matrix, and remodeling of extracellular matrix. Both in chronic vascular rejection (35) and with the occlusion of saphenous vein grafts (36), intimal SMC proliferation occurs in a fashion similar to that seen in coronary artery restenosis. Histopathologic examination of lesions in chronic vascular rejection (37) revealed an extracellular matrix rich in proteoglycans. Although there are no published reports on proteoglycans in occlusion of saphenous vein grafts, histopathologic analysis shows an extracellular matrix rich in glycosaminoglycans, similar to the prominent “ground substance” of restenotic coronary arteries and of transplant vascular disease (chronic vascular rejection). We would speculate that proteoglycans, particularly versican, are more numerous in the extracellular matrix of intimal hyperplasia associated with the most rapidly changing vessels exposed to injurious stimuli, including restenosis, chronic vascular rejection, and occlusion of saphenous vein graft. Thus, although the temporal events and pathways leading to intimal thickening, luminal narrowing, and remodeling may certainly be distinct, the accumulation of matrix that includes substantial proteoglycans probably is an incoming pathway for such responses to injury. In summary, PG deposition in coronary arteries is a prominent feature in both primary atherosclerosis and restenotic arteriopathy. In particular, versican deposition contributes to the extent of early neointima after balloon angioplasty. Differing patterns of PG accumulation in restenotic arteriopathy and in primary lesions support a distinctive pathogenesis for the two diseases despite the eventual similarity of lipid and PG overload (1).
The authors wish to thank Mr. Michael Iagallo and Mrs. Yoshiko Nonaka for excellent histochemical assistance and Dr. Akihiko Nakano for providing atherectomized tissues. We gratefully acknowledge Dr. Larry Fisher, Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD, for providing the biglycan and decorin antibodies and Dr. Erkki Ruoslahti, Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA, for providing the versican antibody. This study was supported by a grant-in-aid (#5-53973) from the Heart and Stroke Foundation of British Columbia and Yukon to B. M. McManus, and a postdoctoral fellowship from the Heart and Stroke Foundation of Canada to H. Lin.
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