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Biol. Pharm. Bull. 27(11) 1763—1768 (2004)
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Characterization of Chondroitin/Dermatan Sulfate Proteoglycans Synthesized by Bovine Retinal Pericytes in Culture Toshiyuki KAJI,*,a Shigeru SAKURAI,a,b Chika YAMAMOTO,a Yasuyuki FUJIWARA,a Sho-ichi YAMAGISHI,b Hiroshi YAMAMOTO,b Michael G. KINSELLA,c and Thomas N. WIGHTc a Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University; Ho-3 Kanagawa-machi, Kanazawa 920–1181, Japan: b Department of Biochemistry, Kanazawa University School of Medicine; 13–1 Takaramachi, Kanazawa 920–8640, Japan: and c Department of Vascular Biology, The Hope Heart Institute; 1124 Columbia St, No. 783, Seattle, Washington 98104–2046, U.S.A. Received August 5, 2004; accepted September 6, 2004; published online September 9, 2004
Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [3H]glucosamine, [35S]sodium sulfate or 35S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high Mr subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a Kav ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1—0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately Mr 45000 in biglycan and decorin, approximately Mr 48000 in the small amount of HSPGs and approximately Mr 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells. Key words
pericyte; proteoglycan; extracellular matrix; biglycan; decorin; versican
Microvessels are composed of endothelial cells and pericytes. Pericytes are elongated cells of mesodermal origin that wrap around and along endothelial cells in small vessels.1) Co-culture of endothelial cells with pericytes results in an inhibition of the proliferation and movement of endothelial cells.2—4) Although endothelial cells promote the growth of pericytes by secretion of endothelin-1,5) the inhibition of endothelial cell growth of pericytes is achieved by close apposition of these two cell types, suggesting that the extracellular matrix synthesized by pericytes may be involved in modulating endothelial cell behavior. Pericytes synthesize and secrete extracellular matrix components including proteoglycans (PGs), thrombospondin, fibronectin, laminin, tenascin and several different collagens.6—8) Early studies showed that bovine retinal microvascular pericytes synthesized glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid9,10) but the type of core protein to which these GAGs are attached remains largely unknown, although production of decorin was observed in tissue-specific pericytes such as kidney glomular mesangial cells and liver fat-string cells.11) However, it has been shown that bovine brain microvascular pericytes synthesize aggrecan,12) a chondroitin sulfate PG normally found in cartilage. Such findings raise the possibility that pericytes, under certain conditions, can assume a chondrogenic phenotype. However, characteris∗ To whom correspondence should be addressed.
tics of PGs synthesized by pericytes have been incompletely understood. Since PGs are known to influence the activity of growth factors such as fibroblast growth factor-2 and transforming growth factor-b ,13—15) it may be that PGs synthesized by pericytes could regulate endothelial cell proliferation, migration and angiogenesis. In fact, previous studies have shown that decorin, a small dermatan sulfate PG, inhibits endothelial cell migration16) but promotes new vessel growth in vitro.17) Furthermore neovascularization that occurs in vascular disease is associated with increased decorin expression.18,19) Such studies highlight the importance of defining the types of PGs synthesized by pericytes. In the present study, bovine retinal pericytes were cultured and their PGs were characterized by biochemical techniques. We demonstrate that retinal pericytes predominantly synthesize CS/DS PGs, which are versican, biglycan and decorin, as well as a small amount of HSPGs. MATERIALS AND METHODS Materials Pericytes were isolated from bovine retina by the method of Capetandes and Gerritsen20) and identified as pericytes by their characteristic size, contour, lack of “hill and valley” growth pattern, positive stain for a isoform of smooth muscle actin21) and inhibitory effect on endothelial
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© 2004 Pharmaceutical Society of Japan
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cell growth when co-cultured with endothelial cells.2,4) Vascular endothelial and smooth muscle cells from bovine aorta were kindly provided by Drs. Yutaka Nakashima and Katsuo Sueishi (Division of Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan). RPMI 1640 medium and fetal bovine serum were from Nissui Pharmaceutical (Tokyo, Japan) and Summit Biotechnology (Ft. Collins, CO, U.S.A.), respectively; tissue culture dishes and plates were from Iwaki (Chiba, Japan); D-[63 H(N)]Glucosamine hydrochloride (2738 GBq/mmol), [35S]Na2SO4 (carrier free) and Tran35S-label metabolic labeling reagent which consists of ca. 70% L-[35S]methionine, ca. 15% 35 35 L-[ S]cysteine and other S-labeled compounds were from MP Pharmaceuticals (Irvine, CA, U.S.A.); Sepharose CL-4B, Sepharose CL-6B, PD-10 columns (disposable Sephadex G-25M), enzyme-linked cheomoluninescence Western blotting detection reagents, horseradish peroxidase-linked protein A, nitrocellurose membranes (Hybond ECL) and Hyperfilm ECL were from Amersham Biosciences (Piscataway, NJ, U.S.A.); DEAE-Sephacel, benzamidine, Tris base, dextran blue, phenylmethansulfonyl fluoride, bovine serum albumin fraction V, papain (1.7 U/mg) and heparitinase (EC 4.2.2.8. derived from Flavobacterium heparinum) were from Sigma Aldrich (St. Louis, MO, U.S.A.); urea, phenol ical and sodium dodecyl sulfate were from Wako Pure Chemical Industries (Osaka, Japan); chondroitin ABC lyase (EC 4.2.2.4. derived from Proteus vulgaris) and chondroitin sulfate A were from Seikagaku Kogyo (Tokyo, Japan); pronase was from Boehringer-Mannheim (Germany); a rabbit antibody against bovine and human versican core was kindly provided by Richard LeBaron (Division of Life Sciences, Cell and Molecular Biology, University of Texas at San Antonio, TX, U.S.A.); rabbit antibodies against bovine decorin (LF-94) and biglycan (LF-96) were kindly provided by Dr. Larry Fisher (National Institute of Dental Research, Bore Research Branch, Bethesda, MD, U.S.A.); XAR 5 films, developer and fixer were from Eastman Kodak (Rochester, NY, U.S.A.); cetylpyridinium chloride and other reagents were from Nacalai Tesque (Kyoto, Japan). Accumulation of GAGs Pericytes were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum in 6-well culture plates at 37 °C in a humid atmosphere of 5% CO2 in air until confluent. They were incubated at 37 °C for 3, 6, 12, 24, 48 and 72 h in RPMI 1640 medium supplemented with 10% fetal bovine serum in the presence of [3H]glucosamine (100 kBq/ml). After incubation, the medium was harvested and the cell layer was washed with calcium and magnesium free phosphate-buffered saline (CMF-PBS); the wash was combined with the corresponding medium. The cell layer was incubated at 37 °C for 5 min with CMF-PBS containing 0.2% trypsin and 0.02% EDTA and the cell suspension was collected. The culture well was washed with CMF-PBS and the wash was combined with the cell suspension. The cell suspension was centrifuged at 1500 g for 20 min to obtain the supernatant. The cell supernatant includes GAGs derived from the cell surface and the solubilized extracellular matrix.22) The cell supernatant and the harvested medium were used for the determination of GAGs by the method of Wasteson et al.23) as follows: the cell supernatant and the medium were incubated with 3 mg/ml pronase
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at 50 °C for 3 h. The pronase digest was mixed with 4 mg/ml carrier chondroitin sulfate A and 0.5% cetylpyridinium chloride. After incubation at 37 °C for 30 min, the mixture was centrifuged at 1500 g for 10 min to obtain the precipitated GAGs. The precipitate was dissolved in 4 M NaCl and re-precipitated by addition of 80% aqueous ethanol. The precipitate was collected by centrifugation at 1500 g for 10 min and dissolved in distilled water. The incorporated radioactivity was measured by liquid scintillation counting. Dissociative Extraction of PGs Confluent cultures of pericytes, vascular endothelial and smooth muscle cells were incubated at 37 °C for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum in the presence of [35S]sulfate (740 kBq/ml) or 35S-labeled amino acids (1 MBq/ ml) in 100-mm dishes. The conditioned medium was harvested and solid urea was added at a concentration of 8 M (“the medium extract”). The cell layer was washed with CMF-PBS and PGs were extracted with 8 M urea buffer (pH 7.5) containing 0.1 M 6-aminohexanoic acid, 5 mM benzamidine, 10 mM N-ethylmaleimide, 2 mM EDTA, 0.1 M NaCl, 50 mM Tris base, 1 mM phenylmethansulfonyl fluoride and 2% Triton X-100 at 4 °C for 15 min. The cell layer was harvested with a rubber policeman and the dish was washed with the urea buffer; the wash was combined with the harvested cell layer (“the cell layer extract”). The cell layer and medium extracts were chromatographed on PD-10 columns equilibrated in 8 M urea buffer (pH 7.5) containing 2 mM EDTA, 0.1 M NaCl, 0.5% Triton X-100 and 50 mM Tris base to obtain high molecular weight (3 kDa) macromolecules. DEAE-Sephacel Ion Exchange Chromatography To separate PGs on the basis of differences in charge density, the macromolecules labeled with [35S]sulfate or 35S-labeled amino acids were applied to a DEAE-Sephacel column (5 ml of resin) in 8 M urea buffer (pH 7.5) containing 2 mM EDTA, 0.1 M NaCl, 0.5% Triton X-100 and 50 mM Tris base. Unbound radioactivity was removed from the column by washing with 30 ml of the buffer. Bound radioactivity was eluted from the column with a linear gradient of 0.1—0.7 M NaCl in the urea buffer (total volume of 50 ml). Analysis of GAGs [35S]Sulfate-labeled PGs separated by DEAE-Sephacel chromatography were digested with 30 m g/ml papain in 0.1 M acetate buffer (pH 7.0) containing 5 mM EDTA and 5 mM cysteine at 65 °C for 4 h or with 1 U/ml heparitinase in 0.1 M Tris–HCl buffer (pH 7.0) containing calcium acetate at 37 °C for 4 h or with 1.7 U/ml chondroitin ABC lyase in 50 mM Tris–HCl buffer (pH 7.4) at 37 °C for 4 h. The digests were chromatographed on a Sepharose CL-6B column (0.990 cm) equilibrated in 0.2 M Tris–HCl buffer (pH 7.0) containing 0.2 M NaCl. The void and total volumes were estimated by the elution position of dextran blue and phenol red, respectively. Estimates of GAG chain size were made by comparison of Sepharose CL-6B elution Kav, determined experimentally, with a previously published curve of log Mr versus Kav on Sepharose CL-6B for chondroitin sulfate chains of various known Mr.24) Sepharose CL-4B Molecular Sieve Chromatography and Analysis of PG Core Proteins Subclasses of [35S]sulfate-labeled CS/DS PGs from the medium that were separated by DEAE-Sephacel chromatography were concentrated and chromatographed on a Sepharose CL-4B column (0.9
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90 cm) in 8 M urea buffer containing 0.25 M NaCl to estimate their hydrodynamic size. In another experiment, PGs labeled with 35S-labeled amino acids were separated by DEAESephacel chromatography and digested with chondroitin ABC lyase as described above. The CS/DS PG core proteins were separated by SDS-PAGE according to the procedure of Laemmli25) on acrylamide 4—12% gradient slab gels with a 3% stacking gel. The radioactive PG cores were visualized by fluorography of dried gels previously treated with Enlightening Enhancer and exposed to Kodak XAR-5 films at 70 °C. For Western blot analysis, the SDS-PAGE gels were equilibrated in 25 mM Tris transfer buffer (pH 9.5) with 20% methanol and transferred to nitrocellurose membrane for 90 min with a semidry transfer apparatus (Atoo, AE-6677). The membrane was blocked and exposed to primary antibodies against versican, biglycan and decorin (each diluted 1 : 1000) overnight at 4 °C. After incubation of the blots with horseradish peroxidase-linked protein A, bands that bound to the primary antibodies were visualized by an enzyme-linked chemiluminescence procedure.
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Fig. 1. Accumulation of [3H]Glucosamine-Labeled GAGs in the Cell Layer and the Conditioned Medium of Cultured Pericytes Confluent cultures of bovine retinal pericytes were incubated at 37 °C for 3, 6, 12, 24, 48 and 72 h in the presence of [3H]glucosamine.
RESULTS Pericytes Synthesize and Actively Secrete GAGs Figure 1 shows the accumulation of GAGs in the cell layer and the medium of pericytes. Incorporation of [35S]sulfate into PGs within the pericyte cell layer plateaued by 24 h while those released into the medium increased steadily during a 72 h labeling period. By 72 h, approximately 74% of the newly synthesized PGs were present in the medium with the remainder, 26%, associated with the cell layer. PGs Synthesized by Pericytes Resemble Those Synthesized by Vascular Smooth Muscle Cells but Differ from Those Synthesized by Vascular Endothelial Cells The cell layer and medium extracts of [35S]sulfate-labeled pericyte cultures were submitted to DEAE-Sephacel ion exchange chromatography to separate the PGs based on charge density. In the cell layer, radiolabeled peaks eluted at NaCl concentrations of 0.4 M (C-I), 0.5 M (C-II) and 0.6 M (C-III) (Fig. 2A). In the conditioned medium, radiolabeled peaks eluted at NaCl concentrations of 0.4 M (shoulder, M-I) 0.5 M (M-II) and 0.6 M (M-III) (Fig. 2B). The predominant peak in the cell layer was C-III (approximately 40% of the total) and that from the conditioned medium was M-III (approximately 63% of the total). When these profiles were compared to those obtained from cultures of vascular smooth muscle cells radiolabeled under the same conditions, both major peaks from the cell layer and medium arterial smooth muscle cell cultures corresponded to C-III and M-III in the pericyte cultures (Figs. 2C, D), suggesting similarities in the synthesis of PGs by these two cell types. However, these patterns differed dramatically from the DEAE profiles of radiolabeled PGs isolated from cultured endothelial cells which lacked radiolabeled peaks corresponding to the elution position of C-III and M-III in the pericyte cultures (Figs. 2E, F). These results suggest that PGs synthesized by endothelial cells differ from those synthesized by pericytes and vascular smooth muscle cells. Pericytes Predominantly Synthesize CS/DS PGs The GAGs bound to all PGs synthesized by pericytes were characterized by Sepharose CL-6B chromatography and the
Fig. 2. DEAE-Sephacel Chromatography of [35S]Sulfate-Labeled PGs Extracted from the Cell Layers (A, C and E) and the Conditioned Media (B, D and F) of Pericytes (A and B), Vascular Smooth Muscle Cells (C and D) and Endothelial Cells (E and F) with a Linear Gradient of 0.1—0.7 M NaCl in 8 M Urea Buffer Confluent cultures of bovine retinal pericytes, bovine aortic smooth muscle cells and bovine aortic endothelial cells were incubated at 37 °C for 24 h in the presence of 35 [ S]sulfate. Horizontal bars indicate the fractions that were pooled, and chromatographed on Sepharose CL-6B (see Fig. 3), Sepharose CL-4B columns (see Fig. 4) and used for core protein analysis (see Fig. 5).
results are shown in Fig. 3. In the Sepharose CL-6B profiles, C-I was shifted from the void volume to Kav of 0.33 and 0.75/0.85 by digestion with papain and heparitinase, respectively, indicating that C-I is composed of HSPGs and the length of heparan sulfate chains is Mr ca. 48000. Since C-II and M-II were shifted from the void volume to Kav of 0.34 and 0.79 by digestion with papain and chondroitin ABC lyase, respectively, both C-II and M-II are composed of CS/DS PGs and the length of chondroitin/dermatan sulfate chains is Mr ca. 45000. Similarly, it was indicated that C-III and M-III are also composed of CS/DS PGs with chondroitin/dermatan sulfate chains of Mr ca. 66000. However, M-I could not be analyzed because of low radioactivity. Pericytes Synthesize Both Large and Small CS/DS PGs The CS/DS PGs in DEAE peaks C-II, C-III, M-II and M-III were the major PGs synthesized by pericytes. Differences in the hydrodynamic sizes of these populations were deter-
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mined by Sepharose CL-4B chromatography. As shown in Fig. 4, C-II/M-II and C-III/M-III eluted at Kav of 0.43 and at the void volume, respectively. Large CS/DS PGs Are Versican Isoforms and Small CS/DS PGs Are Biglycan and Decorin To assess the size of the core proteins present in C-II, C-III, M-II and M-III, PGs synthesized by pericytes were labeled with 35S-labeled
amino acids, separated by DEAE-Sephacel chromatography and their core proteins analyzed by SDS-PAGE and Western blot analysis (Fig. 5). Digestion of C-II and M-II with chondroitin ABC lyase generated a single band at approximately 50 kDa. Both C-II and M-II were immunoactive with antisera to biglycan and decorin, indicating that C-II and M-II contained a mixture of decorin and biglycan. On the other hand, digestion of C-III and M-III with chondroitin ABC lyase generated a doublet at Mr ca. 450 and 550 kDa. These bands showed positive immunoactivity to antisera against versican. Although separation of PGs by DEAE-Sephacel chromatography is incomplete, it is indicated that the dominating component of C-I, C-II/M-II and C-III/M-III is HSPGs, biglycan/decorin and versican, respectively. DISCUSSION In the present study, PGs synthesized by bovine retinal pericytes were characterized and classified by biochemical techniques. We found that: (a) Pericytes predominantly synthesize and secrete CS/DS PGs rather than HSPGs. (b)
Fig. 3. Characterization of GAG Chains Bound to Peaks C-I, C-II, M-II, C-III and M-III Isolated by DEAE-Sephacel Ion Exchange Chromatography (See Fig. 2) Peaks were chromatographed on a Sepharose CL-6B column before (open circles) and after digestion with papain (closed circles), heparitinase (closed triangles), and chondroitin ABC lyase (open triangles). Vertical broken lines indicate the position of peaks after papain digestion. The chain size of GAGs was estimated by comparison with a published chondroitin sulfate calibration.26)
Fig. 5.
Fig. 4. Sepharose CL-4B Molecular Sieve Chromatography of HSPG and CS/DS PG Populations Isolated by DEAE-Sephacel Chromatography The populations were chromatographed on a Sepharose CL-4B column in 8 M urea buffer.
Fluorographs of SDS-PAGE of 35S-Labeled Amino Acids-Labeled CS/DS PGs Separated by DEAE-Sephacel Chromatography
The PG populations derived from the cell layer and the conditioned medium of bovine retinal pericytes isolated and digested with or without chondroitin ABC lyase were run on a 4—12% gradient slab gels. Separately, chondroitin ABC lyase-generated core proteins accumulated in the cell layer and the conditioned medium were probed with an antibody specific for versican, biglycan and decorin.
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There are two subclasses of CS/DS PGs synthesized by pericytes; one is the high Mr subclass with a high charge density, which is mainly composed of versican and the other is the low Mr subclass with a lower charge density, which is composed of biglycan and decorin. (c) Small amounts of HSPGs with heparan sulfate chains of Mr ca. 48000 are synthesized by the cells. (d) Versican and biglycan/decorin bear chondroitin/dermatan sulfate chains of Mr ca. 66000 and Mr ca.45000, respectively. The overall profile of PG synthesis by retinal cells bears a close resemblance to that of vascular smooth muscle cells26—28) but differs from that of endothelial cells. It was shown that [35S]sulfate-labeled PGs synthesized by pericytes are separated into three populations by DEAESephacel chromatography whereas those synthesized by endothelial cells have only two populations; endothelial cell PGs are lacking in the population with the highest charge density eluted by NaCl at approximately 0.6 M, suggesting that this cell type, unlike pericytes and vascular smooth muscle cells, synthesizes only a very small amount of versican, although a versican-like CSPG was expressed.29) The synthesis of decorin by pericytes, but not by nonsprouting endothelial cells is of interest. For example, Schor et al.8) found that pericytes form nodules in vitro and those nodules undergo calcification. Recent studies suggest that decorin influences calcification.30) Thus pericyte synthesis of decorin may be an important determinant in the chondrogenic and osteogenic differentiation associated with pericytes.8,27) It is also of interest that increased decorin is associated with the formation of new vessels during the process of angiogenesis.17—19,31) For example, decorin inhibits endothelial migration in vitro32) and may assist in stabilizing the extracellular matrix in promoting new vessel formation. In addition, it has been shown that glucose effects the production of decorin by mesangial cells and this increase appears to be regulated by a high glucose response element in the promotor region of the decorin gene.33) Glucose has been shown to influence retinal pericyte cell associated PGs as well.34) Since excess new vessel formation is one of the characteristics in diabetic retinopathy, it may be that decorin synthesized by pericytes plays a critical role in diabetic retinopathy. Pericytes also synthesize biglycan as do vascular smooth muscle and endothelial cells. The exact role that biglycan serves in the structure and properties of microvessels is not certain but our results show that both pericytes and endothelial cells contribute to biglycan composition of the microvessels. Furthermore, biglycan expression by large vessel endothelial cells is increased when exposed to fibroblast growth factor-2 and when these cells are stimulated to migrate in a scratch wound assay in vitro.32) The principal PG synthesized by retinal pericytes is versican. Versican is also a principal PG synthesized by vascular smooth muscle cells,28,35—37) but is present in low amounts in endothelial cultures.32) Versican inhibits the adhesion of various cell types to substrates coated with fibronectin, vitronectin, or collagen38) and suppresses their migration.39) Versican is also upregulated as cells are stimulated by growth factors such as transforming growth factor-b 1 and plateletderived growth factor27) and is increased around cells that are actively proliferating and/or migrating.40) The absence of versican appears to influence the shape of arterial smooth
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muscle cells in vitro.36) Thus, this PG may influence pericyte shape and the ability of the cell type to proliferate and migrate as blood vessels grow. In summary, the present data demonstrate that pericytes predominantly synthesize CS/DS PGs such as versican, biglycan and decorin and small amounts of HSPGs. These PGs resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells. Regulation of PG synthesis by pericytes is incompletely understood. Given the importance these molecules in regulating endothelial cell phenotype, a more complete understanding of the metabolism of these molecules by pericytes should aid in the elucidation of those events that control new blood vessel formation. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (TK), the Specific Research Fund of Hokuriku University (TK) and a grant from the National Institute of Health, U.S.A. (TW). REFERENCES 1) Sims D. E., Can. J. Cardiol., 7, 431—443 (1991). 2) Orlidge A., D’Amore P. A., J. Cell Biol., 105, 1455—1462 (1987). 3) Sato Y., Rifkin D. B., J. Cell Biol., 109, 309—315 (1989). 4) Yamagishi S., Kobayashi K., Yamamoto H., Biochem. Biophys. Res. Commun., 190, 418—425 (1993). 5) Yamagishi S., Hsu C. C., Kobayashi K., Yamamoto H., Biochem. Biophys. Res. Commun., 191, 840—846 (1993). 6) Canfield A. E., Allen T., Grant M., Schor S. L., Schor A. M., J. Cell Sci., 96, 158—169 (1990). 7) Canfield A. E., Schor A. M., FEBS Lett., 286, 171—175 (1991). 8) Schor A. M., Canfield A. E., Sloan P., Schor S. L., In Vitro Cell Dev. Biol., 27A, 651—659 (1991). 9) Stramm L. E., Li W., Aguirre G. D., Rockey J. H., Exp. Eye Res., 44, 17—28 (1987). 10) Fisher E. J., McLennan S. V., Yue D. K., Turtle J. R., Microvasc. Res., 48, 179—189 (1994). 11) Marra F., Bonewald L. F., Park-Snyder S., Park I. S., Woodruff K. A., Abboud H. E., J. Cell. Physiol., 166, 537—546 (1996). 12) Diefenderfer D. L., Bringhton C. T., Biochem. Biophys. Res. Commun., 269, 172—178 (2000). 13) Aviezer D., Hecht D., Safran M., Eisinger M., David G., Yayon A., Cell, 79, 1005—1013 (1994). 14) Hildebrand A., Romaris M., Rasmussen L. M., Heinegard D., Twardzik D. R., Border W. A., Ruoslahti E., Biochem. J., 302, 527— 534 (1994). 15) Sato Y., Rifkin D. B., J. Cell Biol., 107, 1199—1205 (1988). 16) Kinsella M. G., Fischer J. W., Mason D. P., Wight T. N., J. Biol. Chem., 275, 13924—13932 (2000). 17) Schönherr E., O’Connell B. C., Schittny J., Robenek H., Fastermann D., Fisher L. W., Plenz G., Vischer P., Young M. F., Kresse H., Eur. J. Cell Biol., 78, 44—55 (1999). 18) Gutierrez P., O’Brien K. D., Ferguson J., Nikkari S. T., Alpers C. E., Wight T. N., Cardiovasc. Pathol., 6, 271—278 (1997). 19) Nelimarkka L., Salminen H., Kuopio T., Nikkari S., Esfors T., Laine J., Pelliniemi L., Järveläinen H. T., Amer. J. Pathol., 158, 345—353 (2000). 20) Capetandes A., Gerritsen M. E., Invest. Ophthalmol. Vis. Sci., 31, 1738—1744 (1990). 21) Herman I. M., D’Amore P. A., J. Cell Biol., 101, 43—52 (1985). 22) Gill P. J., Alder J., Silbert C. K., Silbert J. E., Biochem. J., 194, 299— 307 (1981). 23) Wasteson Å., Uthene K., Westermark B., Biochem. J., 136, 1069— 1074 (1973). 24) Wasteson Å., J. Chromatogr., 59, 87—97 (1971). 25) Laemmli U. K., Nature (London), 227, 680—685 (1970). 26) Wight T. N., Hascall V. C., J. Cell Biol., 96, 167—176 (1983).
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