The role of proteoglycans in cell adhesion, migration and proliferation.

Proteoglycans comprise a part of the extracellular matrix that participates in the molecular events that regulate cell adhesion, migration and proliferation. Their structural diversity and tissue distribution suggest a functional versatility not generally encountered for other extracellular matrix components. This versatility is mainly dictated by their molecular interactions and their ability to regulate the activity of key molecules involved in several biological events. This molecular cooperativity either promotes or inhibits cell adhesion, migration and proliferation. A growing number of studies indicate that proteoglycans can play a direct role in these cellular events by functioning either as receptors or as ligands for molecules that are required for these events to occur. Such studies support a role for proteoglycans as important effectors of cellular processes that constitute the basis of development and disease.

Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration.

The rates of 35S-sulfate incorporation into proteoglycan were compared in multi-scratch wounded and confluent cultures of bovine aortic endothelial cells to determine whether proteoglycan synthesis is altered as cells are stimulated to migrate and proliferate. Incorporation was found to be stimulated in a time-dependent manner, reaching maximal levels 44-50 h after wounding, as cells migrated into wounded areas of the culture dish. Quantitative autoradiography of 35S-sulfate-labeled single-scratch wounded cultures demonstrated a 2-4-fold increase in the number of silver grains over migrating cells near the wound edge when compared to cells remote from the wound edge. Furthermore, when cell proliferation was blocked by inhibition of DNA synthesis, the increase in 35S-sulfate incorporation into proteoglycan after wounding was unaffected. These data indicate that cell division is not required for the modulation of proteoglycan synthesis to occur after wounding. Characterization of the newly synthesized proteoglycan by ion-exchange and molecular sieve chromatography demonstrated that heparan sulfate proteoglycan constitutes approximately 80% of the labeled proteoglycan in postconfluent cultures, while after wounding, chondroitin sulfate proteoglycan and/or dermatan sulfate proteoglycan (CS/DSPG) increases to as much as 60% of the total labeled proteoglycan. These results suggest that CS/DSPG synthesis is stimulated concomitant with the stimulation of endothelial cell migration after wounding.

Origin of cushion tissue in the developing chick heart: cinematographic recordings of in situ formation.

Differential interference microscopy and time-lapse cinematography were used to determine unequivocally the origin of cushion tissue cells migrating in situ in the atrioventricular region of the embryonic chick heart. These studies have verified the presumed endocardial origin of cushion tissue mesenchyme.

Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.

The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers.

Retroviral overexpression of decorin differentially affects the response of arterial smooth muscle cells to growth factors.

Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by arterial smooth muscle cells (ASMCs). This proteoglycan accumulates in topographically defined regions of atherosclerotic lesions and may play a role in the development of this disease. However, little is known about whether decorin has specific effects on the cellular events that contribute to atherosclerotic lesion formation. In the present study, rat ASMCs were transduced with a retroviral vector (LDSN) that carries the bovine decorin gene. Compared with vector control cells (LXSN), these cells constitutively overexpress decorin, as verified by Northern and Western analysis and by metabolic labeling. Experiments were performed to examine the responsiveness of decorin-overexpressing rat ASMCs to platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1), 2 growth factors that affect cell proliferation and extracellular matrix production in atherosclerosis. Decorin-overexpressing cells had decreased [(3)H]thymidine incorporation into DNA and increased the levels of the cyclin-dependent kinase inhibitors p21 and p27 in the first 24 hours of response to serum and PDGF-BB. However, these effects of decorin were not apparent at 48 or 72 hours after plating and did not result in reduced growth of decorin-overexpressing cells in response to serum and PDGF-BB. In contrast, the growth response of decorin-overexpressing ASMCs to TGF-beta1, as well as the expression of TGF-beta1-responsive genes, such as plasminogen activator inhibitor-1 and versican (an extracellular matrix proteoglycan), was diminished. These results indicate that decorin selectively inhibits the responsiveness of rat ASMCs to TGF-beta1 and suggests that the induction of constitutive decorin overexpression by ASMCs in vivo may have therapeutic value in the inhibition of TGF-beta1-mediated effects on the development of atherosclerotic lesions.

Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of perlecan gene expression in aortic smooth muscle cells.

Heparan sulfate proteoglycans (HSPGs) are believed to act as potent endogenous regulators of vascular smooth muscle cell (SMC) replication, migration, gene expression and differentiation. Here we describe the pattern of expression of perlecan, the predominant basement membrane HSPG, during aortic development in the rat. Expression of perlecan mRNA and protein in the aortic SMC was first significantly observed at day e19 (day 19 of embryonic development), a time which marks a dramatic switch in SMC replication rate and growth phenotype. Expression of perlecan message and protein was high throughout fetal and early neonatal life, and it remained readily detectable in the adult aorta. Using a double-labeling technique (in situ hybridization for perlecan message coupled with bromodeoxyuridine immunohistochemistry), we determined the relationship between DNA synthesis and perlecan mRNA expression in individual SMC at days e17-e21; we found that perlecan gene expression was largely limited to non-replicating cells. Consistent with the in vivo data, perlecan mRNA was undetectable in cultured e17 SMC by Northern or RT-PCR analysis, while in cultured adult SMC, perlecan mRNA was significantly higher in non-replicating (serum-starved) cultures compared to replicating cultures. Treatment of growth-arrested adult SMC cultures with heparin caused a further accumulation in perlecan mRNA levels. The data suggest that the expression of perlecan by vascular SMC is regulated by apparent developmental age as well as by cellular growth state. The developmentally times expression of perlecan in the aortic wall may contribute to the establishment and/or maintenance of vascular SMC differentiation and quiescence.

Isolation and characterization of dermatan sulfate proteoglycans synthesized by cultured bovine aortic endothelial cells.

Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.

Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures.

Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of high molecular weight dermatan sulfate proteoglycan in bovine aortic endothelial cell cultures. Evidence for transglutaminase-catalyzed cross-linking to fibronectin.

Three glucuronate-rich dermatan sulfate proteoglycan (DS-PG) subclasses were isolated and previously characterized from bovine aortic endothelial cell cultures (Kinsella, M. G., and Wight, T. N. (1988) J. Biol. Chem. 263, 19222-19231). In the present study, pulse-chase experiments indicate that the DS-PG of highest apparent Mr (approximately 1 x 10(6)), denoted previously as HMW-DS, is a relatively stable component of the endothelial extracellular matrix and is formed at the expense of lower Mr DS-PG species. The formation of HMW-DS is reduced in a dose-dependent manner in the presence of dansylcadaverine, an inhibitor of transglutaminase-catalyzed protein cross-linking, but not when the activity of other cross-linking enzymes such as lysyl oxidase is inhibited. The putative DS-PG precursor to HMW-DS accumulates during inhibition of cross-linking only when lysosomal degradation is also inhibited by ammonium chloride, suggesting that the precursor is degraded rapidly in the absence of cross-linking. HMW-DS is precipitable from endothelial cell monolayer extracts with antibodies against fibronectin, a known transglutaminase substrate. Thus, we conclude that the stability of HMW-DS in the subendothelial matrix in culture depends upon the cross-linking of a low Mr DS-PG precursor to matrical protein(s), including fibronectin, resulting in the formation of a DS-PG subclass of high apparent molecular mass.

Degradation of endothelial cell matrix heparan sulfate proteoglycan by elastase and the myeloperoxidase-H2O2-chloride system.

The degradation of the heparan sulfate proteoglycans of subendothelial matrix by neutrophil elastase and the myeloperoxidase-H2O2-chloride system added separately, sequentially, or together at pH 4.5 to 7.5 was determined by the release of lower molecular weight 35S-labeled material. Elastase alone and the myeloperoxidase system alone caused degradation, and when 4-hour exposure to elastase was followed by 15 minutes of exposure to the myeloperoxidase system, the effect was greater than additive. A greater than additive effect was not observed when elastase followed the myeloperoxidase system or the two were added together. Chloride (or sulfate) alone increased the release of 35S-labeled material from elastase-treated matrix, although the effect of 0.1 M chloride was not as great as that observed when an equivalent concentration of chloride was combined with myeloperoxidase and H2O2. The release of these systems at sites of adherence of neutrophils to glomerular basement membrane may contribute to neutrophil-associated proteinuria.

Genistein selectively inhibits platelet-derived growth factor-stimulated versican biosynthesis in monkey arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) stimulates not only the proliferation and migration of arterial smooth muscle cells (ASMCs), but also the transcription, translation, and posttranslational processing of versican, a large chondroitin sulfate proteoglycan present in the extracellular matrix of blood vessels. PDGF receptor tyrosine kinase activity is required for signaling events associated with mitogenic and motogenic stimulation of cells by PDGF. Therefore, we have asked if inhibiton of tyrosine kinase activity by genistein also blocks the stimulation of both versican core protein synthesis and glycosaminoglycan (GAG) chain modifications induced by PDGF in ASMCs. The tyrosine kinase inhibitor, genistein, in a dose-dependent manner, reversibly inhibits PDGF-stimulated ASMC cell proliferation and RNA and core protein expression of versican, without affecting the expression of decorin and biglycan. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. This suggests that different aspects of the biosynthesis of versican are differentially regulated. To determine if such differential regulation involves downstream activation of protein kinase C, ASMCs were treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to directly activate this kinase. In comparison to PDGF stimulation, TPA has little effect on expression of versican mRNA expression, nor does TPA stimulate ASMC cell proliferation. However, like PDGF, TPA increases [35S]sulfate incorporation into proteoglycans and GAG chain elongation. These results indicate that PDGF-induced GAG chain elongation, which is not inhibited by genistein treatment and is stimulated by protein kinase C activation, involves signaling pathways different from those that regulate PDGF-stimulated versican mRNA and protein expression.

Alterations in nonhuman primate (M. nemestrina) lung proteoglycans during normal development and acute hyaline membrane disease.

Proteoglycans (PGs) and lung hyaluronan (HA) are important components of the lung matrix both during normal development and in response to injury. We combined morphologic and biochemical techniques to study changes in PG and HA in a developmental series of Macaca nemestrina lungs ranging from 62% gestation to 3 mo post-term (n = 16), in adult lungs (n = 6), and from prematurely delivered, mechanically ventilated monkeys with hyaline membrane disease (HMD) (n = 7). Three groups of cuprolinic blue-positive (CuB) precipitates, identified by size, location, and susceptibility to enzyme digestion were found in lungs from all animals. Immature alveolar interstitium is characterized by loosely woven collagen bundles and an abundance of large (100 to 200 nm) stained filaments representing chondroitin sulfate proteoglycans (CSPGs). As maturation proceeds, the interstitial matrix appears increasingly organized, with large collagen bundles associated with 20 nm CuB-stained deposits (dermatan sulfate proteoglycans, DSPGs), and fewer large CSPGs. Fetal alveolar basement membrane contains CuB-stained heparin sulfate proteoglycans (HSPGs) (10 nm) scattered throughout. Lung matrix from animals with HMD appeared to have a disruption of the collagen-DSPG relationship, in addition to an enrichment in large CSPG. Complementary biochemical analysis of lung PGs and HA was done. Minced lung parenchyma was cultured with [3H]-glucosamine and [35S]-sulfate for 24 h; PGs and HA were extracted and analyzed. While PG synthesis during development tended to be highest at 80% gestation, animals with HMD showed greatly increased synthesis, approximately 2.5-fold higher than comparable fetal animals. In the developmental series, [3H]-glucosamine incorporation into HA was maximal at term, falling abruptly thereafter. HMD animals, however, showed a 2.3-fold increase over controls in net HA synthesis. Extracted PGs were separated according to buoyant density by dissociative cesium chloride density gradient ultracentrifugation. Two peaks of 35S-labeled PGs were separated from each density gradient fraction by chromatography on Sepharose CL-4B. A large CSPG was the principal PG eluting in the voiding volume, while the second broad peak (K(av) = 0.42) contained a mixed population of CSPG, DSPG, and HSPGs, the proportions of which varied with age. Both ultrastructural and biochemical analyses indicate that production of a large, high buoyant density CSPG predominates in fetal lung tissue, and diminishes with developmental age. Synthesis of large CSPG is greatly increased in lung explants from prematurely delivered animals with HMD.(ABSTRACT TRUNCATED AT 400 WORDS)

Vascular cell proteoglycans: evidence for metabolic modulation.

Proteoglycans accumulate in the intimal layer of blood vessels during the early stages of atherosclerosis and predispose the vessel wall to further complications of this disease. Arterial endothelial and smooth muscle cell cultures have been used to study the metabolism of vessel wall proteoglycans in an attempt to determine whether cellular events associated with the genesis of this disease, such as cellular proliferation, ageing, migration and interaction with components of the extracellular matrix, influence the metabolism of arterial proteoglycans. Proteoglycan analyses of vascular cells reveal that endothelial cells synthesize multiple species of heparan sulphate proteoglycan while smooth muscle cells synthesize little heparan sulphate proteoglycan but significant quantities of chondroitin and dermatan sulphate proteoglycan. Each family of proteoglycans synthesized by each cell type differs with regard to charge density, hydrodynamic size, glycosaminoglycan type and size, oligosaccharide content and ability to form high molecular weight aggregates. A monoclonal antibody has been generated against the chondroitin sulphate proteoglycan and used to immunolocalize this antigen to the interstitial matrix of normal and diseased blood vessels. Experiments are presented to indicate that proteoglycan metabolism is modulated when cultured arterial cells are stimulated to proliferate and migrate. Other factors shown to influence proteoglycan metabolism include the age of the cell and the nature of the substratum upon which the cells are grown. These culture systems provide useful models with which to study the factors involved in the regulation of proteoglycan synthesis by vascular cells.

Platelet-derived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells.

Platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1), two growth-regulatory peptides with opposite effects on arterial smooth muscle cell (ASMC) proliferation, were examined for their influence on the synthesis of two small chondroitin sulfate/dermatan sulfate proteoglycans (CS/DS PGs) called biglycan and decorin. Quiescent ASMCs treated with either PDGF or TGF-beta 1 for 24 hours increased [35S]sulfate incorporation into biglycan 3.3- and 2.9-fold, respectively, whereas the incorporation of [35S]sulfate into decorin was not significantly affected. Treatment with TGF-beta 1 but not PDGF more than doubled the steady-state level of messenger RNA (mRNA) transcripts hybridizing to a complementary DNA (cDNA) encoding biglycan. Both growth factors had little or no effect on steady-state levels of mRNA transcripts hybridizing to a decorin cDNA. Incorporation of [35S]sulfate into biglycan glycosaminoglycan (GAG) was maximal by 12 to 18 hours after either PDGF or TGF-beta 1 addition. Both PDGF and TGF-beta 1 increased the molecular sizes of biglycan and decorin. This increase was a result of the synthesis of longer GAG chains substituted on the core proteins of both PGs. PDGF but not TGF-beta 1 led to an increase of more than twofold in the ratio of 6'- to 4'-sulfated disaccharides in these newly synthesized GAG chains. These results indicate that PDGF and TGF-beta 1 have specific but different effects on the synthesis of small CS/DS PGs by monkey ASMCs in culture.

Selective expression and processing of biglycan during migration of bovine aortic endothelial cells. The role of endogenous basic fibroblast growth factor.

Repair of the vascular lumenal surface after injury requires a controlled endothelial cell response that includes cell migration, proliferation, and remodeling of the extracellular matrix. These cellular processes are modulated by growth factors that are released or activated following cell injury. When endothelial cell migration is stimulated in response to monolayer wounding in vitro, cells increase synthesis of small leucine-rich dermatan sulfate proteoglycans (PGs) (Kinsella, M. G., and Wight, T. N. (1986) J. Cell Biol. 102, 679-687). However, the identity of the PGs that are increased during cell migration and the factors that affect this modulation have not been identified. We now report that basic fibroblast growth factor (bFGF) is responsible for the transient increase of [35S]sulfate incorporation into PGs following monolayer wounding. SDS-polyacrylamide gel electrophoresis analysis revealed that bFGF-treated and wounded cultures increase both biglycan core protein synthesis and biglycan proteolytic processing, which results in the accumulation of a approximately 20-kDa N-terminal biglycan fragment in the culture media. Biglycan RNA steady-state levels also selectively increase 2- to 3-fold after wounding or bFGF treatment. Finally, immunocytochemical staining localizes biglycan to the tips and edges of lamellopodia on migrating cells, indicating that biglycan is found at loci at which the formation and dissolution of adhesion plaques occurs, consistent with hypotheses that predict involvement of biglycan in the control of cell migration. Taken together, these results suggest that release of endogenous bFGF is primarily responsible for altered biglycan expression, synthesis, and proteolytic processing as endothelial cells migrate after wounding.

Retrovirally mediated expression of decorin by macrovascular endothelial cells. Effects on cellular migration and fibronectin fibrillogenesis in vitro.

Decorin is a member of the widely expressed family of small leucine-rich proteoglycans. In addition to a primary role as a modulator of extracellular matrix protein fibrillogenesis, decorin can inhibit the cellular response to growth factors. Decorin expression is induced in endothelial cells during angiogenesis, but not when migration and proliferation are stimulated. Thus, decorin may support the formation of the fibrillar pericellular matrix that stabilizes the differentiated endothelial phenotype during the later stages of angiogenesis. Therefore, we tested whether constitutive decorin expression alone could modify endothelial cell migration and proliferation or affect pericellular matrix formation. To this end, replication-defective retroviral vectors were used to stably express bovine decorin, which was detected by Northern and Western blotting. The migration of endothelial cells that express decorin is significantly inhibited in both monolayer outgrowth and microchemotaxis chamber assays. The inhibition of cell migration by decorin was not accompanied by decreased proliferation. In addition, endothelial cells that express decorin assemble an extensive fibrillar fibronectin matrix more rapidly than control cells as assessed by immunocytochemical and fibronectin fibrillogenesis assays. These observations suggest that cell migration may be modulated by the influence of decorin on the assembly of the cell-associated extracellular matrix.

Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells.

Migration of vascular smooth muscle cells (SMCs) is a key step in vascular remodeling and formation of pathological lesions in diseased arteries and may be controlled by extracellular matrix (ECM) and by factors that regulate ECM composition, such as platelet-derived growth factor (PDGF). In culture, PDGF-AB and -BB enhance but PDGF-AA (although having no effect alone) suppresses SMC migration stimulated by other PDGF isoforms. To determine whether the migration-inhibitory mechanism of PDGF-AA was mediated by ECM composition, we examined baboon SMC migration in a Boyden chamber assay using filters coated with different ECM proteins. PDGF-AA suppressed the PDGF-BB-induced migration of baboon SMCs on a filter coated with basement membrane proteins (Matrigel) and fibronectin but failed to inhibit cell migration on a type I collagen (Vitrogen)-coated filter. Fibronectin and fibronectin fragments that contain heparin-binding domains permitted PDGF-AA inhibition of cell migration, but a fragment lacking heparin-binding domains did not. Treatment of SMCs with heparin lyases II and III, but not with chondroitin ABC lyase, diminished the PDGF-AA-mediated inhibition of migration. PDGF-AA stimulated accumulation of proteoglycan (PG) in the cell layer more potently than did PDGF-BB, whereas the turnover of cell layer PG was unaffected by either PDGF-AA or -BB. Northern blot analysis revealed that PDGF-AA increased syndecan-1 mRNA expression more than did PDGF-BB, whereas both PDGF isoforms decreased perlecan expression. The changes in cell migration and PG synthesis induced by PDGF-AA were accompanied by changes in the morphology of SMCs. PDGF-AA dramatically induced the spreading of SMCs, whereas the heparin lyase treatment of PDGF-AA-stimulated cultures diminished cell spreading. The data suggest that PDGF-AA selectively modifies heparan sulfate PG accumulation on SMCs and thereby influences the interactions of SMCs with heparin-binding ECM proteins. These interactions, in turn, generate signals that suppress SMC migration.

Differential expression of small chondroitin/dermatan sulfate proteoglycans, PG-I/biglycan and PG-II/decorin, by vascular smooth muscle and endothelial cells in culture.

Cultured bovine aortic smooth muscle (BASM) and endothelial (BAE) cells, like articular chondrocytes, synthesize two populations of small chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) of similar size as PG-I/biglycan and PG-II/decorin. However, Northern blot analyses demonstrated that BAE cells express detectable amounts of mRNA transcripts only for PG-I/biglycan, whereas BASM cells and articular chondrocytes express mRNA transcripts for both PG-I/biglycan and PG-II/decorin. Endothelial cells from human umbilical vein also expressed detectable amounts of mRNA transcripts only for PG-I/biglycan, and not PG-II/decorin. Antiserum raised against bovine PG-II/decorin immunoprecipitated an apparent single PG species with relative molecular mass (Mr) of approximately 120,000-180,000 from BASM cell and articular chondrocyte cultures but failed to immunoprecipitate an equivalent PG species from BAE cell cultures, consistent with the results from Northern blot analysis. In contrast, immunoprecipitations by antisera to PG-I/biglycan indicated that cultured endothelial cells synthesize two forms of PG-I/biglycan with Mr values slightly larger than 200,000 and 120,000-140,000. It is likely, based on the magnitude of the size difference, that these two forms of PG-I/biglycan differ in the number of glycosaminoglycan chains. Additionally, BASM but not BAE cells were found to express detectable amounts of mRNA transcripts for type I collagen. The above results indicate that the two main cell types of the vascular wall, endothelial and smooth muscle cells, express different sets of small interstitial CS/DS PGs and that the synthesis of PG-II/decorin by these cells correlates with the expression of type I collagen, a collagen known to interact specifically with this PG. These differences in the expression of extracellular matrix molecules may be important in regulating the cell type-specific functions of endothelial and smooth muscle cells within the vascular tissue.