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Endocrinology Vol. 143, No. 4 1441-1450
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

The Angiogenic Factor Cysteine-Rich 61 (CYR61, CCN1) Supports Vascular Smooth Muscle Cell Adhesion and Stimulates Chemotaxis through Integrin {alpha}6ß1 and Cell Surface Heparan Sulfate Proteoglycans

Tatiana M. Grzeszkiewicz, Volkhard Lindner, Ningyu Chen, Stephen C.-T. Lam and Lester F. Lau

Departments of Molecular Genetics (T.M.G., N.C., L.F.L.) and Pharmacology (S.C.-T.L.), University of Illinois at Chicago, Chicago, Illinois 60607-7170; and Center for Molecular Medicine (V.L.), Maine Medical Center Research Institute, Scarborough, Maine 04074

Address all correspondence and requests for reprints to: Lester F. Lau, Department of Molecular Genetics, University of Illinois at Chicago College of Medicine, 900 South Ashland Avenue, Chicago, Illinois 60607-7170. E-mail: . lflau{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cysteine-rich 61 (CYR61, CCN1) is a heparin-binding, extracellular, matrix-associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. CYR61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor-stimulated mitogenesis in fibroblasts and endothelial cells. Although the expression of CYR61 has been observed in arterial walls, its function in vascular smooth muscle cells (VSMCs) has not been examined to date. Here we show that purified CYR61 supports VSMC adhesion in a dose-dependent, saturable manner through integrin {alpha}6ß1 with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin {alpha}6ß1 and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CCN FAMILY of proteins consists of cysteine-rich 61 (CYR61; CCN1), connective tissue growth factor (CTGF; CCN2), nephroblastoma overexpressed (Nov; CCN3), Wnt-induced secreted protein-1 (WISP-1; CCN4), WISP-2 (CCN5), and WISP-3 (CCN6) (1, 2, 3, 4). CCN proteins are comprised of four conserved modular domains that share sequence similarities with IGF-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and growth factor cysteine knots. WISP-2 is unique by lacking precisely the carboxyl-terminal cysteine knot domain (5, 6). Because each domain is encoded by a separate exon, the CCN gene family probably arose through exon shuffling, and the overall functions of CCN proteins may be programmed by the combinatorial activities of each domain acting independently and interdependently.

CYR61 is a secreted, extracellular matrix (ECM)-associated heparin-binding protein. In fibroblasts and endothelial cells, CYR61 mediates cell adhesion, stimulates cell migration, and potentiates growth factor-induced DNA synthesis (7, 8, 9). CYR61 induces angiogenesis in vivo (10), and its expression enhances the tumorigenicity of human tumor cells in immunodeficient mice by increasing tumor size and vascularization (10, 11). CYR61 also regulates the expression of genes involved in cutaneous wound healing, including up-regulation of angiogenic and inflammatory cytokines, matrix metalloproteinases, cell adhesion receptors, and down- regulation of type I collagen (12). These activities of CYR61 are consistent with its expression in angiogenic cell types during development and granulation tissue during cutaneous wound healing (12, 13).

At least some of the activities of CYR61 are mediated through its interaction with integrin receptors. Integrins are heterodimeric cell surface receptors that interact with extracellular molecules and regulate a broad spectrum of cellular functions, including cell adhesion, proliferation, migration, differentiation, and survival (14, 15). CYR61 is a ligand of, and binds directly to, integrins {alpha}vß3, {alpha}vß5, and {alpha}IIbß3 (9, 10, 16, 17). Interaction between these integrins and CYR61 mediates fibroblast proliferation and endothelial cell adhesion and migration ({alpha}vß3), blood platelet adhesion ({alpha}IIbß3), and fibroblast migration ({alpha}vß5). In addition, both CYR61 and CTGF support the adhesion of primary human fibroblasts through integrin {alpha}6ß1 with cell surface heparan sulfate proteoglycans (HSPGs) (18, 19). Fibroblast adhesion to CYR61 or CTGF results in adhesive signaling manifested by persistent formation of filopodia and lamellipodia, formation of integrin subunit {alpha}6- and ß1-containing focal complexes, and activation of focal adhesion kinase and mitogen-activated protein kinases (19). Moreover, CYR61 activates a genetic program for wound healing, culminating in the expression of genes that regulate angiogenesis, inflammation, ECM remodeling, and cell-matrix interactions (12). Recently, several members of the CCN protein family have been implicated in vascular smooth muscle cell (VSMC) function and vascular pathology (20, 21, 22). For example, CTGF is expressed in advanced atherosclerotic lesions (21). VSMCs transfected with a CTGF expression vector showed enhanced cell proliferation and migration in culture (23). Paradoxically, CTGF has also been reported to induce apoptosis in cultured aortic smooth muscle cells via caspase-3 (24, 25, 26). At present, the cell surface receptors mediating CTGF actions in VSMCs are unknown.

CYR61 is strongly expressed in smooth muscle cells of arterial walls during embryonic development (27). Although the activities of CYR61 have been examined in endothelial cells and fibroblasts (8, 16, 17, 18), they have not been examined in VSMCs to date. In this study, we showed that purified CYR61 supports VSMC adhesion through integrin {alpha}6ß1 and cell surface HSPGs. Both receptors are also involved in CYR61-induced chemotaxis. Although integrins {alpha}vß3 and {alpha}vß5 are known receptors for CYR61-stimulated migration in other cell types (9, 10), they apparently play no role in CYR61-promoted VSMC adhesion or chemotaxis. Furthermore, we employed a balloon angioplasty model to show that CYR61 is strongly up-regulated during proliferative restenosis in the media and neointima following vascular injury. Together, our data demonstrate the activities of CYR61 on VSMCs, identify the cell surface receptors that mediate its actions and underscore the potential importance of CYR61 in vascular responses to injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies, peptides, and reagents
Function-blocking monoclonal antibodies (mAbs) against integrins were purchased from Chemicon, Inc. (Temecula, CA), including LM609 (anti-{alpha}Vß3), P1F6 (anti-{alpha}Vß5), and JB1A (anti-ß1). Additionally, P4C10 (anti-ß1) was acquired from Life Technologies, Inc. (Rockville, MD), JBS5 (anti-{alpha}5ß1) from Serotec, Inc. (Raleigh, NC), and NKI-GoH3 (anti-{alpha}6) from Immunotech (Wesbtrook, ME). Polyclonal anti-CYR61 antibodies used for immunoblotting were raised in rabbits as previously described (28). GRGDSP and GRGESP synthetic peptides were purchased from Life Technologies, Inc. Heparin (sodium salt, from porcine intestinal mucosa), heparinase I, chondroitinase ABC, avidin/biotin complex, and ß-tubulin mAbs were from Sigma (St. Louis, MO). Horseradish peroxidase-conjugated donkey antirabbit antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

For immunohistochemistry, an antibody was designed to specifically recognize CYR61 without cross-reacting with other family members. Accordingly, an area of minimal homology at the very carboxyl terminal of the protein was chosen (1). A peptide to the most C-terminal 15 residues of CYR61 (FPFYRLFNDIHKFRD, corresponding to amino acids 367–381) was synthesized and used to raise polyclonal anti-CYR61 antibodies in rabbits (29) and affinity purified against the peptide as previously described (28). The University of Illinois at Chicago Protein Research Laboratory completed all steps of antibody production. Antibodies were tested using immunoblotting and found to recognize as little as 10 ng CYR61 but not cross-reacting with as much as 1 µg purified recombinant CTGF.

CYR61, CYR61DM, CYR61{Delta}CT, and matrix proteins
Recombinant CYR61DM was produced in a serum-free baculovirus system using SF9 cells as previously described (18). Likewise, recombinant human CYR61 and CYR61{Delta}CT were produced in a serum-free baculovirus system using High5 cells as detailed (9). Human vitronectin, fibronectin, and recombinant platelet-derived growth factor (PDGF) were purchased from Life Technologies, Inc.

Cell culture and adhesion assay
Primary bovine aortic smooth muscle cells were obtained from Clonetics (San Diego, CA) and maintained in their smooth muscle basal medium (SmBm) medium supplemented with SmGm-2 (5% FBS, 0.5 ng/ml human endothelial growth factor, 2 ng/ml human fibroblast growth factor, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 5 µg/ml insulin). Adhesion assays were conducted as previously described (16). Briefly, VSMCs were harvested in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, pH 7.3) with 2.5 mM EDTA, resuspended in serum-free F12K medium (Life Technologies, Inc.) containing 0.5% BSA to 2.5 x 105 cells/ml. To each well, 50 µl cell suspension was plated and allowed to adhere for 20 min at 37 C. Where indicated, cells were first treated with reagents for 30 min or function blocking mAbs for 1 h at room temperature. After incubation, cells were rinsed once each with plating medium and PBS, adherent cells were fixed with 10% formalin, stained with methylene blue, and quantified by dye extraction with measurement of absorbance at 620 nm.

Cell migration assay
A modified Boyden chamber was used to determine VSMC migration as previously described (30). Test proteins were diluted in SmBm serum-free medium containing 0.5% BSA and loaded in the lower well of the chamber in quadruplicate. The lower well was then covered with a polycarbonate filter (5 µm pore diameter, Nucleopore), which was treated with 2.9% (vol/vol) glacial acetic acid overnight, and rinsed three times in PBS for 1 h immediately before use. VSMCs were trypsinized, resuspended in serum-free SmBm medium containing 0.5% BSA, and applied to the upper wells (5 x 104 cells/well). Where indicated, VSMCs were treated with function-blocking mAbs for 1 h before chamber loading. After a 6-h incubation at 37 C, 5% CO2, the membrane was removed and stained with Diff-Quik (Dade-Behring, Deerfield, IL). The chemotactic response was determined by counting the total number of cells migrated in 10 randomly selected microscope fields at 400x magnification.

Rat carotid artery balloon angioplasty
Male Sprague Dawley rats (400 g, 3–4 months old) were purchased from Taconic (Germantown, NY). All surgical procedures were carried out under general anesthesia by ip injection of xylazine (2.2 mg/kg, AnaSed, Lloyd Laboratories, Shenandoah, IA) and ketamine (50 mg/kg body weight, Ketaset, Aveco Co., Inc., Fort Dodge, IA). The left and right carotid artery and the aorta were denuded with a 2F balloon catheter as recently described (31). Deendothelialized segments of arteries were identified by iv injection of Evans blue (0.3 ml of 5% solution in saline) 10 min before killing. For Western blotting and immunostaining, animals were perfused with ice-cold lactated Ringer’s solution to remove blood and plasma proteins followed by excision and removal of periadventitial fat. Vessels were embedded in OCT (optimal cutting temperature) compound (Miles, Torrance, CA) for preparation of frozen sections or snap frozen in liquid nitrogen for immunoblotting. Rats were killed at the indicated times after injury. All animal studies were approved by the Institutional Animal Care and Use Committee.

Western blotting and immunohistochemistry
Left and right common carotid arteries were harvested from rats at the indicated time points after balloon injury to prepare vessel wall extract samples. The vessels were pulverized in liquid nitrogen using mortar and pestle and resuspended in buffer containing 20 mM HEPES, pH 7.9; 1.5 mM MgCl2; 420 mM NaCl; 0.2 mM EDTA; 1 mM DTT; 0.5 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 1.5 µg/ml pepstatin A; 40 µM calpain inhibitor I (Roche, Burlington, NC); 1 mM Na3VO4; and 1 mM NaF. A protein extract was generated by freeze thawing the suspension three times and then removing the insoluble material by centrifugation at 14,000 rpm for 10 min at 4 C. Protein concentrations were determined by the Coomassie protein assay (Pierce Chemical Co., Rockford, IL). For immunoblotting, 70 µg total cell lysate proteins from each sample was loaded per well, electrophoresed on a 10% SDS-PAGE polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti-CYR61 antibodies using standard protocol (29). Membranes were subsequently stripped and reprobed with ß-tubulin as a loading control. For immunohistochemistry, tissue samples were postfixed in acetone at 4 C for 10 min, quenched in 0.3% hydrogen peroxide (Sigma) in methanol (Fisher, Pittsburgh, PA), and rinsed in PBS prior to treatment with the Histomouse kit reagents (Zymed Laboratories, Inc., San Francisco, CA) and anti-CYR61 antibodies. For antibody saturation experiments, anti-CYR61 antibodies were incubated in PBS containing 2 µg/ml recombinant CYR61 for 1 h at room temperature before use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CYR61 supports VSMC adhesion through integrin {alpha}6ß1 and cell surface HSPGs
CYR61 is highly expressed in VSMCs during embryonic development (27). To understand its activities in VSMCs, we first examined the ability of CYR61 to support cell adhesion. Microtiter wells were coated with purified CYR61, and VSMCs were allowed to adhere under serum-free conditions. CYR61 supported VSMC adhesion in a dose-dependent and saturable manner, with optimal adhesion achieved at a coating concentration of approximately 5 µg/ml CYR61 (Fig. 1AGo). To gain further insight into the mechanism of CYR61 action, we examined the ability of mutant CYR61 proteins to support cell adhesion. A mutant CYR61 (CYR61{Delta}CT) (9) with the carboxyl-terminal heparin-binding domain deleted was unable to support VSMC adhesion at any concentration tested (Fig. 1AGo). Likewise, CYR61DM (18), a mutant protein harboring amino acid substitutions that destroyed the heparin-binding sites, was also defective in supporting VSMC adhesion. These results suggest that the cell adhesive activity CYR61 in VSMCs requires its ability to bind heparin.



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  		Figure 1. CYR61 supports VSMC adhesion in a dose-dependent and heparin-sensitive manner. Cell adhesion assays were performed with VSMCs detached with 2.5 mM EDTA and resuspended in serum-free F12K medium at 2.5 x 105 cells/ml. Then 50-µl cell suspensions were plated on each microtiter well coated with protein. Unless otherwise indicated, wells were coated with 1% BSA, 3 µg/ml fibronectin (FN), 1.5 µg/ml vitronectin (VN), or 5 µg/ml CYR61. After incubation at 37 C for 20 min, adherent cells were fixed and stained with methylene blue, and extracted dye was quantified by absorbance at 620 nm. A, Wells coated with the indicated concentration of CYR61, CYR61DM, and CYR61{Delta}CT. B, The indicated amount of heparin was included in the cell suspension before plating and during the adhesion assay. C, Cells were treated with 2 U/ml heparinase I or chondroitinase ABC at 37 C for 30 min before plating. Data shown for all panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
A plausible interpretation of the above data is that CYR61 may bind to cell surface HSPGs, which help to mediate cell adhesion. This model predicts that the presence of soluble heparin, at a concentration that saturates the heparin-binding sites of CYR61, may prevent CYR61 from mediating cell adhesion. To test this possibility, we added soluble heparin to cell suspensions before their plating on various protein substrates. Whereas soluble heparin had no effect on VSMC adhesion to fibronectin or vitronectin, it strongly inhibited cell adhesion to CYR61 in a dose-dependent manner. VSMC adhesion to CYR61 (5 µg/ml) was completely abrogated by 0.5 µg/ml of heparin in the medium (Fig. 1BGo). To evaluate whether cell surface HSPGs are indeed required for this process, we treated VSMCs with heparinase I and chondroitinase ABC before plating. Cells treated with heparinase I were unable to adhere to CYR61, but chondroitinase ABC had no effect. Neither heparinase I nor chondroitinase ABC had any effect on cell adhesion to fibronectin or vitronectin (Fig. 1CGo). These results show that cell surface HSPGs are necessary for VSMCs to adhere to CYR61.

Because CYR61 supports adhesion of fibroblasts and endothelial cells through integrin receptors, we next investigated the role of integrins in CYR61-supported VMSC adhesion. An examination of the effects of divalent cations showed that VSMC adhesion to CYR61 was completely blocked by the presence of 10 mM EDTA and restored by the addition of 20 mM Mg2+ or Ca2+ (Fig. 2AGo). Both Mg2+ and Mn2+ supported cell adhesion to CYR61, whereas the presence of Ca2+ was completely inhibitory. Mn2+, but not Mg2+, was able to overcome the Ca2+ inhibition. This divalent cation sensitivity profile is consistent with, and indicative of, an integrin as the adhesion receptor for CYR61 in VSMCs.



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  		Figure 2. VSMC adhesion to CYR61 is not mediated by {alpha}v integrin receptors. Cell adhesion assays were performed with VSMCs plated on microtiter wells coated with 1% BSA, 2 µg/ml fibronectin (FN), 0.5 µg/ml vitronectin (VN), or 4 µg/ml CYR61. A, EDTA (10 mM) and/or Ca2+, Mg2+, and Mn2+ (20 mM) was added to the cell suspension before plating. B, GRGDSP or GRGESP peptide (0.2 mM) was incubated with the cells for 30 min before plating. C, Cells were incubated with 50 µg/ml LM609 (integrin {alpha}Vß3 mAb) for 1 h before plating. D, Cells were incubated with 50 µg/ml P1F6 (integrin {alpha}Vß5 mAb) for 1 h before plating. Data shown for all panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
A number of integrin receptors expressed on smooth muscle cells, particularly the {alpha}v integrins, are sensitive to inhibition by arginine-glycine-aspartate (RGD)-containing peptides (32). We thus examined the inhibition of cell adhesion to CYR61 by RGD-containing peptides. Because vitronectin is known to bind {alpha}v integrins, the observation that GRGDSP, but not the control peptide GRGESP, completely abolished adhesion to vitronectin was as expected (Fig. 2BGo). By contrast, GRGDSP had no effect on VSMC adhesion to CYR61, suggesting that the {alpha}v integrins do not mediate VSMC adhesion to CYR61. Consistently, mAbs against integrin {alpha}vß3 (LM609) or integrin {alpha}vß5 (P1F6) had no effect on cell adhesion to CYR61 (Figs. 2Go, C and D), whereas cell adhesion to vitronectin (which binds {alpha}v integrins), but not fibronectin (which binds integrin {alpha}5ß1), was inhibited. Because fibroblast adhesion to CYR61 and CTGF is mediated through integrin {alpha}6ß1 and HSPGs (18, 19), we examined the involvement of integrin {alpha}6ß1. As shown in Fig. 3Go, A and B, mAbs against either integrin {alpha}6 (GoH3) or ß1 (JB1a) completely abolished VSMC adhesion to CYR61. By contrast, a mAb against integrin {alpha}5ß1 (JBS5) had no effect on CYR61-mediated VSMC adhesion but inhibited adhesion to fibronectin (Fig. 3CGo). Taken together, these results showed that VSMC adhesion to CYR61 is mediated through integrin {alpha}6ß1 and HSPGs working as coreceptors.



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  		Figure 3. Integrin {alpha}6ß1 mediates VSMC adhesion to CYR61. VSMCs were treated with integrin mAbs where indicated for 1 h at room temperature and then plated on 1% BSA, 2 µg/ml fibronectin (FN), 0.5 µg/ml vitronectin (VN), and 4 µg/ml CYR61. A, GoH3 (anti-integrin {alpha}6). B, JB1A (anti-integrin ß1). C, JBS5 (anti-integrin {alpha}5ß1). Data shown for all panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
Integrin {alpha}6ß1 and HSPGs mediate VMSC chemotaxis to CYR61
To investigate the effects of CYR61 on VSMCs further, we next examined its ability to stimulate cell migration. Using a modified Boyden chamber, we examined the migration of VSMCs loaded on the upper chamber through a membrane to the lower chamber, in which the chemoattractant is placed. As shown in Fig. 4AGo, VSMCs migrated to soluble CYR61 in a dose-dependent manner, with maximal migration occurring at 2 µg/ml. To determine whether CYR61 stimulates chemotaxis (directed migration) or chemokinesis (random motion), a checkerboard analysis was performed. Maximal VSMC migration was achieved when CYR61 was added to the lower chamber, whereas no migration over background was observed when CYR61 was added to the upper chamber (Fig. 4CGo). When CYR61 was added to both upper and lower chambers, migration was reduced by 37%. Thus, CYR61 stimulates chemotaxis, but not chemokinesis, in VSMCs.



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  		Figure 4. CYR61-induced VSMC migration is dose dependent, heparin sensitive, and directional. A modified Boyden chamber was used to measure smooth muscle cell migration. VSMCs were detached with trypsin and resuspended in serum-free SmBm, and 5 x 104 cells were loaded per well. Cells were allowed to migrate for 6 h at 37 C before being fixed and stained. Unless otherwise noted, the protein concentrations were 0.5% BSA, 5 ng/ml PDGF, and 2 µg/ml CYR61. A, Serially diluted CYR61, CYR61{Delta}CT, and CYR61DM at indicated concentrations were used as the chemoattractant. B, The proteins were incubated with the indicated heparin concentration 15 min before chamber loading at room temperature. C, Checkerboard migration assay, proteins were added to the cells (upper), as the chemoattractant (lower), or placed in both chambers as indicated. Data shown for all panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
Interestingly, heparin-binding mutants of CYR61 still maintained the ability to stimulate VSMC chemotaxis, albeit at a decreased level, compared with the wild-type. Both CYR61{Delta}CT and CYR61DM stimulated VSMC chemotaxis at a level approximately 40% of wild-type CYR61 (Fig. 4AGo), indicating that the heparin-binding activity of CYR61 is necessary for achieving maximal activity. To test this notion, we preincubated wild-type CYR61 with various amounts of heparin for 15 min before application. As evident in Fig. 4BGo, the presence of only 100 ng/ml heparin was sufficient to cause a 60% decrease in cell migration. At higher heparin concentrations (>1 µg/ml), more than 80% of cell migration to CYR61 was inhibited, but no effect on PDGF-induced migration was observed (Fig. 4BGo). These results show that the heparin-binding capability of CYR61 contributes significantly to its chemotactic activity.

Because cell surface HSPGs can function as coreceptors of integrins (33, 34) (Figs. 1 through 3GoGoGo), we examined the role of integrin receptors in CYR61-stimulated VSMC chemotaxis. In particular, the findings that integrins {alpha}vß3 and {alpha}vß5 mediate CYR61-induced cell migration in endothelial cells and fibroblasts, respectively (9, 10), prompted us to investigate the role of {alpha}v integrins in VSMC migration. When cells were treated with the peptide GRGDSP, a known antagonist of several integrin receptors including integrins {alpha}vß3 and {alpha}vß5 (32), no effect on CYR61-stimulated cell migration was observed (Fig. 5AGo). However, vitronectin-induced migration was reduced to background level because the RGD-sensitive {alpha}vß3 and {alpha}vß5 are the major vitronectin receptors (35). The control peptide GRGESP had no effect on vitronectin- or CYR61-mediated VSMC migration. Consistent with these results, neither LM609 (anti-{alpha}vß3) nor P1F6 (anti-{alpha}vß5) was able to inhibit VSMC migration to CYR61, whereas cell migration to vitronectin was diminished as expected (Fig. 5BGo). These results establish that VSMC migration is not mediated through {alpha}v integrins.



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  		Figure 5. VSMC migration to CYR61 is not mediated by {alpha}v integrin receptors. Migration assays were performed using VSMCs in a modified Boyden chamber with 0.5% BSA, 5 µg/ml fibronectin (FN), 1 µg/ml vitronectin (VN), and 2 µg/ml CYR61 as indicated. A, Cells were treated with GRGDSP or GRGESP peptide (0.2 mM) for 1 h at room temperature before chamber loading. B, Cells were treated with 50 µg/ml LM609 (integrin {alpha}Vß3 mAb) or P1F6 (integrin {alpha}Vß5 mAb) for 1 h at room temperature before chamber loading. Data shown for both panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
Treatment of VSMC with a mAb against integrin {alpha}6 (GoH3) resulted in approximately 30% decrease in migration to CYR61, whereas migration to vitronectin was unaffected (Fig. 6AGo). However, GoH3 completely inhibited VSMC migration to CYR61{Delta}CT, which lacks the heparin-binding domain. Similar results were obtained with the anti-ß1 mAb P4C10. In controls, an anti-{alpha}5ß1 mAb (JBS5) had no effect on VSMC chemotaxis to either wild-type CYR61 or CYR61{Delta}CT (data not shown). Thus, heparin-binding activity and interaction with integrin may both contribute to CYR61-mediated VSMC chemotaxis. To test this interpretation, we used heparin in conjunction with mAbs against integrin {alpha}6ß1. Although heparin, GoH3 (anti-{alpha}6), or P4C10 (anti-ß1) each had only partial inhibitory effect, the combination of heparin with either GoH3 or P4C10 completely abrogated CYR61-induced chemotaxis (Fig. 6BGo). As expected, these antagonists had no significant effect on vitronectin-stimulated migration. Taken together, these data show that VSMC migration to CYR61 is mediated through integrin {alpha}6ß1 and HSPGs.



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  		Figure 6. VSMC migration to CYR61 is mediated by integrin {alpha}6ß1 and heparan sulfate proteoglycan receptors. Migration assays were performed using VSMCs in a modified Boyden chamber with 0.5% BSA, 5 µg/ml fibronectin (FN), 1 µg/ml vitronectin (VN), and 2 µg/ml CYR61 as indicated. A, Cells were treated with 50 µg/ml GoH3 (integrin {alpha}6 mAb) for 1 h at room temperature before chamber loading. B, As indicated, protein was treated with 0.1 µg/ml heparin (Hep) for 15 min before chamber loading and addition of cells preincubated for 1 h with GoH3 (integrin {alpha}6 mAb) or P4C10 (integrin ß1 mAb). Data shown for all panels are mean ± SD of triplicate determinations and are representative of three experiments.

 
CYR61 is expressed during proliferative restenosis in the vessel media and neointima. We have demonstrated above that CYR61 supports cell adhesion and induces chemotaxis in VSMCs through integrin {alpha}6ß1 and cell surface HSPGs. These activities may be important in a number of vascular processes, including responses to injury. To explore this possibility, we employed a balloon angioplasty-induced proliferative restenosis model to investigate the roles of CYR61 in vascular injury (36). Total cell lysates were prepared from rat carotid artery obtained at the indicated times following balloon angioplasty and analyzed by immunoblotting (Fig. 7AGo). In the uninjured vessel (d 0), CYR61 was detected at a low level by immunoblotting. Following balloon angioplasty, the level of CYR61 started to increase at d 4 (more than 7-fold of control), peaked, and remained elevated from about d 7 through at least d 28 (more than 15-fold of control) (Fig. 7AGo). During this period, VSMCs in the media degrade the surrounding ECM, migrate out from the media to form the neointima, reorganize the ECM, and proliferate within the neointima (36, 37, 38, 39).



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  		Figure 7. CYR61 expression during vascular injury following rat carotid artery balloon angioplasty. A, Seventy micrograms of rat carotid artery total cell lysate from the days indicated were loaded per lane and electrophoresed on an SDS-polyacrylamide gel, followed by immunoblotting with anti-CYR61 antibodies. The nitrocellulose membrane was subsequently stripped and reprobed with anti-ß-tubulin antibodies. B, Immunohistochemistry of rat carotid artery frozen sections before (d 0) and after (d 7 and d 14) balloon angioplasty. Sections were probed with anti-CYR61 and counterstained with hematoxylin (a, c, e). As a control, CYR61-specific antibodies were first saturated with 2 µg/ml recombinant CYR61 before tissue application (b, d, f). In the normal carotid artery, only the intima stains positively for CYR61 expression (a), and the staining is diminished in the control sample (b). The marked elevation in CYR61 by d 7 is accompanied by its expression surrounding the medial smooth muscle cells (c), and the inhibition of this staining by saturating the antibody demonstrated specificity (d). CYR61 expression remains elevated in the media by d 14 but is also associated with proliferating smooth muscle cells in the neointima (e). This staining is abrogated in the control section (f). A, Adventitia; I, intima; M, media; N, neointima. Bar, 20 µm.

 
The pattern of vascular CYR61 accumulation was determined by immunohistochemistry performed using antibodies generated against a peptide corresponding to the last 15 amino acid residues of CYR61 (Fig. 7BGo). Because this region is significantly divergent among CCN family members, our antibodies do not cross-react with other CCN family members (data not shown). Whereas there was no detectable CYR61 in smooth muscle cells in the media of the uninjured vessel (Fig. 7BaGo), CYR61 was detected in endothelial cells of the intima (13, 27). When the antibody was saturated with 2 µg/ml recombinant CYR61 protein before application, the intimal staining was blocked (Fig. 7BbGo), although some level of nonspecific staining can be observed in the adventitia. At d 7 post angioplasty, CYR61 was markedly up-regulated and detectable in the media. At this stage, activated VSMCs had not yet migrated from the media to form the neointima (Fig. 7BcGo). No medial staining was observed with the neutralized antibody, demonstrating the specificity of the CYR61 staining (Fig. 7BeGo). Between d 7 and d 14, VSMCs migrated from the media to form the neointima in which they continue to proliferate. By d 14, CYR61 was associated with both VSMCs of the media and proliferating neointima (Fig. 7BeGo). As expected, pretreating the antibodies with CYR61 abolished this staining (Fig. 7BfGo). These results show that CYR61 is up-regulated following rat carotid artery balloon angioplasty and is associated with VSMC migration, proliferation, and matrix remodeling during proliferative restenosis.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have established that the angiogenic factor CYR61 supports VSMC adhesion and stimulates chemotaxis and that both processes are mediated though integrin {alpha}6ß1 and cell surface HSPGs. Furthermore, CYR61 is up-regulated in the media and neointima during restenosis following rat carotid artery balloon angioplasty. These findings demonstrate the activities of CYR61 in VSMCs, identify the cellular receptors mediating its actions, and suggest that CYR61 may be involved in vascular responses to injury.

Although the expression of CYR61 has been noted in smooth muscle cells of arterial walls (27), its functions in VSMCs have not been investigated. The finding that the dose-dependent adhesion of VSMC to purified CYR61 is through integrin {alpha}6ß1 and cell surface HSPGs is based on several lines of evidence (Figs. 1–3GoGoGo). First, mAbs against integrin {alpha}6 and ß1 effectively and specifically blocked CYR61-supported VSMC adhesion. Second, removal of cell surface HSPGs with heparinase abrogated cell adhesion. Third, soluble heparin completely blocks CYR61-supported VSMC adhesion, most likely because of saturation of the CYR61 heparin-binding sites. Finally, heparin-binding defective mutants of CYR61 are unable to support VSMC adhesion. These results indicate that integrin {alpha}6ß1 and cell surface HSPGs function as coreceptors for VSMC adhesion to CYR61 and that CYR61 interacts directly with cell surface HSPGs. The absolute requirement of HSPGs for integrin-mediated cell adhesion appears to be a unique property of CYR61 as an adhesive substrate and this requirement is associated with utilization of integrin {alpha}6ß1 (18). For example, heparin-binding defective CYR61 mutants can still mediate adhesion of endothelial cells through integrin {alpha}vß3 (18).

A checkerboard analysis showed that CYR61 stimulated VSMC chemotaxis, rather than chemokinesis (Fig. 4CGo). CYR61 has been shown to stimulate endothelial cell chemotaxis through integrin {alpha}vß3 in endothelial cells and fibroblast migration through integrin {alpha}vß5, with no apparent role for HSPGs (9, 10). Therefore, our finding that CYR61-stimulated VSMC chemotaxis is mediated through both integrin {alpha}6ß1 and cell surface HSPGs is somewhat unexpected (Figs. 4–6GoGoGo). These observations support the contention that integrin receptor utilization of CYR61 is both cell type and function specific (9). Thus, CYR61-supported cell adhesion, migration, and proliferation in fibroblasts are mediated through integrin {alpha}6ß1, {alpha}vß5, and {alpha}vß3, respectively (9, 18), whereas the activities of CYR61 in endothelial cells are largely dependent on integrin {alpha}vß3 (10, 16). Although the principal known substrate for integrin {alpha}6ß1 is laminin (40), the abundance of CYR61 in arterial smooth muscle cells after injury makes it a suitable substrate for this integrin (Fig. 7Go). To our knowledge, this study provides the first evidence showing that integrin {alpha}6ß1 cooperates with cell surface HSPGs to mediate chemotaxis in any cell type, although it has been shown that integrin {alpha}6ß1 can support cell motility and integrin {alpha}6 subunit mediates chemotaxis to laminin (41, 42, 43).

Cell surface HSPGs can modulate VSMC migration to many factors, either positively or negatively (44, 45, 46). Heparin has also been used to inhibit proliferative restenosis and neointimal formation in vivo, although its exact mechanism of action is not known (47, 48, 49, 50). It is noteworthy that CYR61-induced VSMC chemotaxis is 100-fold more sensitive to inhibition by heparin than other chemoattractants such as PDGF (Refs. 45 and 51 and Fig. 4Go). Thus, whereas approximately 60% inhibition of CYR61-stimulated migration was observed at 100 ng/ml heparin, similar inhibition of PDGF-stimulated migration requires 10 µg/ml heparin, suggesting a mechanistic difference in the inhibitory role of heparin. Integrin {alpha}6ß1 and cell surface HSPGs appear to act cooperatively, with contributions from both receptors being required for maximal chemotaxis to CYR61 (Fig. 4Go). However, heparin-binding defective CYR61 mutants, CYR61DM and CYR61{Delta}CT, can still stimulate {alpha}6ß1-dependent chemotaxis in VSMCs (Figs. 4Go and 6Go). Thus, the integrin {alpha}6ß1-dependent chemotactic activity of CYR61 can be dissociated from the HSPG mediated activities. Furthermore, the finding that CYR61{Delta}CT support integrin {alpha}6ß1-dependent VSMC chemotaxis indicate that an integrin {alpha}6ß1 binding site resides within the first three domains of CYR61.

Although CYR61 may play an important role during embryonic development (27), its activities in the adult have been associated with tissue responses to injury. CYR61 is expressed during bone fracture repair, liver regeneration, and granulation tissue formation during cutaneous wound healing (13, 52, 53). Furthermore, CYR61 up-regulates expression of genes that control processes related to wound repair, including angiogenesis, inflammation, ECM remodeling, and cell-matrix interactions (12, 19). These findings and the ability of CYR61 to modulate VSMC adhesion and migration prompted us to examine its role in vascular injury using a rat carotid artery model for studying restenosis following balloon angioplasty (36, 54, 55). Immunoblot and histological analyses showed that CYR61 accumulates at high levels in the vessel media and neointima from d 7 to d 14 after balloon angioplasty (Fig. 7Go), corresponding to the period of migratory and proliferative activity of smooth muscle cells in restenosis (36, 37, 54). ECM remodeling follows vascular injury, such as that induced by plaque rupture or balloon angioplasty, concomitant with increased matrix metalloproteinase activity and new ECM protein synthesis. These activities create an environment that facilitates VSMC migration from the vascular media to the intima and supports VSMC anchorage, survival, and proliferation (56, 57). In this regard, it is interesting to note that CYR61 can up-regulate the expression of matrix metalloproteinases I and III as an adhesion substrate and in soluble form (12, 19). CYR61 appears to be part of the arsenal of new matrix proteins synthesized by VSMCs during restenosis (Fig. 7Go), and its ability to promote VSMC adhesion and migration is consistent with a role in the response to vascular injury.

Despite recent therapeutic advances, proliferative restenosis continues to be the major problem limiting long-term efficacy of angioplasty procedures. ECM proteins that promote matrix remodeling or stimulate VSMC chemotaxis and proliferation have increasingly become the target of therapeutic interventions (37, 56, 58). CYR61, through its interaction with integrin {alpha}6ß1 and HSPGs, may play a critical role in the cellular response to vascular injury and thus presents a potential target of therapeutic intervention that warrants further investigation.


   Footnotes
 
This work was supported by NIH Grants CA-46565 and CA-80080 (to L.F.L.) and HL-41793 (to S.C.-T.L.).

Abbreviations: ABC, Avidin/biotin complex; CTGF, connective tissue growth factor; CYR61, cysteine-rich 61; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycan; mAb, monoclonal antibody; PDGF, platelet-derived growth factor; RGD, arginine-glycine-aspartate; SmBm, smooth muscle basal medium; VSMC, vascular smooth muscle cell; WISP-1, Wnt-induced secreted protein-1.

Received October 12, 2001.

Accepted for publication December 12, 2001.


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 Introduction
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 Results
 Discussion
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