This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ichii, T. Articles by Nishizawa, Y. Search for Related Content PubMed PubMed Citation Articles by Ichii, T. Articles by Nishizawa, Y. Related Collections Gene expression Smooth muscle proliferation and differentiation

(Circulation Research. 2001;88:460.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Fibrillar Collagen Specifically Regulates Human Vascular Smooth Muscle Cell Genes Involved in Cellular Responses and the Pericellular Matrix Environment

Takuya Ichii, Hidenori Koyama, Shinji Tanaka, Shokei Kim, Atsushi Shioi, Yasuhisa Okuno, Elaine W. Raines, Hiroshi Iwao, Shuzo Otani, Yoshiki Nishizawa

From the Departments of Biochemistry (T.I., S.O.), Internal Medicine (H.K., S.T., Y.N.), Pharmacology (S.K., H.I.), and Cardiovascular Medicine (A.S., Y.O.), Osaka City University Medical School, Osaka, Japan, and Department of Pathology (E.W.R.), University of Washington, Seattle, Wash.

Correspondence to Hidenori Koyama, MD, PhD, Second Department of Internal Medicine, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail hidekoyama{at}med.osaka-cu.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Proliferation and _vß₃ integrin–dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including -actinin; (3) signaling molecules; (4) protein synthesis–associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix–specific gene modulation. Moreover, in vivo balloon catheter–mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1–mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased -actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.

Key Words: thrombospondin-1 • platelet-derived growth factor • -actinin • filamin • balloon injury

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Migration of vascular smooth muscle cells (SMCs) from the media into the intima contributes to progression of atherosclerotic lesions and restenosis after balloon angioplasty.¹ SMCs in the normal media are surrounded by extracellular matrix (ECM) molecules, including collagen type I, III, and IV and laminin. SMC interaction with matrix components can significantly influence their ability to respond to growth factors and/or chemoattractants and can promote the modulation of SMCs from a contractile to a synthetic phenotype.² Breakdown of the matrix may be necessary for initiation of SMC migration.³

A majority of cell-cell and cell-matrix interactions are mediated by specific transmembrane adhesion receptors of the integrin family of proteins.^{4 5} These molecules assemble as heterodimers through the noncovalent association of and ß subunits.^{4 5} Integrins serve as transmembrane links between the ECM and the cell surface, resulting in increased adhesion. Occupancy and clustering of integrins can activate intracellular signaling pathways and induce transcription factors and subsequent gene expression.^{4 6} Thus, integrins can influence cell migration not only by the regulation of adhesion and spreading but also by modulation of intracellular signaling events.

We have recently demonstrated that SMCs are arrested in G₁ on polymerized type I collagen fibrils in vitro, whereas monomer collagen supports SMC proliferation. Analysis of molecular mechanisms has revealed that on polymerized collagen, cyclin E–cyclin-dependent kinase 2 activity is suppressed through upregulation of the cyclin-dependent kinase inhibitor p27^Kip1.⁷ Moreover, in an animal model of atherosclerosis, collagen expression is clearly associated with upregulation of p27^Kip1 expression and inhibition of cell replication,⁸ indicating the significant role of fibrillar collagen and integrins on the regulation of SMC phenotype in the progression of atherosclerosis. We have also shown that polymerized collagen suppresses _vß₃ integrin–dependent SMC migratory activity stimulated by platelet-derived growth factor (PDGF).⁹ Moreover, SMCs cultured on polymerized collagen mimic many of the characteristics of medial SMCs in vivo.¹⁰

The present study was designed to identify genes specifically regulated by polymerized collagen in human SMCs. Using suppressive subtraction hybridization, we show that polymerized collagen suppresses expression of many genes, including ECM molecules and actin binding proteins, many of which are induced in carotid arteries after balloon catheter–mediated injury. Examination of one of the modulated matrix genes, thrombospondin-1, after culture of SMCs on polymerized collagen, demonstrates that in addition to altered gene expression, the response of the SMCs to a given matrix molecule is also modulated.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
We purchased the following reagents: type I collagen (Vitrogen 100), (Collagen Corp); human vitronectin (Takara Biomedicals); human thrombospondin-1 and RGE and cyclic Pen-RGD (cRGD) peptides (GIBCO BRL, Life Technologies); antibodies against human fibronectin, human thrombospondin, human ß₁ integrin (P4C10), and human _vß₃ integrin (LM609) (GIBCO BRL, Life Technologies); antibody against human CD36 (NeoMarkers); antibody against human CD47 (Pharmingen); antibody against chicken vinculin (Calbiochem-Novabiochem Corp); antibody against -actinin (ICN Biomedicals, Inc); phycoerythrin (PE)–conjugated anti-human thrombospondin-1 (Immunotech); and recombinant PDGF-BB (Genzyme). Human recombinant osteopontin was kindly provided by Dr. C.M. Giachelli, University of Washington (Seattle, Wash).¹¹

Cell Culture
Human SMCs were obtained and cultured as previously described.¹² The SMCs were isolated from umbilical artery and express SMC markers including smooth muscle (SM) -actin, calponin, and SM22. SMCs were cultured on the surface of collagen preparations (polymerized collagen fibrils and monomer collagen) as described.⁷ All experiments were repeated at least twice, and results were reproducible.

Animal Models
All procedures conformed with institutional guidelines for animal research. Male Sprague-Dawley rats 10 to 11 weeks old (Clea Japan, Tokyo, Japan) were used in the present study and fed standard laboratory chow (MF, Oriental Kobo) and given tap water ad libitum. Balloon injury of the carotid artery and isolation of RNA were performed as previously described.¹³ Carotid arteries from 10 to 14 animals were pooled for indicated time points, and 30 to 90 µg of RNA was isolated.

Suppressive Subtraction Hybridization
Poly A⁺ RNA was isolated, by use of the FastTrack 2.0 kit (Invitrogen), from human SMCs that had been cultured on monomer or polymerized collagen for 24 hours. Suppressive subtraction hybridization was performed with the PCR-Select cDNA subtraction kit (Clontech) as directed by the manufacturer, with the modification that a 5-fold greater than recommended amount of driver cDNA was added to the second hybridization.

Cloning and Sequencing of cDNAs and Northern Blot Hybridization
cDNAs isolated by suppressive subtraction hybridization were cloned into the PCR2.1 vector by way of the TA cloning kit (Invitrogen) and sequenced by the SQ-5500 DNA sequencer (Hitachi) with Thermo sequenase (Amersham Pharmacia Biotech Inc). Sequenced cDNA fragments were analyzed by Advanced Blast (National Center for Biotechnology Information; provided by the National Institutes of Health). Northern blot analysis was performed as described previously.¹⁴

Protein Analysis
Preparation of cell lysates, Western blot analysis, immunocytochemistry, and flow cytometric analysis were performed as previously described.⁷

Chemotaxis/Migration Assay
Chemotaxis/migration assays were performed in a modified Boyden chamber as described previously.¹⁵ Polycarbonate filters (Nuclepore, 5-µm pores; obtained from Costar Science Corp) were coated overnight with 10 µg/mL human vitronectin, 20 µg/mL human osteopontin, or 100 µg/mL bovine type I collagen. A solution of chemoattractant or vehicle diluted in 0.15% BSA/DMEM was placed in the bottom chamber. When the effects of blocking reagents were examined, suspended cells were preincubated with 100 µmol/L cRGD peptide or 20 µg/mL anti-integrin antibodies. Maximally blocking concentrations of reagents were determined from cell attachment assays.

Statistical Analysis
Statistical analysis was done by using the Student t test or ANOVA. These analyses were carried out using Stat View V software.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Eighteen Genes Are Suppressed and Four Genes Are Induced in Human SMCs on Polymerized Collagen Compared With SMCs on Monomer Collagen
Suppressive subtraction hybridization identifies differential suppression of 18 mRNAs when human SMCs are cultured on polymerized collagen for 24 hours compared with SMCs on monomer collagen. Differential expression of these genes was confirmed by Northern blot analyses (Figure 1). Sequence analyses revealed that the following 5 of the suppressed genes are ECM molecules: fibronectin, thrombospondin-1, tenascin-C, cysteine-rich protein 61 (Cyr61), and collagen type 1 (Table 1). Of 13 other cDNA fragments, 5 genes encode molecules involved in cytoskeletal organization, 3 encode signaling molecules, 2 are associated with protein synthesis, and 3 encode 2 known genes and a novel gene (M24-1), for which functions have not been elucidated (Table 1). The following 4 mRNAs are induced in SMCs on polymerized collagen: microfibril-associated glycoprotein 4 (MAGP-4), tissue-type plasminogen activator (tPA), spermidine/spermine N₁-acetyltransferase, and angiopoietin-related protein-2.

View larger version (60K): [in this window] [in a new window]   Figure 1. Kinetics of mRNA changes after plating cells on monomer versus polymerized collagen. Twenty-two cDNAs identified by suppressive subtraction hybridization in human SMCs on polymerized collagen compared with monomer collagen were analyzed by Northern blot (a representative Northern blot is shown, n=3) using total RNA isolated from human SMCs cultured on monomer (M) or polymerized (P) collagen for indicated times. Before plating on monomer or polymerized collagen, SMCs were maintained in 0.15% BSA/DMEM for 48 hours.

View this table: [in this window] [in a new window]   Table 1. Summary of cDNAs Regulated by Polymerized Collagen Identified by Subtraction Hybridization

Analyses of the kinetics of SMC mRNA regulation revealed that several of the ECM molecules, including fibronectin, thrombospondin-1, and tenascin-C, show similar kinetics of regulation by monomer and polymerized collagen. On monomer collagen, they were slowly induced in serum-free conditions with maximal levels observed 24 hours after plating the cells (Figure 1). Induction of fibronectin and thrombospondin-1 by PDGF or FCS was more modest with monomer collagen, whereas tenascin-C was strongly induced by FCS (Table 2). Cyr61 mRNA was differentially regulated by ECM and growth factors. In contrast to FCS, which strongly induced Cyr61 mRNA levels, monomer collagen slightly suppressed and polymerized collagen strongly suppressed Cyr61 mRNA levels (Table 2). Five genes involved in cytoskeletal organization showed similar kinetics of mRNA regulation in response to ECM and growth factors. All of the genes were markedly induced in cells plated on monomer collagen or in response to growth factors but not on polymerized collagen (Figure 1, Table 2).

View this table: [in this window] [in a new window]   Table 2. Patterns of mRNA Regulation by Collagens and Growth Factors in Human SMCs

Importantly, growth factor regulation of gene expression was dramatically modulated in cells cultured on polymerized collagen. Neither PDGF nor FCS was able to upregulate ECM molecules on polymerized collagen, except the slight FCS-induced upregulation of Cyr61 (Figure 2A). In contrast, MAGP-4 and tPA, both of which were upregulated on polymerized collagen, were downregulated after stimulation with growth factors. Interestingly, molecules involved in cytoskeletal organization were upregulated both by PDGF and by FCS in cells cultured on polymerized collagen (Figure 2A). Among signaling molecules, both PDGF and FCS were able to strongly induce calmodulin, but not calcium-independent phospholipase A2.

View larger version (62K): [in this window] [in a new window]   Figure 2. A, Effect of growth factors on gene expression of molecules, isolated with subtraction hybridization, on polymerized collagen in human SMCs. After SMCs were cultured on polymerized collagen in 0.15% BSA/DMEM for 24 hours, PDGF (10 ng/mL) or 10% FCS was added, and total RNA was isolated at indicated times. Northern blot analyses were performed using cDNA fragments isolated by suppressive subtraction hybridization. PLA2 indicates phospholipase A2. B, Expression of molecules isolated with subtraction hybridization in balloon catheter–injured rat carotid. At the indicated time after balloon injury of the rat carotid artery, the artery was removed; RNA was isolated and analyzed by Northern blot using cDNA isolated from the screen of human SMCs.

Many of the Genes Suppressed on Polymerized Collagen Are Induced in the Vascular Injury Model In Vivo
Human SMCs cultured on polymerized collagen mimic many of the characteristics of medial SMCs in vivo.¹⁰ To test the in vivo regulation of genes identified in our screen, we used the balloon catheter–injured rat carotid model of acute injury in vivo and examined regulation of the molecules suppressed on polymerized collagen in SMCs (Figure 2B). ECM molecules, including fibronectin, thrombospondin, tenascin-C, and Cyr61, were all upregulated as early as 6 hours after balloon injury. Similar upregulation of molecules involved in cytoskeletal organization, except myosin light chain, was also observed. Calmodulin was also upregulated after balloon injury in vivo.

To examine the polymerized collagen regulation of ECM molecules at the protein level, Western blot and flow cytometric analyses for thrombospondin-1 were performed. Cellular abundance of thrombospondin-1 protein was decreased on polymerized collagen as early as 6 hours, and this suppression was maintained for 24 hours (Figure 3A). A similar suppression of cellular fibronectin protein levels was also observed on polymerized collagen. Moreover, thrombospondin-1 accumulation on the cell surface was also markedly inhibited on polymerized collagen as determined by flow cytometric analyses (Figure 3B). Thrombospondin-1 was known to exert its effect through various receptors, including _vß₃ integrin, ₂ß₁ integrin, ₃ß₁ integrin, CD36, and CD47.¹⁶ Surface levels of _vß₃ integrin, CD36, and CD47 were comparable between cells cultured on monomer or polymerized collagen for 24 hours (Figure 3B). The _vß₃ integrin level was not altered on polymerized collagen, and surface expression of ₂ß₁ integrin was increased on polymerized collagen as previously described (data not shown).⁷ Thus, cellular abundance and surface accumulation of thrombospondin-1 were inhibited by polymerized collagen, and this regulation was likely due to suppression of mRNA expression.

View larger version (33K): [in this window] [in a new window]   Figure 3. Thrombospondin-1 protein abundance and accumulation on the cell surface are suppressed in SMCs cultured on polymerized collagen. A, Western blot analysis for thrombospondin-1. After 48 hours in 0.15% BSA/DMEM, SMCs were plated on monomer (M) or polymerized (P) collagen for indicated times. Cell lysates were prepared and analyzed by Western blot. B, Flow cytometric analyses for cell surface thrombospondin-1. Quiescent human SMCs were cultured on monomer (thin line) or polymerized (thick line) collagen for 24 hours and suspended by collagenase digestion. Flow cytometric analyses for thrombospondin and 3 known thrombospondin receptors (vß3, CD36, and CD47) were performed.

To understand which type of receptor is involved in accumulation of thrombospondin-1 on the SMC surface on monomer or polymerized collagen, we have examined the effect of blocking reagents on the cell surface thrombospondin-1 levels determined by flow cytometry. SMCs arrested in serum-free medium for 24 hours were suspended by trypsinization, incubated with antibodies or peptides, and then cultured on monomer or polymerized collagen for 24 hours in the presence of the reagents. Cells were resuspended with collagenase digestion, incubated with PE-labeled anti–thrombospondin-1 antibody, and analyzed by flow cytometry. As shown in Figure 4, cell surface accumulation of thrombospondin-1 was significantly suppressed by cRGD peptide or anti–_vß₃ integrin antibody, which suggests that _vß₃ integrin is one of the major thrombospondin-1 receptors for cell surface accumulation. Anti–ß₁ integrin antibody also significantly affects accumulation of thrombospondin-1 on cell surface.

View larger version (24K): [in this window] [in a new window]   Figure 4. vß3 Integrins are involved in cell surface accumulation of thrombospondin-1. Quiescent human SMCs were suspended by brief trypsinization, washed with 0.15% BSA/DMEM, and incubated for 30 minutes with 100 µmol/L peptide or 20 µg/mL purified antibody as indicated. Thereafter, cells were cultured for 24 hours on monomer (open columns) or polymerized (closed columns) collagen in 0.15% BSA/DMEM with the same peptide or antibody. After cells were suspended by collagenase digestion, levels of cell surface thrombospondin-1 were analyzed with flow cytometry using PE-conjugated anti–thrombospondin-1 antibody. Ordinate axis shows % of cell surface thrombospondin-1 level compared with RGE peptide–treated or IgG-treated SMCs cultured on monomer collagen expressed as 100%. Two-way ANOVA was performed with usage of inhibitor and matrix conditions (monomer or polymer) as 2 variables. a indicates P<0.05 vs monomer; b, P<0.05 vs control reagents against inhibitors; and c, P<0.05 vs monomer and control reagents.

Polymerized Collagen Suppresses Thrombospondin-1–Stimulated _vß₃ Integrin–Dependent Chemotaxis in Human SMCs
Thrombospondin-1 is a mitogen and chemoattractant for SMCs.^{17 18} We examined the effect of polymerized collagen on thrombospondin-mediated migration of human SMCs. Thrombospondin stimulated SMC migration on vitronectin, and its effect is somehow additive to PDGF (Figure 5A). Thrombospondin-stimulated SMC migration on vitronectin was inhibited by a blocking anti–_vß₃ integrin antibody and cRGD peptide (Figure 5B). Thrombospondin-1 acts as a chemoattractant; when it was added to the upper and lower chambers at the same concentration, we did not observe a migration of SMCs (Figure 5B). After culture of SMCs on polymerized collagen for 24 hours, the chemotactic effect of thrombospondin-1 on vitronectin was dramatically suppressed compared with cells cultured on monomer collagen (Figure 5C). Interestingly, this suppressive effect of polymerized collagen was only observed for vitronectin- and osteopontin-supported, but not for collagen-supported, SMC migration (Figure 5C). Because vitronectin and osteopontin were mainly recognized by _vß₃ integrin,^{9 19} polymerized collagen could suppress thrombospondin-mediated chemotaxis, at least partly, through inhibition of _vß₃ integrin function.

View larger version (20K): [in this window] [in a new window]   Figure 5. A, Thrombospondin-1 (TSP-1) is a potent stimulant of human SMC migration. Quiescent SMCs were trypsinized and washed with 0.15% BSA/DMEM. Migration was analyzed using a Boyden chamber system. A polycarbonate filter (5-µm pores) was precoated with 10 µg/mL human vitronectin. Indicated concentrations of human thrombospondin-1 with (filled bars) or without (open bars) 10 ng/mL PDGF-BB were added to the lower chamber. B, vß3 Integrins are involved in thrombospondin-1–mediated SMC chemotaxis on vitronectin. Quiescent SMCs were suspended and preincubated with 20 µg/mL antibody, 100 µmol/L peptide (RGE and cRGD), or 30 µg/mL thrombospondin-1 for 30 minutes. Cells were analyzed for migration (30 µg/mL thrombospondin-1 at lower chamber) on vitronectin. The chemotactic nature of the stimulation is demonstrated by the lack of migratory response after preincubation with thrombospondin-1. *P<0.05 vs IgG, 1-way ANOVA with multiple comparison (Scheffé type); P<0.05 vs RGE (Student t test). C, Polymerized collagen suppresses thrombospondin-1–mediated SMC chemotaxis on vitronectin and osteopontin. Quiescent SMCs were cultured on monomer (open column) or polymerized (filled column) collagen for 24 hours in 0.15% BSA/DMEM and suspended by collagenase digestion. Thrombospondin-1 (30 µg/mL)–mediated migration on vitronectin, osteopontin, or type I collagen was analyzed using a Boyden chamber system. *P<0.05 vs monomer collagen (Student t test). HPF indicates high-power field.

Polymerized Collagen Suppresses -Actinin Accumulation at Focal Adhesions on Vitronectin
-Actinin is one of the actin cross-linking and integrin binding proteins.²⁰ Modulation of -actinin levels affects cell motility and confers tumorigenicity.²¹ Because -actinin levels are potently suppressed by polymerized collagen (Figure 1), we examined whether altered -actinin distribution on vitronectin was associated with decreased _vß₃ integrin–dependent chemotaxis after culture on polymerized collagen. As shown in Figure 6, accumulation of -actinin at focal adhesions is potently suppressed in cells that had been cultured on polymerized collagen. This suppression is associated with less focal adhesion formation as determined by vinculin immunostaining (Figure 6). Thus, suppressed expression and focal accumulation of -actinin by polymerized collagen could be involved in decreased _vß₃ integrin–dependent chemotaxis stimulated by thrombospondin-1.

View larger version (121K): [in this window] [in a new window]   Figure 6. Polymerized collagen suppresses accumulation of -actinin at focal adhesions on vitronectin. Serum-deprived SMCs were cultured on monomer or polymerized collagen for 24 hours, suspended by collagenase digestion, and replated on vitronectin for 4 hours. Immunofluorescent and confocal microscopic analyses were performed to evaluate localization of -actinin and vinculin at the same laser intensity. Only the bottom surface of the cells was scanned by confocal microscopy. Bars=20 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we analyze genes specifically regulated by polymerized collagen by suppressive subtraction hybridization. We show that other ECM molecules and actin binding proteins are primary targets after SMC culture on polymerized collagen fibrils. By focusing on thrombospondin-1, we show that one matrix molecule, polymerized collagen, can dynamically regulate another matrix molecule’s expression and its function in vascular SMCs.

Suppressive subtraction hybridization identifies differential suppression of 18 mRNAs when human SMCs are cultured on polymerized collagen for 24 hours compared with SMCs on monomer collagen. Five of the suppressed genes are ECM molecules; the following 3 of these are implicated in the pathogenesis of atherosclerosis: fibronectin,²² thrombospondin-1,²³ and tenascin-C.²⁴ Moreover, fibronectin, thrombospondin, tenascin-C, and Cyr61 are all upregulated in the carotid artery immediately after the ballooning in vivo. Induction of fibronectin mRNA,²⁵ tenascin-C protein,²⁶ and thrombospondin-1 protein²⁷ in the carotid artery after balloon injury has been reported, and these genes are implicated in injury-induced phenotypic alteration of SMCs. Thus, polymerized collagen suppresses several ECM molecules that are dynamically regulated in the process of vascular injury in vivo. Our data also imply that the alteration of SMC ECM production in vivo may be regulated through an integrin-dependent mechanism.

Our screen also identified proteins involved in cytoskeletal organization. Filamin^{28 29} and -actinin²⁰ are known to interact with the cytoplasmic domain of integrins. Both -actinin and filamin mRNAs are significantly induced in SMCs plated on monomer collagen but not on polymerized collagen (Figure 1). Because cells on polymerized collagen do not form efficient focal adhesions,⁷ regulation of mRNAs involved in actin organization may correlate with the formation of focal adhesions. Moreover, these molecules are dynamically upregulated after arterial injury in vivo, suggesting their significant roles in vascular remodeling. WDR1 cDNA, which was among the regulated genes identified with unknown function, encodes a 67-kDa protein containing 9 WD40 repeat motifs that mediate protein-protein interactions.³⁰ The highly homologous yeast and slime mold WDR1 proteins bind actin, which suggests that WDR1 protein may be an actin binding protein as well.

The kinetics of SMC mRNA regulation in response to growth factors and culture on polymerized collagen suggest that modulation of many of the identified genes are specifically dependent on ECM. Cyr61 is potently downregulated by polymerized collagen, whereas 10% FCS markedly upregulates Cyr61 mRNA levels and PDGF modestly increases Cyr61 mRNA levels. This suggests that suppression of Cyr61 transcription may be unique to ECM (Figure 2). Moreover, neither PDGF nor FCS is able to efficiently induce matrix molecules (fibronectin, thrombospondin-1, tenascin-C, and Cyr61) on polymerized collagen, suggesting that the suppressive effect of integrin signaling is not bypassed by growth factors and could be independent of growth factor signaling. Although efforts are being made to identify specific signaling through ECM receptors, an integrin-specific signaling system or promoter element has not been identified.^{31 32} Thus, the genes that are inversely regulated by ECM and growth factors, including Cyr61, are attractive candidates for exploring the transcriptional mechanism specifically regulated by an integrin signaling system.

Polymerized collagen not only suppresses ECM molecule expression but also can suppress its function in SMCs. We have used a chemotactic assay system to examine the effect of polymerized collagen on thrombospondin-1–mediated function. By using selective matrices, this system is useful for examining the interaction of specific integrins, _vß₃ integrin–dependent migration through vitronectin and osteopontin^{9 19} and ₂ß₁ integrin–dependent migration on type I collagen.³³ By using these matrices, we are able to show that polymerized collagen can suppress thrombospondin-1–stimulated SMC chemotaxis on ligands recognized by _vß₃ integrin. Thus, polymerized collagen may directly, or indirectly by altering the SMC phenotype, suppress _vß₃ integrin function, which results in decreased thrombospondin-1–stimulated SMC migration.

In this study, we have shown that expression and accumulation of -actinin at focal adhesions are dramatically suppressed in cells cultured on polymerized collagen. Because -actinin is one of the actin cross-linking and integrin binding proteins²⁰ and is involved in cell motility and tumorigenicity,²¹ dynamic suppression of -actinin by polymerized collagen could be one of the mechanisms for decreased _vß₃ integrin–dependent SMC chemotaxis. _vß₃ integrins are shown to be expressed on some SMCs.^{19 33} _vß₃ integrin expression is upregulated in intimal lesions at various stages of atherosclerosis,³⁴ and _vß₃ blocking peptides effectively inhibit the vascular fibroproliferative response after balloon catheter injury.^{35 36} Our results demonstrate that ligand levels and functions of _vß₃ integrins are dynamically regulated by polymerized collagen fibrils.

In summary, we show that other ECM molecules and many actin binding proteins are primary targets of polymerized collagen fibril–regulated gene expression. By focusing on thrombospondin-1, we also show that one matrix molecule, polymerized collagen, can dynamically regulate another matrix molecule’s function in vascular SMCs. Finally, our data raise the possibility that polymerized collagen fibrils regulate SMC phenotype in the progression of atherosclerosis, and this effect may be partly mediated by regulation of _vß₃ integrin function and expression of its ligands.

	Acknowledgments

 
This work was supported by grants for scientific research (Grant 11838014 to H.K. and 11694307 to H.K., Y.N., A.S., E.W.R.) from the Ministry of Education, Science and Culture of Japan; a Japan Heart Foundation/Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease (to H.K.); a grant from Osaka Medical Research Foundation for Incurable Diseases (to H.K.); and National Institutes of Health Grant HL18645 (to E.W.R.). We thank Masayo Monden for excellent technical assistance and Barbara Droker for editorial assistance.

	Footnotes

 
Original received May 25, 2000; revision received December 28, 2000; accepted January 17, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]

2. Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol. 1988;107:307–319.[Abstract/Free Full Text]

3. Carragher NO, Levkau B, Ross R, Raines EW. Degraded collagen fragments promote rapid disassembly of smooth muscle focal adhesions that correlates with cleavage of pp125(FAK), paxillin, and talin. J Cell Biol. 1999;147:619–630.[Abstract/Free Full Text]

4. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11–25.[Medline] [Order article via Infotrieve]

5. Ruoslahti E. Integrins. J Clin Invest. 1991;87:1–5.

6. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549–599.[Medline] [Order article via Infotrieve]

7. Koyama H, Raines EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of Cdk2 inhibitors. Cell. 1996;87:1069–1078.[Medline] [Order article via Infotrieve]

8. Tanner FC, Yang ZY, Duckers E, Gordon D, Nabel GJ, Nabel EG. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res. 1998;82:396–403.[Abstract/Free Full Text]

9. Tanaka S, Koyama H, Ichii T, Shioi A, Mori K, Yamakawa K, Raines EW, Ross R, Morii H, Hosoi M, Nishizawa Y. Suppression of integrin alpha v beta 3-dependent smooth muscle cell migration by fibrillar type I collagen: involvement of plasminogen activator inhibitor-1. Circulation. 1999;100(suppl I):I-694. Abstract.

10. Raines EW, Koyama H, Carragher NO. The extracellular matrix dynamically regulates smooth muscle cell responsiveness to PDGF. Ann N Y Acad Sci. 2000;902:39–51.[Medline] [Order article via Infotrieve]

11. Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem. 2000;275:20197–20203.[Abstract/Free Full Text]

12. Fukumoto S, Koyama H, Hosoi M, Yamakawa K, Tanaka S, Morii H, Nishizawa Y. Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation. Circ Res. 1999;85:985–991.[Abstract/Free Full Text]

13. Kim S, Kawamura M, Wanibuchi H, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Iwao H. Angiotensin II type 1 receptor blockade inhibits the expression of immediate-early genes and fibronectin in rat injured artery. Circulation. 1995;92:88–95.[Abstract/Free Full Text]

14. Koyama H, Nishizawa Y, Hosoi M, Fukumoto S, Kogawa K, Shioi A, Morii H. The fumagillin analogue TNP-470 inhibits DNA synthesis of vascular smooth muscle cells stimulated by platelet-derived growth factor and insulin-like growth factor-I: possible involvement of cyclin-dependent kinase 2. Circ Res. 1996;79:757–764.[Abstract/Free Full Text]

15. Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest. 1994;93:1266–1274.

16. Frazier WA. Thrombospondins. Curr Opin Cell Biol. 1991;3:792–799.[Medline] [Order article via Infotrieve]

17. Yabkowitz R, Mansfield PJ, Ryan US, Suchard SJ. Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells. J Cell Physiol. 1993;157:24–32.[Medline] [Order article via Infotrieve]

18. Patel MK, Lymn JS, Clunn GF, Hughes AD. Thrombospondin-1 is a potent mitogen and chemoattractant for human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997;17:2107–2114.[Abstract/Free Full Text]

19. Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, Giachelli CM. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins: role of _vß₃ in smooth muscle cell migration to osteopontin in vitro. J Clin Invest. 1995;95:713–724.

20. Otey CA, Pavalko FM, Burridge K. An interaction between -actinin and the ß₁ integrin subunit in vitro. J Cell Biol. 1990;111:721–729.[Abstract/Free Full Text]

21. Gluck U, Ben-Ze’ev A. Modulation of -actinin levels affects cell motility and confers tumorigenicity on 3T3 cells. J Cell Sci. 1994;107:1773–1782.[Abstract]

22. Stenman S, von Smitten K, Vaheri A. Fibronectin and atherosclerosis. Acta Med Scand Suppl. 1980;642:165–170.[Medline] [Order article via Infotrieve]

23. Wight TN, Raugi GJ, Mumby SM, Bornstein P. Light microscopic immunolocation of thrombospondin in human tissues. J Histochem Cytochem. 1985;33:295–302.[Abstract]

24. Wallner K, Li C, Shah PK, Fishbein MC, Forrester JS, Kaul S, Sharifi BG. Tenascin-C is expressed in macrophage-rich human coronary atherosclerotic plaque. Circulation. 1999;99:1284–1289.[Abstract/Free Full Text]

25. Bauters C, Marotte F, Hamon M, Oliviero P, Farhadian F, Robert V, Samuel JL, Rappaport L. Accumulation of fetal fibronectin mRNAs after balloon denudation of rabbit arteries. Circulation. 1995;92:904–911.[Abstract/Free Full Text]

26. Hedin U, Holm J, Hansson GK. Induction of tenascin in rat arterial injury: relationship to altered smooth muscle cell phenotype. Am J Pathol. 1991;139:649–656.[Abstract]

27. Raugi GJ, Mullen JS, Bark DH, Okada T, Mayberg MR. Thrombospondin deposition in rat carotid artery injury. Am J Pathol. 1990;137:179–185.[Abstract]

28. Loo DT, Kanner SB, Aruffo A. Filamin binds to the cytoplasmic domain of the ß₁-integrin: identification of amino acids responsible for this interaction. J Biol Chem. 1998;273:23304–23312.[Abstract/Free Full Text]

29. Pfaff M, Liu S, Erle DJ, Ginsberg MH. Integrin ß cytoplasmic domains differentially bind to cytoskeletal proteins. J Biol Chem. 1998;273:6104–6109.[Abstract/Free Full Text]

30. Adler HJ, Winnicki RS, Gong TW, Lomax MI. A gene upregulated in the acoustically damaged chick basilar papilla encodes a novel WD40 repeat protein. Genomics. 1999;56:59–69.[Medline] [Order article via Infotrieve]

31. Danen EH, Lafrenie RM, Miyamoto S, Yamada KM. Integrin signaling: cytoskeletal complexes, MAP kinase activation, and regulation of gene expression. Cell Adhes Commun. 1998;6:217–224.[Medline] [Order article via Infotrieve]

32. Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999;285:1028–1032.[Abstract/Free Full Text]

33. Skinner MP, Raines EW, Ross R. Dynamic expression of ₁ß₁ and ₂ß₁ integrin receptors by human vascular smooth muscle cells: ₂ß₁-integrin is required for chemotaxis across type I collagen-coated membranes. Am J Pathol. 1994;145:1070–1081.[Abstract]

34. Hoshiga M, Alpers CE, Smith LL, Giachelli CM, Schwartz SM. _vß₃ integrin expression in normal and atherosclerotic artery. Circ Res. 1995;77:1129–1135.[Abstract/Free Full Text]

35. Choi ET, Engel L, Callow AD, Sun S, Trachtenberg J, Santoro S, Ryan US. Inhibition of neointimal hyperplasia by blocking _vß₃ integrin with a small peptide antagonist GpenGRGDSPCA. J Vasc Surg. 1994;19:125–134.[Medline] [Order article via Infotrieve]

36. Matsuno H, Stassen JM, Vermylen J, Deckmyn H. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation. 1994;90:2203–2206. [Abstract/Free Full Text]

This article has been cited by other articles:

				N. Ferri, E. Roncalli, L. Arnaboldi, S. Fenu, O. Andrukhova, S. Aharinejad, M. Camera, E. Tremoli, and A. Corsini Fibrillar Collagen Inhibits Cholesterol Biosynthesis in Human Aortic Smooth Muscle Cells Arterioscler Thromb Vasc Biol, October 1, 2009; 29(10): 1631 - 1637. [Abstract] [Full Text] [PDF]

				E. Adiguzel, P. J Ahmad, C. Franco, and M. P Bendeck Collagens in the progression and complications of atherosclerosis Vascular Medicine, February 1, 2009; 14(1): 73 - 89. [Abstract] [PDF]

				C. Franco, G. Hou, P. J. Ahmad, E. Y.K. Fu, L. Koh, W. F. Vogel, and M. P. Bendeck Discoidin Domain Receptor 1 (Ddr1) Deletion Decreases Atherosclerosis by Accelerating Matrix Accumulation and Reducing Inflammation in Low-Density Lipoprotein Receptor-Deficient Mice Circ. Res., May 23, 2008; 102(10): 1202 - 1211. [Abstract] [Full Text] [PDF]

				J. Sottile, F. Shi, I. Rublyevska, H.-Y. Chiang, J. Lust, and J. Chandler Fibronectin-dependent collagen I deposition modulates the cell response to fibronectin Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1934 - C1946. [Abstract] [Full Text] [PDF]

				E. Adiguzel, G. Hou, D. Mulholland, U. Hopfer, N. Fukai, B. Olsen, and M. Bendeck Migration and Growth Are Attenuated in Vascular Smooth Muscle Cells With Type VIII Collagen-Null Alleles Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 56 - 61. [Abstract] [Full Text] [PDF]

				A. A. Arslan, L. I. Gold, K. Mittal, T.-C. Suen, I. Belitskaya-Levy, M.-S. Tang, and P. Toniolo Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review Hum. Reprod., April 1, 2005; 20(4): 852 - 863. [Abstract] [Full Text] [PDF]

				T. T.-B. Nguyen, J. P. T. Ward, and S. J. Hirst {beta}1-Integrins Mediate Enhancement of Airway Smooth Muscle Proliferation by Collagen and Fibronectin Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 217 - 223. [Abstract] [Full Text] [PDF]

				S. D. Rybalkin, C. Yan, K. E. Bornfeldt, and J. A. Beavo Cyclic GMP Phosphodiesterases and Regulation of Smooth Muscle Function Circ. Res., August 22, 2003; 93(4): 280 - 291. [Abstract] [Full Text] [PDF]

				T. Morioka, H. Koyama, H. Yamamura, S. Tanaka, S. Fukumoto, M. Emoto, H. Mizuguchi, T. Hayakawa, I. Kojima, K. Takahashi, et al. Role of H1-Calponin in Pancreatic AR42J Cell Differentiation Into Insulin-Producing Cells Diabetes, March 1, 2003; 52(3): 760 - 766. [Abstract] [Full Text] [PDF]

				Q. J. Zhang, M. Goddard, C. Shanahan, L. Shapiro, and M. Bennett Differential Gene Expression in Vascular Smooth Muscle Cells in Primary Atherosclerosis and In Stent Stenosis in Humans Arterioscler Thromb Vasc Biol, December 1, 2002; 22(12): 2030 - 2036. [Abstract] [Full Text] [PDF]

				D. E. Vaughan PAI-1 and Cellular Migration: Dabbling in Paradox Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1522 - 1523. [Full Text] [PDF]

				S. Tanaka, H. Koyama, T. Ichii, A. Shioi, M. Hosoi, E. W. Raines, and Y. Nishizawa Fibrillar Collagen Regulation of Plasminogen Activator Inhibitor-1 Is Involved in Altered Smooth Muscle Cell Migration Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1573 - 1578. [Abstract] [Full Text] [PDF]

				T. Ichii, H. Koyama, S. Tanaka, A. Shioi, Y. Okuno, S. Otani, and Y. Nishizawa Thrombospondin-1 Mediates Smooth Muscle Cell Proliferation Induced by Interaction With Human Platelets Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1286 - 1292. [Abstract] [Full Text] [PDF]

				A. C. Newby Vitronectin is implicated as the matrix takes control of neointima formation Cardiovasc Res, March 1, 2002; 53(4): 779 - 781. [Full Text] [PDF]

				S. D. Rybalkin, I. Rybalkina, J. A. Beavo, and K. E. Bornfeldt Cyclic Nucleotide Phosphodiesterase 1C Promotes Human Arterial Smooth Muscle Cell Proliferation Circ. Res., February 8, 2002; 90(2): 151 - 157. [Abstract] [Full Text] [PDF]

				N. Anilkumar, D. S. Annis, D. F. Mosher, and J. C. Adams Trimeric assembly of the C-terminal region of Thrombospondin-1 or Thrombospondin-2 is necessary for cell spreading and fascin spike organisation J. Cell Sci., January 6, 2002; 115(11): 2357 - 2366. [Abstract] [Full Text] [PDF]

				S. Sartore, A. Chiavegato, E. Faggin, R. Franch, M. Puato, S. Ausoni, and P. Pauletto Contribution of Adventitial Fibroblasts to Neointima Formation and Vascular Remodeling: From Innocent Bystander to Active Participant Circ. Res., December 7, 2001; 89(12): 1111 - 1121. [Abstract] [Full Text] [PDF]

				K. R. Stenmark Cell-, age-, and phenotype-dependent differences in the control of gene expression Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L762 - L765. [Full Text] [PDF]

				J. G. Pickering Regulation of Vascular Cell Behavior by Collagen : Form Is Function Circ. Res., March 16, 2001; 88(5): 458 - 459. [Full Text] [PDF]

				S. D. Rybalkin, I. Rybalkina, J. A. Beavo, and K. E. Bornfeldt Cyclic Nucleotide Phosphodiesterase 1C Promotes Human Arterial Smooth Muscle Cell Proliferation Circ. Res., February 8, 2002; 90(2): 151 - 157. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ichii, T. Articles by Nishizawa, Y. Search for Related Content PubMed PubMed Citation Articles by Ichii, T. Articles by Nishizawa, Y. Related Collections Gene expression Smooth muscle proliferation and differentiation