Fibrillar Collagen Regulation of Plasminogen Activator Inhibitor-1 Is Involved in Altered Smooth Muscle Cell Migration
Objective— Vascular smooth muscle cells (SMCs) cultured on polymerized type I collagen fibrils are arrested in the G1 phase of the cell cycle, and their phenotypic markers and pattern of expressed genes are markedly altered. In this study, we examined polymerized collagen regulation of plasminogen activator inhibitor (PAI)-1 and its involvement in SMC migration.
Methods and Results— We demonstrate that secretion and cell surface accumulation of PAI-1 are suppressed in SMCs cultured on polymerized collagen compared with SMCs cultured on monomer collagen. SMCs replated on vitronectin after culture on monomer collagen result in PAI-1 accumulation at focal adhesions and colocalization with αvβ3 integrins. In contrast, polymerized collagen inhibits PAI-1 accumulation at focal adhesions when the SMCs are replated on vitronectin. Furthermore, for SMCs cultured on polymerized collagen, platelet-derived growth factor-stimulated migration on vitronectin is enhanced by PAI-1, with its function counteracted by urinary plasminogen activator. Finally, exogenous addition of PAI-1 appears to partly restore platelet-derived growth factor-stimulated αvβ3-dependent SMC migration that is specifically suppressed by polymerized collagen.
Conclusions— Polymerized type I collagen fibrils dynamically regulate PAI-1, which may be involved in altered αvβ3 integrin-dependent SMC migration.
Migration and proliferation of vascular smooth muscle cells (SMCs) from the media into the intima contribute to lesion progression in atherosclerosis and restenosis after balloon angioplasty.1 SMCs in the normal media are surrounded by extracellular matrix molecules, including collagen types I, III, and IV and laminin. SMC interaction with these 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.2,3⇓
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Generation of pericellular plasmin by urinary plasminogen activator (uPA) with subsequent direct or indirect proteolysis of the extracellular matrix is thought to contribute to matrix remodeling and cellular migration.4 The local tissue levels of active uPA and plasmin are regulated by plasminogen activator inhibitor (PAI) types 1 and 2 (PAI-1 and PAI-2, respectively). Active uPA avidly binds to specific glycosylphosphatidylinositol-anchored cell surface receptors (uPARs),5 also promoting focused pericellular proteolysis. Inhibition of active uPA by complex formation with PAI-1 or PAI-2 results in the removal of urokinase-PAI-uPAR complexes from the cell surface,6 which may be associated with a dynamic regulatory role of PAI-1 in uPAR-mediated cell adhesion and release.7 PAI-1, independent of its ability to inhibit plasminogen activators, binds to vitronectin or αvβ3 integrins and directly modulates vitronectin receptor-mediated cell adhesion and migration.8–11⇓⇓⇓ Moreover, PAI-1 levels in SMCs are shown to be upregulated in arteries after balloon injury,12,13⇓ and PAI-1 is known to be abundantly expressed in human atherosclerotic lesions.14,15⇓ Thus, PAI-1 expression may be involved in phenotypic alteration of the SMCs in the progression of atherosclerosis.
We demonstrate that SMCs are arrested in the G1 phase on polymerized type I collagen fibrils in vitro, whereas monomer collagen supports SMC proliferation. On polymerized collagen, cyclin E-cyclin-dependent kinase 2 activity is suppressed through the upregulation of p27Kip1, a cyclin-dependent kinase inhibitor.16 In vivo, collagen expression is associated with the upregulation of p27Kip1 expression and inhibition of cell replication in an animal model of lesion formation,17 suggesting a potential role of fibrillar collagen in the regulation of SMC phenotype in the progression of atherosclerosis. Moreover, we have recently shown that a culture of SMCs on polymerized collagen mimics many of the features of SMCs in normal media18 and modulates SMC gene expression.19 In the present study, we have examined the effect of polymerized collagen on PAI-1 regulation and cell migration in human SMCs. We demonstrate that polymerized collagen regulation of PAI-1 is involved in altered αvβ3 integrin-dependent SMC migration stimulated by platelet-derived growth factor (PDGF).
Immunochemically prepared human PAI-1 (No. 1090, 2850±264 uPA U/mg), a recombinant human metabolically stable mutant form of PAI-1 (No. 1094, 11 600±850 uPA U/mg), and recombinant inactive human PAI-1 (No. 1096, <500 uPA U/mg) were purchased from American Diagnostica Inc. The activity of each PAI-1 preparation was determined as described below (PAI-1 and uPA assay). Chemically purified PAI-1 (No. 1090) was 66.4±18.7% (mean±SD) as effective as recombinant stable PAI-1 (No. 1094) in stimulating SMC migration. Thus, we used the stable form of PAI-1 for most of the experiments to examine the role of active PAI-1. Anti-PAI-1 monoclonal antibody (Nos. 379 and 380, which recognize active and inactive PAI-1, respectively), uPA (No. 124), and blocking anti-uPAR antibody (No. 3936) were also purchased from American Diagnostica Inc. Type I collagen (Vitrogen 100) was obtained from Collagen Corp; human vitronectin, from Takara Biomedicals; human fibronectin, from GIBCO-BRL, Life Technologies Inc; and anti-human αvβ3 integrin (LM609), from Chemicon International Inc. Recombinant human osteopontin20 was kindly provided by Dr C.M. Giachelli (University of Washington, Seattle). Recombinant PDGF-BB was purchased from Genzyme. Human angiotensin II was purchased from Sigma Chemical Co.
Cells and Cell Culture
Human SMCs (umbilical artery origin), obtained from Cell System Co, were cultured as described.21 SMCs were cultured on the surface of the indicated collagen preparations, polymerized collagen fibrils, and monomer collagen film, prepared as described.16
Chemotaxis/migration assays were performed in a modified Boyden chamber, as described previously,19 with the use of matrix-coated filters (10 μg/mL human vitronectin, 20 μg/mL human osteopontin, 100 μg/mL bovine type I monomer collagen, or 10 μg/mL human fibronectin) and 10 ng/mL PDGF-BB or 100 nmol/L angiotensin II as a chemoattractant.
Cell Adhesion Assay
SMCs were plated for 30 minutes under the same conditions used for the migration assay. Attached cells were fixed in 3.7% formaldehyde and stained with 0.5% toluidine blue/3.7% formaldehyde. Adherent cells were directly counted or solubilized in 2% sodium dodecyl sulfate for measurement of 650-nm absorption with a spectrophotometer.
PAI-1 and uPA Assay
Release of PAI-1 and uPA into culture media was measured by a PAI-1 ELISA kit (TintElize PAI-1, Biopool) and a uPA ELISA kit (IMUBIND uPA ELISA kit, American Diagnostica Inc). The PAI-1 ELISA kit detects active and inactive forms of PAI-1, as indicated in the manufacturer’s instructions. PAI-1 activity was measured by titrating samples with increasing amounts of uPA into a fixed volume of SMC-conditioned media, as originally described.22 The excess of uPA activity was quantified by a uPA assay kit (Chemicon International). PAI-1 activity was calculated from the intersection of the asymptote of the titration curve with the x-axis and was expressed as units of uPA inhibited.
Flow Cytometry, Immunocytochemistry, and Confocal Microscopy
Flow cytometric analysis, immunocytochemistry, and confocal microscopic analysis were performed as described previously.16 For flow cytometry, SMCs were suspended with collagenase digestion. In preliminary experiments, 30 minutes of collagenase treatment did not affect the surface level of PAI-1 or uPAR in our system. For cell surface PAI-1 determination, No. 380 anti-PAI-1 antibody, which equally recognizes active and inactive PAI-1, was used.
All the experiments were repeated at least twice. Statistical analysis was performed by the Student t test or ANOVA combined with a multiple comparison (Scheffé-type) test for comparing groups. These statistical analyses were carried out with the use of Stat View IV software (SAS Institute).
Polymerized Collagen Fibrils Dynamically Regulate uPA, uPAR, and PAI-1 Expression in Human SMCs
We have demonstrated that the culture of SMCs on polymerized collagen fibrils arrests cells in the G1 phase of the cell cycle16 and alters the pattern of expressed genes.19 In Figure 1, we examined possible regulation of PAI-1 in human SMCs cultured on different forms of type I collagen. Compared with SMCs cultured on monomer collagen, SMCs cultured on polymerized collagen secreted less PAI-1 into the culture media (Figure 1a). PAI-1 activity in conditioned media at 24 hours on monomer collagen (0.26±0.04 U/mL) was significantly (P<0.05, Student t test) higher than that on polymerized collagen (0.18±0.03 U/mL). Surface accumulation of PAI-1 was also significantly suppressed by culture on polymerized collagen, as determined by flow cytometry (Figure 1b). In our SMC system, compared with PAI-1 secretion, uPA secretion was less abundant (Figure 1a). Moreover, flow cytometric analyses showed that only 5% of our SMCs were positive for uPAR expression (Figure 1b). Polymerized collagen increased the expression of uPAR, but only ≈20% to 25% of the SMCs were positive for the receptor (Figure 1b). Thus, PAI-1 dominates uPA expression in this SMC system, and its expression level is suppressed by polymerized collagen.
PAI-1 is known to interact with vitronectin8 and may modulate the function of αvβ3 integrin, a cell surface vitronectin receptor.10 Because levels of PAI-1 are dynamically altered in SMCs cultured on polymerized collagen, we examined its distribution in SMCs on vitronectin after the culture of SMCs on monomer or polymerized collagen for 24 hours. Analysis of PAI-1 distribution after culture on monomer collagen demonstrated the accumulation of PAI-1 close to the leading edge and in focal adhesion sites, together with diffuse staining at the bottom surface of the SMCs on vitronectin (Figure 1c). However, after 24 hours on polymerized collagen, SMCs failed to accumulate PAI-1 at focal adhesion sites, and PAI-1 was granularly distributed at the bottom of the cells.
PAI-1 Induces PDGF-Stimulated SMC Migration on Vitronectin
PAI-1 has been reported to affect cell migration in vitro in various experimental systems with different results.10,11,23⇓⇓ We examined the effects of PAI-1 on SMC migration after the culture of cells on polymerized collagen for 24 hours, a condition under which cells are arrested in the G1 phase and mimic many of the characteristics of medial SMCs in vivo.16,18⇓ Vitronectin-coated filters were used for migration; thus, SMC motility was dependent on αvβ3 integrin24 (data not shown). Exogenous addition of PAI-1 protein to SMCs dose-dependently increased PDGF-induced chemotaxis (Figure 2). A similar effect of PAI-1 was also observed on filters coated with osteopontin, another αvβ3 integrin-dependent ligand (Figure 2). As previously described,23 PAI-1 (10 μg/mL) also accelerated cellular motility on vitronectin in U937 monocytic cells by 2.55±1.2-fold (mean±SD).
To understand the mechanism underlying PAI-1-stimulated SMC migration, we first examined the effect of PAI-1 on SMC adhesion to vitronectin. As shown in Figure 3a, PAI-1 did not affect the adhesion of SMCs to vitronectin. However, focal adhesion formation on vitronectin, determined by immunostaining of αvβ3 integrins, was suppressed by treatment with 10 μg/mL PAI-1 (Figure 3b). Thus, PAI-1 appears to inhibit the integrity of SMC adhesion to vitronectin.
We next examined whether uPA could counteract the action of PAI-1 on PDGF-stimulated SMC migration. After culture of the cells on polymerized collagen, the ability of PAI-1 (10 μg/mL, equal to 232 nmol/L) to stimulate SMC migration on vitronectin was suppressed by the simultaneous addition of uPA, and 300 nmol/L uPA almost completely abrogated the effect of PAI-1 (see Figure I, which can be accessed online at http://www.ahajournals.org/). In accordance with the low expression level of uPAR in our experimental system, blocking the anti-uPAR antibody did not influence the effects of PAI-1 on PDGF-stimulated SMC migration.
Colocalization of PAI-1 With αvβ3 Integrin in SMCs Cultured on Vitronectin
Given the accumulation of PAI-1 at focal adhesions and its ability to enhance PDGF-stimulated SMC migration on vitronectin, we sought to determine whether PAI-1 associates with αvβ3 integrins on vitronectin. Double immunostaining with anti-αvβ3 integrin and PAI-1 antibody revealed that both molecules are colocalized at focal adhesions on vitronectin after culture on monomer collagen for 24 hours (Figure 4a). Moreover, when SMCs were plated on PAI-1 (50 μg/mL)-coated filters, cell adhesion was significantly inhibited by an anti-αvβ3 integrin-blocking antibody (Figure 4b). Thus, PAI-1 may interact with αvβ3 integrin and modulate its migratory function.
Polymerized Collagen Fibrils Suppress αvβ3 Integrin-Dependent SMC Migration, and Its Effect Is Partly Reversed by Exogenous PAI-1
Because polymerized collagen modulates the levels of PAI-1 that affect SMC migration on vitronectin, we sought to determine whether polymerized collagen might modulate αvβ3 integrin-dependent SMC migration. After 24 hours on polymerized collagen, PDGF-stimulated migration on vitronectin was suppressed by >50% compared with SMCs cultured on monomer collagen (Figure 5a). A similar inhibitory effect was also observed for osteopontin-coated filters, another ligand for αvβ3 integrin, but was not observed with monomer type I collagen or fibronectin as support matrices (Figure 5a), implying a suppressive effect of polymerized collagen specifically on αvβ3 integrin-dependent SMC migration. In cells that had been cultured on polymerized collagen, exogenous addition 30 minutes before the migration assay of active PAI-1, but not the latent and inactive form, which also lacks vitronectin binding activity, significantly restored PDGF-stimulated chemotaxis on vitronectin (Figure 5b). In contrast, PAI-1 did not affect SMC migration on vitronectin in cells cultured on monomer collagen. Thus, polymerized collagen fibrils specifically suppress αvβ3 integrin-dependent SMC migration, and altered regulation of PAI-1 may, at least partly, be involved in this process. The effect of PAI-1 may be specific to PDGF-stimulated signals, inasmuch as PAI-1 failed to recover polymerized collagen suppression of angiotensin II-induced SMC migration (Figure II, which can be accessed online at http://www.ahajournals.org/).
Polymerized Type I Collagen Suppresses PAI-1 Expression in Human SMCs
We have demonstrated that the phenotype of SMCs is markedly modulated by polymerized type I collagen fibrils in vitro. This modulation includes cell cycle arrest with alteration in the levels of cell cycle molecules,16 changes in phenotypic markers,18 and an altered pattern of expressed genes.19 Collagen expression is also associated with the upregulation of p27Kip1, the cell cycle inhibitor, and with the inhibition of cell replication in an animal model of lesion formation.17 Thus, fibrillar collagen appears to be involved in the regulation of the SMC phenotype in the progression of atherosclerosis. In the present study, we show that PAI-1 expression is dynamically suppressed by polymerized collagen compared with monomer collagen. PAI-1 is induced in arterial SMCs by balloon injury12,13⇓ and is known to be abundantly expressed in atherosclerotic lesions.14,15⇓
PAI-1 Interacts With αvβ3 Integrins and Enhances PDGF-Stimulated SMC Migration on Vitronectin
In addition to its strong protease inhibitory action against uPA, PAI-1 binds to vitronectin or αvβ3 integrins and may play a role in vitronectin receptor-mediated cell adhesion and migration.8–11,25⇓⇓⇓⇓ PAI inhibits cell migration through blocking the binding of αvβ3 integrin to vitronectin in rabbit SMCs.10 In vivo, PAI-1-null mice exhibit excessive intimal thickening in blood vessels after transluminal mechanical injury.26 Accordingly, local overexpression of PAI-1 into an injured artery is shown to inhibit neointimal formation.26,27⇓ However, by using atherosclerosis-prone apoE-null mice crossbred with PAI-1-null mice, the presence of the PAI-1 gene is shown to dramatically promote neointimal formation after oxidative vascular injury.28 Thus, the effects of PAI-1 on SMC motility may be dependent on the experimental system. In the present study, we demonstrated that in SMCs cultured on polymerized collagen, PAI-1 appears to enhance PDGF-stimulated SMC migration after plating on vitronectin. These data are in contrast to the observations of Stefansson and Lawrence10 involving rabbit SMCs, in which PAI-1 potently inhibited the attachment of SMCs to vitronectin, thus abrogating the migration of the cells. In human SMCs, adhesion to vitronectin was not suppressed by PAI-1. However, focal adhesion formation on vitronectin (detected by αvβ3 integrin immunostaining) appeared to be markedly suppressed by exogenous PAI-1. Thus, PAI-1 does not inhibit adhesion but decreases the stringency of cell attachment to vitronectin in our system, which may result in an increase in cellular migratory response to PDGF. It is known that relative cell adhesion to matrix can modulate SMC migration.29 It has been reported that the antiadhesive effect of PAI-1 is not dependent on its ability to inhibit uPA.10,11⇓ In our system, immunochemically prepared PAI-1, which was 24.5% as active in uPA inhibition, was 66% as effective as recombinant active PAI-1. This weak relationship between the activity of PAI-1 and the capacity to stimulate SMC migration may be explained by the fact that both PAI-1 preparations are capable of binding vitronectin. Accordingly, latent inactive PAI-1, which is incapable of binding vitronectin, fails to affect migration in human SMCs on vitronectin. We show that exogenous uPA counteracts the action of PAI-1, even though uPAR expression was low in our SMCs, potentially because uPA-PAI-1 complexes have little affinity for vitronectin and rapidly release PAI-1 from vitronectin.30,31⇓
Polymerized Collagen Specifically Suppresses αvβ3 Integrin-Dependent SMC Migration
The present study suggests that interaction of SMCs with polymerized collagen suppresses a distinct group of integrins, the vitronectin receptors. After culture on polymerized collagen, PDGF-directed SMC migration on vitronectin or osteopontin, not type I collagen and fibronectin, is significantly suppressed. The major vitronectin receptors identified to date are αvβ1, αvβ3, and αvβ5 integrins.32 The blocking antibody against αvβ3 integrin, not anti-β1 integrin antibody, can inhibit SMC migration on vitronectin, and αvβ5 integrins are not detected by flow cytometry in this SMC system (data not shown). Moreover, β1 integrins do not cluster to form focal adhesion on vitronectin (data not shown). uPAR is also known as an alternative receptor for vitronectin.33 uPA alters the conformation of uPAR34 and increases its affinity for vitronectin35 with its effect independent of proteolytic activity.33,36⇓ In our SMCs, uPA secretion is 10-fold less than PAI-1 secretion on a molar/molar ratio, and only trace amounts of uPAR are detected by flow cytometric analysis. Furthermore, an inhibitor of αv integrin, cyclic Pen-RGD peptide, completely inhibits SMC attachment to vitronectin, whereas blocking anti-uPAR antibody has no effect (data not shown). Thus, uPAR appears not to be a major vitronectin receptor in our SMCs. Taken together, αvβ3 integrin appears to be a specific target of polymerized collagen that could be involved in altered αvβ3 integrin-dependent SMC migration.
In summary, polymerized type I collagen fibrils regulate PAI-1 expression, which may modulate αvβ3 integrin function. Our data further suggest the possibility that within the normal media, inhibitory conditions for SMC migration on vitronectin induced by surrounding type I collagen fibrils may be dynamically modulated by local release of uPA and PAI-1 from infiltrating macrophages and inflammatory cells present at all stages of atherosclerotic lesion development.
This work is supported by grants for scientific research (No. 11838014 to H.K., No. 13671197 to H.K., and NO. 11694307 to H.K., Y.N., A.S., and E.W.R.) from the Ministry of Education, Science, and Culture, Japan; a grant from ONO Medical Research Foundation (to H.K.); a grant from Osaka Medical Research Foundation for Incurable Diseases (to H.K.); and a grant from the National Institutes of Health (HL-18645 to E.W.R.). The authors thank M. Monden, Osaka City University, for excellent technical assistance, and Dr C.M. Giachelli, University of Washington, Seattle, for providing us with osteopontin.
Received January 31, 2002; revision accepted June 14, 2002.
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