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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:998-1005

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:998.)
© 2000 American Heart Association, Inc.

Vascular Biology

Matrix Metalloproteinase and {alpha}vß3 Integrin–Dependent Vascular Smooth Muscle Cell Invasion Through a Type I Collagen Lattice

Shigeru Kanda; Masafumi Kuzuya; Miguel A. Ramos; Teruhiko Koike; Kohichiro Yoshino; Shoji Ikeda; Akihisa Iguchi

From the Department of Geriatrics (S.K., M.K., M.A.R., T.K., A.I.), Nagoya University Graduate School of Medicine, Nagoya; and R&D Laboratories (K.Y., S.I.), Nippon Organon, Osaka, Japan.

Correspondence to Shigeru Kanda, MD, Department of Geriatrics, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail kanda3{at}spice.or.jp


*    Abstract
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 *Abstract
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Abstract—Smooth muscle cell (SMC) migration from the tunica media to the intima is a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. SMCs require not only migratory but also degradative abilities that enable them to migrate through extracellular matrix proteins, which surround and embed these cells. We used a collagen type I lattice as a coating on top of a porous filter as a matrix barrier in a chamber to test the invasive behavior of SMCs in response to a chemoattractant (invasion assay) and compared that behavior with simple SMC migration through collagen type I–coated filters (migration assay). Inhibitors of matrix metalloproteinase, KB-R8301, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2, and peptide 74, attenuated platelet-derived growth factor-BB (PDGF-BB)–directed SMC invasion across the collagen lattice, whereas no effect was seen with these inhibitors on simple SMC migration through collagen-coated filters. RGD peptide inhibited SMC invasion but did not affect SMC migration. Anti-{alpha}vß3 integrin antibody attenuated PDGF-BB–directed SMC invasion, whereas other antibodies against RGD-recognizing integrins, namely {alpha}vß5 and {alpha}5, had no effect. None of these antibodies had any effect on simple SMC migration. RGD peptide and anti-{alpha}vß3 antibody inhibited the attachment and spreading of SMCs on denatured collagen but not on native collagen. These findings indicate that there is a difference in the mechanisms between simple SMC migration across a collagen-coated filter and SMC invasion through a fibrillar collagen barrier. A proteolytic process is required for SMC invasion, and the degradation of matrix proteins alters the relationship between matrix protein molecules and SMC surface integrins.


Key Words: smooth muscle cells • integrins • collagen • matrix metalloproteinases • cell migration


*    Introduction
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 *Introduction
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During vessel remodeling in arterial disorders such as atherosclerosis, hypertension, and after balloon angioplasty, migration of medial smooth muscle cells (SMCs) to the subendothelial region plays a major role in the lesion formation of intimal thickening.1 2 To understand the molecular mechanisms of SMC movement into the subendothelial space in the vascular wall, in vitro models of chemoattractant-directed SMC migration across a porous filter have frequently been used.3 4 5 These studies have revealed that growth factors such as platelet-derived growth factor (PDGF) are essential for the directed migration of SMCs and that the interaction between matrix proteins and SMC surface receptors, or integrins, also regulates chemoattractant-directed SMC migration across the matrix protein–coated membrane.3 4 5

Because SMCs in large vessels are usually surrounded by and embedded in extracellular matrix proteins, including collagens, elastin, and proteoglycans,6 7 8 the migration of SMCs and the remodeling of tissues during intimal thickening require controlled degradation of the extracellular matrix and the activation or release of growth factors. During the processes of SMC migration from the tunica media to the intima, SMCs must degrade and breach the extracellular matrix proteins surrounding each cell. The process can be described in 3 steps: a phenotypic change from the contractile to the synthetic state, proteolytic dissolution of extracellular matrix proteins, and cell migration through the digested matrixes, a process that resembles tumor cell invasion.9 10 Because SMC invasion into the vascular wall intima seems to consist of complex processes, an in vitro SMC migration assay does not always reflect actual SMC–extracellular matrix protein interactions.

It has been known that SMCs can produce proteolytic enzymes such as matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes, and plasminogen activators, including tissue-type and urokinase-type plasminogen activators.11 12 In fact, recent evidence supports the concept that these enzymes play an important role in SMC migration into the intima in the balloon-injured carotid artery.13 14 15

The interactions of cells with extracellular matrix proteins that are mediated through a family of cell-surface receptors, or integrins, regulate a variety of cell behaviors, including migration, proliferation, proteinase production, and differentiation.16 17 18 The intracellular domains of integrin subunits are connected to cytoskeletal proteins and signal transduction pathways, which regulate cell behavior on the matrix proteins. During SMC movement from the tunica media to the intima, it is likely that SMCs interact with the denatured extracellular matrix protein molecules, because SMCs need to digest surrounding matrix proteins to allow their migration into the vascular wall. The interaction between SMCs and denatured matrix proteins may have a different influence on SMC behavior, because proteolysis releases cryptic activities of extracellular matrix protein ligands that may signal differently compared with those of the intact proteins.19

To understand the molecular mechanisms of SMC migration during the process of intimal thickening, in the present study we used a collagen type I lattice as a coating on top of a porous filter in a chamber to examine the invasive behavior of SMCs and to compare it with simple SMC migration across a collagenous thinly coated filter.


*    Methods
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 *Methods
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Cell Culture
SMCs were isolated from calf aortic media by the explant method and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nissui Pharmaceutical) supplemented with 10% fetal bovine serum and antibiotics. For the experiments, subconfluent SMCs between the seventh and 12th passage were used.

Reagents
Recombinant human PDGF-BB was obtained from Pepro Tech. Peptide 74, an MMP inhibitor20 that is derived from the prosegment region of MMPs and contains an unpaired cysteine residue, was purchased from Bachem (Feinchemikalien AG). GRGDSP and GRGESP peptides were purchased from GIBCO BRL. Rat tail collagen type I was obtained from Collaborative Research Inc. Bovine tissue inhibitor of MMP-1 (TIMP-1) and TIMP-2, both of which are intrinsic inhibitors of MMP,21 22 were purchased from Fuji Chemical Industries, Ltd. A hydroxamic acid–based metalloproteinase inhibitor, [4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide (KB-R8301), and a nonhydroxamate derivative of KB-R8301, 4-(N-hydroxy-2R-isobutyl-3S-methylsuccinyl-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide (KB-R8845), were synthesized by Kanebo Ltd.23 24 These compounds were dissolved in dimethyl sulfoxide at 10 mmol/L as stock solutions. {alpha}vß3 integrin–specific blocking monoclonal antibody (mAb; LM609), the {alpha}vß5 integrin–specific blocking mAb P1F6, and the {alpha}5 integrin–specific blocking mAb P1D6 were purchased from Chemicon International, Inc. The F(ab')2 fragment of FITC-conjugated anti-mouse IgG antibody was obtained from Vector Laboratories Inc.

Migration and Invasion Assays
The migration assay was performed with Transwell (Costar) 24-well tissue-culture plates composed of a polycarbonate membrane containing 5-µm pores. The membrane was coated with 50 µg/mL collagen type I solution in 0.02N acetic acid for 12 hours at 4°C and then rinsed well with Dulbecco’s PBS (Nissui Pharmaceutical). SMCs were seeded on the inner chamber of the Transwell at 106 cells in 100 µL of DMEM containing 0.3% bovine serum albumin (BSA). In some experiments, SMCs were pretreated in the DMEM containing 0.3% BSA in the presence or absence of GRGDSP, GRGESP, or anti-integrin mAbs, including LM609, P1F6, and P1D6 at the indicated concentrations for 30 minutes at room temperature. The cell suspension was then seeded on the inner chamber. To examine the role of MMPs, an SMC suspension containing MMP inhibitors, including KB-R8301 (1 to 10 µmol/L), TIMP-1 (2.5 to 10 µg/mL), TIMP-2 (2.5 to 10 µg/mL), and peptide 74 (100 µmol/L), was plated in the upper chambers. The inner chamber was placed into the outer chamber, which contained 600 µL of DMEM containing 0.3% BSA supplemented with recombinant human PDGF-BB (10 ng/mL), and was incubated for the indicated periods of time at 37°C in a CO2 incubator. Cells that migrated onto the outer side of the membrane were fixed and stained with Dif-Quick stain (American Hospital Supply Corp). The number of migrated cells was counted in 6 to 8 randomly chosen fields of duplicate chambers at x200 magnification for each sample.

The invasion assay was conducted in similar fashion but with a coating of fibrillar collagen. In brief, 1.0 mg/mL collagen type I solution in 0.02N acetic acid and a 1/10 volume of 10x DMEM were mixed and neutralized with 1N NaOH at 4°C. Twenty microliters of the mixture was added to an inner-chamber membrane and polymerized at 37°C for 6 hours. The SMC suspension in DMEM containing 0.3% BSA was added to the inner chamber as described above.

Gelatin Zymography
For gelatin zymography, each aliquot of the medium conditioned for 12 hours in the upper chamber was mixed with an equal volume of lysis buffer containing 0.25 mol/L Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, and 10 µg/mL bromophenol blue without a reducing agent. Each sample was then loaded onto a 10% SDS-polyacrylamide gel containing 1 mg/mL gelatin as a substrate. After electrophoresis, the gels were incubated with 2 changes of 2.5% Triton X-100, followed by overnight incubation at 37°C with substrate buffer (50 mmol/L Tris, pH 8.0, 50 mmol/L NaCl, and 10 mmol/L CaCl2). The gels were then stained with 0.1% Coomassie Brilliant Blue, and gelatinolytic activity was revealed as clear bands against a blue-stained background.

Attachment Assay
To prepare heat-denatured collagen, type I collagen solution (50 µg/mL in 0.02N acetic acid) was heated at 100°C for 15 minutes. Protease-denatured collagen type I was prepared as follows. Collagen type I solution (1 mg/mL) in 0.02N acetic acid was incubated with 200 U/mL pepsin at 4°C for 20 hours, precipitated with 1.7 mol/L NaCl, and then centrifuged to remove any remaining pepsin. The precipitate was redissolved in 0.02N acetic acid. The precipitation step was repeated 3 times. A 96-well plate was coated with 50 µg/mL native or denatured collagen type I for 16 hours at 4°C and washed twice with PBS, and uncoated parts of the plate were blocked with DMEM containing 1% BSA for 1 hour. The SMC suspension in serum-free DMEM containing 0.3% BSA was preincubated with blocking agents for 30 minutes and plated at a density of 2.0x104 cells/well. After 1-hour incubation at 37°C, unattached cells were carefully washed away with PBS, and the number of attached cells was counted in 8 randomly chosen fields of duplicate wells at x200 magnification for each sample.

Flow Cytometry
Subconfluent SMCs were preincubated with DMEM containing 0.3% BSA in the presence or absence of PDGF-BB (10 ng/mL) for 12 hours. The cells were trypsinized, washed once with PBS, and resuspended at 106 cells/mL in PBS. Primary antibody against {alpha}vß3 (LM609, 10 µg/mL) was incubated with the cell suspension for 30 minutes at room temperature, and the cells were washed twice with PBS. The cell suspension was then incubated with 200 µg/mL FITC-conjugated anti-mouse IgG for 15 minutes at room temperature, washed twice with PBS, and analyzed for fluorescence by using a flow cytometer (Coulter Epics).

Statistical Evaluation
Values were expressed as mean±SD. Analysis by ANOVA was used, followed by post hoc testing (Scheffe’s test). A value of P<0.05 was considered to be statistically significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Time Course of SMC Migration and Invasion
The time-course experiments of PDGF-BB–directed SMC migration across collagenous thinly coated filters and SMC invasion through a collagen gel barrier were examined (see FigureDown I; the TableDown and Figures I through IV can be found online at http://atvb.ahajournals.org/cgi/content/full/20/4/998/DC1). The number of cells that passed the filters increased in a time dependent manner up to 24 hours in both migration and invasion assays. At every time point, the number of migrated cells was more than that of invaded cells. From 1 through 16 hours of incubation, a significant difference was observed between the number of migrated and invaded cells. We selected a 12-hour assay for SMC invasion and a 6-hour assay for SMC migration for further investigation.



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  		Figure 1. Effect of KB-R8301 and peptide 74, MMP inhibitors, on SMC migration and invasion. PDGF-BB–directed SMC migration (A) and invasion (B) were assessed in the presence or absence of KB-R8301, an MMP inhibitor; KB-R8845, a control compound; or peptide 74 in the inner chamber. *P<0.01, **P<0.0001 vs control (n=6).


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  		Table 1. Summary of the Effect of Inhibitors, Peptides, and Antibodies on SMC Migration and Invasion

In the absence of PDGF-BB in the outer chamber and with or without PDGF-BB in the inner chamber, a few migrated SMCs and no invading SMCs were observed on the outer side of the filters over periods of 6 and 12 hours, respectively (Figures IIA and IIB online). When PDGF-BB was added to both the inner and outer chambers, a moderate number of cells were found on the filters in both migration and invasion assays but many fewer than in the presence of a steep gradient of chemoattractant (Figures IIA and IIB online), suggesting that both SMC invasion through the fibrillar collagen barrier and SMC migration across the filter were mainly dependent on the presence of a gradient of chemoattractant.

Involvement of MMPs in SMC Invasion
To examine whether proteolytic processes are required for SMC migration across collagen-coated filters and for SMC invasion through a collagen gel barrier, migration and invasion assays were performed in the presence or absence of KB-R8301, a hydroxamic acid–based MMP inhibitor, and KB-R8845, a nonhydroxamic acid control compound.23 24 As shown in Figure 1BUp, KB-R8301 inhibited PDGF-BB–directed SMC invasion in a concentration-dependent manner, although this synthetic MMP inhibitor had no effect on SMC migration (Figure 1AUp). Neither SMC invasion nor SMC migration was inhibited in the presence of KB-R8845, a control compound. Peptide 74, a synthetic peptide containing a highly conserved peptide sequence of the MMP family,21 also blocked SMC invasion but not SMC migration (Figures 1AUp and 1BUp). We also examined the effect of TIMP-1 and TIMP-2, inhibitors of MMP,21 22 on PDGF-BB–directed SMC migration and invasion (Figures 2ADown and 2BDown). Although these inhibitors had no effect on SMC migration on collagen-coated filters, TIMP-1 partially but statistically significantly attenuated SMC invasion through a collagen gel barrier in a concentration-dependent manner. TIMP-2 inhibited SMC invasion more effectively than did TIMP-1 at a higher concentration. It should be noted that even when the migration assay was extended to 12 hours, no effect of MMP inhibitors was observed on SMC migration, suggesting that the ineffectiveness of these inhibitors on SMC migration was not due to shorter assay times. These MMP inhibitors did not alter SMC attachment to collagen-coated filters as well as to collagen gels (data not shown).



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  		Figure 2. Effect of TIMP-1 and TIMP-2 on SMC migration and invasion. PDGF-BB–directed SMC migration (A) and invasion (B) were assessed in the presence or absence of TIPM-1 or TIPM-2 in the inner chamber. *P<0.01, **P<0.0001 vs control (n=6).

As shown in Figure 3ADown, gelatin zymographic analysis of the conditioned medium from invasion assays revealed that major gelatinolytic activity was observed at a molecular mass of 72 kDa, which most likely corresponds to either the inactive form of MMP-2, on the basis of its size and inhibition of activity on incubation with EDTA, or 1,10-phenanthroline. A band at 62 kDa was also detected in the conditioned medium, which was assigned as the activated form of MMP-2. In addition, the 62-kDa activated form of MMP-2 was not observed in the medium conditioned in the presence of MMP inhibitors KB-R8301 and TIMP-2, but it was detected in the presence of control compound KB-R8845 and TIMP-1 (Figure 3ADown). It should be noted that there were no differences in the amounts of inactive and activated forms of MMP-2 in the invasion assay between those with and without PDGF-BB in the outer chamber as a chemoattractant. Neither was MMP-9 detected in these zymographic analyses. Although the 72-kDa band, an inactive form of MMP-2, was detected in the conditioned medium from the migration assay, only a faint band of 62 kDa, corresponding to the activated form of MMP-2, was found in the gelatin zymogram. When SMC migration-assay time was extended to 12 hours, no increase in the 62-kDa band was observed (Figure 3BDown). Therefore, the different patterns of zymography are not due to the different intervals that we used between migration and invasion assays. MMP-1, which induces faint gelatinolytic activity at a molecular mass of 50 kDa, was not detected under any of our experimental conditions.



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  		Figure 3. Gelatin zymographic analysis of conditioned medium from SMC invasion and migration assays. A, Gelatin zymography of conditioned medium from SMC invasion assays in the presence or absence of KB-R8301 (10 µmol/L), KB-R8845 (10 µmol/L), TIMP-1 (10 µg/mL), or TIMP-2 (10 µg/mL). B, Gelatin zymography of conditioned medium from invasion assay and assays. Migration assay time was extended to 12 hours to exclude the influence of assay time.

Inhibition of SMC Invasion by RGD Peptide and Anti-{alpha}vß3 mAb
Adhesion of SMCs to extracellular matrix proteins through cell-surface receptors, or integrins, appears to be essential for SMC motility. The RGD sequence is present in many adhesive molecules, and short, synthetic, RGD-containing peptides also inhibit ligand binding to integrins. Early studies showed that RGD-containing peptides inhibit neointima formation in damaged arteries and prevent SMC migration into the vascular wall.25 26 Therefore, we examined whether RGD sequence recognition is involved in SMC migration across a collagen-coated filter and in SMC invasion through fibrillar collagen. Addition of RGD peptide during the course of the experiments inhibited SMC invasion in a concentration-dependent manner (Figure 4BDown) but had no effect on SMC migration (Figure 4ADown). RGE peptide, the control peptide of RGD, had no effect on both SMC migration and invasion (Figures 4ADown and 4BDown).



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  		Figure 4. Effect of RGD and RGE peptides on SMC migration and invasion. PDGF-BB–directed SMC migration (A) and invasion (B) were performed in the presence or absence of RGD or RGE peptides. SMCs were pretreated with RGD or RGE for 30 minutes and then placed into inner chambers. *P<0.05, **P<0.01, ***P<0.001 vs control (n=8).

To evaluate which kind of integrin-recognizing RGD sequence is involved in SMC invasion through a collagen gel barrier, the effect of 2 kinds of {alpha}v integrin–blocking mAb, LM609 (anti-{alpha}vß3 integrin mAb) and P1F6 (anti-{alpha}vß5 integrin mAb), and the {alpha}5 integrin–blocking mAb P1D6 was examined. These integrins have been reported to recognize the RGD sequence and to exist in several kinds of mammalian SMCs.5 27 28 29 As shown in Figure 5BDown, when SMCs were pretreated and exposed to LM609, the anti-{alpha}vß3 mAb, during assay, SMC invasion through the collagen lattice barrier was significantly attenuated in a concentration-dependent manner. However, this antibody had no effect on SMC migration on collagen-coated filters (Figure 5ADown). Neither P1F6, the anti-{alpha}vß5 mAb, nor P1D6, the anti-{alpha}5 mAb, had any effect on SMC invasion and migration (Figures 5ADown and 5BDown).



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  		Figure 5. Effect of blocking integrin antibodies on SMC migration and invasion. SMC migration (A) and invasion (B) assays were conducted in the presence or absence of LM609, an anti-{alpha}vß3 mAb; P1D6, an anti-{alpha}5 mAb; or P1F6, an anti-{alpha}vß5 mAb in inner chambers. SMC suspension was pretreated with integrin mAb for 30 minutes and then seeded onto the inner chamber. *P<0.01 vs control (n=8).

To confirm the existence of an {alpha}vß3 integrin on the bovine SMC surface, flow cytometry analysis was performed. The mean intensity value of FITC fluorescence was remarkably increased in the cell population stained with LM609, suggesting the existence of integrin {alpha}vß3 on bovine SMC surfaces (FigureUp III online). It should be noted that pretreatment of SMCs with PDGF-BB for 12 hours did not affect LM609 binding, indicating that at least a 12-hour treatment with PDGF-BB had no effect on {alpha}vß3 expression on SMCs.

SMC Attachment to and Spreading on Native or Denatured Type I Collagen
To examine the possible role of denatured collagen molecules on SMC invasion through a collagen gel barrier, an SMC attachment assay was performed by using native collagen–coated and denatured collagen–coated wells. Within 1 hour of incubation, SMCs became attached to and spread on both native type I collagen and denatured type I collagen, which had been induced by heating or pepsin digestion. No difference in the morphology and number of attached cells was observed between native and denatured collagen (Figures 6ADown, 6EDown, and online FigureUp IV). When the SMC suspension was preincubated with RGD peptide, SMC attachment and spreading were significantly inhibited on denatured collagen (Figure 6FDown and online FigureUp IV), whereas no effect of RGD peptide was detected on SMC attachment to and spreading on native type I collagen (Figure 6BDown and online FigureUp IV). Control peptide RGE did not alter SMC attachment and spreading on both native and denatured collagen surfaces (Figures 6CDown, 6GDown, and online FigureUp IV). Although the anti-{alpha}vß3 mAb LM609 also did not inhibit SMC attachment to and spreading on native type I collagen (Figure 6DDown and online FigureUp IV), this mAb attenuated the attachment to and spreading on denatured type I collagen (Figure 6HDown and online FigureUp IV). The anti-{alpha}vß5 mAb P1F6 and the combination of LM609 and P1F6 failed to show a difference in SMC attachment to and spreading on denatured collagen from control and LM609 alone, respectively (data not shown).



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  		Figure 6. SMC attachment to and spreading on native and denatured collagen type I. SMC suspension was pretreated with RGD (B, F), RGE (C, G), peptide (1 mmol/L), or {alpha}vß3 mAb (10 µg/mL, D, H) and then seeded onto native (A through D) or heat-denatured (E through H) collagen type I–coated wells. A and B show control SMC attachment on native collagen type I (A) and denatured collagen (B). Photographs were taken after 1 hour of incubation. Magnification x200.


*    Discussion
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 *Discussion
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In the present study, we observed the different processes between SMC migration across collagen-coated filters and SMC invasion through fibrillar collagen. The effects of various kinds of inhibitor and function-blocking antibodies on SMC migration and invasion are summarized in the TableUp (online).

The kinetic study showed that SMCs crossed the collagenous thinly coated filters more rapidly than they penetrated the collagen lattice barrier in response to PDGF-BB. The different time course of SMC motility between the 2 assays may be explained by the different processes of SMC migration across collagen-coated filters and of invasion through a collagen lattice barrier. The SMC invasion assay measures the ability of these cells to attach to the collagen molecule, degrade collagen fibrils, and migrate toward a chemoattractant, PDGF-BB. The role of a proteolytic process is supported by the observations of an effect of MMP inhibitors on SMC invasion. KB-R8301, a synthetic inhibitor of MMP,23 24 blocked the penetration of SMCs, and TIMP1 and TIMP-2, naturally occurring inhibitors of MMPs,21 22 attenuated SMC invasion. In addition, peptide 74, a synthetic peptide that has been demonstrated to effectively inhibit MMP-2 activation and tumor cell invasion,20 almost completely inhibited SMC invasion through a collagen lattice barrier. These inhibitors had no effect on SMC migration on collagen-coated filters, indicating that MMPs play an important role in SMC invasion but not in SMC migration, at least in our system. MMP-2 has been implicated in a variety of invasive process, including tumor invasion30 and SMC migration through a reconstituted basement membrane protein complex10 and from aortic explants.31 In addition, MMP-2 has been shown to be present in human atherosclerotic lesions and is upregulated during the course of neointimal lesion formation after balloon angioplasty.32 33 34 35 Zymographic analysis of the conditioned medium revealed that the major gelatinolytic MMP derived from SMCs during the invasion assay was MMP-2, which degrades a variety of extracellular matrix proteins, including type IV collagen as well as denatured collagen of all types.30 36 An activated form of MMP-2 was also detected in the conditioned medium from invasion assays. Our findings that MMP inhibitor KB-R8301 and TIMP-2 inhibited MMP-2 activation during the course of SMC invasion and that TIMP-1 had no effect on MMP-2 activation are consistent with a previous report that a high concentration of TIMP-2 inhibits MMP-2 activation but that TIMP-1 has no effect.37 38 TIMP-1 and TIMP-2 form specific complexes with both the zymogen and active forms of MMP-9 and MMP-2 and inhibit their respective activities.22 Our result that TIMP-2 was much more efficient at inhibiting SMC invasion than was TIMP-1 and the fact that TIMP-1 and TIMP-2 have a similar inhibitory activity against MMP-121 raise the possibility that MMP-2 together with interstitial collagenase may play a key role in SMC invasion through collagen fibrils. Recently, the active form of MMP-2 was found to be capable of degrading type I collagen to a degree comparable to that of MMP-1,39 indicating that interstitial collagenase and MMP-2 are required for the complete dissolution of stromal collagen during cellular invasion.

We could not find any difference in the levels of latent and activated MMP-2 between media conditioned in the presence and absence of PDGF-BB in lower chambers as a chemoattractant, suggesting that PDGF-BB has no effect on MMP-2 activation. Because it has been reported that fibrillar collagen activates tumor cell–derived MMP-2 through a membrane-type MMP,40 a recently discovered MMP that has the ability to activate MMP-2,41 upregulation of this membrane-type MMP through the interaction of SMCs with fibrillar collagen might be involved in MMP-2 activation.

Although RGD-containing peptides have been demonstrated to suppress neointimal formation after arterial injury,25 26 the exact mechanisms of this effect remain unknown. In the present study, we showed that RGD peptide attenuated SMC invasion through a collagen lattice, whereas this peptide had no effect on SMC migration on collagen. It should be noted that RGD peptide did not affect either SMC attachment to or spreading on a collagen lattice, suggesting that the effect of RGD peptide is not due to inhibition of SMC attachment to and spreading on a collagen lattice. SMC invasion was also inhibited by treatment with a neutralizing antibody against {alpha}vß3 integrin, LM609, but not with antibodies against {alpha}vß5 and {alpha}5 integrins, which also recognize RGD sequences.42 However, LM609 had no effect on both directed migration of SMCs across collagen type I–coated filters and on SMC adhesion to and spreading on a collagen lattice. These results suggest that RGD peptide and LM609 do not alter the initial interaction between SMCs and collagen molecules.

Although RGD sequences exist in native collagen molecules, they are masked within the triple helix, and integrin {alpha}vß3 ligates poorly to it.43 44 We observed that RGD peptide and LM609 interfered significantly with SMC attachment to and spreading on denatured collagen, supporting previous findings that melanoma cells adhere to denatured versus native type I collagen through different integrins, namely {alpha}vß3 versus ß1 integrins, respectively,43 and that melanoma cells degrade collagen molecules (mediated by proteinases) and expose cryptic {alpha}vß3 binding sites when cultured in a 3-dimensional collagen lattice.44 These observations together with the results of MMP inhibitor experiments indicate that SMCs seem to recognize RGD sites on denatured type I collagen with {alpha}vß3 integrin after transforming collagen molecules to the denatured form, a process mediated by MMPs derived from SMCs themselves. It should be noted that the inhibitory effect of LM609 on SMC attachment to denatured collagen was less than that of RGD peptide, suggesting that RGD-recognizing integrins other than {alpha}vß3 might take part in the recognition of denatured type I collagen. Our findings may indicate that the inhibitory effect of RGD-containing peptides on neointimal lesion formation after arterial injury may be due, at least in part, to a reduction in SMC invasion through the matrix barrier. Coleman et al45 have reported that systemic administration of vitaxin, an mAb to {alpha}vß3 integrin, inhibits neointimal formation after balloon injury. In addition, these authors also demonstrated that this antibody inhibits SMC migration through vitronectin-coated filters, inconsistent with our result that {alpha}vß3 integrin was not involved in SMC migration through collagen type I–coated filters. Vitronectin has been known to react with {alpha}vß3 integrin through RGD sites without denaturation. On the other hand, as described above, denaturation is necessary for collagen type I to be recognized by {alpha}vß3 integrin. Therefore, there is considerable difference between vitronectin and type I collagen in terms of the necessity of a denaturation step for recognition by {alpha}vß3 integrin.

It has been demonstrated that {alpha}vß3 integrin is detectable in intimal lesions associated with various stages of atherosclerosis but is absent in the normal artery,46 suggesting that {alpha}vß3 integrin may play a fundamental role in the formation of intimal thickening. In the present study, we confirmed that bovine SMCs have {alpha}vß3 integrins on their surfaces, on the basis of results from the attachment assay as well as flow cytometric analysis. It should be noted that exposure of SMCs to PDGF-BB for 12 hours did not alter {alpha}vß3 integrin expression on SMCs. This may indicate that PDGF-BB–directed SMC invasion is independent of the upregulation of {alpha}vß3 integrin expression on SMCs.

Recently, it has been found that ligation of the {alpha}vß3 integrin enhances PDGF-BB–induced cell proliferation and migration.47 48 The activated PDGF-ß receptor forms a complex with {alpha}vß3 integrin, and several signaling molecules that are known to be associated with the activated PDGF receptor are present in {alpha}vß3 integrin complexes. Therefore, it is conceivable that ligation of the {alpha}vß3 integrin with denatured collagen may enhance the PDGF-BB gradient–stimulated invasion of SMCs.

The finding presented in this report demonstrates the difference in the mechanisms between SMC migration across collagen-coated filters and SMC invasion through a fibrillar collagen barrier. We showed that a proteolytic process is required for SMC penetration through matrix proteins and that the degradation of matrix protein alters the relationship between matrix molecules and cell-surface integrins. Our study supports a role for MMPs and the {alpha}vß3 integrin in SMC migration from the tunica media to the intima during vessel remodeling in arterial disorders.

Received February 17, 1999; accepted November 3, 1999.


*    References
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 *References
 

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