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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1467.)
© 2000 American Heart Association, Inc.

Vascular Biology

Smooth Muscle Cell Matrix Metalloproteinase Production Is Stimulated via _vß₃ Integrin

Michelle P. Bendeck; Colleen Irvin; Michael Reidy; Laura Smith; Diane Mulholland; Michael Horton; Cecilia M. Giachelli

From the Terrence Donnelly Research Laboratories (M.P.B., D.M.), Division of Cardiology, St. Michael’s Hospital, and the Departments of Medicine and Laboratory Medicine and Pathobiology (M.P.B., D.M.), University of Toronto, Toronto, Ontario, Canada; the Department of Pathology (C.I., M.R., L.S., C.M.G.), University of Washington, Seattle; and the Bone and Mineral Centre (M.H.), Department of Medicine, Royal Free and University College Medical School, London, UK.

Correspondence to Dr Michelle Bendeck, Department of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Building, Room 6315, 1 King’s College Circle, Toronto, Ontario, Canada M55 1A8. E-mail bendeckm{at}smh.toronto.on.ca

	Abstract

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Abstract—This study tests the hypothesis that _vß₃ integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that ß₃ integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the ß₃ integrin–blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8±30.8 cells/mm² in control rats to 10.29±7.03 cells/mm² in F11-treated rats (P=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed _vß₃ integrin receptors, whereas adult SMCs did not. Stimulation of newborn (_vß₃+) SMCs with osteopontin, a matrix ligand for _vß₃, increased MMP-1 production from 114.4±35.8 ng/mL at 0 nmol/L osteopontin to 232.5±57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC _vß₃ integrin receptors play an important role in regulating migration by stimulating SMC MMP production.

Key Words: smooth muscle cells • matrix metalloproteinases • _vß₃ integrins • migration • arterial injury

	Introduction

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Smooth muscle cell (SMC) migration and proliferation are critical steps in atherosclerosis and restenosis and are controlled by interactions between cells and the extracellular matrix. Of particular interest, SMC migration has been linked to the _vß₃ integrin receptor. The receptor and several matrix ligands are elevated in human atherosclerotic plaque and after experimental arterial injury.¹ In vitro, SMC migration^{2 3} and proliferation^{3 4} are blocked after treatment with _vß₃ integrin–blocking antibodies or Arg-Gly-Asp (RGD) peptides, and recent studies have shown that blocking the _vß₃ integrin retards the development of intimal thickening and restenosis after vascular injury.^{5 6 7 8 9 10 11} Taken together, these studies suggest that _vß₃ integrins play important roles in regulating the migration and proliferation of vascular SMCs.

We and other investigators have shown that matrix metalloproteinase (MMP) activity is necessary for SMC migration; MMP-2, MMP-3, MMP-9, and membrane-type MMP-1 (MT-MMP-1) are upregulated coincident with SMC migration during the first week after balloon injury in several species,¹² and treatment with specific MMP inhibitors dramatically attenuates SMC migration in vivo.^{13 14} Studies with fibroblasts, keratinocytes, and melanoma cells have demonstrated that MMP production is regulated by feedback from the extracellular matrix through integrin receptor signaling.¹⁵ We have investigated the possibility that SMCs are susceptible to similar feedback from the matrix, particularly by osteopontin, a ligand for the _vß₃ receptor.

We demonstrated that ß₃ integrin mRNA was upregulated after balloon catheter injury of the rat carotid artery and that MMP-1 protein in the vessel wall was increased in parallel with ß₃ integrin expression. Treatment with the ß₃ integrin–blocking antibody (F11)¹⁶ caused a reduction in SMC migration, but not proliferation, 4 days after balloon catheter injury. Finally, we showed that osteopontin alone and in coordination with platelet-derived growth factor (PDGF)-BB stimulated MMP activity in SMCs, and we also demonstrated the critical importance of the _vß₃ integrin receptor in mediating this response.

	Methods

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All chemicals were obtained from Sigma Chemical Co unless stated otherwise.

Surgery and Antibody Treatment
In vitro adhesion assays were performed to determine whether F11 blocked _vß₃ in rat carotid SMCs. Tissue culture plates were coated with 20 nmol/L osteopontin, and rat SMCs were preincubated with 0 or 10 µg/mL F11 and then plated on the osteopontin-coated dishes. After 90 minutes, nonadherent cells were washed from the plate, the remaining adherent cells were stained with toluidine blue, and adhesion was quantified by measuring optical density at 595 nm in a spectrophotometer (Molecular Devices).

A total of 122 male Sprague-Dawley rats (3 to 4 months old, Charles River, Constant, Quebec, Canada) were used in all experiments. Balloon catheter injury of the left common carotid artery was performed as previously described.¹⁷ Purified F11 immunoglobulin to the rat ß₃ integrin¹⁶ was administered daily via tail vein injection at a dose of 1 mg/d, starting 18 hours before balloon catheter injury and continuing daily until the rats were euthanized. Control rats were injected with equivalent doses of a nonimmune mouse monoclonal antibody. To label all cells entering S phase, a 50 mg pellet of 5-bromo-2'-deoxyuridine (Boehringer Mannheim Corp) was implanted subcutaneously at the nape of the neck in all rats 24 hours before they were euthanized. Rats were killed at 4 days after injury by intravenous injection of T-61, which contains (in 1 mL) 200 mg of N-[2-(m-methoxyphenyl)-2 ethylbutyl-(1)]-hydroxy-butyramide, 50 mg of 4,4'-methylene-bis-(cyclohexyl,trimethylammonium iodide), and 5 mg of tetracaine hydrochloride with dimethyl formamide (Hoechst Roussel Veterinarian). The carotid arteries were perfusion-fixed at physiological pressure with 0.1 mol/L phosphate-buffered 4% paraformaldehyde. A 1-cm length was excised from the middle of the common carotid artery and used for the SMC migration assay as previously described.¹⁷ Adjacent sections 5 mm in length were embedded in paraffin, and cross sections were cut and immunostained for 5-bromo-2'-deoxyuridine as previously described.¹⁸

In a subset of rats, the balloon catheter was advanced into the thoracic aorta, inflated, and withdrawn 3 times to denude the aorta. Just before euthanasia, the rats were injected with 0.5% Evans blue dye via the tail vein. Evans blue binds to plasma albumin, and the dye-albumin complex penetrated the vessel wall only in the absence of an intact endothelial monolayer; therefore, denuded areas of the vessel were stained blue, and endothelialized areas were white. The percentage of vessel surface area that was white was measured and used as an index of regeneration.

Platelet Parameters
Scanning electron microscopy was used to assess platelet deposition on the vessel wall 4 hours after balloon catheter injury. The arteries were perfusion-fixed with 2% glutaraldehyde and 1% paraformaldehyde in phosphate buffer and then prepared for scanning electron microscopy as previously described.¹⁹ Platelet deposition (number of platelets per square millimeter of surface area) was determined from photographs by counting platelets on several randomly selected fields along the length of the carotid intimal surface.

Northern Blots for _vß₃ and Western Blots for MMP-1 After Balloon Catheter Injury
Carotid arteries were harvested at 1 hour, 4 hours, 24 hours, 4 days, 7 days, and 14 days after balloon catheter injury (6 to 8 rats per time point); total cellular RNA was extracted; and Northern blots were prepared. Blots were hybridized with a cDNA probe for rat ß₃ integrin²⁰ or for 18S ribosomal RNA labeled with [³²P]dCTP by random primer extension (Multi-Prime, Amersham). The hybridized blots were used for autoradiographic analysis by the PhosphorImager Facility of the Markey Molecular Center at the University of Washington. The signal for ß₃ integrin mRNA was normalized with the use of 18S rRNA as a loading control. Changes in ß₃ mRNA expression are shown relative to the uninjured control. Carotids were harvested at 6-hour, 24-hour, 2-day, 4-day, 7-day, and 14-day time points (4 rats per time point) for Western blots, which were probed with an antibody against MMP-1.²¹ Western blots were quantified by using scanning densitometry of the blots with Molecular Analyst software and a GS700 Imaging Densitometer (Bio-Rad). Changes in MMP-1 production are shown relative to the uninjured control carotid.

SMC Culture
Human adult aortic SMCs were obtained from heart transplant donor specimens, and human newborn aortic SMCs were derived from an autopsy specimen (2-day-old infant).² The SMCs derived from the newborn were shown to express significant levels of _vß₃ integrin by flow cytometry, whereas the adult SMCs did not² ; thus, we refer to these cells as _vß₃+ (newborn) or _vß₃- (adult) SMCs. For the MMP-1 ELISA assays and gelatin zymograms, _vß₃+ SMCs were harvested from the aorta of an aborted human fetus.² Cells were confirmed as smooth muscle by immunofluorescence staining with an anti-smooth muscle actin–specific antibody, SM-1.

MMP-1 Production Stimulated by Osteopontin
_vß₃+ or _vß₃- SMCs were suspended in DMEM supplemented with 10% FCS and 2% penicillin-streptomycin (Canadian Life Technologies), plated in 96-well plates at a density of 30 000 cells per well, and allowed to attach for 16 hours. Nonadherent cells were removed by washing 3 times with 100 µL of DMEM. Then 100 µL of DMEM containing 200 µg/mL BSA and osteopontin (50 nmol/L, recombinant protein obtained from C.M.G.), fibronectin (50 nmol/L), or phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; positive control for induction of MMP-1) was added to the cells, and they were incubated at 37°C for 48 hours. Conditioned medium (10 µL) from each well was combined 1:1 with 2x Laemmli sample buffer and subjected to gel electrophoresis and Western blotting. MMP-1 protein in the conditioned medium from human SMC cultures was detected by probing Western blots with a rabbit polyclonal antibody raised against human MMP-1.²² Blots were quantified by use of Molecular Analyst software and a GS700 Imaging Densitometer (Bio-Rad). The percentage increase in MMP-1 production by cells stimulated with PMA, osteopontin, or fibronectin was estimated by comparison with MMP-1 levels in medium from cells that were cultured in serum-free medium alone. These experiments were performed in triplicate and repeated 3 times.

ELISAs and Zymograms for MMPs
MMP-1 production was quantified by ELISA. _vß₃+ (fetal) SMCs were cultured as described above, and conditioned medium was collected from the wells and assayed for MMP-1 production by a 2-antibody sandwich ELISA with the protocol provided by the manufacturer (Amersham). These experiments were performed in triplicate and repeated 3 times.

MMP-2 and MMP-9 activity in the conditioned medium was determined by gelatin zymography, as described in our previous publication.¹⁷ The MMPs were identified by their molecular weights, by inhibition with EDTA or phenanthroline, and by comparison with Western blots bound with antibodies specific for human MMP-2 or MMP-9 (Oncogene Sciences). Activity on the zymograms was quantified by scanning densitometric analysis by use of a Bio-Rad Gel 1000 documentation system and Molecular Analyst software (Bio-Rad). These experiments were performed in triplicate and repeated 3 times.

Statistical Analysis
Values are expressed as mean±SEM. Group means were compared by the 2-tailed Student t test for independent samples.

	Results

Top Abstract Introduction Methods Results Discussion References

 
ß₃ Integrin mRNA Expression Was Increased in Coincidence With MMP-1 Production in Injured Rat Carotid Arteries
ß₃ integrin mRNA expression was increased between 4 and 24 hours after injury but returned to control levels at later time points, as shown on Northern blots containing total RNA extracted from control and balloon-injured rat carotid arteries (Figure 1A) Production of MMP-1 was dramatically increased in the injured rat carotid artery between 6 hours and 2 days after balloon injury, as shown by a Western blot containing protein extracts from the injured rat carotid, which was bound with an antibody against MMP-1 (Figure 1B).

View larger version (55K): [in this window] [in a new window]   Figure 1. A, Northern blot probed with a cDNA against rat ß3 integrin. Bar graph shows changes in normalized ß3 signal at various times in injured vessels compared with uninjured control (C) vessels. All values were normalized to 18S rRNA. B, Western blot probed with antibody against MMP-1. Bar graph shows changes in MMP-1 protein in injured carotid arteries compared with uninjured control (C) carotid arteries. A.U. indicates arbitrary units.

Treatment With F11 Inhibited SMC Migration but Not Proliferation in Injured Rat Carotid Arteries
Carotid vascular SMCs attached to 20 nmol/L osteopontin, and pretreatment of cells with 10 µg/mL F11 (the dose achieved in rat plasma after in vivo treatment) resulted in a 42% reduction in SMC attachment to osteopontin. Attachment was reduced from an optical density of 0.062±0.004 to 0.036±0.006.

Treatment of rats with 1 mg/d F11 resulted in plasma antibody levels of 10 µg/mL. SMC migration from the media to the intima was reduced by 90% in F11-treated rats compared with control (NIgG) rats at 4 days after balloon catheter injury (Figure 2A). The number of intimal SMCs was decreased from 110.8±30.8 cells/mm² in control rats to 10.3±7.0 cells/mm² in F11-treated rats (P=0.008). By contrast, neither the medial SMC proliferation rate (Figure 2B) nor the medial SMC number (Figure 2C) was significantly reduced in the F11-treated rats. Medial SMC number was determined by counting SMC nuclei on vessel cross sections.

View larger version (20K): [in this window] [in a new window]   Figure 2. A, SMC migration. B, Medial SMC replication rate. C, Medial SMC number measured 4 days after carotid artery injury in control and F11-treated rats. D, Endothelial regeneration in the balloon-injured thoracic aorta. Values are mean±SEM; n=9 for F11-treated rats, and n=10 for NIgG-treated control rats.

F11 Treatment Did Not Affect Endothelial Regeneration
Endothelial regeneration was 1.25±0.07 mm² in F11-treated rats, a value not significantly different from 1.16±0.07 mm² in control rats (Figure 2D). Endothelial regeneration rates were determined by denuding the thoracic aorta and then measuring the surface area covered by endothelial cells that migrated from the ostia of the intracostal arteries by 4 days after injury.

F11 Did Not Affect Early Platelet Deposition in Injured Rat Carotid Arteries
F11 binds to the platelet integrin _IIbß₃, which shares an identical ß₃ subunit with _vß₃, and some antiplatelet antibodies induce thrombocytopenia or inhibit platelet deposition. However, we found that platelet deposition in F11 rats (Figure 3B) was equivalent to the deposition in control rats (Figure 3A). There were 49.2±7.6 platelets/mm² in F11 rats, which was not significantly different from 45.0±10.3 platelets/mm² in control rats. Forty-eight hours after initiation of treatment, there were 332 000±292 078 platelets/µL plasma in F11 rats compared with 1 552 500±392 906 platelets/µL plasma in control rats (P=0.012). However, at 96 hours (4 days), the platelet counts in F11-treated rats were not significantly different from counts in control rats.

View larger version (121K): [in this window] [in a new window]   Figure 3. Scanning electron micrographs of platelets deposited on denuded carotid surface 48 hours after balloon injury in control (A) and F11-treated (B) rats (n=6 rats per group).

Finally, we performed experiments in which the administration of F11 was delayed until 24 hours after injury to allow sufficient time for platelet adhesion and release of granules at the vessel wall before blocking SMC ß₃ integrin function. In these experiments, the delayed administration of F11 still caused a significant (59%) reduction in SMC migration from 120.5±16.6 cells/mm² in control rats to 49.5±17.7 cells/mm² in F11-treated rats (P=0.014).

Osteopontin Stimulated MMP-1 Synthesis, Dependent on _vß₃
When _vß₃+ (newborn) and _vß₃- (adult) SMCs were incubated with serum-free medium, only the _vß₃+ SMCs produced MMP-1 (Figure 4). Addition of PMA to the medium stimulated a 53% increase in MMP-1 production compared with MMP-1 levels in control _vß₃+ cells in serum-free medium. Osteopontin (50 nmol/L) stimulated a 40% increase in MMP-1, whereas fibronectin had no effect. PMA stimulated MMP-1 production by the _vß₃- cells, but neither osteopontin nor fibronectin had any effect on these cells. The amount of MMP-1 secreted into the media was estimated semiquantitatively by using scanning densitometry of the Western blots.

View larger version (55K): [in this window] [in a new window]   Figure 4. Western blot containing conditioned medium (CM) from vß3+ (newborn) and vß3- (adult) SMCs probed with an antibody against MMP-1. PMA was used at a concentration of 50 ng/mL, and osteopontin and fibronectin were used at concentrations of 50 nmol/L.

MMP-1 production by _vß₃+ SMCs stimulated with osteopontin increased from 114.4±35.8 ng/mL at 0 nmol/L osteopontin, to 138.8±43.4 ng/mL at 50 nmol/L osteopontin, and to 232.5±57.5 ng/mL at 100 nmol/L osteopontin. Addition of PDGF-BB or fibroblast growth factor (FGF)-2 together with 50 nmol/L osteopontin did not increase the amount of MMP-1 further (data not shown).

PDGF-BB and Osteopontin Coordinately Increase MMP-9 Activity
Conditioned medium from _vß₃+ SMCs contained 2 bands of lytic activity on gelatin zymogram gels, with molecular weights of 83 and 72 kDa. On the basis of comparison with Western blots with anti-MMP antibodies, these bands represent active MMP-9 (83 kDa) and latent MMP-2 (72 kDa). Adding osteopontin alone to _vß₃+ SMCs did not affect the MMP-2 or MMP-9 activity released into the culture medium (Figure 5, top; with 0 and 50 nmol/L osteopontin). However, the addition of either 5 ng/mL PDGF-BB and 50 nmol/L osteopontin (Figure 5, middle) or 10 ng/mL PDGF-BB and 50 nmol/L osteopontin (Figure 5, bottom) resulted in the appearance of a third band, latent MMP-9 (109 kDa), and a slight increase in active MMP-9 (83 kDa, Figure 5). There was no change in latent MMP-2. By contrast, FGF-2 did not affect MMP production or activity, either alone or in combination with osteopontin (data not shown).

View larger version (41K): [in this window] [in a new window]   Figure 5. Gelatin zymograms showing activity of latent MMP-2 (72 kDa) and MMP-9 (109 kDa latent and 83 kDa active) after stimulating vß3+ SMCs with 0 or 50 nmol/L osteopontin in combination with 0 (top), 5 (middle), or 10 (bottom) ng/mL PDGF-BB added to the medium.

	Discussion

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We have shown coincident increases in ß₃ integrin and MMP-1 expression that correlate with the period of SMC proliferation and migration from the media to the neointima in the balloon-injured rat carotid artery. Furthermore, blocking the ß₃ receptor by administration of antibodies inhibited SMC migration after injury. In vitro, human newborn SMCs produced MMP-1, and production was upregulated by osteopontin; this correlated with the presence of _vß₃ integrin on the surface of these cells. By contrast, adult SMCs lacked _vß₃ expression and did not produce MMP-1.

ß₃ integrin receptor mRNA was upregulated early after injury of the rat carotid artery, with a time course correlated with SMC proliferation and migration to the intima. Early increases in ß₃ immunostaining have also been reported in primates and rabbits after vascular injury.^{4 23 24} The ß₃ integrin–blocking antibody F11¹⁶ almost completely blocked SMC migration from the media to the intima. By contrast, neither medial SMC replication rate nor total medial SMC number in vessel cross sections at 4 days was reduced by the treatment, suggesting that ß₃ integrin blockade specifically inhibited SMC migration. Slepian et al¹¹ also measured a decrease in SMC migration in the rat carotid artery after treatment with cyclic RGD peptides. However, they did not measure proliferation, and cyclic RGD peptides are not specific to _vß₃ but instead block all _v-containing integrins. Intimal thickening was inhibited after treatment with various agents that act against _vß₃, including cyclic RGD, c7E3, and small molecule–selective _vß₃ antagonists, but none of these studies measured migration in vivo.^{5 6 7 8 9 10 11 25} Deitch et al²⁶ failed to see an effect of c7E3 in reducing postangioplasty or in-stent restenosis in monkeys, but the negative results in that study may have been due to differences in dose, administration, or species. The present study suggests that a decrease in SMC migration and not proliferation is the principle mechanism of action of the _vß₃ antagonist.

_vß₃ is expressed by endothelial cells migrating at the leading edge of a denuded artery²⁷ ; however, F11 did not block endothelial cell regeneration on the surface of the thoracic aorta. This is consistent with a report from van der Zee et al,¹⁰ in which administration of LM609 failed to inhibit reendothelialization in the injured rabbit iliac artery. Regenerating endothelial cells migrate in a sheet on the 2D luminal surface of the vessel, whereas SMCs invade through the 3D matrix of the vessel media; therefore, it is reasonable to hypothesize that migration may involve different mechanisms in these 2 situations. _vß₃ plays an essential role in endothelial cell angiogenesis, by binding and activating MMP-2 at the cell surface, a process that is necessary for proteolytic invasion of a 3D matrix.²⁸ SMCs may use similar mechanisms to invade the 3D matrix of the vessel media.

F11 cross-reacts with platelet _IIbß₃ receptors; therefore, we cannot rule out the possibility that the change in SMC migration was due to an antiplatelet effect of F11. However, we think that this is unlikely because we observed no change in platelet deposition after treatment. In the rat carotid artery injury model, a monolayer of platelets is deposited on the denuded vessel surface within 4 hours after injury, with little fibrin deposition and no thrombus formation at any time after injury.¹⁹ We did observe a transient thrombocytopenia 48 hours after initiation of treatment with F11; but by 96 hours (4 days), platelet counts had recovered. Even when we delayed the administration of F11 until 24 hours after injury to ensure adequate time for platelet deposition and release, treatment still caused a significant reduction in SMC migration compared with no treatment. This suggests that blocking SMC _vß₃, even in the presence of platelet factors, is sufficient to significantly inhibit SMC migration.

SMCs require proteinase activity to migrate from the media to the intima, and the present study shows that _vß₃ plays an important role in determining the SMC proteolytic phenotype by mediating increases in MMP activity stimulated by the matrix ligand osteopontin. Newborn SMCs, which were positive for _vß₃ integrin expression, produced MMP-1, and we were able to increase MMP-1 production further by stimulating these cells with osteopontin. By contrast, adult SMCs, which were _vß₃ negative, did not produce MMP-1 and were not responsive to osteopontin. One limitation of this experiment is the use of newborn and adult SMCs, because a wide variety of genes is likely to be differentially expressed in the 2 cell types,²⁹ and we cannot rule out the possibility that differences in cell phenotype other than _vß₃ expression may have accounted for the differences in MMP production.

There is a growing body of literature suggesting that interactions with matrix molecules can regulate proteinase production. In a previous study, we showed that another matrix molecule produced after vascular injury, type VIII collagen, stimulated SMC production of MMP-2 and MMP-9.³⁰ In addition, studies with several other cell types have demonstrated that MMP production is regulated by the extracellular matrix through integrin receptor signaling.¹⁵

We next asked whether 2 growth factors expressed in abundance after injury, PDGF-BB and FGF-2, could potentiate SMC responses to osteopontin and further increase MMP production. We found that treatment with osteopontin and PDGF-BB together increased MMP-9 activity in SMCs. This was particularly interesting in light of previous studies showing that PDGF-BB stimulates the expression of ß₃ integrin on SMCs^{31 32} and that _vß₃ ligation results in association with and phosphorylation of the PDGF receptor.³³ By contrast, FGF-2 did not potentiate the matrix-driven increase in MMP activity. Our data suggest coordinate signaling between osteopontin and PDGF, but not FGF-2, in the regulation of SMC proteinase production.

Finally, our results are consistent with findings from recent clinical studies examining agents with inhibitory activity against _IIbß₃ and SMC _vß₃. In the Evaluation of Platelet IIb/IIIa Inhibition for Prevention of Ischemic Complications (EPIC) trial, c7E3 was effective in limiting the need for late coronary revascularization after PTCA for at least 3 years after treatment.³⁴ More recently, studies show that c7E3 treatment reduces in-stent restenosis, ischemic complications, and late mortality, particularly in diabetic patients.³⁵ In contrast, the recent Integrilin to Minimise Platelet Aggregation and Coronary Thrombosis (IMPACT) II trial, which used eptifibatide, an agent with anti-_IIbß₃ activity but without specific _vß₃ inhibitory activity, was not effective.³⁶ These clinical results suggest that the _vß₃ inhibitory effects of c7E3 could be useful in the prevention of the SMC response in restenosis.

In conclusion, we have shown that increased expression of ß₃ integrin in the rat carotid artery correlates with the expression of MMP-1 and early SMC proliferation and migration from the media to the neointima. Administration of F11, a ß₃ integrin inhibitory antibody, inhibited SMC migration but not proliferation in the carotid artery. Finally, we demonstrated that human newborn SMCs stimulated with osteopontin upregulated their production of MMP-1, that this was dependent on the presence of _vß₃, and that in coordination with stimulation by PDGF-BB, MMP-9 activity was also increased. These data suggest that _vß₃ integrins are important in mediating SMC migration from the media to the neointima and that feedback through _vß₃ receptors may upregulate MMP activity, which is a necessary correlate of SMC invasion and migration in vivo.

	Acknowledgments

 
This work was supported by Medical Research Council of Canada grant MT 13180 to M.P. Bendeck, National Institutes of Health grant HL-18645 to C.M. Giachelli, and a grant from The Wellcome Trust to M. Horton. M.P. Bendeck is a Research Scholar of the Heart and Stroke Foundation of Canada, and C.M. Giachelli is an Established Investigator of the American Heart Association. The authors acknowledge the generous donation of recombinant PDGF-BB from Dr Charles Hart, Zymogenetics, Seattle, Wash.

Received February 8, 2000; accepted March 17, 2000.

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