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(Circulation. 1998;97:907-915.)
© 1998 American Heart Association, Inc.

Basic Science Reports

ß₃ Integrins Are Upregulated After Vascular Injury and Modulate Thrombospondin- and Thrombin-Induced Proliferation of Cultured Smooth Muscle Cells

George A. Stouffer, MD; Zhaoyong Hu, MD; Mansoor Sajid, MD; Husong Li, MD; Guifang Jin, MD; Marian T. Nakada, PhD; Stephen R. Hanson, PhD; ; Marschall S. Runge, MD, PhD

From the Sealy Center for Molecular Cardiology (G.A.S., Z.H., M.S., H.L., G.J., M.S.R.), University of Texas Medical Branch, Galveston; Centocor, Inc (M.T.N.), Malvern, Pa; and Yerkes Regional Primate Research Center and Department of Medicine (S.R.H.), Emory University School of Medicine, Atlanta, Ga.

Correspondence to George A. Stouffer, Route 1064, Medical Research Bldg, 301 University Blvd, University of Texas Medical Branch, Galveston, TX 77555-1064. E-mail stouffer{at}cardiology.utmb.edu

	Abstract

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Background—Treatment with an antibody that binds ß₃ integrins (abciximab; c7E3 Fab) at the time of coronary angioplasty decreases the need for repeat revascularization. Two potential mechanisms have been proposed to explain this effect: (1) inhibition of platelet aggregation or (2) interruption of ligand binding to ß₃ integrins on the smooth muscle cell (SMC) surface. We examined the latter hypothesis by determining (1) if ß₃ integrin expression is upregulated after vascular injury in the baboon, (2) if 7E3 binds ß₃ integrins on cultured SMC, and (3) if ß₃ integrin activation plays a role in proliferation of cultured SMC.

Methods and Results—Results demonstrated that immunostaining for ß₃ integrins was present in the neointima 1 week after balloon withdrawal injury of baboon brachial arteries and that ß₃ integrin expression colocalized with -actin–positive cells. In contrast, staining for ß₃ integrins was undetectable in contralateral uninjured brachial arteries. 7E3 bound to cultured human aortic SMC with an affinity (K_D=3.3 nmol/L) similar to 7E3 binding to endothelial cells or platelets. Cotreatment with 7E3 partially inhibited thrombospondin-induced or -thrombin–induced proliferation but not PDGF-induced or serum-induced proliferation.

Conclusions—In summary, these studies demonstrate that vascular cell ß₃ integrin expression is increased after injury, that 7E3 binds to cultured SMC with high affinity, and that ß₃ activation is important for thrombospondin-induced or -thrombin–induced proliferation. These results support the hypothesis that ß₃ integrins play a role in SMC growth responses after balloon injury.

Key Words: receptors • muscle, smooth • angioplasty • signal transduction

	Introduction

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Long-term success of percutaneous transluminal coronary revascularization remains limited by luminal renarrowing (ie, restenosis) that occurs within 6 months in 20% to 30% of patients. Pathologically, restenosis is characterized by SMC proliferation and extracellular matrix production. Of the numerous pharmacological agents that have been evaluated in an attempt to reduce the rate of restenosis, only an antigen-binding fragment derived from a human/mouse chimeric monoclonal antibody directed against ß₃ integrin subunits (abciximab; c7E3 Fab) has proven effective in a large clinical trial. In the EPIC trial,¹ 2099 patients who were undergoing balloon angioplasty or directional coronary atherectomy and who were considered high risk for ischemic complications because of recent acute coronary syndrome or unfavorable coronary anatomic features were randomized to receive either bolus+12-hour infusion of abciximab, bolus of abciximab+infusion of placebo, or bolus+infusion of placebo. Six months after the initial intervention, there was a 26% reduction in the need for target vessel revascularization in the group receiving bolus+infusion of abciximab compared with the group that received placebo.

One potential mechanism for the clinical beneficial effects of abciximab is by binding to, and thereby preventing activation of, platelet _IIbß₃ (glycoprotein IIb/IIIa), the receptor that mediates the final common pathway of platelet aggregation. According to this hypothesis, interruption of local thrombus formation and release of platelet-derived mitogens would decrease vascular cell proliferation that ultimately leads to restenosis. Previous trials have, however, failed to demonstrate any reduction in the rate of restenosis in patients treated with hirudin,² aspirin,³ dipyridamole,³ ticlopidine,⁴ prostacyclin,^{5 6} thromboxane synthetase inhibitor,⁷ or serotonin-receptor antagonists.⁸ Although it is likely that at least some of the effects of abciximab on restenosis are mediated through interruption of thrombus formation, these data are consistent with the hypothesis that mechanisms independent of thrombus formation may also play a role in neointimal growth.

One thrombus-independent potential mechanism by which abciximab could reduce restenosis is through interactions with ß₃ integrins expressed by vascular cells in the injured artery. Previous studies⁹ have shown that c7E3 binds _vß₃ expressed on the surface of cultured endothelial cells. Recently, Hoshiga et al¹⁰ reported that _vß₃ was expressed in the intima of atherosclerotic coronary arteries from explanted human hearts and that this expression colocalized with SMC. Using four different monoclonal antibodies directed against _vß₃ or ß₃, these investigators found that _vß₃ was expressed, in both atherosclerotic arteries and in arteries with diffuse intimal thickening, along the lumen and throughout the intima and media. Although _vß₃ activation in cultured SMC has not been linked directly to cell growth, it has been implicated in osteopontin-induced or vitronectin-induced migration. Liaw et al¹¹ demonstrated that human aortic SMC deficient in _vß₃ would migrate in response to fibronectin but failed to migrate in response to osteopontin. Furthermore, osteopontin-induced migration in SMC expressing _vß₃ was inhibited by LM609, an antibody that specifically binds _vß₃. Brown et al¹² showed that antisera to _vß₃ inhibited vitronectin-induced migration of human aortic SMC. Taken together, these studies demonstrate that _vß₃ is expressed by SMC in diseased coronary arteries and that activation leads to changes in SMC phenotype necessary for cell migration.

Characterization of the role, if any, that _vß₃ plays in postintervention restenosis would enable the design of more targeted therapy. Therefore, the purpose of the present studies was to determine (1) whether expression of ß₃ integrins is upregulated after vascular injury in the baboon, (2) whether 7E3 binds ß₃ integrins on cultured SMC, and (3) whether ß₃ integrin activation plays a role in proliferation of cultured SMC.

	Methods

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Smooth Muscle Cell Culture
Human aortic SMC were purchased from Clonetics Corp (San Diego, Calif). The cells were cultured in Clonetics SmGM-2 medium containing hEGF 0.5 ng/mL, insulin 5 µg/mL, hFGF 2 ng/mL, 5% FBS, gentamicin 50 µg/mL, and amphotericin B 50 ng/mL. Cells were harvested for passaging at subconfluence with a 0.025% trypsin/0.01% EDTA solution. Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO₂/95% air. SMC between passages 4 and 15 were plated at 3x10³ cells/cm² in SmGM-2 medium. They were grown to confluence and then growth-arrested in Clonetics SmBM medium containing 0.5% FBS.

Growth Curves
SMC were grown in culture and growth-arrested as described above. The 0.5% serum–containing medium was changed and then TSP with and without antibodies was added. Five days after treatment, the cultures were washed with PBS, harvested with trypsin-EDTA, diluted with 0.9% NaCl, and counted with a Coulter counter.

Baboon Arterial Injury Model
Balloon catheter–denuding injury of the left brachial artery was performed on juvenile male baboons (Papio anubis) weighing 8 to 12 kg, as previously described.¹³ Briefly, the animals were anesthetized with ketamine and halothane. An incision was made over the medial aspect of the forearm and a side branch of the brachial artery was isolated and controlled with vessel loops. A 3F Fogarty embolectomy catheter was passed through the branch to a distance of 10 cm, inflated to a diameter of 4 mm by filling with sterile saline, and withdrawn the length of the vessel by a gentle twisting motion. A moderate resistance to the passage of the balloon was achieved in all cases. The procedure was repeated three times; the branch vessel was then ligated and the incision closed. The right brachial artery was not injured and served as a control. One week after injury, both brachial arteries were harvested, cleaned of periadventitia, fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed, and rehydrated.

Immunohistochemistry
Slides were preincubated in 50 µg/mL trypsin for 15 minutes at 37°C and then incubated in PBS containing 3% H₂O₂/methanol for 30 minutes and 10% normal rabbit serum in PBS for 30 minutes. A 1:100 dilution of Y2/51 (Dako Corp), 10E5 (Centocor), or -actin (Sigma) in PBS containing 0.1% BSA was applied to slides for 12 hours at 4°C. The slides were washed and then incubated with rabbit anti-mouse antibody conjugate biotin for 30 minutes. The slides were then washed, incubated with ABC solution, washed again, and developed in Vector black substrate solution.

For the double staining (Fig 2), a 1:100 dilution of Y2/51 in PBS containing 0.1% BSA was applied to slides for 12 hours at 4°C. The slides were washed three times with PBS containing 0.1% Tween 20 (Sigma). ABC staining was performed with the use of alkaline phosphatase Vectastain ABC kit and visualized with Vector red substrate. The slides were then washed with stripping buffer (50 mmol/L Tris-HCl, pH 7.4, 10 mmol/L ß-mercaptoethanol, 1% SDS) at 42°C for 30 minutes and then three times with PBS containing 0.1% Tween 20. A 1:50 dilution of anti–-smooth muscle actin (clone asm-1, Novacastra Immunohistochemistry, Newcastle on Tyne, UK) was applied. ABC staining was performed with a peroxidase Vectastain ABC kit and visualized with 3,3'-diaminobenzidine plus nickel (Vector).

View larger version (135K): [in this window] [in a new window]   Figure 2. Expression of ß3 integrins and -actin by neointimal cells in injured baboon brachial arteries. Baboon brachial arteries were harvested 1 week after balloon withdrawal injury. ß3 integrin immunoreactivity was detected utilizing Y2/51 and Vector red substrate (red staining) (A). The slides were washed with stripping buffer (which removes antibodies but not substrates) and -actin immunoreactivity in the same section was detected with asm-1 as the primary antibody and 3,3'-diaminobenzidine (DAB) substrate (gray/black staining) (B).

MAP Kinase Activity
Quiescent human aortic SMC were treated with TSP with and without antibodies for the indicated times. After washing once with ice-cold PBS, the cells were lysed with RIPA lysis buffer. The extracts were then centrifuged at 14 000 rpm at 4°C for 10 minutes. Aliquots of extracts (400 µg of protein) were incubated in 400 µL of lysis buffer with 5 µg of anti–ERK 1 and 5 µg of anti–ERK 2 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif) at 4°C for 24 hours and immunoprecipitated with 50 µL of protein A–agarose slurry. The immunoprecipitates were assayed for kinase activity by adding to reaction buffer (25 mmol/L HEPES, pH 7.5, 10 mmol/L magnesium acetate, 50 µmol/L ATP) containing 2 µCi of [³²P]ATP and 10 µg of substrate (PHAS-1, Stratagene, La Jolla, Calif) and incubated at 30°C for 10 minutes. The reaction was stopped with the addition of 2x SDS sample buffer and boiling for 5 minutes. The protein was resolved on a 12.5% SDS–polyacrylamide gel and subjected to autoradiography. Densitometric analysis was done on a Molecular Dynamics Personal Densitometer SI, with Image QuaNT software.

Receptor Binding Studies
m7E3 IgG was radiolabeled to a specific activity of 7 µCi/µg with Na ¹²⁵I (Amersham) with Iodobeads (Pierce Chemicals). SMC were seeded into 96-well Removacell tissue culture plates (Dynatech) and grown and growth-arrested as described elsewhere in "Methods." For saturation binding, ¹²⁵I-m7E3 IgG was diluted in growth-arrest media containing 0.02% azide to prevent internalization. A 100-fold excess of unlabeled m7E3 IgG was used to define nonspecific binding. Specific binding was defined as total binding (in the absence of unlabeled competitor) minus nonspecific binding. The cells were incubated with antibodies for 4 hours at 37°C and then washed twice with 200 µL of media. The wells were removed and radioactivity bound was quantitated with a gamma counter. The number of cells per well was quantitated by trypsinization of sample wells and counting of cells with a hemacytometer. Assays (n=3) were performed with duplicate determinations. Nonlinear regression with GraphPad (Prism) was used to calculate the K_D and B_max values.

Reagents
TSP was isolated from outdated human platelets as described by Santoro and Frazier.¹⁴ m7E3 used in cell culture studies was provided by Centocor (Malvern, Pa). This antibody has the same murine variable IgG regions as does the chimeric human/murine Fab fragment c7E3 that was used in the EPIC trial.¹ PDGF-AA and PDGF-BB were obtained from Genzyme (Cambridge, Mass), and -thrombin was the kind gift of Dr John Fenton II (Albany, NY).

Statistical Analysis
Results of cell proliferation assays are presented as mean±SD unless otherwise stated. One-way ANOVA followed by the Newman-Keuls multiple range test was used to analyze data. A value of P.05 was considered statistically significant. Triplicate wells were analyzed for each experiment, and each experiment was performed independently a minimum of three times. Data plotted are from representative experiments.

	Results

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ß3 Integrin Expression Was Increased 1 Week After Balloon Injury of Baboon Brachial Arteries
ß₃ integrins are expressed by SMC in atherosclerotic coronary arteries harvested from explanted human hearts; however, little is known about regulation of ß₃ integrin expression after vascular injury. In the present study, a baboon vascular injury model was used to allow comparison of ß₃ integrin expression in uninjured brachial arteries and 1 week after balloon withdrawal injury. ß₃ integrin expression was detectable at the lumen surface and throughout the neointima in injured arteries from all three animals examined with Y2/51 antibody (representative section is shown in Fig 1). Y2/51 is a mouse monoclonal -IgG1 that reacts with ß₃ integrin subunits complexed with either _v or _IIb. Previously, Hoshiga et al¹⁰ demonstrated that ß₃ integrin expression as determined by Y2/51 correlated with results obtained by staining with anti-_vß₃ antibodies anti-VnR1 and LM609 and anti-ß₃ integrin monoclonal antibody SZ21 in human coronary arteries.

View larger version (125K): [in this window] [in a new window]   Figure 1. ß3 integrin expression in injured baboon brachial arteries. Baboon brachial arteries were harvested 1 week after balloon withdrawal injury. Immunostaining was done as described in "Methods" with a monoclonal antibody directed against ß3 integrins (Y2/51). Original magnification: x100 (A) or x200 (B).

Double-staining technique was used to determine whether ß₃ integrins were expressed by SMC within the neointima. A section from an injured baboon artery was initially stained with Y2/51 to detect ß₃ expression. The same section was then stained with an anti–-actin monoclonal antibody to look for SMC. As shown in Fig 2, there are -actin–positive cells within the neointima in areas that show large amount of staining for ß₃ integrins. ß₃ integrin expression was undetectable in uninjured arteries from three baboons (Fig 3).

View larger version (137K): [in this window] [in a new window]   Figure 3. ß3 integrin expression in uninjured baboon brachial arteries. Uninjured baboon brachial arteries were harvested 1 week after balloon withdrawal injury of the contralateral brachial artery. Immunostaining was done as described in "Methods" with a monoclonal antibody directed against ß3 integrins (Y2/51). Original magnification x200.

Additional studies were performed to exclude the possibility that Y2/51 was binding to _IIbß₃ on platelets. Using light microscopy, we were unable to find any morphological evidence of platelets (S.R. Hanson, Yerkes Regional Primate Research Center, 1990, unpublished observations). Additionally, there was no immunostaining seen along the lumen or within the neointima using 10E5, a monoclonal antibody with high specificity for _IIbß₃ in both humans and primates (data not shown).¹⁵ Together these data provide strong evidence that at least some SMC within the neointima are expressing ß₃ integrins after vascular injury.

m7E3-Bound Cultured Human Aortic Smooth Muscle Cells With High Affinity
The next aim of these studies was to determine if m7E3 bound to cultured SMC. This antibody has the same murine variable IgG regions as does the chimeric human/murine Fab fragment c7E3 that was used in the EPIC trial. Results demonstrated that SMC bound ¹²⁵I-m7E3 in a specific and saturable manner (Fig 4). The K_D was 3.33 nmol/L and the B_max was 100 000±12 000 sites/cell (Fig 5). Previous studies have demonstrated that m7E3 binds bivalently and therefore 200 000 _vß₃ molecules are expressed per cell. This high-affinity binding is similar to that observed for m7E3 binding to endothelial cells (K_D=2.46 nmol/L).¹⁶

View larger version (12K): [in this window] [in a new window]   Figure 4. Specific binding of m7E3 IgG to human aortic SMC. Cells were growth-arrested, and the ability of 125I-m7E3 IgG to bind to the cells was determined by saturation binding. Specific binding was defined as total binding minus nonspecific binding, which was determined by a 100-fold excess of unlabeled m7E3 IgG. Data points are the mean±SD of triplicate determinations from a representative experiment performed three times.

View larger version (11K): [in this window] [in a new window]   Figure 5. Scatchard transformation and linear regression of m7E3 binding to SMC. Cells were growth-arrested, and the ability of 125I-m7E3 IgG to bind to the cells was determined by saturation binding. Specific binding was defined as total binding minus nonspecific binding, which was determined by a 100-fold excess of unlabeled m7E3 IgG. Data points are the mean±SD of triplicate determinations from a representative experiment performed three times.

Cotreatment With m7E3 Partially Inhibited TSP-Induced SMC Proliferation and MAP Kinase Activation
The next aim of these studies was to determine if activation of ß₃ integrins influences SMC growth responses. To examine this question, SMC were treated with TSP with or without m7E3. TSP was chosen because it is released by degranulating platelets, produced by SMC at sites of vascular injury,^{17 18} and binds _vß₃ in cell culture.¹⁹ Treatment of cultured human aortic SMC with human platelet–derived TSP resulted in a proliferative response, with cell number increasing from 45% to 155% when measured, in multiple experiments, 5 days after treatment. Treatment with m7E3 alone had no effect on quiescent SMC (cell number at 5 days was 105±5% of control in groups treated with 7E3 and 104±6% in groups treated with 10E5; neither value is statistically different from untreated control groups). In contrast, cotreatment with m7E3 reduced TSP-induced proliferation by 37% to 60% in various experiments, whereas cotreatment with 10E5, a monoclonal antibody that binds _IIbß₃ at an epitope distinct from m7E3, had no effect (a representative experiment is shown in Fig 6).

View larger version (45K): [in this window] [in a new window]   Figure 6. TSP-induced proliferation of SMC. SMC were grown to confluence and growth-arrested as described in "Methods." The serum-free media was changed and TSP (50 µg/mL) or vehicle plus or minus m7E3 (30 µg/mL) or 10E5 (30 µg/mL) added. Five days later the cells were trypsinized and counted.

A potential pathway by which ß₃ integrin activation could lead to cell growth is through activation of MAP kinase. MAP kinase has been shown to be activated during transition from G₀/G₁ to S phase after growth factor stimulation and to lead to transcriptional activity. To measure MAP kinase activity, the ability of proteins immunoprecipitated from SMC lysates by affinity-purified antibodies directed against specific regions within the carboxy terminus of two MAP kinase isoforms, ERK 1 (C-14) or ERK 2 (C-16), to phosphorylate exogenous PHAS-I was measured. Recent studies have demonstrated that PHAS-I can be phosphorylated in SMC by MAP kinase–independent mechanisms²⁰ ; however, it has not been shown that PHAS-I can be phosphorylated by proteins other than ERK 1 or ERK 2 under the conditions of our experiments.

As shown in Fig 7, TSP-treated SMC had a marked increase in MAP kinase activity 10 minutes after treatment. Cotreatment with m7E3 but not 10E5 partially inhibited this response, whereas treatment with m7E3 or 10E5 alone had no effect on MAP kinase activity. These data demonstrated that maximal TSP-induced increases in MAP kinase activity were dependent on _vß₃ activation, or, stated another way, that _vß₃ transduces signals leading to SMC proliferation.

View larger version (32K): [in this window] [in a new window]   Figure 7. TSP-induced MAP kinase activation. SMC were grown to confluence and growth-arrested as described in "Methods." The serum-free media was changed and TSP (50 µg/mL) or vehicle plus or minus m7E3 (30 µg/mL) or 10E5 (30 µg/mL) added. Ten minutes later the cells were lysed. The lysates were immunoprecipitated on protein A beads with anti–ERK 1 and anti–ERK 2 rabbit polyclonal antibodies. The immunoprecipitates were then assayed for kinase activity (with recombinant PHAS-I as substrate) as described in "Methods."

Cotreatment With m7E3 Partially Inhibited Thrombin-Induced But Not PDGF-AA–, PDGF-BB–, or Serum-Induced SMC Proliferation
To determine whether interruption of ligand binding to _vß₃ influenced proliferative responses to agents that do not bind _vß₃ directly, we examined the effects of m7E3 on thrombin-induced, PDGF-induced, or serum-induced proliferation. Thrombin²¹ and PDGF²² have been implicated in mediating arterial responses after balloon injury and bind specific cell surface receptors expressed by SMC. Thrombin has been shown to stimulate SMC proliferation by binding to receptors belonging to the seven-transmembrane, G-protein–coupled superfamily,²³ whereas PDGF-induced proliferation is mediated by binding to receptors with intrinsic tyrosine kinase activity.²⁴ Our results demonstrated that 7E3 inhibited 40% of -thrombin–induced proliferation but had no effect on PDGF-AA–induced or PDGF-BB–induced proliferation (Fig 8). Furthermore, we found that treatment with 7E3 did not influence proliferative responses to 2% or 5% FBS (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 8. Effect of m7E3 on -thrombin–induced, PDGF-AA–induced, or PDGF-BB–induced proliferation of SMC. SMC were grown to confluence and growth-arrested as described in "Methods." The serum-free media was changed and thrombin (10 nmol/L), PDGF-AA (1.4 nmol/L), PDGF-BB (1.4 nmol/L) or vehicle plus or minus m7E3 (30 µg/mL) or 10E5 (30 µg/mL) added. Five days later the cells were trypsinized and counted.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Arterial restenosis after percutaneous revascularization is a clinical problem of enormous significance. Approximately 20% to 30% of patients treated percutaneously undergo a repeat procedure within 6 months, greatly increasing the morbidity and healthcare expenditures associated with this treatment. Because of the clinical significance of vascular responses after mechanical injury, there is great interest in understanding factors that regulate neointimal growth. The results of the EPIC trial demonstrate that treatment with abciximab, an antibody that interrupts ligand binding to ß₃ integrins, reduces the need for target vessel revascularization at 6 months. This antibody is a powerful inhibitor of platelet aggregation, and it has been speculated that the beneficial effects observed are due to inhibition of thrombus formation and release of platelet-derived growth factors.¹ Results of the present studies demonstrating that (1) ß₃ integrin subunits were upregulated after vascular injury, (2) m7E3 bound to ß₃ integrins on the surface of cultured human SMC, and (3) m7E3 inhibits TSP and -thrombin–induced SMC proliferation suggest that inhibition of activation of ß₃ integrins on the surface of vascular SMC may also play an important role in regulating restenosis.

The present studies demonstrate that vascular SMC expression of ß₃ integrin subunits is regulated after mechanical injury. One week after balloon withdrawal injury of baboon arteries, immunostaining for ß₃ integrins was detected along the lumen and throughout the neointima. It is very unlikely that this staining represents residual platelet activity because there were no platelets observed by microscopy; furthermore, there was no staining observed with the monoclonal antibody 10E5 that binds _IIbß₃ with high specificity. Using serial sections from injured arteries, we found that areas of neointima with staining for ß₃ integrins also demonstrated staining with antibodies against -actin. Taken together these data strongly suggest that ß₃ integrins are expressed by neointimal SMC after injury and extend the findings of Hoshiga et al,¹⁰ who reported that _vß₃ was expressed by SMC in the intima of diseased coronary arteries from explanted human hearts in patients undergoing heart transplantation.

ß₃ integrin expression was undetectable in normal baboon brachial arteries. In apparent contrast to our findings, Hoshiga et al¹⁰ reported detection of ß₃ integrin expression in "normal" human coronary arteries. Their studies, however, used arteries from patients who were chronically ill (awaiting cardiac transplantation) and that demonstrated intimal thickening on morphological study, consistent with an early stage of nonmechanical arterial injury. Although we cannot rule out species differences or differences in immunohistochemical technique, our observations of a lack of integrin expression in normal baboon arteries is not inconsistent with the observations of Hoshiga et al and may simply reflect the fact that we were using arteries from young, healthy baboons in contrast to chronically ill humans.

Results of the present studies demonstrated that m7E3 binds to cultured human SMC with an affinity similar to that observed for m7E3 binding to platelets or endothelial cells. Abciximab is unique among the _IIbß₃ inhibitors in clinical trials in that it binds _IIbß₃ and _vß₃ with similar affinity. Eptifibatide (integrilin), a synthetic KGD (Lys-Gly-Asp)- containing peptide, binds _IIbß₃ 100- to 1000-fold more avidly than it binds _vß₃. Results of the recently concluded IMPACT-II study showed that a bolus+infusion of eptifibatide during coronary intervention reduced acute thrombotic complications but failed to reduce angiographically defined restenosis in a subset of patients that underwent repeat catheterization at 6 months. Another _IIbß₃ inhibitor that is in phase III clinical trials is tirofiban (MK-383). This nonpeptide synthetic inhibitor binds human umbilical vein endothelial cells (which express _vß₃ but not _IIbß₃) 24 000-fold less avidly than it binds platelets. We await the results of clinical studies (RESTORE trial) examining the effect of tirofiban on restenosis.

Data that m7E3 partially inhibited TSP-induced proliferation and MAP kinase activation strongly suggest that m7E3 binds to the cell surface in a manner that blocks activation of _vß₃ by agonists. This finding is consistent with previous reports of Coller et al²⁵ that 7E3 prevented binding of fibrinogen to the surface of activated platelets. The agonist that we used in these studies, TSP, was chosen because of previous studies demonstrating that TSP is present at sites of vascular injury,^{17 18 26} enhances proliferative responses of cultured rat SMC to epidermal growth factor²⁷ and binds _vß₃ on the surface of cultured cells.¹⁹ Results of the present studies demonstrate that human platelet-derived TSP stimulates proliferation of cultured human SMC. Maximal TSP-induced proliferation was dependent on _vß₃ activation, thus providing evidence that _vß₃ transduces signals that stimulate SMC proliferation.

There are several potential reasons to explain why m7E3 only partially inhibited TSP-induced proliferation. First, _vß₃ molecules may be constitutively activated by binding to vitronectin or other components of extracellular matrix and TSP may regulate these responses through a mechanism independent of binding to _vß₃. In particular, Gao et al²⁸ have reported that TSP modulates _vß₃ function by binding to integrin-associated peptide (IAP). They found that TSP-induced spreading of C32 human melanoma cells was inhibited by LM609 or the anti-IAP monoclonal antibody B6H12. Second, m7E3 may degrade faster than TSP in the culture medium leading to a delayed proliferative response that is measured by our assay (cell counts at 5 days). Last, TSP has been shown to bind a variety of receptors other than _vß₃, and one or more of these may transduce signals leading to proliferation. Yabkowitz et al²⁹ reported that TSP-induced migration of calf pulmonary artery SMC was mediated by the COOH terminus of TSP and did not involve _vß₃. TSP also interacts with lipoprotein receptor related protein, which has been shown to mediate TSP internalization by SMC,^{30 31} and in other cell lines, TSP has been shown to elicit effects by binding to CD-36³² or cell surface heparan proteoglycans.

Abciximab could also directly influence SMC responses after mechanical injury through mechanisms independent of thrombospondin. First, results of the present studies demonstrate that m7E3 partially inhibited -thrombin–induced proliferation. Since -thrombin elicits SMC proliferation through activation of specific cell surface receptors, the data are surprising and suggest that _vß₃ activation may play a role in proliferative responses to other SMC agonists. Data that m7E3 does not inhibit PDGF-AA–induced proliferation, however, demonstrate that interruption of ligand binding to _vß₃ does not have nonspecific inhibitory effect on entry of SMC into the cell cycle. Second, several studies have implicated _vß₃ in mediating SMC migratory responses to growth factors or _vß₃-binding ligands. In human SMC, treatment with LM609 or a specific RGD-containing peptide inhibited PDGF-induced migration,³³ and treatment with LM609 or kistrin, a disintegrin that binds _vß₃, inhibited 80% of insulin-like growth factor-I (IGF-I)-induced migration.³⁴ Activation of _vß₃ has also been shown to mediate human SMC migratory responses to vitronectin¹² or osteopontin,¹¹ rat SMC migratory responses to osteopontin,³⁵ and porcine SMC migratory responses to IGF-I.³⁴

Studies of the effects of _vß₃ activation on SMC growth responses have been limited by controversy over whether _vß₃ integrins are consistently expressed on cultured human SMC. Skinner et al³⁶ found that cultured human SMC expressed _v but not ß₃. In a subsequent study,¹¹ these investigators and their colleagues reported that some isolates of aortic SMC from human transplant donor specimens expressed high levels of _vß₃, whereas other isolates were _vß₃ deficient. Brown et al¹² reported that _v and ß₃ were present in cultured human aortic SMC and that vitronectin-induced migration could be blocked by antisera to _vß₃. These investigators reported that increased levels of _vß₃ were found after treatment with TGFß or thrombin. Similarly, other investigators have found increased levels of _vß₃ after TGFß treatment in cultured rabbit³⁷ or bovine¹² SMC. Our data demonstrate that the number of _vß₃ molecules expressed by the cultured human SMC line used in these studies is greater than the number of PDGF ß-receptors or thrombin receptors expressed by other primary human cell lines as reported in previous studies. Taken together, these studies suggest that _vß₃ expression varies with the cell line studied, culture conditions, and presence of particular agonists. More importantly, the immunocytochemistry data demonstrate that phenotypically modulated SMC (ie, neointimal) express _vß₃ at much higher levels than medial SMC. Regulation of expression of _vß₃ by cultured cells is poorly understood but warrants further study to provide insight into regulation of _vß₃ expression in vivo.

The phenotype of SMC in culture varies with cell density, growth state, presence or absence of serum, and many other known and unknown variables. SMC express different receptors and respond differently to agonists, on the basis of culture conditions (eg, TGFß inhibits proliferation of actively growing, subconfluent rat aortic SMC but stimulates proliferation of growth-arrested, confluent rat aortic SMC).^{38 39} As noted above, _vß₃ integrin expression varies, depending on the primary cell line studied, the culture conditions, the proliferative state, and/or the presence of agonists that induce _v coupling with ß₃. In the present study, SMC that were growth-arrested were used to study effects of _vß₃ activation on entry into the cell cycle. The use of cells that are subconfluent and/or actively growing may provide a "purer" system in which to study cell cycle entry; however, there is little relation between cells under these conditions in culture and SMC within the artery before angioplasty. Because the phenotype of the SMC in culture may not represent the phenotype of neointimal SMC, all results from cultured SMC must be interpreted cautiously.

The mechanisms by which activation of _vß₃ elicits cell growth are likewise incompletely understood. Recent evidence has demonstrated that integrins can activate many of the intracellular signaling pathways elicited by growth factors, including activation of MAP kinase.^{40 41} Unlike growth factor receptors, however, the short cytoplasmic domains of the - and ß-integrin subunits do not have any intrinsic enzymatic activity and thus integrins appear to function by coupling with cytoplasmic proteins that nucleate the formation of large protein complexes containing both cytoskeletal and catalytic signaling proteins. Several protein tyrosine kinases have been implicated in integrin signaling events by virtue of their integrin-dependent activation or their localization to focal contacts. One of these, focal adhesion kinase (FAK), is localized to focal contacts and autophosphorylates when cell surface integrins adhere to ligands. FAK in turn links integrin activation to phosphorylation of various proteins including Src substrates.⁴² Preliminary studies in our laboratory have shown that nonmuscle myosin heavy chain-A, a 206-kD protein that is tightly regulated on the basis of the position in the cell cycle^{43 44 45} and that has been implicated in SMC proliferation,⁴⁶ associates with _vß₃ and with FAK after TSP treatment.

Results of the present studies demonstrate that 7E3 partially inhibits proliferative responses of cultured SMC to TSP or -thrombin but not to PDGF (AA or BB) or to serum. Because TSP binds to _vß₃, these data suggest that activation of _vß₃ by ligand binding can elicit SMC proliferation. Mechanisms by which 7E3 would inhibit -thrombin–induced proliferation are less obvious. -Thrombin binds specific cell surface receptors on SMC that belong to the seven-transmembrane, G-coupled superfamily, and data suggest that binding to these receptors accounts for the vast majority, if not all, of the effects of -thrombin on SMC proliferation. Results of the present studies suggest that there are interactions between _vß₃ and the thrombin receptor at the cell surface or between signaling pathways activated by these receptors. 7E3 had no effect on PDGF- or serum-induced proliferation, demonstrating that interruption of ligand binding to _vß₃ does not have a generalized effect on proliferation of SMC in culture under the conditions used in these experiments.

In summary, results of the EPIC trial demonstrate that treatment with antibody directed against ß₃ integrins at the time of angioplasty reduced the rate of target vessel revascularization within 6 months. In the present studies we have shown that ß₃ integrins are upregulated after vascular injury in the baboon, that m7E3 binds _vß₃ on the surface of cultured SMC with high affinity, and that m7E3 partially inhibits TSP-induced proliferation and MAP kinase activation. These data support the hypothesis that a potential mechanism contributing to the beneficial effects of abciximab on clinical restenosis is through binding to _vß₃ on vascular SMC and thereby preventing activation by ligands present at sites of mechanical injury.

	Selected Abbreviations and Acronyms

 

ABC = avidin:biotinylated enzyme complex EPIC = Evaluation of c7E3 to Prevent Ischemic Complications trial MAP = mitogen-associated protein PDGF = platelet-derived growth factor SMC = smooth muscle cell(s) TGFß = transforming growth factor-ß TSP = thrombospondin

	Acknowledgments

 
This study was supported in part by an American Heart Association Texas Affiliate Grant-in-Aid (96G-631), Public Health Service grant HL-48667 from the National Institutes of Health, and RR-00165 from Kirin Brewery Co, Ltd. We gratefully acknowledge the technical help of Azita Reger and Patricia Michini.

	Footnotes

 
Presented in part during the Young Investigator Awards Competition at the 46th Annual Scientific Sessions of the American College of Cardiology, Anaheim, Calif.

Received July 7, 1997; revision received September 27, 1997; accepted October 20, 1997.

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