{alpha}v{beta}3-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells

M. Sajid, R. Zhao, A. Pathak, S. S. Smyth, and G. A. Stouffer

Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599

Submitted 10 October 2002 ; accepted in final form 10 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}v{beta}3-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because {alpha}-thrombin contributes to neointimal formation, we examined the hypothesis that {alpha}v{beta}3-integrins influence {alpha}-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed {alpha}v{beta}3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to {alpha}-thrombin were partially inhibited by anti-{beta}3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that {alpha}-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by {alpha}v{beta}3 antagonists. {beta}3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. {alpha}-Thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). {alpha}v{beta}3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from {beta}3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, {alpha}v{beta}3-integrins play an important role in {alpha}-thrombin-induced proliferation and focal adhesion formation in RASMC.

receptors; vitronectin; integrins; focal adhesions


PERCUTANEOUS CORONARY INTERVENTION (PCI) involves application of a controlled injury to a coronary artery to relieve focal obstruction. The injury stimulates a healing response, which, when overexuberant, leads to re-narrowing of the artery in a process labeled "restenosis." Animal models have implicated {alpha}v{beta}3-integrins in playing an important role in vascular healing responses. Studies in baboons, rats, rabbits, and pigs have shown that balloon angioplasty is a stimulus for {beta}3-integrin expression by vascular smooth muscle cells (SMC) (6, 21, 23) and that treatment with {alpha}v{beta}3 antagonists reduces (neo)intima formation after vascular injury (reviewed in Ref. 19).

{alpha}v{beta}3-Integrins influence responses of cultured SMC to many extracellular agonists that may be important in healing responses in injured blood vessels. In addition to inhibiting responses to agents that directly bind {alpha}v{beta}3, such as thrombospondin and osteopontin (23, 24, 27), {alpha}v{beta}3 antagonists also regulate SMC responses to platelet-derived growth factor (3, 4), epidermal growth factor (10), insulin-like growth factor-I (9), transforming growth factor-{beta} (18), and {alpha}-thrombin (24). {alpha}-Thrombin is of particular interest because it is concentrated at sites of vascular injury and because thrombin inhibition reduces intimal formation and luminal narrowing in many different animal models of vascular injury (14). In addition to its well-known hemostatic effects, {alpha}-thrombin directly stimulates SMC proliferation via activation of protease-activated receptor-1 (PAR-1), a member of the seven-transmembrane-domain, G protein-coupled superfamily of receptors.

Focal adhesions (also called focal complexes) in differentiated smooth muscle are membrane-associated dense plaques that include integrins, cytoskeletal and signaling proteins. They enable bidirectional communication with the extracellular environment and are important in cellular processes of migration, spreading, and contraction. Ishida et al. (8) showed that in serum-starved rat aortic smooth muscle cells (RASMC), there were few focal adhesion patches and that these were small in area. Treatment with thrombin resulted in a marked increase in focal adhesion formation as indicated by increased F-actin bundling, vinculin staining, and phosphotyrosine staining. Subsequently, Ballestrem et al. (1) showed that {alpha}v{beta}3 movement and clustering regulated focal adhesion formation in fibroblasts and melanoma cells. Because antagonists interfere with integrin dynamics, the present studies examined the hypothesis that {alpha}v{beta}3 antagonists inhibit thrombin-induced focal adhesion formation and proliferation in RASMC.


    METHODS
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 METHODS
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 DISCUSSION
 REFERENCES
 
SMC culture. SMC isolated from the aortas of Sprague-Dawley rats were cultured as previously described (11, 18). Briefly, RASMC, passages 3-10, were grown to 70% subconfluence in serum-containing medium. The cells were then growth-arrested for 48-72 h in a quiescent medium (0.5% FBS) before treatment. Proliferation assays, immunocomplex kinase assays for c-Jun NH2-terminal kinase-1 (JNK1) activity, cell adhesion assays, and fluorescence-activated cell sorting (FACS) analysis were performed as previously described (11, 18).

The generation of {beta}3-integrin-deficient mice by homologous recombination in embryonic stem cells and their phenotype have been described previously (7). Wild-type ({beta}3+/+) and {beta}3-/- mice were backcrossed on a C57Bl/6 background. SMC from wild-type and {beta}3-integrin null mice were isolated from the medial layer of the thoracic aorta and cultured using standard techniques.

Apoptosis assays. TdT-mediated dUTP nick-end labeling (TUNEL) assays were performed using the In Situ Cell Death Detection Kit (Boehringer-Mannheim). Briefly, 2 x 104 cells were cultured and then growth-arrested on multi-well chamber slides (Nunc). At the specified time points, cells were washed with PBS, briefly dried to air, fixed with 3% paraformaldehyde, blocked with 1% BSA-PBS containing 0.5% Triton X-100, incubated with the TUNEL reaction mix containing fluorescence-tagged dUTP for 2 h at room temperature, washed, and then evaluated with a fluorescent microscope. To quantitate an apoptosis index, we counted the percentage of TUNEL-positive cells from six to eight mid-power fields of each well.

Apoptosis was also determined using the Annexin V-FITC Kit (Bender Med Systems, Vienna, Austria). Briefly, cells were washed with ice-cold PBS twice and resuspended in 195 µl of binding buffer. FITC-labeled purified recombinant annexin V (5 µg) was then added, gently vortexed, and incubated for 15 min at room temperature. Cells were washed with 1 ml of binding buffer. Dilute propidium iodide (PI) in 200 µl of binding buffer was added to the cell pellet. Binding buffer (400 µl) was added to each tube, and cells were analyzed by FACS within 5 min.

Immunofluorescence microscopy. Immunofluorescence microscopy was performed by using methods similar to those described by Ishida et al. (8) Briefly, RASMC were grown to confluence and then growth-arrested for 48 h using a LabTek II glass chamber (Nalge Nunc, Naperville, IL). After exposure to {alpha}-thrombin for the indicated time, cells were washed, fixed with a 1:1 methanol-acetone mixture, and then blocked using PBS containing 10% goat serum and 5% BSA. Cells were stained with the primary antibody and then incubated with Texas Red-goat anti-mouse (1:100) and/or FITC-goat anti-rabbit (1:60).

Reagents. Reagents were obtained from the following sources. {alpha}-Thrombin and echistatin were from Sigma (St. Louis, MO). Anti-FAK, PY20, and PY69 were from Upstate Biotechnology (Lake Placid, NY); anti-FAK was from Santa Cruz Biotechnology (Santa Cruz, CA), F11 was from Pharmin-Gen (San Diego, CA), and polyclonal anti-{beta}3 antibodies AB1932 were from Chemicon (Temecula CA). 10E5 was a gift from Susan Tam (Centocor, Malvern PA). GPenGRGDSPCA and GRGESP peptides were from GIBCO BRL (Grand Island, NY).

Data analysis. Numerical data are presented as means ± SE unless otherwise stated. Experiments were performed in duplicate and repeated at least three times. One-way analysis of variance followed by Dunn's multiple range test were used to analyze for significant differences (P <= 0.05).


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment with monoclonal anti-{beta}3-integrin antibody F11 or cyclic RGD peptides inhibited proliferative responses to {alpha}-thrombin. FACS analysis demonstrated binding of F11, a monoclonal anti-{beta}3-integrin antibody that binds {alpha}v{beta}3-integrins with high specificity (27), to quiescent RASMC. RMV7, an anti-mouse {alpha}v antibody that does not recognize rat proteins, was used as a control and did not bind RASMC (Fig. 1A).



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Fig. 1. Effect of {alpha}v{beta}3 antagonists on {alpha}-thrombin-induced proliferation of rat aortic smooth muscle cells (RASMC). A: fluorescence-activated cell sorting (FACS) analysis was performed without antibody, by using F11, a monoclonal anti-{beta}3-integrin antibody that binds {alpha}v{beta}3-integrins on RASMC, or by using RMV7, an anti-mouse {alpha}v antibody that does not recognize rat proteins, as a control. B and C: RASMC were treated with {alpha}-thrombin (1 U/ml) or vehicle ± F11, cRGD peptides (10 µM), or the disintegrin echistatin at the indicated concentrations. A monoclonal antibody directed against {alpha}IIb{beta}3 (10E5; 20 µg/ml) and RGE peptides (10 µM) were used as controls. Three days later, the cells were counted. Results are means ± SE from 5 independent experiments. *P < 0.05 compared with Thr + 10E5 (B) or Thr + RGE (C). Cnt, control; Thr, thrombin; echi, echistatin. Cnt, control; Thr, thrombin; echi, echistatin.

 

{alpha}-Thrombin, when added to growth-arrested RASMC, elicited a proliferative response measured by increases in cell number. F11, which has function-blocking activity both in vitro (12, 18) and in vivo (2), inhibited proliferative responses to {alpha}-thrombin in a dose-dependent manner (Fig. 1B). At a concentration of 20 µg/ml, F11 inhibited ~50% of the proliferative response elicited by {alpha}-thrombin. Treatment with a monoclonal antibody directed against {alpha}IIb{beta}3 (10E5), used as a control, had no effect on {alpha}-thrombin-induced proliferation.

Cyclic RGD peptides (GPenGRGDSPCA; cRGD) have been shown to block {alpha}v{beta}3-mediated migration of RASMC and neointimal formation following rat carotid artery balloon injury (21). Treatment with cRGD at a concentration of 10 µM completely inhibited {alpha}-thrombin-induced proliferation (Fig. 1C). Echistatin is a function-blocking disintegrin found in the venom of Echis carinatus that binds {alpha}v{beta}3-integrins on SMC (9). Echistatin, at a concentration of 10 nM, had a non-statistically significant inhibitory effect on the proliferative response elicited by {alpha}-thrombin. Peptides containing an RGE sequence (GRGESP) had a small, statistically insignificant inhibitory effect.

Effect of {alpha}v{beta}3-integrin antagonists on SMC detachment and apoptosis. At the concentrations used in these studies, F11 had no effect on cell adhesion (Fig. 2A). This is consistent with our prior studies (18) and also with studies from Clyman et al. (5) showing that adherence of SMC to standard tissue culture plates is mediated primarily by {beta}1-integrins. Echistatin at 10 nM and cRGD at 10 µM had no effect on adhesion. At 100 nM, echistatin caused detachment of ~30% of cells. This detachment was statistically significant and consistent in four separate experiments.



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Fig. 2. Effect of {alpha}v{beta}3 antagonists on cell adhesion and apoptosis. To determine cell adhesion, RASMC in suspension were incubated with echistatin (various concentrations), cRGD peptides (10 µM), RGE peptides (10 µM), F11 (20 µg/ml), or 10E5 (20 µg/ml) and then added to noncoated plates (A). To determine apoptosis, annexin (B) and TUNEL staining (C) were determined 24, 48, 72, and 120 h after RASMC were placed in quiescent media. At 72 h, echistatin (10 nM), cRGD peptides (10 µM), RGE peptides (10 µM), F11 (20 µg/ml), or 10E5 were added for an additional 48 h. *P < 0.05 compared with control.

 

Because {alpha}v{beta}3-integrins regulate apoptosis in certain cell types, we examined apoptosis using TUNEL assays and annexin staining (Fig. 2, B and C). SMC maintained in quiescent medium for 120 h showed stable amounts of cells with positive TUNEL or annexin staining and at levels consistent with other studies (17). The number of cells staining positive for TUNEL was consistently higher than for annexin, possibly because the TUNEL assay, in addition to identifying apoptotic cells, also stains nuclei with high RNA synthetic activity that are not in the execution phase of apoptosis. Treatment with F11 (or the control antibody 10E5) for 48 h had no effect on apoptotic indexes. Treatment with peptide inhibitors of {alpha}v{beta}3-integrins, as well as RGE, caused an increase in TUNEL staining without an increase in annexin staining.

{alpha}v{beta}3-Integrin antagonists inhibit thrombin-induced focal adhesion formation. As shown in Fig. 3, we found that focal adhesions as identified by staining with anti-vinculin formed within 5 min of exposure to {alpha}-thrombin, consistent with previous studies (8). After exposure to {alpha}-thrombin, {beta}3-integrin expression remained diffuse and did not show concentration at focal adhesions.



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Fig. 3. Immunofluorescence microscopy of {beta}3-integrins and vinculin. RASMC were treated with {alpha}-thrombin (1 U/ml) for 10 min (A) or 20 min (B and C) and subjected to immunofluorescence microscopy using anti-{beta}3-integrin polyclonal antibodies (AB1932; left images) or anti-vinculin monoclonal antibody (right images).

 

To test the hypothesis that {alpha}v{beta}3-integrin antagonists inhibit focal adhesion formation, we treated RASMC with thrombin or vehicle with or without F11 and then counted focal adhesions as defined by staining with anti-vinculin antibody. In RASMC maintained for 72 h in quiescent medium and then exposed to vehicle for 10 min, 2% of the cells had >60 focal adhesions and 57% of the cells had >30 focal adhesions (Fig. 4). After a 10-min exposure to thrombin, 72% of the cells had >60 focal adhesions and 90% of the cells had >30 focal adhesions.



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Fig. 4. Effect of {alpha}v{beta}3 antagonists on focal adhesion formation in RASMC. Growth-arrested RASMC were pretreated with F11 (20 µg/ml) or 10E5 (20 µg/ml) for 1 h. The cells were then treated with vehicle (A) or {alpha}-thrombin (1 U/ml; B) for 10 min and subjected to immunofluorescence microscopy using anti-vinculin monoclonal antibody. To quantify the effect, we counted the number of focal adhesions in at least 50 cells in each group. Note that the legends are different. *P < 0.05 compared with no antibody and 10E5.

 

The number of focal adhesions was decreased after pretreatment with F11. In RASMC pretreated with F11 for 1 h, 32% of vehicle-treated SMC had >30 focal adhesions. In RASMC pretreated for 1 h with F11, 29% of thrombin-treated SMC had >60 focal adhesions and 56% had >30 focal adhesions. In SMC pretreated with 10E5, 62% had >60 focal adhesions after exposure to thrombin (Fig. 4).

To further test the hypothesis that {beta}3-integrins function in focal adhesion formation in thrombin-treated SMC, we utilized SMC isolated from {beta}3-integrin null mice ({beta}3-/-) and SMC isolated from wild-type littermates ({beta}3+/+) (22). Focal adhesions formed in 65 ± 8% of {beta}3+/+ SMC that were growth-arrested and then treated with thrombin (2 U/ml) for 1 h (Fig. 5). In contrast, focal adhesions were observed in <20% of {beta}3-/- SMC treated in the same manner. However, whereas focal adhesions (elongated areas of vinculin staining localized to the ends of actin stress fibers) were more common in thrombin-treated {beta}3+/+ SMC, low-density adhesions (small pointlike vinculin staining localized to sites of lamellipodia induction) were more common in {beta}3-/- SMC (58 ± 9% of {beta}3-/- SMC vs. <20% of {beta}3+/+ SMC). Treatment with sphingosine-1-phosphate, a potent activator of Rho in {beta}3-/- and {beta}3+/+ SMC (unpublished observations) stimulated robust focal adhesion formation in both cell types, demonstrating that focal adhesion formation does occur in {beta}3-/- SMC. These results were consistently observed in two experiments, each with three different SMC lines from wild-type mice and three different SMC lines from {beta}3-/- mice.



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Fig. 5. Focal adhesion formation in SMC isolated from wild-type and {beta}3-integrin null ({beta}3-/-) mice. SMC from wild-type or {beta}3-/- mice were treated with {alpha}-thrombin (2 U/ml) or sphingosine-1-phosphate (SIP; 200 nM) for 60 min and then subjected to immunofluorescence microscopy using an anti-vinculin monoclonal antibody. Representative images are shown from 2 experiments, each with 3 different SMC lines from wild-type and 3 different SMC lines from {beta}3-/- mice.

 

Thrombin-induced tyrosine phosphorylation of focal adhesion kinase was not inhibited by {alpha}v{beta}3 antagonists. Previous studies in quiescent RASMC (15, 26) demonstrated that {alpha}-thrombin stimulates focal adhesion kinase (FAK) phosphorylation on tyrosine. Because FAK is involved in focal adhesion signaling, we sought to determine whether {alpha}v{beta}3 antagonists inhibited {alpha}-thrombin-induced FAK phosphorylation. Immunoprecipitation with an anti-human FAK antibody raised in rabbits immunized with a pGEX-derived fusion protein containing residues 748-1053 of human FAK demonstrated that FAK was present and constitutively phosphorylated on tyrosine in cultured RASMC (Fig. 6, A-C). Levels of FAK phosphorylated on tyrosine increased after treatment with {alpha}-thrombin. This effect was time dependent with maximal levels of phosphorylated FAK observed 5 min after exposure to {alpha}-thrombin. Similar results were observed by using two different monoclonal anti-phosphotyrosine antibodies (PY20 or PY69) followed by Western analysis with anti-FAK. Treatment with functionally active concentrations of {alpha}v{beta}3 antagonists (F11, cRGD, and echistatin) did not inhibit {alpha}-thrombin-induced tyrosine phosphorylation of FAK (Fig. 6D).



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Fig. 6. Effect of {alpha}v{beta}3 antagonists on {alpha}-thrombin-induced focal adhesion kinase (FAK) tyrosine phosphorylation. Growth-arrested RASMC were pretreated with echistatin (10 nM), cRGD peptides (10 µM), RGE peptides (10 µM), F11 (20 µg/ml), or 10E5 (20 µg/ml) for 1 h. The cells were then treated with {alpha}-thrombin (1 U/ml) or vehicle for various time periods. Cell extracts were immunoprecipitated (IP) with anti-phosphotyrosine antibody (PY69) or anti-FAK (B). The immunoprecipitates were then Western immunoblotted (IB) with anti-phosphotyrosine antibody (PY20) or anti-FAK (A and D). C: activity was quantified using densitometry. Data are means ± SE from 3 independent experiments. *P < 0.05 compared with control. Blots in A, B, and D are representative of 3 independent experiments.

 

cRGD peptides and echistatin inhibited {alpha}-thrombin-induced increases in JNK1 activity. JNK1 (also known as stress-activated protein kinase-1) is a member of the mitogen-activated protein kinase superfamily that is activated by dual phosphorylation at a Thr-Pro-Tyr motif and, once activated, functions to phosphorylate c-Jun at amino-terminal serine regulatory sites, which increases activity of the transcription factor AP-1. To determine the effect of {alpha}v{beta}3 antagonists on {alpha}-thrombin-induced activation of JNK1, we utilized an in vitro immunocomplex kinase assay with glutathione S-transferase-c-Jun as the substrate. {alpha}-Thrombin-induced effects on JNK1 activation were transient with peak effects observed at 10 min and a return to near-baseline levels by 15 min (Fig. 7A). Others (16) have previously shown, using the same experimental system, that {alpha}-thrombin stimulation of JNK1 activity is associated with subsequent increases in c-Jun expression, AP-1 DNA-binding activity, and AP-1 transactivation activity.



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Fig. 7. Effect of echistatin and cRGD peptides on {alpha}-thrombin-induced c-Jun NH2-terminal kinase-1 (JNK1) activity. RASMC were treated with {alpha}-thrombin (1 U/ml) or vehicle ± echistatin (10 nM in B, various concentrations in C), cRGD peptides (10 µM in B, various concentrations in C), or RGE peptides (10 µM). JNK1 activity was determined at various time points using an in vitro immunocomplex kinase assay with glutathione S-transferase-c-Jun as the substrate. A and B: activity was quantified using densitometry. Data are means ± SE from 3 independent experiments. *P < 0.05 compared with Thr + RGE. Blots in C and D are representative of 3 independent experiments.

 

Pretreatment with echistatin or cRGD peptides for 1 h reduced {alpha}-thrombin-induced JNK1 activity measured 10 min after treatment in a dose-dependent manner (Fig. 7, B and C). RGE peptides had no effect on JNK1 activation in {alpha}-thrombin-treated SMC.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}v{beta}3-Integrin antagonists partially inhibited proliferative responses of RASMC to {alpha}-thrombin. This effect was observed with both peptide and antibody antagonists and was independent of the extracellular matrix on which the cells were grown (unpublished observations). {alpha}v{beta}3-Integrins thus join the long list of molecules that have been implicated in SMC responses to {alpha}-thrombin, including platelet-derived growth factor-AA, basic fibroblast growth factor, epiregulin, heparin-binding epidermal growth factor-mediated transactivation of EGF receptor, flavin-containing oxidases, insulin-like growth factor-I receptor, Rho, pp60c-src, p21ras, nuclear factor-B, G protein receptor kinase-2, and cAMP (reviewed in Ref. 14). More importantly, because {alpha}-thrombin has been implicated in various animal models of restenosis (14), these data suggest one mechanism by which {alpha}v{beta}3 antagonists may inhibit vascular responses following vascular injury.

We found that focal adhesions as delineated by anti-vinculin staining formed rapidly after treatment of quiescent RASMC with {alpha}-thrombin and that {alpha}v{beta}3 antagonists partially inhibited thrombin-induced focal adhesion formation. The importance of {beta}3-integrins in thrombin-induced focal adhesion formation was confirmed by using SMC isolated from mice deficient in {beta}3-integrins. The mechanism underlying this effect require further study to identify, but an intriguing hypothesis involving Src-{alpha}v{beta}3 interactions has been suggested by two recent studies. Ishida et al. (8) demonstrated that focal adhesion formation in SMC was mediated, at least in part, by Src. Subsequently, Obergfell et al. (13) found that Src and Csk, a negative regulator of Src, associated with {alpha}IIb{beta}3 in platelets. Fibrinogen binding to {alpha}IIb{beta}3 caused dissociation of Csk and upregulation of Src activity. It may be that {alpha}v{beta}3 antagonists prevent ligand binding that is needed for Src activation and subsequent focal adhesion formation.

In contrast to the punctate nature of vinculin staining, immunofluorescence microscopy demonstrated diffuse expression of {beta}3-integrins. Because of the diffuse nature of {beta}3 expression, there might have been low levels of {beta}3-integrins that were present in focal adhesions; however, {beta}3-integrins did not concentrate at focal adhesions. The granular, diffuse pattern of {beta}3-integrin staining observed in quiescent RASMC grown on tissue culture plates is distinct from the prominent appearance of focal contacts containing {alpha}v{beta}3-integrins observed in SMC spreading on Del1 or vitronectin (17) or in RASMC overexpressing {beta}3-integrins (unpublished observations).

{alpha}-Thrombin-induced JNK1 activation in RASMC was partially inhibited by echistatin and cRGD peptides. We previously reported that {alpha}v{beta}3 antagonists inhibited TGF{beta}-induced JNK1 activation (18) and have also found that {alpha}v{beta}3 antagonists inhibit H2O2-mediated JNK1 activation (unpublished observations). These results are consistent with either 1) an important role for {alpha}v{beta}3-integrins in JNK1 activation in RASMC in response to a variety of stimuli or 2) a distinct effect of {alpha}v{beta}3 antagonists on this pathway. The inability of echistatin or cRGD peptides to completely inhibit thrombin-induced JNK1 activation may reflect inherent limitations of these inhibitors (although cRGD at the doses used in these experiments completely inhibited thrombin-induced proliferation) or, more likely, that {alpha}v{beta}3-integrin-independent pathway(s) of JNK1 activation exist in RASMC.

Tyrosine phosphorylation of FAK is primarily mediated by {alpha}v- and {beta}1-integrins, and FAK has been proposed as a potential merging point of G protein-coupled receptors and integrin signaling pathways. Upon activation, FAK undergoes autophosphorylation and combines with Src. Src then phosphorylates several sites within FAK (20). We found that {alpha}-thrombin stimulated a rapid and transient increase in tyrosine phosphorylation of FAK but that total tyrosine phosphorylation of FAK in RASMC treated with {alpha}-thrombin for 5 min was not inhibited by functionally active concentrations of echistatin, cRGD peptides, or F11. Because we did not examine phosphorylation of specific tyrosine residues within FAK, we cannot exclude a role for {alpha}v{beta}3 in the phosphorylation of specific tyrosine residues in FAK. Our data, however, do support the hypothesis that FAK phosphorylation in RASMC can be mediated by integrins other than {alpha}v{beta}3, because total levels of phosphorylated tyrosine increased in RASMC after treatment with {alpha}-thrombin and these levels were not reduced by {alpha}v{beta}3 antagonists.

In fibroblasts echistatin has also been shown to bind RGD-containing {beta}1-integrins including {alpha}5{beta}1, {alpha}8{beta}1, {alpha}v{beta}1, and {alpha}3{beta}1 as well as {alpha}v{beta}3 (25). Because SMC attachment to tissue culture plates is primarily mediated by {beta}1-integrins (5), echistatin antagonism of {beta}1-integrins may explain the observation that SMC detachment occurred at higher doses of echistatin. The inhibitory effect of 10 nM echistatin on thrombin-induced JNK1 activation, however, was most likely due to antagonism of {alpha}v{beta}3, because it occurred at a concentration that did not elicit cell detachment and the effect was similar to that observed with other antagonists of {alpha}v{beta}3.

It is well established that thrombin-induced proliferation of cultured SMC is mediated predominantly by activation of PAR-1, although potential roles for PAR-3 and PAR-4 cannot be excluded. Less well understood are the events elicited by activation of PAR-1 that contribute to progression through the cell cycle. Previous studies have implicated hydrolysis of inositol phosphates, secretion of secondary growth factors, and tyrosine kinase activity (14). The present studies demonstrate that {alpha}v{beta}3-integrin-mediated events are important in proliferative responses of SMC to {alpha}-thrombin and that cellular responses to thrombin may be regulated by {alpha}v{beta}3 antagonists.


    ACKNOWLEDGMENTS
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-70213-1.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. A. Stouffer, Division of Cardiology, Univ. of North Carolina, Chapel Hill, NC 27599-7075 (E-mail: rstouff{at}med.unc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Ballestrem C, Hinz B, Imhof BA, and Wehrle-Haller B. Marching at the front and dragging behind: differential {alpha}v{beta}3-integrin turnover regulates focal adhesion behavior. J Cell Biol 155: 1319-1332, 2001.[Abstract/Free Full Text]

2. Bendeck MP, Irvin C, Reidy M, Smith L, Mulholland D, Horton M, and Giachelli CM. Smooth muscle cell matrix metalloproteinase production is stimulated via {alpha}v{beta}3 integrin. Arterioscler Thromb Vasc Biol 20: 1467-1472, 2000.[Abstract/Free Full Text]

3. Bilato C, Curto KA, Monticone RE, Pauly RR, White AJ, and Crow MT. The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the {alpha}v{beta}3 integrin complex is reversed by activated calcium/calmodulin-dependent protein kinase II. J Clin Invest 100: 693-704, 1997.[Abstract/Free Full Text]

4. Choi ET, Engel L, Callow AD, Sun S, Trachtenberg J, Santoro S, and Ryan US. Inhibition of neointimal hyperplasia by blocking {alpha}v{beta}3 integrin with a small peptide antagonist Gpen-GRGDSPCA. J Vasc Surg 19: 125-134, 1994.[ISI][Medline]

5. Clyman RI, Mauray F, and Kramer RH. {beta}1 and {beta}3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix. Exp Cell Res 200: 272-284, 1992.[ISI][Medline]

6. Corjay MH, Diamond SM, Schlingmann KL, Gibbs SK, Stoltenborg JK, and Racanelli AL. {alpha}v{beta}3, {alpha}v{beta}5, and osteopontin are coordinately upregulated at early time points in a rabbit model of neointima formation. J Cell Biochem 75: 492-504, 1999.[ISI][Medline]

7. Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullere M, Ross FP, Coller BS, Teitelbaum S, and Hynes RO. {beta}3-Integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J Clin Invest 103: 229-238, 1999.[Abstract/Free Full Text]

8. Ishida T, Ishida M, Suero J, Takahashi M, and Berk BC. Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src. J Clin Invest 103: 789-797, 1999.[Abstract/Free Full Text]

9. Jones JI, Prevette T, Gockerman A, and Clemmons DR. Ligand occupancy of the {alpha}v{beta}3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor. Proc Natl Acad Sci USA 93: 2482-2487, 1996.[Abstract/Free Full Text]

10. Jones PL, Crack J, and Rabinovitch M. Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the {alpha}v{beta}3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 139: 279-294, 1997.[Abstract/Free Full Text]

11. Lele M, Sajid M, Wajih N, and Stouffer GA. Eptifibatide and 7E3, but not tirofiban, inhibit {alpha}v{beta}3 integrin-mediated binding of smooth muscle cells to thrombospondin and prothrombin. Circulation 104: 582-587, 2001.[Abstract/Free Full Text]

12. Li G, Chen YF, Kelpke SS, Oparil S, and Thompson JA. Estrogen attenuates integrin-{beta}3-dependent adventitial fibroblast migration after inhibition of osteopontin production in vascular smooth muscle cells. Circulation 101: 2949-2955, 2000.[Abstract/Free Full Text]

13. Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, and Shattil SJ. Coordinate interactions of Csk, Src, and Syk kinases with {alpha}IIb{beta}3 initiate integrin signaling to the cytoskeleton. J Cell Biol 157: 265-275, 2002.[Abstract/Free Full Text]

14. Patterson C, Stouffer GA, Madamanchi NR, and Runge MS. New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology. Circ Res 88: 987-997, 2001.[Abstract/Free Full Text]

15. Polte TR, Naftilan AJ, and Hanks SK. Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II. J Cell Biochem 55: 106-119, 1994.[ISI][Medline]

16. Rao GN, Katki KA, Madamanchi NR, Wu Y, and Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells. J Biol Chem 274: 6003-6010, 1999.[Abstract/Free Full Text]

17. Rezaee M, Penta K, and Quertermous T. Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin {alpha}v{beta}3. Am J Physiol Heart Circ Physiol 282: H1924-H1932, 2002.[Abstract/Free Full Text]

18. Sajid M, Lele M, and Stouffer GA. Autocrine thrombospondin partially mediates TGF-beta-induced proliferation of vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 279: H2159-H2165, 2000.[Abstract/Free Full Text]

19. Sajid M and Stouffer GA. The role of {alpha}v{beta}3 integrins in vascular healing. Thromb Haemost 87: 187-193, 2002.[ISI][Medline]

20. Schlaepfer DD, Hauck CR, and Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71: 435-478, 1999.[ISI][Medline]

21. Slepian MJ, Massia SP, Dehdashti B, Fritz A, and White-sell L. {beta}3-Integrins rather than {beta}1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration. Circulation 97: 1818-1827, 1998.[Abstract/Free Full Text]

22. Smyth SS, Reis ED, Zhang W, Fallon JT, Gordon RE, and Coller BS. {beta}3-Integrin-deficient mice but not P-selectin-deficient mice develop intimal hyperplasia after vascular injury. Circulation 103: 2501-2507, 2001.[Abstract/Free Full Text]

23. Srivatsa SS, Fitzpatrick LA, Tsao PW, Reilly TM, Holmes DR Jr, Schwartz RS, and Mousa SA. Selective {alpha}v{beta}3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury: evidence for the functional importance of integrin {alpha}v{beta}3 and osteopontin expression during neointima formation. Cardiovasc Res 36: 408-428, 1997.[ISI][Medline]

24. Stouffer GA, Hu Z, Sajid M, Li H, Jin G, Nakada MT, Hanson SR, and Runge MS. {beta}3 Integrins are upregulated following vascular injury and mediate proliferation of cultured smooth muscle cells. Circulation 97: 907-915, 1998.[Abstract/Free Full Text]

25. Thibault G, Lacombe MJ, Schnapp LM, Lacasse A, Bouzeghrane F, and Lapalme G. Upregulation of {alpha}8{beta}1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-{beta}1. Am J Physiol Cell Physiol 281: C1457-C1467, 2001.[Abstract/Free Full Text]

26. Turner CE, Pietras KM, Taylor DS, and Molloy CJ. Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells. J Cell Sci 108: 333-342, 1995.[Abstract/Free Full Text]

27. Yue TL, McKenna PJ, Ohlstein EH, Farach-Carson MC, Butler WT, Johanson K, McDevitt P, Feuerstein GZ, and Stadel JM. Osteopontin-stimulated vascular smooth muscle cell migration is mediated by {beta}3 integrin. Exp Cell Res 214: 459-464, 1994.[ISI][Medline]