Inhibition of vascular smooth muscle cell adhesion and migration by c7E3 Fab (abciximab): a possible mechanism for influencing restenosis

OBJECTIVES: Brief intravenous administration of chimeric antibody c7E3 Fab during coronary angioplasty has been shown in some studies to provide long term protection against coronary events. Smooth muscle cell (SMC) adhesion and migration are key initial steps in the development of restenosis. The purpose of this study was to investigate the effect of c7E3 Fab on adhesion and migration of SMC to the extracellular matrix (ECM) proteins osteopontin (Opn) and vitronectin (Vn). METHODS: Adhesion of human vascular SMCs to ECM proteins was quantified using a CyQUANT assay kit. Migration of SMCs to Vn, Opn and PDGF was studied using a modified Boyden's chamber migration assay. Integrin expression was determined by immunoprecipitation. RESULTS: c7E3 Fab reduced SMC adhesion on Vn and Opn to 69.2+/-3.3% (P<0.001) and 52.5+/-4.8% (P<0.001) respectively, compared to adhesion without antibody present. This reduction was the same as that for anti-alpha(v)beta(3) integrin antibody LM609 (P=0.5). The combination of anti-alpha(v)beta(5) integrin antibody and c7E3 Fab had a greater effect than either antibody alone (P<0.001). c7E3 Fab reduced SMC migration to Vn and Opn to 51.6+/-8.9% (P<0.001) and 20.3+/-6.1% (P<0.001) respectively, compared to migration in the absence of antibodies. Again, similar results were seen with LM609. PDGF-induced SMC migration was also inhibited by c7E3 Fab (P=0.004) and LM609 (P=0.001), but to much less an extent. The migration SMCs from a culture found not to express the alpha(v)beta(3) integrin was unaffected by these antibodies, strengthening the argument that c7E3 Fab inhibits SMC function via this integrin. CONCLUSIONS: c7E3 Fab inhibits the adhesion and migration of SMCs via the alpha(v)beta(3) integrin. The inhibition, however, is partial, and varied depending on type of ECM protein and alpha(v)beta(3) integrin expression. Some of the clinical benefits of c7E3 Fab may be due to its effect on SMCs.     

Regional differences in integrin expression: role of alpha(5)beta(1) in regulating smooth muscle cell functions

There is increasing evidence to suggest that coronary smooth muscle cells (SMCs) differ from noncoronary SMCs. As integrin adhesion molecules regulate many SMC functions, we hypothesized that differences in integrin expression on coronary and noncoronary SMCs may account for cellular differences. Analysis of integrin expression on freshly isolated porcine coronary and noncoronary SMCs revealed that coronary SMCs express significantly less alpha(5)beta(1) than noncoronary SMCs, whereas the expression of total beta(1) and that of alpha(v)beta(3) are similar. Consistent with these findings, coronary SMCs demonstrated significantly less adhesion to fibronectin, compared with carotid artery SMCs. As alpha(5)beta(1)-mediated signaling has been associated with cellular proliferation, the effects of differential alpha(5)beta(1) expression on cell proliferation were examined by comparing primary coronary and carotid artery SMC proliferation. Coronary SMC growth was significantly lower than that of carotid artery SMCs when plated on fibronectin or type I collagen. Blocking alpha(5)beta(1) function on carotid artery SMCs produced a significant decrease in cellular proliferation, resulting in growth similar to that of coronary SMCs. Furthermore, blocking alpha(5)beta(1), but not alpha(v)beta(3), inhibited loss of alpha-smooth muscle actin in proliferating SMCs. Proliferating coronary SMCs were found to upregulate alpha(5)beta(1) expression, further indicating a role for alpha(5)beta(1) in SMC growth. These results suggest that dissimilar alpha(5)beta(1) integrin expression may mediate regional differences in phenotype of vascular SMCs.     

Tyrosine kinase activity of discoidin domain receptor 1 is necessary for smooth muscle cell migration and matrix metalloproteinase expression

Smooth muscle cell (SMC) interactions with collagen mediate cell migration during the pathogenesis of atherosclerosis and restenosis. Discoidin domain receptors (DDRs) have been identified as novel collagen receptors. We used aortic SMCs from wild-type and DDR1(-/-) mice to evaluate the function of the DDR1 in regulating migration. DDR1(-/-) SMCs exhibited impaired attachment to and migration toward a type I collagen substrate. Matrix metalloproteinase-2 (MMP-2) and MMP-9 activities were concomitantly reduced in these cells. Transfection of a full-length cDNA for DDR1b rescued these deficits, whereas kinase-dead mutants of DDR1 restored attachment but not migration and MMP production. These results suggest that active DDR1 kinase is a central mediator of SMC migration.     

Vitronectin is up-regulated after vascular injury and vitronectin blockade prevents neointima formation

OBJECTIVE: Smooth muscle cell (SMC) migration involves interactions with extracellular matrix (ECM) and is an important process in response to arterial wall injury. We investigated the expression and the functional role of vitronectin (VN) in the response after vascular injury. METHODS: VN and alpha v beta 3/beta 5 integrin expressions were investigated after balloon carotid injury of Sprague-Dawley rats. Adventitial delivery of blocking antibodies to VN, alpha v beta 5 and beta 3 integrins were performed to assess their roles in neointima formation. In vitro, migration assays were carried out on human SMC. RESULTS: Immunohistochemistry and in situ hybridization for VN showed an upregulation of VN during the early time points of intima formation. alpha v beta 3/beta 5 integrins expression correlated with VN expression. After 7 days, blocking antibodies to VN, alpha v beta 5 and beta 3 induced a significant decrease on intimal area associated with a decrease in intimal cell counts. A slight decrease in intimal cell proliferation without any effect on apoptosis was observed after VN blockade. In vitro, migrating SMC strongly expressed VN after injury and neutralizing anti-VN antibody inhibited SMC migration. Blocking experiment with anti-alpha v beta 5 and -alpha v beta 3 integrin antibodies showed that not only VN-alpha v beta 3 but also VN-alpha v beta 5 interactions are required for SMC migration. CONCLUSION: This study characterizes the VN-ECM interaction in SMC and supports the role of VN in mediating SMC migration and neointimal formation in response to injury.     

Effects of fibrinogen, fibrin and their degradation products on the behaviour of vascular smooth muscle cells

The transition of fibrinogen to fibrin and to their degradation products within the arterial wall has been reported to be accompanied by atherosclerotic progression. A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (SMCs) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. I have been studying the effects of fibrinogen, fibrin and their degradation products on the behaviour, particularly migration, of SMCs. Fibrinogen/fibrin stimulates the adhesion and migration of SMCs and their effects are mediated by both the RGD-containing region of the alpha chain of fibrinogen/fibrin and integrin alpha v beta 3 on the cell surface. SMCs migrate into fibrin gel even with no other chemotactic stimuli. SMCs displayed two-fold increase in migration into crosslinked fibrin gels compared to non-crosslinked gels, suggesting the importance of fibrin crosslinking by factor XIIIa on its three-dimensional structure for the migration of SMCs. Fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, with ACTE, which cleaves only fibrinopeptide B, and with protamine sulfate, which cleaves nothing, but forms a fibrin-like gel, induce migration of SMCs in a manner similar to the gel prepared with thrombin, suggesting that the cleavage of fibrinopeptides is not involved in the migration of SMCs. Both anti-fibrinogen fragment D and E antibodies inhibit the migration of SMCs into fibrin gel, suggesting that both D and E regions of fibrin are involved in the migration of SMCs into fibrin gel. The migration of SMCs into fibrin gel also depends on the RGD-containing region and integrin alpha v beta 3. Both fibrinogen fragments D and E inhibit the migration of SMCs into fibrin gels, suggesting that these fragments may be involved in the regulation of SMC migration into fibrin gel as the result of fibrinolysis. Although subcultured SMCs usually show a synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. We employed an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. alpha v beta 3 integrin and the RGD-containing region are involved in the migration of SMCs into the fibrin gels. SMCs which migrated from the explants showed positive staining with monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from a contractile to synthetic state. These findings show that fibrin (ogen) itself induces adhesion and migration of SMCs without other chemotactic or chemokinetic substances, suggesting a crucial role for fibrin (ogen) in the development and progression of such vascular diseases as atherosclerosis, thrombosis and restenosis following balloon angioplasty.     

The beta3 integrin antagonist m7E3 reduces matrix metalloproteinase activity and smooth muscle cell migration.

Treatment with c7E3 (abciximab, ReoPro) has been associated with a reduction in coronary events and the need for revascularization. Some of these beneficial effects may be due to blockade of the alphavbeta3 integrin receptor on smooth muscle cells (SMCs), however very little is known about the mechanisms involved. The current studies were designed to test the hypothesis that beta3 integrin antagonists inhibit the arterial response to injury by reducing matrix metalloproteinase (MMP) activity in the vessel wall. Male Sprague-Dawley rats were treated with daily intraperitoneal injections of the monoclonal antibody m7E3 at a dose of 6 mg/kg/day. MMP-9 activity was reduced by 73%, and MMP-2 activity by 75%, in the injured carotids of the m7E3-treated rats compared to saline-treated controls. By contrast, tissue inhibitor metalloproteinase (TIMP) activity was not changed. SMC migration assayed at 4 days after injury was reduced from 56.7 +/- 14 cells/mm(2) intimal surface area in controls to 17.5 +/- 5 cells/mm(2) in m7E3-treated rats (p = 0.02). Medial cell replication measured at 4 days and intimal cell replication measured at 7 days were not affected. Intimal cross-sectional area, measured 14 days after injury was reduced by 28% after m7E3 treatment (p = 0.05). Intimal smooth muscle cell number and the ratio of intima/media cross-section area were also reduced. By contrast, intimal SMC density was not affected by m7E3 treatment, indicating no effect on matrix accumulation. We conclude that treatment with m7E3 reduced SMC migration following vascular injury, possibly via an inhibitory effect on MMP activity, and this resulted in a decrease in intimal size at 14 days after injury.     

Galectin 1 modulates attachment, spreading and migration of cultured vascular smooth muscle cells via interactions with cellular receptors and components of extracellular matrix

Galectin 1 (Gal-1), a lactose-binding lectin, is a component of vascular extracellular matrix and secreted by human vascular smooth muscle cells (SMCs). The purpose of this study was to investigate a possible role of Gal-1 in controlling adhesion and migration of cultured human vascular SMCs. Gal-1 co-localised with laminin and cellular fibronectin in extracellular matrix (ECM) secreted by cultured human vascular SMCs. Recombinant glutathione S-transferase (GST)-Gal-1 fusion protein bound to laminin and cellular fibronectin in ELISA. GST-Gal-1 inhibited SMC attachment to laminin via interactions with both SMCs and laminin. GST-Gal-1 inhibited SMC spreading on plastic or on laminin, but not on cellular fibronectin. GST-Gal-1 modulated SMC migration on laminin and inhibited migration on cellular fibronectin. GST-Gal-1 bound to several 35S-labelled proteins in SMC extracts including laminin and alpha1beta1 integrin, identified by depletion of SMC protein extracts with respective antibodies. We conclude that Gal-1 is able to modulate SMC attachment, spreading and migration via interactions with ECM proteins and alpha1beta1 integrin.     

Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury

Matrix metalloproteinases (MMPs) and, in particular, MMP-9 are important for smooth muscle cell (SMC) migration into the intima. In this study, we sought to determine whether MMP-9 is critical for SMC migration and for the formation of a neointima by using mice in which the gene was deleted (MMP-9(-/-) mice). A denuding injury to the arteries of wild-type mice promoted the migration of medial SMCs into the neointima at 6 days, and a large neointimal lesion was observed after 28 days. In wild-type arteries, medial SMC replication was approximately 8% at day 4, 6% at day 6, and 4% at day 8 and had further decreased to 1% at day 14. Intimal cell replication was 65% at 8 days and had decreased to approximately 10% at 14 days after injury. In MMP-9(-/-) arteries, SMC replication was significantly lower at day 8. In addition, SMC migration and arterial lesion growth were significantly impaired in MMP-9(-/-) arteries. SMCs, isolated from MMP-9(-/-) mouse arteries, showed an impairment of migration and replication in vitro. Thus, our present data indicate that MMP-9 is critical for the development of arterial lesions by regulating both SMC migration and proliferation.     

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.     

Periostin mediates vascular smooth muscle cell migration through the integrins ανβ3 and ανβ5 and focal adhesion kinase (FAK) pathway

Smooth muscle cell (SMC) migration involves interactions of integrin receptors with extracellular matrix (ECM) and is an important process of neointimal formation in atherosclerosis and restenosis after vascular interventions. Previous studies have shown that periostin (PN), a novel ECM protein, is upregulated in rat carotid artery after balloon injury, and growth factor-stimulated expression of PN promotes SMC migration in vitro. Here, we address the mechanism by which PN–integrin interaction mediates SMC migration in vitro. Aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate in vitro. Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN?/? and wildtype SMCs, respectively. In both types of SMCs, adenovirus-mediated overexpression of HA-tagged PN to a similar extent, which induced a robust cell migration concomitantly with an increase in β3-integrin expression and phosphorylation of FAK (Tyr397). Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-ανβ3 and PN-ανβ5, but not PN-β1 integrins, are required for SMC migration. Inhibition of FAK signaling by overexpression of an endogenous FAK inhibitor termed FRNK (FAK-related nonkinase) significantly attenuated FAK (Tyr397) phosphorylation and the SMC migration induced by PN. These results reveal a mechanism whereby PN mediates vascular SMC migration through an interaction with alphaV-integrins (mainly ανβ3) and subsequent activation of FAK pathway.     

Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9

MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the alpha(v)beta5 integrin, a vitronectin receptor. The levels of alpha(v)- and beta5-subunits expressed in MCF-7 cells depend on the IGF-I concentration, which triggers an increase in both of these subunits. Based on these results, we suggest that IGF-I-induced MCF-7 cell migration is mediated by the MMP-9 activity on the cell surface and by alpha(v)beta5 integrin.     

Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl-l-ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 x 10(4) cells/well) were treated with placebo, SNAP (100 microM), or L-NIO (100 microM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 +/- 693 vs 6258 +/- 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 +/- 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the alpha(v)beta(3) integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner alpha(v)beta(3) integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of alpha(v)beta(3) integrin on endothelial cells, a critical mediator of cell-matrix adhesion and migration.     

Expression and cellular distribution of alpha(v)integrins in beta(1)integrin-deficient embryonic stem cell-derived cardiac cells

beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.     

Tissue inhibitor of matrix metalloproteinases-1 impairs arterial neointima formation after vascular injury in mice

The hypothesis that tissue inhibitor of metalloproteinases-1 (TIMP-1) plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (TIMP-1(+/+)) and TIMP-1-deficient (TIMP-1(-/-)) mice. The neointimal area at 1 to 3 weeks after electric injury of the femoral artery was significantly higher in TIMP-1(-/-) as compared with TIMP-1(+/+) mice (0.012+/-0. 0015 versus 0.0033+/-0.0008 mm(2) at 1 week, P<0.005). The medial areas were comparable, resulting in intima/media ratios that were significantly larger in TIMP-1(-/-) as compared with TIMP-1(+/+) arteries (1.2+/-0.22 versus 0.39+/-0.08 at 1 week, P<0.005). Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in TIMP-1(-/-) as compared with TIMP-1(+/+) arteries (138+/-15 versus 69+/-8 at 1 week, P<0.005). Immunocytochemical analysis revealed that alpha-actin-positive smooth muscle cells (SMCs) at 2 weeks after injury were more abundant in the intima of TIMP-1(-/-) arteries than in that of TIMP-1(+/+) arteries, whereas after 3 weeks the intimal cell population consisted mainly of SMCs in both genotypes. In in vitro scrape-wounding assays, SMCs of TIMP-1(-/-) mice migrated faster than those of TIMP-1(+/+) mice. Zymography of arterial extracts revealed a higher active matrix metalloproteinase (MMP)-2 level at 1 to 3 weeks after injury in TIMP-1(-/-) arteries, whereas active MMP-9 was only detected in TIMP-1(-/-) arteries at 1 week after injury. These data are compatible with a role of TIMP-1 in the impairment of SMC migration and neointima formation after vascular injury, as a result of inhibition of MMP activity.     

eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity

Vascular smooth muscle cell (SMC) migration is a critical step in the development of neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and the extracellular matrix, facilitating SMC migration. Transfer of the endothelial nitric oxide synthase (eNOS) gene to the injury site inhibits neointima formation. Neither the signaling pathways leading to NO-mediated inhibition of SMC migration and proliferation nor the alterations in these pathways have been characterized. We hypothesize that NO inhibits SMC migration in part by regulating MMP activity. To test this hypothesis, we transfected cultured rat aortic SMCs with replication-deficient adenovirus containing bovine eNOS gene and analyzed the conditioned medium for MMP activity. We observed that eNOS gene transfer significantly (P<0.05) inhibited SMC migration and significantly (P<0.05) decreased MMP-2 and MMP-9 activities in the conditioned medium. Similarly, addition of the NO donor DETA NONOate and 8-bromo-cGMP to the culture medium significantly decreased MMP-2 and MMP-9 activities in the conditioned medium collected 24 hours after treatment. Furthermore, Western blot analysis of the conditioned medium collected from eNOS gene-transfected SMCs showed a significant increase in tissue inhibitor of metalloproteinases-2 (TIMP-2) levels. Our data suggest that NO decreases MMP-2 and MMP-9 activities and increases TIMP-2 secretion, and this shifts the balance of MMP activity, which may favor the inhibition of cell migration because of inhibition of extracellular matrix degradation.     

Mechanisms of hepatocyte growth factor-mediated vascular smooth muscle cell migration

The migration of vascular smooth muscle cells (SMCs) from the media into the neointima and their subsequent proliferation is important in the pathogenesis of atherosclerosis. This process is regulated by multiple factors, including growth factors, and involves changes in the interaction of SMCs with the extracellular matrix and in intracellular signaling cascades that regulate cell movement. We demonstrated previously that hepatocyte growth factor (HGF) is expressed in human atherosclerotic plaques. Although HGF has been shown to promote SMC migration, the mechanisms involved in this process have not been characterized fully. In this study, inhibitory antibodies were used to determine which integrins mediated HGF-induced SMC migration. Inhibition of beta1 or beta3 integrin resulted in a significant decrease in migration. Subsequent experiments were performed to characterize additional biochemical mechanisms involved in HGF-mediated migration. HGF induced the redistribution of focal adhesions, the activation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and their increased association with beta1 and beta3 integrins, and the production of pro-matrix metalloproteinase-2. Migration levels were significantly reduced by cotreatment of SMCs with the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, UO126, the p38 inhibitor, SB203580, or the phosphatidylinositol-3 kinase inhibitor, LY294002. In HGF-treated SMCs, focal adhesion redistribution and FAK and Pyk2 activation were decreased by ERK1/2 inhibition. Neither SB203580 nor LY294002 inhibited HGF-induced ERK1/2 activation. Thus, ERK1/2 signaling may play an important role in HGF-mediated SMC migration by contributing to focal adhesion redistribution and FAK and Pyk2 activation.     

Different effects of high and low shear stress on platelet-derived growth factor isoform release by endothelial cells: consequences for smooth muscle cell migration

In the present study, we analyzed the effect of conditioned media (CM) from bovine aortic endothelial cells exposed to laminar shear stress (SS) of 5 dyne/cm2 (SS5) or 15 dyne/cm2 (SS15) for 16 hours on smooth muscle cell (SMC) migration. In response to CM from bovine aortic endothelial cells exposed to SS5 (CMSS5) and SS15 (CMSS15), migration was 45 +/- 5.5 and 30 +/- 1.5 cells per field, respectively (P<0.05). Similar results were obtained with SS of 2 versus 20 dyne/cm2 and also when SS of 5 and 15 dyne/cm2 lasted 24 hours. Platelet-derived growth factor (PDGF)-AA levels in CMSS5 and CMSS15 were 9 +/- 7 and 18 +/- 5 ng/10(6) cells for 16 hours, respectively (P<0.05); PDGF-BB levels in CMSS5 and CMSS15 were 38 +/- 10 and 53 +/- 10 ng/10(6) cells for 16 hours, respectively (P<0.05). PDGF receptor alpha (PDGFRalpha) and PDGF receptor beta (PDGFRbeta) in SMCs were phosphorylated by CMSS15>CMSS5. In response to CMSS15, a neutralizing antibody against PDGF-AA enhanced SMC migration to a level comparable to that of CMSS5; in contrast, antibodies against PDGF-BB abolished SMC migration. Transfection of SMCs with a dominant-negative PDGFRalpha or PDGFRbeta increased or inhibited, respectively, SMC migration in response to CMSS15. Overexpression of wild-type PDGFRalpha inhibited SMC migration in response to CMSS5, CMSS15, or recombinant PDGF-BB (P<0.001). These results suggest that the ability of high SS to inhibit arterial wall thickening in vivo may be related to enhanced activation of PDGFRalpha in SMCs by PDGF isoforms secreted by the endothelium.     

Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.