NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis

Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced VSMC DNA synthesis. PDGF-BB also induced cyclin A mRNA levels in VSMC in an NFAT-dependent manner. Cloning and bioinformatic analysis of rat cyclin A promoter revealed the presence of NFAT-binding elements, and PDGF-BB induced the binding of NFATs to these regulatory sequences in a CsA- and VIVIT-sensitive manner. Chromatin immunoprecipitation analysis showed that NFATc1 binds to the cyclin A promoter in response to PDGF-BB in a VIVIT-sensitive manner. Furthermore, PDGF-BB induced cyclin A promoter-luciferase reporter gene activity in VSMC, and it was inhibited by both CsA and VIVIT. Balloon injury induced cyclin A expression and CDK2 activity in rat carotid arteries, and these responses were also blocked by VIVIT. In addition, VIVIT attenuated balloon injury-induced SMC proliferation, resulting in reduced restenosis. Down-regulation of NFATc1 by its small interfering RNA inhibited PDGF-BB-induced cyclin A expression and DNA synthesis both in rat and human VSMC. Together, these findings demonstrate that the cyclin A-CDK2 complex may be a potential effector of NFATs, specifically NFATc1, in mediating SMC multiplication leading to neointima formation. Therefore, NFATs may be used as target molecules for the development of therapeutic agents against vascular diseases such as restenosis.     

Thrombin induces expression of FGF-2 via activation of PI3K-Akt-Fra-1 signaling axis leading to DNA synthesis and motility in vascular smooth muscle cells

To understand the mechanisms by which thrombin induces vascular smooth muscle cell (VSMC) DNA synthesis and motility, we have studied the role of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR)-S6K1 signaling. Thrombin stimulated the phosphorylation of Akt and S6K1 in VSMC in a sustained manner. Blockade of PI3K-Akt-mTOR-S6K1 signaling by LY-294002, and rapamycin suppressed both thrombin-induced VSMC DNA synthesis and migration. Adenovirus-mediated expression of dominant-negative Akt also inhibited thrombin-induced VSMC DNA synthesis and migration. Furthermore, thrombin induced the expression of Fra-1 in a sustained PI3K-Akt-dependent and mTOR-independent manner in VSMC. Suppression of Fra-1 by its small interfering RNA attenuated both thrombin-induced VSMC DNA synthesis and migration. Thrombin also induced the expression of FGF-2 in a PI3K-Akt-Fra-1-dependent and mTOR-independent manner, and neutralizing anti-FGF-2 antibodies inhibited thrombin-stimulated VSMC DNA synthesis and motility. In addition, thrombin stimulated the tyrosine phosphorylation of EGF receptor (EGFR), and inhibition of its kinase activity significantly blocked Akt and S6K1 phosphorylation, Fra-1 and FGF-2 expression, DNA synthesis, and motility induced by thrombin in VSMC. Together these observations suggest that thrombin induces both VSMC DNA synthesis and motility via EGFR-dependent stimulation of PI3K/Akt signaling targeting in parallel the Fra-1-mediated FGF-2 expression and mTOR-S6K1 activation.     

STAT-3-dependent cytosolic phospholipase A2 expression is required for thrombin-induced vascular smooth muscle cell motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).     

A requirement for calcium-independent phospholipase A2 in thrombin-induced arachidonic acid release and growth in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A2 (iPLA2). Without affecting its levels, thrombin increased iPLA2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.     

Cyclin D1 Is a Bona Fide Target Gene of NFATc1 and Is Sufficient in the Mediation of Injury-induced Vascular Wall Remodeling

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G₁ to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide −1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.     

Cytosolic phospholipase A2 is an effector of Jak/STAT signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). To understand its mitogenic and chemotactic signaling events, we studied the role of cytosolic phospholipase A(2) (cPLA(2)) and the Jak/STAT pathway. PDGF-BB induced the expression and activity of cPLA(2) in a time-dependent manner in VSMC. Arachidonyl trifluoromethyl ketone, a potent and specific inhibitor of cPLA(2), significantly reduced PDGF-BB-induced arachidonic acid release and DNA synthesis. PDGF-BB stimulated tyrosine phosphorylation of Jak-2 in a time-dependent manner. In addition, PDGF-BB activated STAT-3 as determined by its tyrosine phosphorylation, DNA-binding activity, and reporter gene expression, and these responses were suppressed by AG490, a selective inhibitor of Jak-2. AG490 and a dominant-negative mutant of STAT-3 also attenuated PDGF-BB-induced expression of cPLA(2,) arachidonic acid release, and DNA synthesis in VSMC. Together, these results suggest that induction of expression of cPLA(2) and arachidonic acid release are involved in VSMC growth in response to PDGF-BB and that these events are mediated by Jak-2-dependent STAT-3 activation.     

NF-kappaB is required for TNF-alpha-directed smooth muscle cell migration.

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor-alpha (TNF-alpha) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor-kappaB (NF-kappaB) in human VSMC migration induced by TNF-alpha. Adenoviral expression of a mutant form of the inhibitor of NF-kappaB, IkappaB-alphaM, suppressed TNF-alpha-triggered degradation of cellular IkappaB-alpha, inhibited activation of NF-kappaB, and attenuated TNF-alpha-induced migration. Further, IkappaB-alphaM suppressed TNF-alpha-stimulated release of interleukin-6 and -8 (IL-6 and IL-8). Neutralization of IL-6 and IL-8 with appropriate antibodies reduced TNF-alpha-induced VSMC migration. Addition of recombinant IL-6 and IL-8 stimulated migration. Collectively, our data provide initial evidence that TNF-alpha-mediated VSMC migration requires NF-kappaB activation and is associated with induction of IL-6 and IL-8 which act in an autocrine manner.     

Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways

BACKGROUND: A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. RESULTS: Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-beta1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-beta1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-beta1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. CONCLUSION: These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.     

Dysregulation of CREB binding protein triggers thrombin-induced proliferation of vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs). CBP has been regarded as a potential therapeutic target on the basis of its ability to affect cell growth. Therefore we hypothesized that CBP mediates thrombin-induced proliferation of VSMCs. We constructed recombinant adenoviral vector that expresses four short hairpin RNA (shRNA) targeting rat CBP mRNA (CBP-shRNA/Ad). VSMCs were infected with CBP-shRNA/Ad and treated with thrombin. CBP level were analyzed by quantitative real-time PCR and Western blot. To evaluate VSMC proliferation, the cell cycle and DNA synthesis were analyzed by flow cytometry and (3)H-thymidine incorporation, respectively. CBP-shRNA/Ad infection inhibited thrombin-induced CBP expression in a dose-dependent manner concomitant with a decrease in the percentage of cells in the S phase and in DNA synthesis. These findings suggest that CBP plays a pivotal role in the S phase progression of VSMCs.     

Thrombin rapidly induces protein kinase D phosphorylation,and protein kinase C delta mediates the activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.     

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.     

Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

IL-17 Stimulates Migration of Carotid Artery Vascular Smooth Muscle Cells in an MMP-9 Dependent Manner via p38 MAPK and ERK1/2-Dependent NF-κB and AP-1 Activation

Inappropriate vascular remodeling is thought to be the main cause of restenosis following angioplasty. Migration of vascular smooth muscle cells (VSMC) into lumina, which is promoted by degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays a causal role in pathological vascular remodeling. The aim of the present research is to explore the effects of a novel cytokine, IL-17, on migration of VSMC and MMP-9 secretion. Carotid artery VSMC was isolated from Sprague–Dawley rats. Expression of MMP-9 and cell migration induced by IL-17 and its related signal pathway were detected. The results showed that IL-17-induced migration of VSMC in an MMP-9-dependent manner. IL-17-induced MMP-9 expression was via p38 MAPK and ERK1/2 dependent NF-κB and AP-1 activation. The present results demonstrated that IL-17 may play a role in vascular remodeling and targeting IL-17 or its specific downstream mediators is a potentially novel therapeutic pathway for attenuating the post-angioplastic restenosis.