The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1α expression

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 alpha (PGC-1α) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1α in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1α expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1α mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1α expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1α by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1α had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1α decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPARγ activation by a PPARγ antagonist GW9662 abolished the suppressive effects of PGC-1α on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1α were enhanced by a PPARγ agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1α expression. PGC-1α suppresses PDGF-induced VSMC migration through PPARγ coactivation and, consequently, p38 MAPK inhibition.     

Thymoquinone suppresses platelet‐derived growth factor‐BB–induced vascular smooth muscle cell proliferation,migration and neointimal formation

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.     

白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4 2丝裂素活化蛋白激酶的表达     

Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs

In this study, we examined the roleof insulin in the control of vascular smooth muscle cell (VSMC)migration in the normal vasculature. Platelet-derived growth factor(PDGF) increased VSMC migration, which was inhibited by pretreatmentwith insulin in a dose-dependent manner. Insulin also caused a 60%decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK)phosphorylation and activation. Insulin inhibition of MAPK wasaccompanied by a rapid induction of MAPK phosphatase (MKP-1), whichinactivates MAPKs by dephosphorylation. Pretreatment with inhibitors ofthe nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase I (cGK I), the downstream effector of cGMPsignaling, blocked the activation of MAPK and prevented PDGF-directedVSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPKphosphorylation. We conclude that insulin inhibition of VSMC migrationmay be mediated in part by NO/cGMP/cGK I induction of MKP-1 andconsequent inactivation of MAPKs.

     

ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells

It has been known that Rho-associated protein kinase (ROCK) signaling regulates the migration of vascular smooth muscle cells (VSMCs). However, the isoform-specific roles of ROCK and its underlying mechanism in VSMC migration are not well understood. The current study thus aimed to investigate the roles of ROCK1/2 and their relationship to the MAPK signaling pathway in platelet-derived growth factor (PDGF)-induced rat aorta VSMC migration by manipulating ROCK gene expression. The results revealed that ROCK1 small interfering ribonucleic acid (siRNA) rather than ROCK2 siRNA decreased PDGF-BB-generated VSMC migration, and upregulation of ROCK1 expression via transfection of constructed pEGFP-C1/ROCK1 plasmid further increased the migration of PDGF-BB-treated VSMCs. In PDGF-treated VSMCs, ROCK1 siRNA did not affect the phosphorylation levels of ERK and p38 in the cytoplasm, but decreased the level of ERK phosphorylation in the nucleus. These findings demonstrate that activated ROCK1 can promote VSMC migration through facilitating phosphorylation and nuclear translocation of ERK protein.     

Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of alpha- and beta-actin, but magunapodia consisted exclusively of beta-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway.     

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.

We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.     

MKP-1 expression and stabilization and cGK Ialpha prevent diabetes- associated abnormalities in VSMC migration

Diabetes mellitus is a major risk factor in the development of atherosclerosis and cardiovascular disease conditions, involving intimal injury and enhanced vascular smooth muscle cell (VSMC) migration. We report a mechanistic basis for divergences between insulin's inhibitory effects on migration of aortic VSMC from control Wistar Kyoto (WKY) rats versus Goto-Kakizaki (GK) diabetic rats. In normal WKY VSMC, insulin increased MAPK phosphatase-1 (MKP-1) expression as well as MKP-1 phosphorylation, which stabilizes it, and inhibited PDGF-mediated MAPK phosphorylation and cell migration. In contrast, basal migration was elevated in GK diabetic VSMCs, and all of insulin's effects on MKP-1 expression and phosphorylation, MAPK phosphorylation, and PDGF-stimulated migration were markedly inhibited. The critical importance of MKP-1 in insulin inhibition of VSMC migration was evident from several observations. MKP-1 small interfering RNA inhibited MKP-1 expression and abolished insulin inhibition of PDGF-induced VSMC migration. Conversely, adenoviral expression of MKP-1 decreased MAPK phosphorylation and basal migration rate and restored insulin's ability to inhibit PDGF-directed migration in GK diabetic VSMCs. Also, the proteasomal inhibitors lactacystin and MG132 partially restored MKP-1 protein levels in GK diabetic VSMCs and inhibited their migration. Furthermore, GK diabetic aortic VSMCs had reduced cGMP-dependent protein kinase Ialpha (cGK Ialpha) levels as well as insulin-dependent, but not sodium nitroprusside-dependent, stimulation of cGMP. Adenoviral expression of cGK Ialpha enhanced MKP-1 inhibition of MAPK phosphorylation and VSMC migration. We conclude that enhanced VSMC migration in GK diabetic rats is due at least in part to a failure of insulin-stimulated cGMP/cGK Ialpha signaling, MKP-1 expression, and stabilization and thus MAPK inactivation.     

Sponge-derived acetylenic alcohols,petrosiols, inhibit proliferation and migration of platelet-derived growth factor (PDGF)-induced vascular smooth muscle cells

Platelet-derived growth factor (PDGF) induces the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to the development of various vascular disorders such as restenosis and atherosclerosis. Therefore, inhibitors of PDGF-induced cellular events would be candidate agents for treating these diseases. During the search for such inhibitors from marine sources, we isolated petrosiols A–D (1–4) and related compounds from the marine sponge Petrosia strongylata. These metabolites, which we previously reported as neurotrophic substances, showed an inhibitory effect on PDGF-induced DNA synthesis at IC₅₀ values of 0.69–2.2 μM. Petrosiol A (1) inhibited PDGF-induced cell proliferation without remarkable cytotoxicity and arrested cell cycle progression from the G0/G1 to S phase by inducing the downregulation of the expression of G1 checkpoint proteins cyclin D1, cyclin E, cyclin-dependent kinases (CDK)2, and CDK4 and the upregulation of the expression of p21 and p27. In addition, petrosiol A (1) inhibited the phosphorylation of PDGF receptor-β and its downstream proteins such as phospholipase C (PLC)-γ1, Akt, and extracellular signal-regulated kinase (ERK)1/2. These results suggest that 1 inhibited PDGF-induced VSMC proliferation by interrupting the phosphorylation of PDGF receptor-β followed by downstream signal transduction. Furthermore, petrosiol A (1) suppressed PDGF-induced actin filament dissociation and cell migration, suggesting that 1 and its derivatives may be used for the prevention and treatment of vascular diseases.     

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

The present study was designed to investigate the role of endogenous sulfur dioxide (SO₂) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO₂ inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO₂ synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO₂ suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO₂ did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO₂ dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO₂ simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO₂ could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO₂-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO₂ exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.     

Genipin Inhibits TNF-α-Induced Vascular Smooth Muscle Cell Proliferation and Migration via Induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.     

17Beta-estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level

Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17beta-estradiol (E2) on beta-PDGF receptor (beta-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 microM-0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 +/- 15.8% and 79.4 +/- 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter beta-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the beta-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-gamma, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the beta-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.     

Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

BackgroundIncreased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration.MethodsThe proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury.ResultsVSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity.ConclusionsMagnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation.General significanceThis study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Insulin receptor substrate-1/SHP-2 interaction, a phenotype-dependent switching machinery of insulin-like growth factor-I signaling in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells.     

Transforming Growth Factor β Inhibits Platelet Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation via Akt-Independent,Smad-Mediated Cyclin D1 Downregulation

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.     

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor‐β

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.