Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cells (VSMCs), endothelial cells, or macrophages after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting growth and endogenous PDGF synthesis in VSMCs after in vitro mechanical injury. We analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation. Berberine significantly suppressed PDGF-stimulated Cyclin D1/D3 and Cyclin-dependent kinase (Cdk) gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to phosphorylation activation of p53 and increased protein levels of the Cdk inhibitor p21(Cip1). Compound C, an AMPK inhibitor, partly but significantly attenuated berberine-elicited growth inhibition. In addition, stimulation of VSMCs with PDGF led to a transient increase in GTP-bound, active form of Ras, Cdc42 and Rac1, as well as VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42 and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we conclude that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21(Cip1) signaling while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Rac1 and Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.     

Sparstolonin B suppresses rat vascular smooth muscle cell proliferation,migration, inflammatory response and lipid accumulation

Vascular smooth muscle cells (VSMCs) play a crucial role in atherosclerotic lesion formation. Sparstolonin B (SsnB) is a TLR2/TLR4 antagonist that inhibits inflammatory responses in multiple cell types. Herein, we investigated if SsnB inhibited VSMC proliferation, migration, inflammatory response and lipid accumulation. We found that SsnB suppressed VSMC proliferation and migration induced by PDGF. SsnB significantly suppressed the expression of MCP-1, TNFα and IL-6 in VSMCs stimulated by either lipopolysaccharide (LPS) or PDGF. Erk1/2 and Akt signaling pathways, which are responsible for the VSMC inflammatory response, were activated by LPS or PDGF stimulation, and SsnB significantly inhibited their activation. SsnB also substantially suppressed the intracellular cholesterol accumulation in VSMCs loaded with acetylated LDL. Mechanistically, SsnB remarkably repressed LPS-induced up-regulation of CD36, which is responsible for lipid uptake, and dramatically reversed LPS-induced inhibition of ABCA1, which promotes the efflux of intracellular free cholesterol. In conclusion, our results indicate that SsnB significantly inhibits VSMC proliferation, migration, inflammatory responses and lipid accumulation. Along with the previously reported anti-inflammatory activities of SsnB on macrophages and vascular endothelial cells, our data strongly suggest that SsnB may be developed as a new anti-atherogenic therapy.     

Therapeutic effect of magnesium lithospermate B on neointimal formation after balloon-induced vascular injury

Vascular smooth muscle cell (VSMC) proliferation and migration in response to platelet-derived growth factor (PDGF) play an important role in the development of atherosclerosis and restenosis. Recent evidence indicates that PDGF increases intracellular levels of reactive oxygen species in VSMCs and that both PDGF-induced VSMC proliferation and migration are reactive oxygen species-dependent. Danshen is a representative oriental medicine used for the treatment of vascular disease. Previously, we reported that magnesium lithospermate B, an active component of Danshen, is a potent antioxidant. Thus we investigated the therapeutic potential of magnesium lithospermate B in neointimal formation after carotid artery injury in rats along with its effects on the PDGF signaling pathway for stimulating VSMC proliferation and migration in vitro. PDGF is dimeric glycoprotein composed of two A or two B chains. In this study, we used PDGF-BB, which is one of the isoforms of PDGF (i.e., PDGF-AA, PDGF-BB, and PDGF-AB). Our results demonstrated that magnesium lithospermate B directly scavenged reactive oxygen species in a xanthine/xanthine oxidase system and reduced PDGF-BB-induced intracellular reactive oxygen species generation in VSMCs. In a rat carotid artery balloon injury model, magnesium lithospermate B treatment (10 mg/kg/day, i.p) showed a significant effect on the prevention of neointimal formation compared with vehicle treatment. In cultured VSMCs, magnesium lithospermate B significantly attenuated PDGF-BB-induced cell proliferation and migration as measured by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay and transwell migration assays, respectively. Further, magnesium lithospermate B inhibited PDGF-BB-induced phosphorylation of phospatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways by scavenging reactive oxygen species. Together, these data indicated that magnesium lithospermate B, a potent reactive oxygen species scavenger, prevented both injury-induced neointimal formation in vivo and PDGF-BB-induced VSMC proliferation and migration in vitro, suggesting that magnesium lithospermate B may be a promising agent to prevent atherosclerosis and restenosis following angioplasty.     

Dominant negative c-Jun inhibits platelet-derived growth factor-directed migration by vascular smooth muscle cells

The mitogen-activated protein (MAP) kinase pathways has been shown to be necessary for mitogen-stimulated proliferation, but its role in cell migration has not been fully understood. In this study, we investigated the possible contribution of signaling pathways through c-Jun in platelet-derived growth factor (PDGF)-BB directed cell migration in rat aortic vascular smooth muscle cells (VSMCs) infected with a recombinant adenovirus containing the dominant-negative c-Jun (Ad-DN-c-Jun). DN-c-Jun protein was expressed dose-dependently in VSMCs infected with Ad-DN-c-Jun. Expression of DN-c-Jun significantly inhibited VSMC migration induced by PDGF-BB. Our results provide the first evidence that signaling pathways through c-Jun participates in cell migration induced by PDGF-BB in addition to other MAP kinase pathways in VSMCs.     

Cepharanthine inhibits in vitro VSMC proliferation and migration and vascular inflammatory responses mediated by RAW264.7

Pathogenesis of atherosclerosis involves vascular smooth muscle cell (VSMC) migration and proliferation followed by an inflammation mediated by activated macrophages in the tunica intima of blood vessels. Cepharanthine (CEP) belongs to bisbenzylisoquinoline alkaloids found in the plant Stephania cepharantha, which has been used for various diseases like cancer, alopecia areata, venomous snakebites, and malaria. In this study, we investigated whether CEP suppresses VSMC migration and proliferation and inhibits inflammatory mediator production in macrophage (RAW264.7). Our results showed that CEP possessed significant DPPH scavenging and metal chelating activities. It also markedly inhibited lipid peroxidation. Similarly, CEP suppressed the nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in RAW264.7 cells. Moreover, the level of prostaglandin E₂ was also suppressed and the formation of macrophage derived foam cell was attenuated in RAW264.7 cells. Likewise, NO production in isolated peritoneal macrophage and VSMC migration in response to LPS stimulated RAW264.7 was also halted by CEP treatment. Also, VSMC migration induced by platelet-derived growth factor (PDGF-BB) was inhibited by CEP dose dependently. The anti-migratory effect of CEP on VSMCs was due to its inhibitory effect on metalloproteinase-9 (MMP-9) expression, preventing the degradation of extracellular matrix (ECM) component. Furthermore, CEP suppressed PDGF-BB induced VSMC proliferation by down-regulation of mitogen activated protein kinase (MAPK) signaling molecules. CEP also inhibited the translocation of NF-κB from cytosol to nucleus. Thus, our results suggest that CEP exerts potent anti-atherosclerotic effect through attenuation of inflammation, lipid peroxidation and VSMC migration and proliferation.     

Sesquiterpene lactone suppresses vascular smooth muscle cell proliferation and migration via inhibition of cell cycle progression

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration are involved in restenosis following percutaneous transluminal angioplasty (PTCA) as well as in the development and progression of atherosclerosis. We investigated the mechanisms underlying the inhibitory effect of the sesquiterpene 3-oxo-5alphaH,8betaH-eudesma-1,4(15),7(11)-trien-8,12-olide (1) on rat VSMC proliferation and migration. VSMCs were isolated from rat aorta, and then the effect of 1 on cell proliferation and migration was examined using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and chemotaxis assays, respectively. Compound 1 had a potent inhibitory effect on fetal calf serum-induced VSMC proliferation. This effect correlated with reduced expression of cyclin D(1). In addition, 1 also inhibited platelet derived growth factor (PDGF)-induced migration of VSMCs. These results indicate that 1 is a promising candidate for additional biological evaluation to further define its potential as an inhibitory modulator of VSMC responses that contribute to restenosis following PTCA and to the development and progression of atherosclerosis.     

TAS-301 blocks receptor-operated calcium influx and inhibits rat vascular smooth muscle cell proliferation induced by basic fibroblast growth factor and platelet-derived growth factor

The purpose of this study was to determine the effect of a recently synthesized drug, TAS-301 [3-bis(4-methoxyphenyl)methylene-2-indolinone], on vascular smooth muscle cell (VSMC) proliferation and the intracellular signal transduction pathways involved in VSMC proliferation. In an in vitro assay, TAS-301 inhibited the proliferation of rat VSMCs stimulated by platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor, or 2% fetal bovine serum in a concentration-dependent manner. TAS-301 dose-dependently inhibited the PDGF-induced Ca2+ influx; the concentration for the inhibition of Ca2+ influx was nearly identical to that for inhibition of VSMC proliferation. The Ca2+ influx induced by PDGF was also attenuated by NiCl2 but not by nifedipine, suggesting that PDGF-induced Ca2+ influx would be mediated by some non-voltage-dependent mechanisms. Furthermore, TAS-301 inhibited PDGF-induced activation of protein kinase C (PKC) and the phorbol 12-myristate 13-acetate-mediated induction of activator protein 1 (AP-1) in a concentration-dependent manner. These findings indicate that TAS-301 inhibited the proliferation of VSMCs by blocking voltage-independent Ca2+ influx and downstream signals such as the Ca2+/PKC signaling pathway, leading to AP-1 induction.     

Naringenin inhibits TNF-α induced VSMC proliferation and migration via induction of HO-1

Vascular smooth muscle cell (VSMC) proliferation and migration, which is triggered by various inflammatory stimuli, contributes importantly to the pathogenesis of atherosclerosis and restenosis. Naringenin is a citrus flavonoid with both lipid-lowering and insulin-like properties. Here, we investigated whether naringenin affects TNF-α-induced VSMC proliferation and migration and if so, whether heme oxygenase-1 (HO-1) is involved. Rat VSMCs were treated with naringenin alone or in combination of TNF-α stimulation. We found that naringenin induced HO-1 mRNA and protein levels, as well as its activity, in VSMCs. Naringenin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. Mechanistic study demonstrated that naringenin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Naringenin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 inhibitor ZnPP IX or HO-1 siRNA partially abolished the beneficial effects of naringenin on VSMCs. These results suggest that naringenin 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.     

扇贝裙边提取物抗动脉粥样硬化作用及其机理研究

目的 探讨扇贝裙边提取物（ESS）抗动脉粥样硬化（AS）作用,并研究其对血管平滑肌细胞（VSMCs）增殖的影响。方法 1.通过喂饲高脂饲料建立鹌鹑AS模型,观察ESS对血脂水平、组织中脂质含量及动脉粥样硬化斑块形成的影响。2.采用20％胎牛血清（FBS）、碱性成纤维细胞生长因子（bFGF）所诱导的大鼠VSMCs增殖模型,通过细胞计数、结晶紫染色及MTT比色法研究ESS抑制VSMCs增殖作用,并运用免疫组织化学技术（LSAB法）观察ESS对增殖VSMCs的增殖细胞核抗原（PCNA）和血小板衍化生长因子（PDGF）表达的影响。结果 1.与模型组比较,ESS组动物主动脉、冠状动脉内膜AS斑块形成程度明显减轻（P<0.05、P<0.01）,血清总胆固醇（TC）、甘油三酯TC、TG含量均显著降低（P<0.05、P<0.01）。2.ESS各剂量组VSMCs数目及吸光度均明显少于模型组（P<0.05、P<0.01）,并能逆转VSMCs增殖时PCNA、PDGF的表达增强（P<0.01）。结论 ESS具有明显调血脂、抗AS作用,并能显著抑制VSMCs增殖。提示ESS的抗AS作用可能与其调血脂、抑制VSMCs增殖有关。     

Apamin inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppressions of activated Akt and Erk signaling pathway

The increased proliferation and migration of vascular smooth muscle cells (VSMC) are key process in the development of atherosclerosis lesions. Platelet-derived growth factor (PDGF) initiates a multitude of biological effects that contribute to VSMC proliferation and migration. Apamin, a component of bee venom, has been known to block the Ca^2 +-activated K⁺ channels. However, the effects of apamin in the regulation PDGF-BB-induced VSMC proliferation and migration has not been identified. In this study, we investigate the inhibitory effect of apamin on PDGF-BB-induced VSMC proliferation and migration. Apamin suppressed the PDGF-BB-induced VSMC proliferation and migration with no apparent cytotoxic effect. In accordance with these findings, apamin induced the arrest of cell cycle progression at G0/G1 phase. Apamin also decreased the expressions of G0/G1 specific regulatory proteins including proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinases (CDK) 4, cyclin E and CDK2, as well as increased the expression of p21^Cip1 in PDGF-BB-induced VSMC. Moreover, apamin inhibited PDGF-BB-induced phosphorylation of Akt and Erk1/2. These results suggest that apamin plays an important role in prevention of vascular proliferation and migration through the G0/G1 cell cycle arrest by PDGF signaling pathway. Thus, apamin may be a promising candidate for the therapy of atherosclerosis.     

Luteolin prevents PDGF-BB-induced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation

Luteolin occurs as glycosylated forms in celery, green pepper, perilla leaf and camomile tea, and has been shown to possess antimutagenic, antitumorigenic, antioxidant and antiinflammatory properties. In this study, we have investigated the antiproliferable effect and its mechanism of luteolin on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (VSMCs). Luteolin significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of rat aortic VSMCs in a concentration-dependent manner. In addition, flow cytometry analysis of DNA content revealed blocking of the PDGF-BB-inducible cell cycle progression by luteolin. Pre-incubation of rat aortic VSMCs with luteolin significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and phospholipase C (PLC)-gamma1 activation as well as c-fos gene expression. Consisted with these findings, luteolin inhibited PDGF-Rbeta phosphorylation induced by PDGF-BB in a concentration-dependent manner. These results suggest that the inhibitory effect of luteolin on the PDGF-BB-induced proliferation of rat aortic VSMCs may be mediated by blocking phosphorylation of PDGF-Rbeta.     

Inhibitory effects of docetaxel on platelet-derived growth factor (PDGF)-BB-induced proliferation of vascular smooth muscle cells through blocking PDGF-receptor β phosphorylation

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is an important pathogenic factor for vascular disorders such as atherosclerosis and restenosis after angioplasty. The present study was designed to investigate the inhibitory effects of docetaxel on VSMC proliferation, as well as the molecular mechanism of this inhibition. Docetaxel at 10, 20 and 40 μM significantly inhibited both the proliferation and the DNA synthesis of fetal bovine serum (FBS)- and platelet-derived growth factor (PDGF)-BB-stimulated VSMCs in a concentration-dependent manner. In accordance with these findings, docetaxel blocked the FBS- and PDGF-BB-induced progression of synchronized cells through the G0/G1 phase of the cell cycle. Docetaxel also decreased the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, retinoblastoma protein, and proliferative cell nuclear antigen in PDGF-BB-stimulated VSMCs. Docetaxel significantly inhibited the phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and phospholipase C-γ1, downstream molecule in the PDGF-BB signaling pathway. Docetaxel suppressed the phosphorylation of PDGF receptor (PDGF-R) β, the upstream molecule in PDGF-BB signaling cascade, suggesting that the inhibitory effect of docetaxel on the proliferation of VSMCs may occur by blocking PDGF-Rβ phosphorylation. Thus, docetaxel may be a potential antiproliferative agent for the treatment of atherosclerosis and angioplasty restenosis.[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10276FP].     

5,8-Dimethoxy-2-Nonylamino-Naphthalene-1,4-Dione Inhibits Vascular Smooth Muscle Cell Proliferation by Blocking Autophosphorylation of PDGF-Receptor β

As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced [³H]-thymidine incorporation, cell cycle progression from G₀/G₁ to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptorβ(PDGF-Rβ) enhanced by PDGF at Tyr⁵⁷⁹, Tyr⁷¹⁶, Tyr⁷⁵¹, and Tyr¹⁰²¹ residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and PLCγ1. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-Rβ autophosphorylation, and subsequently PDGF-Rβ-mediated downstream signaling pathways.     

Thrombospondin-1 inhibition of vascular smooth muscle cell responses occurs via modulation of both cAMP and cGMP

Nitric oxide (NO) drives pro-survival responses in vascular cells and limits platelet adhesion, enhancing blood flow and minimizing thrombosis. The matricellular protein thrombospondin-1 (TSP1), through interaction with its receptor CD47, inhibits soluble guanylyl cyclase (sGC) activation by NO in vascular cells. In vascular smooth muscle cells (VSMCs) both intracellular cGMP and cAMP regulate adhesion, contractility, proliferation, and migration. cGMP can regulate cAMP through feedback control of hydrolysis. Inhibition of the cAMP phosphodiesterase-4 selectively interfered with the ability of exogenous TSP1 to block NO-driven VSMC adhesion but not cGMP accumulation, suggesting that cAMP also contributes to VSMC regulation by TSP1. Inhibition of phosphodiesterase-4 was sufficient to elevate cAMP levels, and inhibiting guanylyl cyclase or phosphodiesterase-3, or adding exogenous TSP1 reversed this increase in cAMP. Thus, TSP1 regulates VSMC cAMP levels in part via cGMP-dependent inhibition of phosphodiesterase-3. Additionally basal cAMP levels were consistently elevated in both VSMCs and skeletal muscle from TSP1 null mice, and treating null cells with exogenous TSP1 suppressed cAMP levels to those of wild type cells. TSP1 inhibited both forskolin and isoproterenol stimulated increases in cAMP in VSMCs. TSP1 also abrogated forskolin and isoproterenol stimulated vasodilation. Consistent with its ability to directly limit adenylyl cyclase-activated vasodilation, TSP1 also limited cAMP-induced dephosphorylation of myosin light chain-2. These findings demonstrate that TSP1 limits both cGMP and cAMP signaling pathways and functional responses in VSMCs and arteries, by both phosphodiesterase-dependent cross talk between these second messengers and by inhibition of adenylyl cyclase activation.     

Luteolin inhibited hydrogen peroxide-induced vascular smooth muscle cells proliferation and migration by suppressing the Src and Akt signalling pathways

Objectives Luteolin is a naturally occurring flavonoid found in many vegetables, fruits and medicinal plants. The migration and proliferation of vascular smooth muscle cells (VSMCs) are the critical pathological processes in various cardiovascular diseases, such as atherosclerosis. In this study, we investigated the effect of luteolin and its latent mechanism on the proliferation and migration of VSMCs stimulated by hydrogen peroxide (H₂O₂). Methods VSMC proliferation and cell viability was assayed using the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) method or by cell counting, and H₂O₂‐elicited migration of VSMCs was measured using a transwell migration assay. The phosphorylation levels of Src, 3‐phosphoinositide‐dependent kinase 1 (PDK1) and Akt (protein kinase B) were analysed by immunoblotting. Key findings This study demonstrated that luteolin showed a particularly inhibitory effect on H₂O₂‐elicited VSMC proliferation and migration. In previous research, we originally explored the function of luteolin in blocking H₂O₂‐triggered Src and Akt signalling pathways. The activation of Src, PDK1, Akt (308), Akt (473) in the luteolin‐treated group was significantly lower than that seen in the H₂O₂ group. Conclusions These findings strongly suggested that luteolin suppresses H₂O₂‐directed migration and proliferation in VSMCs partially due to down‐regulation of the Akt and Src signalling pathways, which are important participants in the processes of migration and proliferation of VSMCs.     

Clitocybin B inhibits rat aortic smooth muscle cell proliferation through suppressing PDGF-Rβ phosphorylation

The increased proliferation of vascular smooth muscle cells (VSMCs) in the arterial wall is a critical pathogenic factor for vascular diseases such as atherosclerosis and restenosis after angioplasty. Clitocybin B was reported to have either a potent free radical scavenging effect or effects that were isolated from the culture broth of mushroom Clitocybe aurantiaca. The present study was designed to investigate the effects of clitocybin B on VSMC proliferation and its possible molecular mechanism. Clitocybin B significantly inhibited the proliferation and the DNA synthesis of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. In agreement with these findings, clitocybin B suppressed the PDGF-BB-induced progression through G0/G1 to S phase of cell cycle. Clitocybin B also down-regulated the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK)2, cyclin E, CDK4, cyclin D1, and proliferative cell nuclear antigen in PDGF-BB-stimulated VSMCs. Clitocybin B significantly inhibited the phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and phospholipase C-γ1, in the PDGF-BB signaling pathway. Clitocybin B suppressed the PDGF-Rβ activation in PDGF-BB signaling cascade. These results suggested that the inhibitory effect of clitocybin B on the proliferation of VSMCs may be associated with suppressing PDGF-Rβ phosphorylation. Thus, clitocybin B may be an effective antiproliferative agent for the prevention of atherosclerosis and restenosis.     

The integrin α2β1 agonist,aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production

Background and purpose:

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

Experimental approach:

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

Key results:

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.     

PPAR gamma-ligands inhibit migration mediated by multiple chemoattractants in vascular smooth muscle cells

The purpose of this study was to determine the effect of the peroxisome proliferator-activated receptor gamma-(PPAR gamma) ligands troglitazone (TRO), rosiglitazone (RSG), and 15-deoxy-delta prostaglandin J2 (15d-PGJ2) on vascular smooth muscle cell (VSMC) migration directed by multiple chemoattractants. Involvement of mitogen-activated protein kinase (MAPK) in migration also was examined, because TRO was previously shown to inhibit nuclear events stimulated by this pathway during mitogenic signaling in VSMCs. Migration of rat aortic VSMCs was induced 5.4-fold by PDGF, 4.6-fold by thrombin, and 2.3-fold by insulin-like growth factor I (IGF-I; all values of p < 0.05). The PPAR gamma ligands 15d-PGJ2, RSG, or TRO all inhibited VSMC migration with the following order of potency: 15d-PGJ2 > RSG > TRO. Inhibition of MAPK signaling with PD98059 completely blocked PDGF-, thrombin-, and IGF-I-induced migration. All chemoattractants induced MAPK activation. PPAR gamma ligands did not inhibit MAPK activation, suggesting a nuclear effect of these ligands downstream of MAPK. The importance of nuclear events was confirmed because actinomycin D also blocked migration. We conclude that PPAR gamma ligands are potent inhibitors of VSMC migration pathways, dependent on MAPK and nuclear events. PPAR gamma ligands act downstream of the cytoplasmic activation of MAPK and appear to exert their effects in the nucleus. Because VSMC migration plays an important role in the formation of atherosclerotic lesions and restenosis, PPAR gamma ligands like TRO and RSG, which ameliorate insulin resistance in humans, also may protect the vasculature from diabetes-enhanced injury.