PDGF-stimulated Vascular Smooth Muscle Cells Research Articles

C-Ski inhibits autophagy of vascular smooth muscle cells induced by oxLDL and PDGF.

Autophagy is increasingly being recognized as a critical determinant of vascular smooth muscle cell (VSMC) biology. Previously, we have demonstrated that c-Ski inhibits VSMC proliferation stimulated by transforming growth factor β (TGF-β), but it is not clear whether c-Ski has the similar protective role against other vascular injury factors and whether regulation of autophagy is involved in its protective effects on VSMC. Accordingly, in this study, rat aortic A10 VSMCs were treated with 40 µg/ml oxidized low-density lipoprotein (oxLDL) or 20 ng/ml platelet-derived growth factor (PDGF), both of which were autophagy inducers and closely related to the abnormal proliferation of VSMCs. Overexpression of c-Ski in A10 cells significantly suppressed the oxLDL- and PDGF- induced autophagy. This action of c-Ski resulted in inhibiting the cell proliferation, the decrease of contractile phenotype marker α-SMA expression while the increase of synthetic phenotype marker osteopontin expression stimulated by oxLDL or PDGF. Inversely, knockdown of c-Ski by RNAi enhanced the stimulatory effects of oxLDL or PDGF on A10 cell growth and phenotype transition. And further investigation found that inhibition of AKT phosphorylation to downregulate proliferating cell nuclear antigen (PCNA) expression, was involved in the regulation of autophagy and associated functions by c-Ski in the oxLDL- and PDGF-stimulated VSMCs. Collectively, c-Ski may play an important role in inhibiting autophagy to protect VSMCs against some harsh stress including oxLDL and PDGF.

Inhibitory effect of a novel naphthoquinone derivative on proliferation of vascular smooth muscle cells through suppression of platelet-derived growth factor receptor β tyrosine kinase

This study was designed to investigate the antiproliferative effect of a novel naphthoquinone derivative, 2‐undecylsulfonyl‐5,8‐dimethoxy‐1,4‐naphthoquinone (2-undecylsulfonyl-DMNQ), on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) and examine the possible molecular mechanism of its antiproliferative action. 2-Undecylsulfonyl-DMNQ significantly inhibited PDGF-stimulated cell number and DNA synthesis, and arrested the PDGF-stimulated progression through G0/G1 to S phase of cell cycle supported by the suppression of pRb phosphorylation and cyclin D1/E, CDK2/4 and PCNA expressions. 2-Undecylsulfonyl-DMNQ dose-dependently inhibited the PDGF-stimulated phosphorylation of phospholipase Cγ (PLCγ), protein kinase B (Akt/PKB), signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK 1/2). In addition, 2-undecylsulfonyl-DMNQ inhibited PDGF-induced PDGF receptor β (PDGF-Rβ) dimerization and the phosphorylation of Tyr579/581, Tyr716, Tyr751 and Tyr1021 in PDGF-Rβ. However, 2-undecylsulfonyl-DMNQ has no antiproliferative effect on epidermal growth factor (EGF)- or fetal bovine serum (FBS)-stimulated VSMCs. In conclusion, these findings suggest that the antiproliferative effects of 2-undecylsulfonyl-DMNQ on PDGF-stimulated VSMCs are due to the blockade of receptor dimerization and autophosphorylation on specific tyrosine residues of PDGF-Rβ, which resulted in the subsequent suppression of signaling cascades and a cell cycle arrest. Our observation may explain an important mechanism to block the integration of multiple signals generated by growth factor receptor activation for prevention of VSMC proliferation in cardiovascular diseases.

Abstract 465: The CB2 Cannabinoid Receptor Mediates the Anti-Proliferative Actions of Angiotensin-(1-7) in Vascular Smooth Muscle Cells

Therapeutic interventions to reduce vascular proliferation are critical for the effective treatment of hypertension-induced end-organ damage, restenosis and atherosclerosis. We showed that angiotensin-(1-7) [Ang-(1-7)] reduces neointimal formation following vascular injury and inhibits vascular growth, through activation of the AT7 receptor mas and production of arachidonic acid derivatives. Endocannaboids (ECs) derived from membrane phospholipids also inhibit vascular proliferation and reduce growth following vascular injury, by stimulation of the CB2 receptor. The impact of CB2 receptor blockade on the anti-proliferative actions of Ang-(1-7) was investigated to assess a potential interaction between the EC and Ang-(1-7)/mas receptor systems. Rat thoracic aortic vascular smooth muscle cells (VSMCs) were treated with platelet-derived growth factor (PDGF) to stimulate growth and incubated with Ang-(1-7), the AT7 receptor antagonist [D-alanine7]-angiotensin-(1-7) [Dala], the CB2 receptor agonist HU308 and/or the CB2 receptor antagonist AM630. PDGF-stimulated VSMC growth was markedly reduced by Ang-(1-7) (74 ± 6% of control, n = 10, p<0.0001) and this effect was blocked by Dala (116 ± 14% of control, n = 4; n.s.). The Ang-(1-7)-mediated reduction in growth was abolished by the CB2 receptor antagonist AM630 (136 ± 16% of control, n = 4; n.s.); AM630 alone had no effect. In contrast, the CB1 receptor antagonist AM281 did not prevent the inhibitory actions of Ang-(1-7) on VSMC growth. CB2 receptor activation by the agonist HU308 also reduced PDGF-stimulated VSMC proliferation to a similar extent as Ang-(1-7) (67 ± 4% of control, n = 5, p<0.001); Dala did not influence the response to HU308 (50 ± 10% of control, n = 5, p<0.005). Ang-(1-7) significantly increased the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) by 48% in VSMC by 24 h (p<0.05, n = 4). We conclude that the growth inhibitory properties of Ang-(1-7) involve a novel pathway culminating in the downstream formation of 2-AG and subsequent activation of the CB2 receptor in VSMC. Thus, Ang-(1-7) and/or CB2 receptor activation may constitute a new and beneficial therapeutic strategy for the prevention of vascular proliferation that is prevalent in cardiovascular disease.

Redox-Sensitive Transcription Factor Nrf2 Regulates Vascular Smooth Muscle Cell Migration and Neointimal Hyperplasia

Reactive oxygen species are important mediators for platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells, whereas excess reactive oxygen species-induced oxidative stress contributes to the development and progression of vascular diseases, such as atherosclerosis. Activation of the redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), is pivotal in cellular defense against oxidative stress by transcriptional upregulation of antioxidant proteins. This study aimed to elucidate the role of Nrf2 in PDGF-mediated vascular smooth muscle cell migration and neointimal hyperplasia. PDGF promoted nuclear translocation of Nrf2, followed by the induction of target genes, including NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, and thioredoxin-1. Nrf2 depletion by small interfering RNA enhanced PDGF-promoted Rac1 activation and reactive oxygen species production and persistently phosphorylated downstream extracellular signal-regulated kinase-1/2. Nrf2 depletion enhanced vascular smooth muscle cell migration in response to PDGF and wound scratch. In vivo, Nrf2-deficient mice showed enhanced neointimal hyperplasia in a wire injury model. These findings suggest that the Nrf2 system is important for PDGF-stimulated vascular smooth muscle cell migration by regulating reactive oxygen species elimination, which may contribute to neointimal hyperplasia after vascular injury. Our findings provide insight into the Nrf2 system as a novel therapeutic target for vascular remodeling and atherosclerosis.

Indole-3-carbinol blocks platelet-derived growth factor-stimulated vascular smooth muscle cell function and reduces neointima formation in vivo

The purpose of this study was to determine the effect and associated cell signaling mechanisms of indole-3-carbinol (I3C) on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of cultured vascular smooth muscle cells (VSMCs) and neointima formation in a carotid injury model. Our data demonstrated that I3C inhibited PDGF-BB-induced proliferation of VSMCs in a dose-dependent manner without causing cell cytotoxicity, as assessed by 5-bromo-2′-deoxyuridine incorporation and WST-1 assays. Further studies revealed that the antiproliferative effect of I3C was caused by the arrest of cells in both the G0/G1 and S phases. Moreover, I3C treatment inhibited migration of VSMCs and partly reversed the expression of smooth-muscle-specific contractile markers. We also demonstrated that I3C-induced growth inhibition was associated with an inhibition of the expression of cyclin D1 and cyclin-dependent kinase 4/6, as well as an increase in p27Kip1 levels in PDGF-stimulated VSMCs. These beneficial effects of I3C on VSMCs appeared to be at least partly mediated by the inhibition of Akt and the subsequent activation of glycogen synthase kinase (GSK) 3β. Furthermore, using a mouse carotid artery injury model, we found that treatment with 150 mg/kg I3C resulted in a significant reduction of the neointima/media ratio and cells positive for proliferating cell nuclear antigen. These results demonstrate that I3C can suppress the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury via inhibition of the Akt/GSK3β pathway and suggest that this might be feasible as part of a therapeutic strategy for vascular proliferative diseases.

3,3′Diindolylmethane Suppresses Vascular Smooth Muscle Cell Phenotypic Modulation and Inhibits Neointima Formation after Carotid Injury

Background3, 3′diindolylmethane (DIM), a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs) remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.Methodology/Principal FindingsDIM dose-dependently inhibited the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK) 4/6 as well as an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptorβ (PDGF-Rβ) and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK)3β, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3). Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.ConclusionThese results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-Rβ and the activities of downstream signaling pathways. The results suggest that DIM has the potential to be a candidate for the prevention of restenosis.

Apigenin attenuates neointima formation via suppression of vascular smooth muscle cell phenotypic transformation

Abnormal proliferation, migration, and phenotypic modulation of vascular smooth muscle cells (VSMCs) are critical factors in neointima formation during restenosis. The purpose of this study is to determine the efficacy and possible cell signaling mechanisms of apigenin in VSMC activation induced by platelet-derived growth factor (PDGF)-BB and injury-induced neointima formation. Our data revealed a dose-dependent apigenin inhibition of PDGF-BB-induced proliferation of VSMCs by arresting cells in G0/G1-phase of the cell cycle as determined using 5-bromo-2'-deoxyuridine incorporation and flow cytometry. This was associated with the inhibition of cyclin-dependent kinase (CDK) 4,6 expression and an increase in p27Kip1 levels in PDGF-stimulated VSMCs. Moreover, apigenin was also found to regulate PDGF-induced migration and expression of smooth-muscle-specific contractile markers. Mechanistically, the PDGF-BB-induced phosphorylation of PDGF-receptor β (PDGF-Rβ), Akt/glycogen synthase kinase(GSK)3β, extracellular signal-regulated kinase1/2 (ERK1/2), and signal transducers and activators of transcription 3 (STAT3) is negatively modulated by apigenin. For the in vivo studies using a mouse carotid arterial injury model, the administration of apigenin resulted in a significant inhibition of the neointima/media ratio and proliferating cell nuclear antigen (PCNA)-positive cells. These results demonstrate that apigenin can suppress PDGF-induced VSMC activation and neointima hyperplasia after vascular injury; these beneficial effects are probably the result of the blockade of PDGF-Rβ phosphorylation and its downstream signal transduction, including the Akt/GSK-3β, ERK1/2, and STAT3 pathways. The results suggest that apigenin may be a potential therapeutic candidate for the prevention of restenosis.

Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the "wound," and platelet-derived growth factor (PDGF)-induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

Effects of Panax notoginseng saponins on proliferation and apoptosis of vascular smooth muscle cells

Atherosclerosis is a common cardiovascular disease, and linked with the development of many cardiovascular complications, such as myocardial ischemia and stroke. Although pathogenesis of atherosclerosis is not completely elucidated, increasing evidence has demonstrated that abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in formation of atherosclerosis. Previous studies showed that saponins from Panax notoginseng (PNS) possess anti-atherosclerotic properties. However, the mechanism of PNS against atherosclerosis is not well understood. Therefore, the present study observed the effects of PNS on proliferation and apoptosis of VSMCs. Rat VSMCs were cultured, and platelet-derived growth factor (PDGF) was used to stimulate cell proliferation. The viability of VSMCs was assessed with the MTT method. VSMCs apoptosis was detected by flow cytometry. Expressions of apoptosis related protein p53, Bax, caspase-3 and Bcl-2 were determined using Western blot. Pretreatment of the cells with PNS (200, 400, 800 μg/mL) significantly inhibited proliferation of PDGF-stimulated VSMCs, and induced apoptosis of the proliferated VSMCs in a concentration-dependent way. Western blot analysis showed that PNS upregulated expressions of pro-apoptotic protein p53, Bax and caspase-3, downregulated expression of anti-apoptotic protein Bcl-2, and enlarged Bax/Bcl-2 ratio in the proliferated VSMCs induced by PDGF. This study demonstrates that PNS both inhibits VSMCs proliferation and induces VSMCs apoptosis through upregulating p53, Bax, caspase-3 expressions and downregulating Bcl-2 expression, which constitute the pharmacological basis of its anti-atherosclerotic action.

Dahuang Zhechong Pill (大黄虫丸) containing serum inhibited platelet-derived growth factor-stimulated vascular smooth muscle cells proliferation by inducing G1 arrest partly via suppressing protein kinase C α-Extracellular regulated kinase 1/2 signaling pathway

To investigate effects of dahuang zhechong pill ( DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology. DNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method. DHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G(1) phase, modulated the protein expressions of cyclin D D(1) and p27, and suppressed the activation of PKCα and ERK1/2. DHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G(1) phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.

Inhibition of MDM2 attenuates neointimal hyperplasia via suppression of vascular proliferation and inflammation

Tumour protein p53 plays an important role in the vascular remodelling process as well as in oncogenesis. p53 is negatively regulated by murine double minute 2 (MDM2). A recently developed MDM2 inhibitor, nutlin-3, is a non-genotoxic activator of the p53 pathway. So far, the effect of MDM2 inhibition on vascular remodelling has not been elucidated. We therefore investigated the effect of nutlin-3 on neointima formation. Nutlin-3 up-regulated p53 and its downstream target p21 in vascular smooth muscle cells (VSMCs). DNA synthesis assay and flow cytometric analysis revealed that nutlin-3 inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation by cell cycle arrest. This inhibitory effect was abrogated in p53-siRNA-transfected VSMCs. Furthermore, nutlin-3 inhibited PDGF-stimulated VSMC migration. Treatment with nutlin-3 attenuated neointimal hyperplasia at 28 days after vascular injury in mice, associated with up-regulation of p53 and p21. BrdU incorporation was decreased at 14 days after injury in nutlin-3-treated mice. TUNEL assay showed that nutlin-3 did not exaggerate apoptosis of the injured vessels. Infiltration of macrophages and T-lymphocytes and mRNA expression of chemokine (C-C motif) ligand-5, interleukin-6, and intercellular adhesion molecule-1 were decreased in the injured vessels of nutlin-3-treated mice. Nutlin-3 suppressed NF-κB activation in VSMCs, but not in p53-siRNA-transfected VSMCs. The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Unexpected Role of the Copper Transporter ATP7A in PDGF-Induced Vascular Smooth Muscle Cell Migration

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

Baicalin inhibits PDGF-BB-stimulated vascular smooth muscle cell proliferation through suppressing PDGFRβ-ERK signaling and increase in p27 accumulation and prevents injury-induced neointimal hyperplasia

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of atherosclerotic lesions. Baicalin, an herb-derived flavonoid compound, has been previously shown to induce apoptosis and growth inhibition in cancer cells through multiple pathways. However, the potential role of baicalin in regulation of VSMC proliferation and prevention of cardiovascular diseases remains unexplored. In this study, we show that pretreatment with baicalin has a dose-dependent inhibitory effect on PDGF-BB-stimulated VSMC proliferation, accompanied with the reduction of proliferating cell nuclear antigen (PCNA) expression. We also show that baicalin-induced growth inhibition is associated with a decrease in cyclin E-CDK2 activation and increase in p27 level in PDGF-stimulated VSMCs, which appears to be at least partly mediated by blockade of PDGF receptor β (PDGFRβ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. In addition, baicalin was also found to inhibit adhesion molecule expression and cell migration induced by PDGF-BB in VSMCs. Furthermore, using an animal carotid arterial balloon-injury model, we found that baicalin significantly inhibited neointimal hyperplasia. Taken together, our results reveal a novel function of baicalin in inducing growth arrest of PDGF-stimulated VSMCs and suppressing neointimal hyperplasia after balloon injury, and suggest that the underlying mechanism involves the inhibition of cyclin E-CDK2 activation and the increase in p27 accumulation via blockade of the PDGFRβ-ERK1/2 signaling cascade.

Mechanisms of inhibiting proliferation of vascular smooth muscle cells by serum of rats treated with Dahuang Zhechong pill

Dahuang Zhechong pill (DHZCP), a famous and classical Chinese herbal prescription, consists of twelve traditional Chinese drugs: Eupolyphaga sinensis Walker., Rheum officinale Baill., Scutellaria baicalensis Georgi., Glycyrrhiza uralensis Fisch., Prunus persica Batsch., Prunus armeniaca L., Paeonia lactiflora Pall., Rehmannia glutinosa Libosch., Toxicodendron vernicifluum F.A. Barkl., Tabanus bivittatus Mats., Hirudo nipponica Whitman. and Holotrichia diomphalia Bates., and is clinically used to treat hepatic diseases, gynecopathy and atherosclerosis in China. Our previous studies confirm that DHZCP is able to significantly inhibit proliferation of vascular smooth muscle cells (VSMCs) in vivo and in vitro. Aim of the studyTo investigate the mechanisms of inhibition of VSMCs proliferation by DHZCP with the method of Serum Pharmacology. Materials and methodsVSMCs proliferation of rat was assayed by measuring the cell viability with the MTT method, and platelet-derived growth factor (PDGF) expression in VSMCs was examined by the immunocytochemical method. Cycle and apoptosis of VSMCs were evaluated with flow cytometry. ResultsThe serum of DHZCP-treated rats not only inhibited endothelin-1 (ET-1) stimulated cell proliferation and PDGF expression in VSMCs, but also promoted apoptosis of the proliferated VSMCs. Meanwhile, the serum of rats containing DHZCP interfered with the cycle of PDGF-stimulated VSMCs, increasing proportion of the cells in G0/G1 phases and decreasing proportion of the cells in S and G2/M phases. ConclusionThese suggest that the inhibitory effect of DHZCP on VSMCs proliferation is partially attributed to depressing PDGF expression in VSMCs, retarding the cell cycle and to promoting apoptosis of VSMCs.

Distinct Anti-Migratory Effects of Resveratrol on EGF- versus PDGF-Stimulated Vascular Smooth Muscle Cells

In cardiovascular diseases such as atherosclerosis or restenosis, migration of vascular smooth muscle cells (VSMC) from the media into the intima of blood vessels is an early and crucial step.[...]

Inhibitory Effect of Dehydrozingerone on Vascular Smooth Muscle Cell Function

Growth factor and oxidative stress-mediated migration and proliferation of vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of atherosclerosis. The objective of this study was to assess the ability of dehydrozingerone, a structural analog of curcumin, to inhibit PDGF-stimulated vascular functions in VSMCs. VSMCs isolated from adult rats were treated with dehydrozingerone (0 to 50 microM) before challenge with PDGF (10 ng/mL) and migration, proliferation, and collagen synthesis were assayed by transwell-migration, thymidine-, and L-proline-incorporation assays, respectively. Phosphorylation of PDGF-receptor (PDGFR) and Akt were assessed by Western blotting. Cellular protein tyrosine phosphatase (PTP) activity was determined by the extent of p-nitro-phenyl phosphate hydrolysis. Dehydrozingerone elicited a concentration-dependent inhibition of PDGF-stimulated VSMC migration, proliferation, collagen synthesis, and PDGF/H2O2-stimulated phosphorylation of PDGFR-beta and downstream Akt. Dehydrozingerone also inhibited H2O2-mediated oxidation of PTP. Dehydrozingerone is a potent inhibitor of growth factor/ H2O2-stimulated VSMC functions and may play a critical role in regulating these events after vascular injury. Inhibition of oxidation of cellular phosphatases may represent one of the mechanisms by which dehydrozingerone inhibits these VSMC functions. Inability of the structural analog isoeugenol to inhibit PDGF-signaling suggests that the carbonyl side chain may be necessary for activity.

Inhibitory Effect of Dehydrozingerone on Vascular Smooth Muscle Cell Functions

Migration and proliferation of vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of atherosclerosis and re-stenosis following balloon injury. Growth factors and reactive oxygen species released during vascular injury are thought activate VSMCs. We have recently reported that the food-additive curcumin (diferuloyl methane) is a potent inhibitor of PDGF-stimulated VSMC activation and neointima formation following balloon angioplasty. Dehydrozingerone, isolated from ginger (Zingiber officinale), is a structural half-analog and a biosynthetic intermediate of curcumin with antiinflammatory and antioxidant properties. The objective of this study was to assess the ability of dehydrozingerone to inhibit PDGF-stimulated migration, proliferation and collagen synthesis in cultured VSMCs. Dehydrozingerone (1–50 μM) produced a concentration-dependent inhibition of PDGF-elicited VSMC migration, proliferation, and collagen synthesis as assessed by chemotaxis, [3H]thymidine-incorporation and [3H]-L-proline-incorporation, respectively. At the molecular level dehydrozingerone attenuated agonist and hydrogen peroxide-stimulated PDGF-signal transduction and inhibited the hydrogen peroxide-mediated oxidization of phosphotyrosine phosphatase. Equipotency of dehydrozingerone and curcumin in inhibiting PDGF signaling, and lack of activity of a structural analog isoeugenol suggests that the ketone group on the side chain and the phenolic and methoxy groups are a structural necessity for activity. These data suggest that dehydrozingerone is a potent inhibitor of key PDGF-stimulated VSMC functions and may possess therapeutic implications in regulating these events following vascular injury.

Potent Inhibition of Platelet-Derived Growth Factor-Induced Responses in Vascular Smooth Muscle Cells by BMS-354825 (Dasatinib)

Abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are key events in the pathogenesis of restenosis that undermine the long-term benefit of widely performed balloon angioplasty and stenting procedures. Platelet-derived growth factor (PDGF) is a potent mitogen and motogen for VSMCs and is known to play a prominent role in the intimal accumulation of smooth muscle cells. In this study, we analyzed the effects of a novel protein tyrosine kinase inhibitor, BMS-354825 (dasatinib), on PDGF-stimulated VSMCs. BMS-354825 is an orally bioavailable dual Src/Bcr-Abl tyrosine kinase inhibitor currently undergoing clinical trials in cancer patients. We found that BMS-354825 inhibited PDGF-stimulated activation of PDGF receptor (PDGFR), STAT3, Akt, and Erk2 in rat A10 VSMCs and in primary cultures of human aortic smooth muscle cells (AoSMCs) at low nanomolar concentrations. The 50% inhibition of the PDGFRbeta tyrosine kinase activity in vitro by BMS-354825 was observed at 4 nM. Direct comparison of BMS-354825 and another PDGFR inhibitor, imatinib (Gleevec, STI571), in VSMCs indicated that BMS-354825 is 67-fold more potent than imatinib in inhibition of PDGFR activation. BMS-354825 also inhibited Src tyrosine kinase in A10 cells. At the cell level, PDGF stimulated migration and proliferation of A10 cells and human AoSMCs, both of which were inhibited by BMS-354825 in a concentration dependent manner in the low nanomolar range. These results suggest that BMS-354825 is a potent inhibitor of PDGF-stimulated VSMC activities and a potential agent for the development of a new therapy for vascular obstructive diseases such as restenosis.

Curcumin Inhibits Platelet-Derived Growth Factor–Stimulated Vascular Smooth Muscle Cell Function and Injury-Induced Neointima Formation

Vascular smooth muscle cell (VSMC) migration, proliferation, and collagen synthesis are key events involved in the pathogenesis of cardiovascular disease. Growth factors, such as platelet-derived growth factor (PDGF) and fibroblast growth factor, released during vascular injury plays a pivotal role in regulating these events. Curcumin (diferuloyl methane), a major component of the spice turmeric (Curcuma longa), has been shown recently to have beneficial effects in chronic conditions, such as inflammation, cancer, cystic fibrosis, and Alzheimer's disease. The objective of this study was to investigate the ability of curcumin to inhibit PDGF-stimulated migration, proliferation, and collagen synthesis in cultured VSMCs and neointima formation after carotid artery injury in rats. Curcumin (1 to 25 microM) produced a concentration-dependent inhibition of PDGF-elicited VSMC migration, proliferation, and collagen synthesis assessed by chemotaxis, [3H]thymidine incorporation, and [3H]-L-proline incorporation, respectively. Curcumin blocked PDGF-induced VSMC actin-cytoskeleton reorganization, attenuated PDGF signal transduction, and inhibited the binding of PDGF to its receptors. Carotid artery neointima formation was significantly attenuated by perivascular curcumin compared with vehicle controls 14 days after injury, characterized by reduced DNA synthesis, collagen synthesis, and PDGF receptor phosphorylation. These data suggest that curcumin is a potent inhibitor of key PDGF-stimulated VSMC functions and may play a critical role in regulating these events after vascular injury.

VEGF Inhibits PDGF-Stimulated Calcium Signaling Independent of Phospholipase C and Protein Kinase C

Despite advances in both open and endovascular techniques for treatment of arterial occlusive disease, restenosis because of neointimal hyperplasia continues to be a major cause of graft failure and restenosis. This phenomenon has been attributed to vascular smooth muscle cell (VSMC) activation by several potent mitogens including platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) released at the site of injury. PDGF is known to stimulate calcium influx in VSMC that has been shown to be critical for VSMC migration and proliferation. We have previously shown that VEGF inhibits PDGF-stimulated VSMC proliferation. The objective of this set of experiments was to investigate whether VEGF modulated PDGF-stimulated Ca2+ influx in VSMC. Primary cultured human aortic SMC were grown to subconfluency and assigned to the following groups: no stimulation, stimulation with PDGF-BB (20 ng/ml), stimulation with VEGF165 (40 ng/ml), or a combination of PDGF-BB + VEGF165. Ca2+ influx was measured using a Fura-2 fluorescence assay. The intracellular Ca2+ fraction was assayed with the Fura-2 assay by using Ca2+-free media. Phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC), and Akt phosphorylation was assessed with standard immunoblotting techniques at 1, 5, and 10 min time points. Ca2+-calmodulin kinase II (CaMKII) activity was extrapolated from the phosphorylation of Phospholamban B (PLB), a well-known protein substrate, at 1, 5, and 10 min time points. PDGF stimulation resulted in a 328 +/- 9 nm total calcium influx in VSMC. The combination of VEGF + PDGF resulted in a 273 +/- 21 nm total calcium influx, an amount significantly less than with PDGF alone (P < 0.04). PDGF stimulation resulted in a 72 +/- 35 nm intracellular calcium release. The addition of VEGF to PDGF resulted in an intracellular calcium release of only 15 +/- 11 nm, a significant decrease compared to PDGF alone (P < 0.01). The phosphorylation of PLCgamma1, PKC, and Akt was equivalent at 1, 5, and 10 min between the PDGF and the PDGF + VEGF treatment groups. There was an increase in CaMKII activity at 1 and 5 min time points in both the PDGF and PDGF + VEGF treatment groups suggesting that extracellular calcium influx is sufficient for CaMKII activation. VEGF inhibits PDGF-stimulated total calcium influx and, in particular, PDGF-stimulated intracellular calcium release in VSMC. The equivalent phosphorylation of PLCgamma1, PKC, and Akt suggests that the inhibitory mechanism by VEGF on calcium influx occurs downstream of these proximal mediators. The inhibition of intracellular calcium release did not inhibit CaMKII activity. VEGF may play an important role in modulating PDGF induced VSMC proliferation by specifically inhibiting intracellular calcium release in response to PDGF.