Vascular Smooth Muscle Cell Growth Research Articles

Suppressor of Cytokine Signaling 3 Is a Negative Regulator for Neointimal Hyperplasia of Vein Graft Stenosis

Coronary artery bypass graft (CABG) surgery is one of the most effective treatments for coronary artery disease. However, neointimal hyperplasia and ultimate luminal occlusion that is caused by vascular smooth muscle cell (VSMC) migration, proliferation and inflammatory response impede the long-term prognosis. The SOCS3 protein is involved in modulating various autoimmune and inflammatory diseases. However, the role of SOCS3 in vein graft disease is still unclear. We found that the mRNA and protein expression levels of IL-1β, IL-6, MCP-1, ICAM-1, TNF-α, STAT3, P-STAT3 and SOCS3 were significantly higher in the graft samples compared to normal veins. After transfecting the recombinant adenovirus carrying the rat SOCS3 gene into cultured rat VSMCs or grafting veins in rat, SOCS3 overexpression was found to significantly inhibit VSMC migration and proliferation in vitro and neointimal hyperplasia in vivo, respectively. Furthermore, SOCS3 overexpression inhibited VSMC migration and growth in vitro and alleviated VSMC inflammation in vitro by inhibiting STAT3 activation and phosphorylation. In conclusion, SOCS3 is a crucial physiological negative regulator for vein graft failure and provides a novel target for vein graft stenosis therapy after CABG.

Concentration-dependent differential effects of udenafil on viability, proliferation, and apoptosis in vascular endothelial and smooth muscle cells.

Objectives:Local strategies directed against vascular smooth muscle cell (VSMC) proliferation, such as drug-eluting stents (DES), reduce the occurrence of restenosis. However, these approaches may also inhibit vascular endothelial cell (VEC) proliferation and impair reendothelialization, and hence, increase susceptibility to late thrombosis. In this study we examined the differential effects of various concentrations of the type 5 phosphodiesterase (PDE-5) inhibitor, udenafil, on viability, proliferation, and apoptosis of VEC and VSMC, in order to identify the optimal concentration of udenafil that minimizes inhibition of VEC survival and growth, and maximizes inhibition of VSMC survival and growth.Materials and Methods:VEC from human umbilical veins and VSMC from human aorta were exposed to various concentrations of udenafil (1, 10, and 100 μmol/l and 1 mmol/l) for 24 h, and its effects on cell viability, proliferation, and apoptosis were studied using 5-bromo-2’- deoxyuridine (BrdU), methylthiazoletetrazolium (MTT) assay, trypan blue dye exclusion, and flow cytometry.Results:Udenafil inhibited the survival and growth of VEC and VSMC in a concentration-dependent manner over a range of concentrations. At 100 μmol/l, udenafil, inhibited VEC proliferation significantly less than VSMC proliferation (P < 0.05), and could significantly induce VEC apoptosis less than VSMC apoptosis (P < 0.05).Conclusions:Udenafil has a differential effect on survival and growth in VEC and VSMC. The maximal differential effect, with minimal inhibition of VEC and maximal inhibition of VSMC, occurs at 100 μmol/l. This characteristic suggests that udenafil is a promising agent for use in DES.

Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold

Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC–monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC–monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5ngml−1) or IL-6 (1ngml−1) for 7days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC–monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC–monocyte co-cultures on D-PHI scaffolds for 7days, implying that IL-6 mediates VSMC response in monocyte–VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.

MicroRNA-133a regulates insulin-like growth factor-1 receptor expression and vascular smooth muscle cell proliferation in murine atherosclerosis

ObjectiveMicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. Methods and resultsExpression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE−/− aortas. ApoE−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3′UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE−/− VSMC than in WT cells. ConclusionMiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1-stimulated VSMC survival and growth.

Reactive Oxygen Species Promote Vascular Smooth Muscle Cell Proliferation

Do reactive oxygen species play a role in the pathogenesis of cardiovascular diseases? In 1992, Dr Berk and his group published an article that began to answer this question. During the following years, they performed a series of research projects that established the role of reactive oxygen species in vascular smooth muscle cell proliferation and cardiovascular diseases. It is now widely recognized that production of intracellular reactive oxygen species (ROS) is substantially involved in the pathogenesis of cardiovascular diseases, in part by promoting vascular smooth muscle cell (VSMC) proliferation. However, 20 years ago, it was elusive why VSMCs proliferate in response to arterial injury. Thus, Dr Berk et al hypothesized that ROS generated during arterial injury could be a common mechanism involved. Using xanthine/xanthine oxidase to generate ROS, they were able to demonstrate that ROS stimulate VSMC proliferation in vitro and that H2O2 was primarily responsible for xanthine/xanthine oxidase–induced VSMC DNA synthesis.1 This Circulation Research article was the first milestone article that directly demonstrated the role of ROS in VSMC proliferation, triggering the studies that followed targeting the role of ROS in the pathogenesis of cardiovascular diseases. Until now, this article has been referenced by more than 450 articles. Three years later, they further reported that both O2− and H2O2 stimulate VSMC growth but only O2− rapidly activates MAP kinase, suggesting that additional signal events are required for the mitogenic effects of H2O2.2 Based on these reports, it has been elucidated that the excess amount of ROS (oxidative stress) promotes VSMC proliferation and potentially develops cardiovascular diseases. In the subsequent 5 years, they focused on the interesting finding that ROS stimulates ERK1/2 in a biphasic manner in VSMCs. Finally, they demonstrated that one explanation …

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<i>Circulation</i> Editors’ Picks

Inhibitory effects of suppressor of cytokine signaling 3 on inflammatory cytokine expression and migration and proliferation of IL-6/IFN-γ-induced vascular smooth muscle cells

The main pathogenesis of saphenous vein graft neointimal hyperplasia after coronary artery bypass grafting (CABG) is inflammation-caused migration and proliferation of vascular smooth muscle cells (VSMCs). Janus kinase 2/signal transducer and activators of transcription 3 (JAK2/STAT3) pathway is an important signaling pathway through which VSMCs phenotype conversion occurs. Suppressor of cytokine signaling 3 (SOCS3) is the classic negative feedback inhibitor of JAK2/STAT3 pathway. Growing studies show that SOCS3 plays an important anti-inflammatory role in numerous autoimmune diseases, inflammatory diseases and inflammation-related tumors. However, the effect and mechanism of SOCS3 on vein graft disease is unclear. The purpose of this study was to investigate the effects of SOCS3 on the inflammation, migration and proliferation of VSMCs in vitro and the mechanism. The small interference RNA plasmid targeting rat SOCS3 (SiRNA-rSOCS3) and the recombinant adenovirus vector carrying rat SOCS3 gene (pYrAd-rSOCS3) were constructed, and the empty plamid (SiRNA-control) and vector (pYrAd-GFP) only carrying GFP reported gene were constructed as control. The rat VSMCs were cultured. There were two large groups of A (SOCS3 up-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+pYrAd-rSOCS3 group, IL-6/IFN-γ(+)pYrAd-GFP group; and B (SOCS3 down-regulated): control group, IL-6/IFN-γ group, IL-6/IFN-γ+SiRNA-rSOCS3 group and IL-6/ IFN -γ+SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs induced by IL-6/IFN-γ. After 24 h, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of SOCS3, STAT3 (only by Western blotting), P-STAT3 (only by Western blotting), IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1. The MTT, Transwell assay and flow cytometry were used to examine VSMCs proliferation, migration and cell cycle progression, respectively. As compared with control group, the mRNA and protein expression of SOCS3, STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly up-regulated in VSMCs stimulated by IL-6/IFN-γ. However, in VSMCs transfected with pYrAd-rSOCS3 before stimulation with IL-6/IFN-γ, the expression of SOCS3 mRNA and protein was further up-regulated, and that of STAT3, P-STAT3, IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1 was significantly down-regulated as compared with IL-6/IFN-γ group and IL-6/IFN-γ+pYrAd-GFP group. The expression of those related-cytokines in IL-6/IFN-γ+SiRNA-rSOCS3 group was markedly increased as compared with IL-6/IFN-γ group and IL-6/IFN-γ+SiRNA-control group. The absorbance (A) values, the number of cells migrating to the lower chamber, and percentage of cells in the G2/M+S phase were increased in VSMCs stimulated by IL-6/IFN-γ. In VSMCs incubated with pYrAd-rSOCS3 or SiRNA-rSOCS3 before IL-6/IFN-γ stimulation, the A values, the number of cells migrating to the lower chamber, and the percentage of cells in the G2/M+S phase were significantly decreased, and increased respectively. These results imply that IL-6/IFN-γ, strong inflammatory stimulators, can promote transformation of VSMCs phenotype form a quiescent contractile state to a synthetic state by activating JAK2/STAT3 pathway. Over-expresssed SOCS3 might inhibit pro-inflammatory effect, migration and growth of VSMCs by blocking STAT3 activation and phosphorylation. These data in vitro confirm that SOCS3 may play a negatively regulatory role in development and progression of vein graft failure. These conclusions can provide a novel strategy for clinical treatment of vein graft diseases and a new theoretic clue for related drug development.

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&lt;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&lt;0.001); Dala did not influence the response to HU308 (50 ± 10% of control, n = 5, p&lt;0.005). Ang-(1-7) significantly increased the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) by 48% in VSMC by 24 h (p&lt;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.

An adipokine omentin prevents pathological vascular remodeling

Background: Obese states are associated with the development of atherosclerosis and vascular restenosis after angioplasty. Omentin is an adipocytokine whose concentrations are reduced in obese individuals. Recently, we have reported that circulating omentin levels are down-regulated in association with obesity-linked disorders including carotid atherosclerosis and coronary artery disease. Here, we investigated the role of omentin on neointimal hyperplasia and Vascular Smooth Muscle Cell (VSMC) behavior in vivo and in vitro. Methods and results: An adenoviral vector expressing human Omentin (Ad-OMT) or β-galactosidase as a control was injected into the jugular vein of Wild-Type (WT) mice 3 d prior to vascular injury. Left femoral arteries of mice were injured by a stainless-steel wire inserted from the lumen. Although human omentin protein could not be detected in plasma in control WT mice, plasma human omentin levels were increased to 1353.9±169.6 ng/ml in Ad-OMT-treated WT mice on the 5th day after injection. At 21 days after vascular injury, systemic delivery of Ad-OMT significantly attenuated neointimal thickening compared with control (p<0.01, n=8). Ad-OMT decreased the number of bromodeoxyuridine (BrdU) positive proliferating cells in the neointima at day 7 after vascular injury (p<0.01, n=5). In cultured VSMCs, treatment with recombinant omentin protein attenuated DNA synthesis and chemotactic activity induced by various growth factors as assessed by BrdU incorporation and Boyden chamber assays, respectively. Treatment with omentin suppressed growth factor-induced phosphorylation of ERK in VSMCs. Treatment of VSMCs with omentin stimulated the phosphorylation of AMPK in a time-dependent manner. Furthermore, transduction with dominant-negative AMPK prevented the inhibitory effect of omentin on growth factor-stimulated VSMC proliferation and ERK phosphorylation. Conclusion: Omentin attenuates neointimal hyperplasia after vascular injury and suppresses VSMC growth through its ability to activate AMPK, suggesting that omentin can represent a novel target molecule for the prevention of pathological remodeling.

Angiotensin-converting enzyme 2 attenuates oxidative stress and VSMC proliferation via the JAK2/STAT3/SOCS3 and profilin-1/MAPK signaling pathways

Angiotensin (Ang) II plays a vital role in vascular smooth muscle cell (VSMC) growth and proliferation. Angiotensin-converting enzyme 2 (ACE2) is a specific Ang II-degrading enzyme but its role in VSMC proliferation remains largely unknown. We hypothesized that ACE2 might suppress Ang II-mediated oxidative stress and VSMC proliferation. Human umbilical artery smooth muscle cells (HUASMCs) were pretreated with Ang II (100nM) for 6h and 24h, respectively. Exposure to Ang II resulted in significant increases in suppressor of cytokine signaling 3 (SOCS3) expression and phosphorylation levels of JAK2, STAT3 and ERK1/2 linked with elevated superoxide production and cell proliferation in HUASMCs. These changes were strikingly prevented by administration of ERK1/2 inhibitor PD98059 (10μM) and JAK/STAT inhibitor WP1066 (5μM) but were largely aggravated by ACE2 inhibitor DX600 (0.5μM). More importantly, treatment with human recombinant ACE2 (hrACE2; 1mg/ml) dramatically prevented Ang II-mediated SOCS3 expression and the JAK2–STAT3 and ERK1/2 signaling, and resulted in attenuation of superoxide production and cell proliferation in HUASMCs. Intriguingly, downregulation of profilin-1 with profilin-1 siRNA (50nM) was able to abolish Ang II-induced upregulations of profilin-1 expression, ERK1/2 phosphorylation and superoxide production with attenuation of VSMC proliferation. In conclusion, treatment with hrACE2 prevents Ang II-mediated activation of the JAK2/STAT3/SOCS3 and profilin-1/MAPK signaling pathways, contributing to attenuation of superoxide generation and cell proliferation in HUASMCs, suggesting a protective mechanism of ACE2 against Ang II-mediated oxidative stress and VSMC proliferation. ACE2 may represent a potential candidate to prevent and treat vascular disorders.

In vitro study on the feasibility of magnetic stent hyperthermia for the treatment of cardiovascular restenosis

Thermal treatment or hyperthermia has received considerable attention in recent years due to its high efficiency, safety and relatively few side-effects. In this study, we investigated whether it was possible to utilize targeted thermal or instent thermal treatments for the treatment of restenosis following percutaneous transluminal coronary angioplasty (PTCA) through magnetic stent hyperthermia (MSH). A 316L stainless steel stent and rabbit vascular smooth muscle cells (VSMCs) were used in the present study, in which the inductive heating characteristics of the stent under alternative magnetic field (AMF) exposure, as well as the effect of MSH on the proliferation, apoptosis, cell cycle and proliferating cell nuclear antigen (PCNA) expression of the rabbit VSMCs, were evaluated. The results demonstrated that 316L stainless steel coronary stents possess ideal inductive heating characteristics under 300 kHz AMF exposure. The heating properties were shown to be affected by the field intensity of the AMF, as well as the orientation the stent axis. MSH had a significant effect on the proliferation and apoptosis of VSMCs, and the effect was temperature-dependent. While a mild temperature of 43°C demonstrated negligible effects on the growth of VSMCs, MSH treatment above 47°C effectively inhibited the VSMC proliferation and induced apoptosis. Furthermore, a 47°C treatment exhibited a significant and long-term inhibitory effect on VSMC migration. The results strongly suggested that MSH may be potentially applied in the clinic as an alternative approach for the prevention and treatment of restenosis.

Inhibition of Patched-1 Prevents Injury-Induced Neointimal Hyperplasia

To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. In vitro, human coronary arterial smooth muscle cells were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression while concomitantly decreasing human coronary arterial smooth muscle cell growth to that similar under normal flow conditions. In vivo, ligation injury-induced low flow increased vascular smooth muscle cell growth and vascular remodeling, while increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow-induced increases in Ptc-1/Notch expression and markedly reduced the subsequent vascular remodeling. These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.

Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial injury

Restenosis engenders surgical vascular intervention failure. Zinc finger protein 191 (ZFP191) is a novel member of the SCAN domain family of Krüppel-like zinc finger transcription factors. Previous work reveals that ZFP191 is a pleiotropic factor that plays important roles in hematopoiesis, brain development, and tumor growth. Here, we sought to determine whether intimal hyperplasia was affected by the activity of ZFP191 and to investigate the molecular mechanisms that may underpin the process. Intimal hyperplasia was induced by guidewire injury in mouse femoral arteries. The arteries were harvested for morphometric assessment and determination of ZFP191 expression. Next, ZFP191 knockdown in cultured mouse aortic vascular smooth muscle cells (VSMCs) was achieved by lentiviral transduction of short-hairpin RNA. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, [(3)H]thymidine incorporation assay, scratch assay, and transwell migration assay were used to evaluate the effects of ZFP191 knockdown on VSMC growth and migration. In addition, β-catenin, c-myc, cyclin D1, matrix metalloproteinase (MMP) 9, MMP2, and MMP7 were measured by Western blotting in the absence of ZFP191 in vitro and in vivo. Zymography was used to evaluate MMP activity in cell culture-conditioned media. Lastly, artery injury was performed in wild-type (WT) and heterozygous ZFP191 knockout (KO) mice, and morphometric analysis of the arteries was determined. Guidewire injury was associated with development of intimal hyperplasia, and ZFP191 expression was enhanced by 51% in the injured arteries. Cultured primary VSMCs transfected with lentiviral shZFP191 displayed reduced proliferation and migration compared with controls. Mechanically, ZFP191 knockdown potently decreased the level of β-catenin and its downstream targets c-myc and cyclin D1. ZFP191 knockdown downregulated the expression of MMP9, MMP2, and MMP7, and zymography confirmed that ZFP191 knockdown reduced the activity of MMPs. Consistent with the in vitro data, elevated expression of β-catenin, c-myc, cyclin D1, MMP9, MMP2, and MMP7 accompanied upregulation of ZFP191 after injury in the femoral arteries of mice, and these levels were downregulated in ZFP191 KO vessels. Finally, intimal hyperplasia was greatly blocked in heterozygous ZFP191 KO mice compared with WT mice (intima/media ratio, 0.124 vs 0.412; P < .05). ZFP191 played an essential role in aggressive proliferation and migration of VSMCs, which in turn facilitated intimal hyperplasia. Our findings offer the first genetic evidence of ZFP191 as a potential therapeutic target to prevent restenosis.

Endothelial Dysfunction in Diabetes – Clasic Sources of Vascular Oxidative Stress (Nadph Oxidases, Enos Uncoupling and Xanthine Oxidase)

Abstract Cardiovascular disease is the leading cause of disease / mortality worldwide. It is generally accepted that increased production of reactive oxygen species (ROS) has an important role in cardiovascular pathology, contributing to endothelial dysfunction and to the aggravation of atherosclerosis. Among all cardiovascular risk factors, diabetes mellitus is one of the most important. The worldwide prevalence of diabetes has increased rapidly even in developing countries, doubling the combined risk of cardiovascular events in patients with hypertension. In diabetes, increased reactive oxygen species (ROS) production leads to endothelial dysfunction, recognized by the presence of impaired vascular relaxation, increased vascular smooth muscle cells growth and hypertrophy, all together contributing to atherosclerotic plaque formation. On this basis, the vascular endothelium has emerged as a therapeutic target, with the aim to improve systemic metabolic state by improving vascular function. In this review we have focused on the most important sources of reactive oxygen species generated by vascular endothelium in diabetic patients (NADPH Oxidases, eNOS uncoupling, Xanthine oxidase). The importance of oxidative stress in mediating the vascular complications of diabetes is supported by studies showing that antioxidant therapy correct the vascular function in humans or in experimental models of diabetes. Therefore, understanding the physiological mechanisms involved in vascular disorders resulting from hyperglycemia is essential for the proper use of available therapeutic resources.

Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of atherosclerosis by promoting vascular smooth muscle cell (VSMC) growth, migration, apoptosis, adhesion and the secretion of matrix components. The osteogenic differentiation of VSMCs is essential in the development of vascular calcification. However, the role of CTGF in the transdifferentiation and calcification of VSMCs is unclear. In the present study, we examined whether CTGF stimulates VSMC transdifferentiation. Primary VSMCs were obtained from mouse thoracic aortas by enzymatic digestion and identified by immunostaining for smooth muscle specific α-actin antibody (α-SMA). VSMC calcification was induced by the addition of CTGF to the osteogenic mediaum containing 5-10% FBS in the presence of 0.25 mM ascorbic acid and 10 mM β-glycerophosphate for 14 days. Calcified cells were determined by Alizarin Red S staining. Our results revealed that CTGF induced the expression of several bone markers, including alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and core-binding factor subunit α1 (Cbfα1)/runt-related transcription factor 2 (Runx2), as well as calcification. However, the inhibition of extracellular signal-regulated kinase (ERK) activity using the ERK-specific inhibitor, PD98059, blocked the induction of these proteins and VSMC calcification. Based on these data, we conclude that CTGF stimulates the transdifferentiation of VSMCs into osteoblasts and that the ERK signaling pathway appears to play a critical role in this process.

Increasing our IQ of vascular smooth muscle cell migration with IQGAP1. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

<i>Increasing our IQ of vascular smooth muscle cell migration with IQGAP1</i>. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

Abstract 509: Inhibition of Patched-1 prevents Injury-induced Neointimal Hyperplasia

Intimal remodeling occurs due to alterations in vascular smooth muscle cell (VSMC) proliferation and apoptosis and is a key event in the pathogenesis of vascular disease. Atherosclerosis, vascular rejection, venous graft restenosis and coronary intervention are each characterized by increased VSMC growth and intimal thickening. Objectives To determine the role of Ptc-1 in mediating pulsatile flow-induced changes in SMC growth and neointimal hyperplasia. Methods and Results HCASMC were exposed to “normal“ or pathological “low” pulsatile flow conditions for 24 h by utilizing a perfused transcapillary flow system. In vivo, pathological low flow conditions following carotid artery ligation increased medial SMC growth while increasing Ptc-1/Notch expression. Inhibition of Ptc-1 expression by cyclopamine attenuated low flow-induced increases in VEGF/Notch expression while concomitantly decreasing HCASMC growth to normal flow levels. Perivascular delivery of Ptc-1 siRNA by pluronic gel inhibited the low flow-induced increases in Ptc-1/Notch expression and subsequent increase in medial SMC growth. In addition, low pulsatile flow-induced increases in neointimal hyperplasia and subsequent decreased lumen volume were returned to sham-operated control levels following ptc-1 gene knockdown. Conclusion These results suggest that pathological low flow stimulates SMC growth and neointimal hyperplasia via Ptc-1 regulation of VEGF/Notch signaling.

12S-Lipoxygenase is necessary for human vascular smooth muscle cell survival

Considerable evidence has been published demonstrating the importance of lipoxygenase enzymes for vascular smooth muscle cell (VSMC) growth. The current study sets out to determine whether or not 12-lipoxygenase (12LO) is also important for human placental VSMC survival. Both a pharmacological and two 12LO antisense knockdown approaches were applied. The 12LO inhibitor baicalien induced a 2–2.5-fold increase in cell death, which appeared to result from apoptosis, as indicated by DNA fragmentation, activation of procaspase 3 to caspase 3 and cytochrome C release from the mitochondria to the cytosol. This apoptosis could be prevented by treatment with the 12LO product, 12 hydroxyeicosatetraenoic acid (12HETE). Human platelet-type 12LO-antisense knockdown, by either plasmid transfection or adeno-associated virus (AAV) infection also induced substantial VSMC death over controls, which could also be prevented by treatment with 12HETE, but not 5HETE. Hence, biochemical 12LO inhibition or 12LO-antisense knockdown in VSMC can induce programmed cell death. These observations suggest a previously unrecognized association between human VSMC survivability and 12LO.

Ugonin J, a bioactive compound isolated from Helminthostachys zeylanica (L) Hook. (Helminthostachyaceae), demonstrates inhibitory effects on neointima formation in the rat carotid artery

The clinical value of percutaneous coronary angioplasty (PTCA) for treating arterial obstruction has always been counteracted by neointimal hyperplasia. Development of restenosis has been attributed to abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). The purpose of the present study was to investigate whether Ugonin J, a bioactive compound isolated from Helminthostachys zeylanica (L) Hook. (Helminthostachyaceae), has potential effects to prevent neointimal formation, and to explore its molecular mechanisms. Our current findings demonstrated that at 12, 24 and 36 μM Ugonin J markedly inhibited cell growth and migration of VSMCs (A7r5). Cytometric analysis suggested that Ugonin J arrest VSMCs in the G1 phase at 36 μM. Ugonin J was able to suppress the activities of matrix metalloproteinase (MMP)‐2 and MMP‐9 in a concentration‐dependent manner. Ugonin J was also found to decrease the expression levels of p‐AKT, p‐ERK1/2, p‐FAK, ERP57, PGK1, PI3K and MMP‐2 and MMP‐9, whereas the total protein levels of AKT, ERK1/2 and FAK were not significantly changed. Our data demonstrated that Ugonin J can be a potential regulator to inhibit cell migration and neointimal formation in clinical restenosis after PTCA.

MIR–133A REGULATES EXPRESSION OF INSULIN–LIKE GROWTH FACTOR–1 RECEPTOR AND SMOOTH MUSCLE CELL GROWTH PARTLY BY TARGETING GALACTOSYLTRANSFERASE 1–ASSOCIATING PROTEIN

MiR–133a regulates cardiovascular cell development. However, its impact on vascular smooth muscle cell (VSMC) growth during atherosclerosis remains unclear. Galactosyltransferase 1–associating protein (GTAP), plays a role in stem cell growth. This study will analyze the role of miR–133a in