Promotes Vascular Smooth Muscle Cells Proliferation Research Articles

Cinnamic aldehyde inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in Zucker Diabetic Fatty rats

Atherosclerosis remains the number one cause of death and disability worldwide. Atherosclerosis is treated by revascularization procedures to restore blood flow to distal tissue, but these procedures often fail due to restenosis secondary to neointimal hyperplasia. Diabetes mellitus is a metabolic disorder that accelerates both atherosclerosis development and onset of restenosis. Strategies to inhibit restenosis aim at reducing neointimal hyperplasia by inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. Since increased production of reactive oxygen species promotes VSMC proliferation and migration, redox intervention to maintain vascular wall redox homeostasis holds the potential to inhibit arterial restenosis. Cinnamic aldehyde (CA) is an electrophilic Nrf2 activator that has shown therapeutic promise in diabetic rodent models. Nrf2 is a transcription factor that regulates the antioxidant response. Therefore, we hypothesized that CA would activate Nrf2 and would inhibit neointimal hyperplasia after carotid artery balloon injury in the Zucker Diabetic Fatty (ZDF) rat. In primary ZDF VSMC, CA inhibited cell growth by MTT with an EC50 of 118 ± 7 μM. At a therapeutic dose of 100 μM, CA inhibited proliferation of ZDF VSMC in vitro and reduced the proliferative index within the injured artery in vivo, as well as migration of ZDF VSMC in vitro. CA activated the Nrf2 pathway in both ZDF VSMC and injured carotid arteries while also increasing antioxidant defenses and reducing markers of redox dysfunction. Additionally, we noted a significant reduction of neutrophils (69%) and macrophages (78%) within the injured carotid arteries after CA treatment. Lastly, CA inhibited neointimal hyperplasia evidenced by a 53% reduction in the intima:media ratio and a 61% reduction in vessel occlusion compared to arteries treated with vehicle alone. Overall CA was capable of activating Nrf2, and inhibiting neointimal hyperplasia after balloon injury in a rat model of diabetic restenosis.

Abstract 290: The Transcription Factor Tead1 is Critical for Vascular Development in Mouse by Promoting Differentiation of Vascular Smooth Muscle Cells

Background: Our previous study has shown that the Hippo signaling effector YAP plays a crucial role in the phenotypic modulation of vascular smooth muscle cells (VSMCs) in response to arterial injury and cardiovascular development in mice. However, the role of TEAD1, a co-factor of YAP in VSMC development is unknown. Objective: The goal of this study to investigate the functional role of TEAD1 in cardiovascular development in mice. Methods and Results: TEAD1 was specifically ablated in cardiomyocytes and VSMCs by crossing TEAD1 flox mice with SM22alpha-Cre transgenic mice. Cardiac/VSMC-specific deletion of TEAD1 led to embryonic lethality by E14.5 in mice due to cardiovascular defects including severe hypoplastic cardiovascular wall, resulting from impaired cardiac/VSMC proliferation. Whole transcriptomic analysis revealed deletion of TEAD1 in mouse cardiomyocytes and VSMCs significantly down-regulated expression of cardiac/VSMC differentiation genes and key transcription factors including myocardin and pitx2c that are critical for cardiovascular development. In vitro studies demonstrated that myocardin and pitx2c rescued TEAD1-defiency induced defects of VSMC differentiation. Furthermore, we identified pitx2c was a direct transcriptional target of TEAD1 and pitx2c synergistically promoted VSMC differentiation with myocardin. Conclusions: This study revealed a critical role of TEAD1 in cardiovascular development by promoting VSMC proliferation and differentiation and provided novel insights into the regulatory mechanisms for smooth muscle development.

Aneurysm Severity is Increased by Combined Mmp-7 Deletion and N-cadherin Mimetic (EC4-Fc) Over-Expression

There is an unmet need for treatments to reduce abdominal aortic aneurysm (AAA) progression. Vascular smooth muscle cell (VSMC) apoptosis precipitates AAA formation, whereas VSMC proliferation repairs the vessel wall. We previously demonstrated that over-expression of EC4-Fc (truncated N-cadherin), or deletion of matrix-metalloproteinase-7 (Mmp-7) reduced VSMC apoptosis in mouse atherosclerotic plaques. Additionally, MMP-7 promotes VSMC apoptosis by cleavage of N-cadherin. We investigated their combined effect on AAA formation. Increased apoptosis and proliferation were observed in human AAA (HAAA) sections compared to normal aortae (HA). This coincided with increased MMP-7 activity and reduced N-cadherin protein levels in HAAA sections compared to HA. Using a mouse model of aneurysm formation, we showed that the combination of Mmp-7 deletion and EC4-Fc overexpression significantly increased AAA severity. Medial apoptosis and proliferation were both significantly reduced in these mice compared to control mice. In vitro, MMP-7 inhibition and EC4-Fc administration significantly supressed human aortic VSMC apoptosis (via activation of PI-3 kinase/Akt signalling) and proliferation. In conclusion, combined Mmp-7 deletion and systemic over-expression of EC4-Fc reduced both proliferation and apoptosis. Reduced proliferation-mediated repair over-rides any benefit of reduced apoptosis, increasing aneurysm severity. Future studies should therefore focus on retarding VSMC apoptosis whilst promoting VSMC proliferation.

SRSF1 promotes vascular smooth muscle cell proliferation through a \u0394133p53/EGR1/KLF5 pathway

Though vascular smooth muscle cell (VSMC) proliferation underlies all cardiovascular hyperplastic disorders, our understanding of the molecular mechanisms responsible for this cellular process is still incomplete. Here we report that SRSF1 (serine/arginine-rich splicing factor 1), an essential splicing factor, promotes VSMC proliferation and injury-induced neointima formation. Vascular injury in vivo and proliferative stimuli in vitro stimulate SRSF1 expression. Mice lacking SRSF1 specifically in SMCs develop less intimal thickening after wire injury. Expression of SRSF1 in rat arteries enhances neointima formation. SRSF1 overexpression increases, while SRSF1 knockdown suppresses the proliferation and migration of cultured human aortic and coronary arterial SMCs. Mechanistically, SRSF1 favours the induction of a truncated p53 isoform, Δ133p53, which has an equal proliferative effect and in turn transcriptionally activates Krüppel-like factor 5 (KLF5) via the Δ133p53-EGR1 complex, resulting in an accelerated cell-cycle progression and increased VSMC proliferation. Our study provides a potential therapeutic target for vascular hyperplastic disease.

MiR-448 promotes vascular smooth muscle cell proliferation and migration in through directly targeting MEF2C.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a critical process in various cardiovascular diseases such as coronary artery disease (CAD), atherosclerosis, stroke, and hypertension. MicroRNAs (miRNAs) are small, short, and noncoding RNAs that inhibit gene expression through binding to the 3'-UTR (3' untranslated regions) of target gene mRNAs. We showed that the expression of miR-448 was upregulated in VSMCs from coronary atherosclerotic plaques compared with normal coronary artery tissues. We also found that PDGF-bb promoted VSMCs proliferation and could induce miR-448 expression. Ectopic miR-448 expression induced VSMCs proliferation. Overexpression of miR-448 induced ki-67 mRNA and protein expression. Moreover, we identified MEF2C was a direct target of miR-448 in VSMCs. Overexpression of miR-448 promoted VSMCs migration. Furthermore, overexpression of MEF2C decreased miR-448-induced VSMCs proliferation and migration. These evidences suggested that miR-448 played an important role in the proliferation and migration of VSMCs.

Glycation of high-density lipoprotein triggers oxidative stress and promotes the proliferation and migration of vascular smooth muscle cells.

BackgroundIn type 2 diabetes mellitus (T2DM), high-density lipoprotein (HDL) impairs its anti-atherogenic properties and even develops to a pro-inflammatory and pro-atherogenic phenotype because of abnormal compositions and modifications. In this study, we examined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells (VSMCs).Methods & ResultsGlycated HDL (G-HDL) was modified with D-glucose (25 mmol/L) in vitro. Diabetic HDL (D-HDL) was isolated from T2DM patients. Rat VSMCs were isolated from the thoracic aortas. Human VSMCs were obtained from ScienCell Research Laboratories. Alpha-actin was detected through immunofluorescence. VSMC proliferation was assayed by Cell Count. VSMC migration was determined by transwell chamber and scratch-wound assay. Intracellular reactive oxygen species (ROS) was detected based on ROS-mediated 2′,7′-dichlorofluorescein (DCFH-DA) fluorescence. Compared to native HDL (N-HDL), G-HDL remarkably promoted VSMC proliferation and migration in the dose and time-dependent manners. In addition, G-HDL enhanced ROS generation in VSMCs. However, the ROS scavenger, N-acetylcysteine, efficiently decreased ROS production and subsequently inhibited the proliferation of VSMCs induced by G-HDL. Similarly, D-HDL from T2DM patients also promoted ROS release and VSMC proliferation and migration.ConclusionsHDL either glycated in vitro or isolated from T2DM patients triggered VSMC proliferation, migration, and oxidative stress. These results might partly interpret the higher morbidity of cardiovascular disease in T2DM patients.

VEGF-A Stimulates STAT3 Activity via Nitrosylation of Myocardin to Regulate the Expression of Vascular Smooth Muscle Cell Differentiation Markers

Vascular endothelial growth factor A (VEGF-A) is a pivotal player in angiogenesis. It is capable of influencing such cellular processes as tubulogenesis and vascular smooth muscle cell (VSMC) proliferation, yet very little is known about the actual signaling events that mediate VEGF-A induced VSMC phenotypic switch. In this report, we describe the identification of an intricate VEGF-A-induced signaling cascade that involves VEGFR2, STAT3, and Myocardin. We demonstrate that VEGF-A promotes VSMC proliferation via VEGFR2/STAT3-mediated upregulating the proliferation of markers like Cyclin D1 and PCNA. Specifically, VEGF-A leads to nitrosylation of Myocardin, weakens its effect on promoting the expression of contractile markers and is unable to inhibit the activation of STAT3. These observations reinforce the importance of nitric oxide and S-nitrosylation in angiogenesis and provide a mechanistic pathway for VEGF-A-induced VSMC phenotypic switch. In addition, Myocardin, GSNOR and GSNO can create a negative feedback loop to regulate the VSMC phenotypic switch. Thus, the discovery of this interactive network of signaling pathways provides novel and unexpected therapeutic targets for angiogenesis-dependent diseases.

Abstract 210: Milk Fat Globule Epidermal Growth Factor VIII Mediates Vascular Remodeling by Increasing Inflammation and Smooth Muscle Activation

Vascular remodeling, defined as a change in the geometry of the vessel wall, occurs in the pathological process of vascular diseases, like atherosclerosis, hypertension and restenosis. The resulting neointimal formation is a part of a reparative response including thrombosis, inflammatory cell infiltration, vascular smooth muscle cell (VSMC) proliferation and migration, which lead to the stenosis of blood vessels and the restricted blood flow. Milk fat globule epidermal growth factor VIII (Mfge8), a secreted glycoprotein, is well-characterized for its capacity of assisting the clearance of apoptotic cells in vascular system. Recently, Mfge8 has been identified as a pivot relay between pro-inflammatory signals and activated VSMCs, contributing to intima-media thickening of the vessel wall by promoting VSMC proliferation and migration in aged arteries. We have noted intense Mfge8 expression in the endothelial cells and VSMCs of the carotid artery following ligation injury in mice, suggesting that Mfge8 may regulate the two characteristics of vascular remodeling, inflammatory cell infiltration and VSMC activation, in response to low blood flow. To elucidate the functions of Mfge8 in a flow-induced model of vascular remodeling, a complete carotid ligation was conducted in wild-type (WT) or Mfge8 knockout (KO) mice. Morphometric analysis demonstrated that genetic deletion of Mfge8 in mice reduces carotid intima and media thickening compared to WT mice. Deficiency of Mfge8 prevented VSMC phenotypic modulation, as evidenced by the decreased expression of smooth muscle myosin heavy chain and attenuated cell proliferation in tunica media after ligation injury. VSMCs transfected with SiRNA against Mfge8 migrated slower than in controls as early as 0.5 days post-platelet-derived growth factor (PDGF) stimulation. Further, Mfge8-null mice showed a dramatic decrease in leukocyte infiltration into the vessel wall. Collectively, in a flow-induced model of vascular remodeling, Mfge8 plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating neointimal formation.

Connexin37 reduces smooth muscle cell proliferation and intimal hyperplasia in a mouse model of carotid artery ligation.

Intimal hyperplasia (IH) is an abnormal response to vessel injury characterized by the dedifferentiation, migration, and proliferation of quiescent vascular smooth muscle cells (VSMC) to form a neointima layer. Vascular connexins (Cx) are involved in the pathophysiology of various vascular diseases, and Cx43, the main Cx expressed in VSMC, has been shown to promote VSMC proliferation and IH. The aim of this study was to investigate the participation of another Cx, namely Cx37, in the formation of the neointima layer. Wild-type (WT) and Cx37-deficient (Cx37-/-) C57BL/6J mice were subjected to carotid artery ligation (CAL), a model of vessel injury and IH. The neointima developed linearly in WT until 28 days post surgery. In contrast, the neointima layer was almost absent 14 days after surgery in Cx37-/- mice, and twice as more developed after 28 days compared to WT mice. This large neointima formation correlated with a two-fold increase in cell proliferation in the media and neointima regions between 14 and 28 days in Cx37-/- mice compared to WT mice. The CAL triggered Cx43 overexpression in the media and neointima layers of ligated carotids in WT mice, and selectively up-regulated Cx37 expression in the media layer, but not in the neointima layer. The de novo expression of Cx37 in human primary VSMC reduced cell proliferation and P-Akt levels, in association with lower Cx43 levels, whereas Cx43 overexpression increased P-Akt levels. The presence of Cx37 in the media layer of injured arteries restrains VSMC proliferation and limits the development of IH, presumably by interfering with the pro-proliferative effect of Cx43 and the Akt pathway.

Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases.

Vascular smooth muscle cells (VSMCs) proliferation is involved in vascular atherosclerosis and restenosis. Recent studies have demonstrated that lipopolysaccharide (LPS) promotes VSMCs proliferation, but the signalling pathways which are involved are not completely understood. The purpose of this review was to summarize the existing knowledge of the role and molecular mechanisms involved in controlling VSMCs proliferation stimulated by LPS and mediated by toll-like receptor 4 (TLR4) signalling pathways. Moreover, the potential inhibitors of TLR4 signalling for VSMCs proliferation in proliferative vascular diseases are discussed.

MiR-29b Downregulation Induces Phenotypic Modulation of Vascular Smooth Muscle Cells: Implication for Intracranial Aneurysm Formation and Progression to Rupture

Background/Aims: Our previous microarray results identified numerous microRNAs (miRNAs), including miR-29b, that were differentially expressed in the serum of intracranial aneurysm (IA) patients. The current study aimed to investigate whether miR-29b downregulation in IA could promote the phenotypic modulation of vascular smooth muscle cells (VSMCs) involved in the pathogenesis of aneurysm by activating ATG14-mediated autophagy. Methods: First, the levels of miR-29b and autophagy related genes (ATGs) between IA patients and normal subjects were compared. Next, we modified the level of miR-29b via lentivirus particles in the VSMCs and examined the effects of miR-29b on proliferation, migration, and phenotypic modulation of VSMCs from a contractile phenotype to a synthetic phenotype, as well as the levels of autophagy. Finally, the binding of miR-29b to the 3’UTR of ATG14 mRNA and its effects on ATG14 expression were analysed by a luciferase reporter assay and Western blot, respectively. Results: The level of miR-29b was decreased, and autophagy markers were increased in the IA patients compared to that of the normal subjects. Knockdown of miR-29b significantly promoted VSMCs proliferation and migration and, more importantly, induced the phenotypic modulation associated with autophagy activation, whereas miR-29b overexpression showed the opposite effects. The luciferase reporter assay demonstrated that ATG14 was a functional target gene of miR-29b. Notably, knockdown of ATG14 by siRNA apparently abrogated miR-29b inhibition-mediated phenotypic modulation. Conclusion: Downregulation of miR-29b induced VSMCs phenotypic modulation by directly activating ATG14-mediated autophagy, which is associated with the formation, growth and rupture of IAs.

HHEX: A Crosstalker between HCMV Infection and Proliferation of VSMCs.

Objective: The study was designed to evaluate the role of Human cytomegalovirus (HCMV) infection on homebox (HOX) gene expression and the effects of overexpression of HOX genes on proliferation and apoptosis of vascular smooth muscle cells (VSMCs).Methods: Viral infection was verified by observation of cytopathic effects through inverted microscopy, viral particles by electron microscopy and HCMV IE gene amplification by RT-PCR. cDNA profiling technology was used to screen expression of HOX genes after HCMV infection in VSMCs. Abnormal expression of Haematopoietically-expressed homeobox (HHEX) was selected to construct over-expressed vector and transfected into VSMCs. The effects of over expression of HHEX on cell proliferation and apoptosis of VSMCs were assayed by flow cytometry. Apoptosis and proliferation-associated genes were also assayed by RT-PCR.Results: Multiple HOX gene expression levels had obvious changes after HCMV infection, among which expression of HHEX gene increased obviously at 24, 48, and 72 h after infection. Over expression of HHEX can promote VSMCs proliferation by promoting G0/G1 phase cells into S phase and inhibit VSMCs apoptosis. HHEX inhibited the expression of apoptosis-associated caspase 2 and caspase3 and promoted the expression of cell cycle-related genes such as CDK2 and CDK6, CyclinB2 and CyclinD2.Conclusion: HHEX over expression induced by HCMV infection closely associated with vascular proliferative diseases.

Effects of Astragaloside IV on tumor necrosis factor-α-induced expressions of matrix metalloproteinases in rat vascular smooth muscle cells

Objective To investigate the effect of Astragaloside IV (AS-IV) on tumor necrosis factor-α (TNF-α)-induced expressions of matrix metalloproteinases (MMPs) in a rat vascular smooth muscle cells (VSMCs) proliferation model and its mechanism. Methods VSMCs were prepared from the thoracoabdominal aorta of rats by using issue-sticking method. Morphology of cells was observed by inverted microscope, and identified by immunohistochemical methods with antibody against SM-α-actin. The model of VSMCs proliferation and migration was established by TNF-α inducer in vitro, and randomly divided into the following groups: the control group, the TNF-α group, the TNF-α+ AS-IV (0.5 μg/ml) group, the TNF-α+ AS-IV (5 μg/ml) group, the TNF-α+ AS-IV (25 μg/ml) group, and the TNF-α+ AS-IV (50 μg/ml) group. The effect of AS-IV on TNF-α-induced VSMCs proliferation activity was detected by the caerulein and cholecystokinin octapeptide (CCK-8) method. The quantitative real-time polymerase chain reaction (real-time PCR) and Western blotting were used to examine the effects of AS-IV on the VSMCs-secreted mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2), respectively. Results The proliferative activity, migratory distance and invasive capacity of VSMCs were obviously increased in TNF-α stimulation group versus in control group (all P<0.01), which suggested that TNF-α can promote VSMCs proliferation and migration, and that the rat model of VSMCs proliferation in vitro was successfully established. The results of CCK-8 tests showed that VSMCs proliferation was obvious and the optical density (OD) value was elevated (P<0.01) after a preset time incubation with TNF-α. VSMCs proliferation was inhibited in each AS-IV treatment group, and the OD value was decreased as compared with the TNF-α group. And the inhibitive effect was increased along with the increments of AS-IV concentration and the acting time, which indicated that AS-IV can inhibit TNF-α-induced VSMCs proliferation in a time- and dose-dependent manner. The results of real-time PCR and Western blotting assays indicated that TNF-α changed the ratio of MMPs to the tissue inhibitors of metalloproteinases (TIMPs) by down-regulating active MMP-2 expression without influencing proMMP-2 and TIMP-2 expressions, and thus promoted the degradation of ECM. AS-IV (0.5-50 μg/ml) inhibited VSMCs proliferation and migration by down-regulating the TNF-α-induced MMP-2 overexpression in a dose-dependent manner, up-regulating the mRNA and protein expressions of TIMP-2, and modulating the ratio of MMPs to TIMPs, thereby inhibited the degradation of ECM. Conclusions AS-IV inhibits VSMCs proliferation and migration in a time- and dose-dependent manner. AS-IV inhibits VSMCs proliferation and migration by down-regulating TNF-α-induced MMP-2 overexpression, up-regulating TIMP-2 expressions, and normalizing the ratio of MMPs to TIMPs. Therefore, AS-IV inhibits the degradation of ECM, which may play a role in the prevention and treatment of in-stents restenosis after PCI. Key words: Astragalus; Muscle, smooth, vascular; Tumor necrosis factor-alpha; Matrix

MiR-599 Inhibits Vascular Smooth Muscle Cells Proliferation and Migration by Targeting TGFB2.

Aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of cardiovascular diseases including coronary heart disease, restenosis and atherosclerosis. MicroRNAs are a class of small, non-coding and endogenous RNAs that play critical roles in VSMCs function. In this study, we showed that PDGF-bb, as a stimulant, promoted VSMCs proliferation and suppressed the expression of miR-599. Moreover, overexpression of miR-599 inhibited VSMCs proliferation and also suppressed the PCNA and ki-67 expression. In addition, we demonstrated that ectopic expression of miR-599 repressed the VSMCs migration. We also showed that miR-599 inhibited type I collagen, type V collagen and proteoglycan expression. Furthermore, we identified TGFb2 as a direct target gene of miR-599 in VSMCs. Overexpression of TGFb2 reversed miR-599-induced inhibition of VSMCs proliferation and type I collagen, type V collagen and proteoglycan expression. In conclusion, our findings suggest miR-599 plays a crucial role in controlling VSMCs proliferation and matrix gene expression by regulating TGFb2 expression.

Tissue factor pathway inhibitor gene transfer prevents vascular smooth muscle cell proliferation by interfering with the MCP-3/CCR2 pathway

Increased vascular smooth muscle cell (VSMC) proliferation substantially contributes to the pathogenesis of atherosclerosis and intimal hyperplasia after vascular injury. The importance of inflammation in VSMC proliferation is now being recognized. Preventing the inflammatory response is one therapeutic strategy that can be used to inhibit atherosclerosis in the clinic. The present study, using RNA interference and gene transfer techniques, was conducted to investigate the effect of monocyte chemotactic protein-3 (MCP-3) on VSMC proliferation that is a result of TNF-α stimulation, and whether overexpression of the tissue factor pathway inhibitor (TFPI) gene could prevent VSMC proliferation by blocking the MCP-3/CC chemokine receptor 2 (CCR2) pathway. Mouse VSMCs were infected in vitro with recombinant adenoviruses containing either mouse MCP-3-shRNA (Ad-MCP-3-shRNA), the TFPI gene (Ad-TFPI), or the negative control, which was shRNA encoding the sequence for EGFP (Ad-EGFP) or DMEM only. The cells were then stimulated with TNF-α for different time periods on the third day after gene transfer. The data show that VSMC proliferation in the Ad-MCP-3-shRNA and Ad-TFPI groups was markedly decreased using BrdU ELISA and MTT assays; MCP-3-shRNA and TFPI inhibited the expression of MCP-3 and CCR2 after long-term stimulation and inhibited the phosphorylation of ERK1/2 and AKT after short-term stimulation, as shown by ELISA and western blot analysis. This study provides convincing evidence that clarifies the effect of the proinflammatory factor MCP-3 in promoting VSMC proliferation. Our data also show, for the first time, that TFPI has an anti-proliferative role in TNF-α stimulated-VSMCs at least partly by interfering with the MCP-3/CCR2 pathway and then via suppression of the ERK1/2 and PI3K/AKT signaling pathways. We conclude that TFPI gene transfer may be a safe and effective therapeutic tool for treating atherosclerosis and intimal hyperplasia.

MiRNA-34a reduces neointima formation through inhibiting smooth muscle cell proliferation and migration

AimsWe have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells in vitro and in vivo. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. Methods and resultsVarious pathological stimuli regulate miR-34a expression in VSMCs through a transcriptional mechanism, and the P53 binding site is required for miR-34a gene regulation by these stimuli. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodeling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1–3′-UTR-luciferase activity in VSMCs, but not mutant Notch1–3′-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, the expression level of miR-34a was significantly down-regulated in injured arteries, and miR-34a perivascular over-expression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. ConclusionOur data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.

MicroRNA-10b Induces Vascular Muscle Cell Proliferation Through Akt Pathway by Targeting TIP30.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes significantly to the pathogenesis of atherosclerosis. MiR-10b has recently emerged as a critical mediator in regulating cell proliferation in many diseases. In our study, miR-10b expression was up-regulated in VSMCs isolated from atherosclerotic plaques, as well as in PDGFstimulated VSMCs.Overexpression of miR-10b promoted cell proliferation of VSMCs. Furthermore, we identified the Tat-interacting protein 30 (TIP30) as a direct target gene of miR-10b. TIP30 was down-regulated in VSMCs isolated from atherosclerosis plaques, as well as in proliferative VSMCs. Knockdown of TIP30 promoted VSMCs proliferation. In addition, miR-10b induced TIP30 down-regulation was accompanied by increased Akt phosphorylation. Akt was critical for miR-10b-mediated VSMCs proliferation. Our results demonstrated that miR-10b contributed to abnormal VSMCs proliferation through inhibiting the Akt pathway by targeting TIP30 in atherosclerosis. The modulation of miR-10b in VSMCs provides a potential target for the therapy of atherosclerosis.

MicroRNA-15b/16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP.

To investigate the functional role of the microRNA (miR)-15b/16 in vascular smooth muscle (SM) phenotypic modulation. We found that miR-15b/16 is one of the most abundant mRs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs get converted to a synthetic phenotype, miR-15b/16 expression is significantly reduced. Knocking down endogenous miR-15b/16 in VSMCs attenuates SM-specific gene expression but promotes VSMC proliferation and migration. Conversely, overexpression of miR-15b/16 promotes SM contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, overexpression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced SM dedifferentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP's 3' untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in upregulation of YAP expression. These results indicate that miR-15b/16 plays a critical role in SM phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.

Study on the action mechanism of basic fibroblast growth factor on vascular smooth muscle cells in spontaneously hypertensive rats

Objective To explore the action mechanism of basic fibroblast growth factor (bFGF) on vascular smooth muscle cells (VSMCs) in spontaneously hypertensive rats (SHR). Methods Ts were obtained from SHR and SD rats. The aortic VSMCs were cultured in vitro by tissue explant method. VSMCs were treated with different concentration of exogenous bFGF (0 ng/ml, 20 ng/ml, 40 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml) for 48 h, then cell proliferation was detected by 3 (4, 5 dimethylthiazol)2, 5 diphenyltetrazolium bromide (MTT) colorimetric assay. VSMCs from SHR in control group were treated with bFGF (100 ng/ml) for 48h. VSMCs from SHR in treatment group were treated with bFGF (100 ng/ml) plus Proteinkinase C(PKC) inhibitor (staurosporine) for 48 h. Results After treatment with different concentration (0 ng/ml, 20 ng/ml, 40 ng/ml, 60 ng/ml, 80 ng/ml, 100 ng/ml) of bFGF for 48 h, the values measured by MTT colorimetric method were 0.402±0.103, 0.605±0.090, 0.696±0.131, 0.812±0.080, 0.901±0.065, 1.056±0.078 respectively in aortic VSMCs from SD rats, and 0.404±0.065, 0.507±0.078, 0.608±0.057, 0.704±0.107, 0.812±0.097, 0.908±0.032 respectively in aortic VSMCs from SHR. Compared with control group, the values measured by MTT colorimetric method were decreased in treatment group (P<0.05). The proliferative effect of bFGF in aortic VSMCs from SHR was attenuated after administration of PKC inhibitor staurosporine. Conclusions Exogenous bFGF administration promotes VSMCs proliferation in SHR and SD rats in a concentration-dependent manner. PKC plays an important role in the signal transduction mechanism in VSMCs proliferation by exogenous bFGF. Key words: Muscle, smooth, vascular; Basic fibroblast growth factor; Protein kinase C

211 Mirna-34a Reduces Neointima fommation through Inhibiting Smooth Muscle Cell Proliferation and Migration

<h3>Background/objective</h3> We have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells <i>in vitro</i> and <i>in vivo</i>. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. <h3>Methods and results</h3> miR-34a was significantly down-regulated in VSMCs upon various pathological stimuli, and in injured arteries. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodelling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1-3’-UTR-luciferase activity in VSMCs, but not mutant Notch1-3’-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, miR-34a perivascular overexpression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. <h3>Conclusions/implications</h3> Our data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.