STUDY ON THE INHIBITORY EFFECT OF ANTISENSE ET_AR OLIGODEOXYNUCLEOTIDES ON THE PROLIFERATION OF VASCULAR SMOOTH CELLS

Objective To study the inhibitory effect of antisense endothelin receptor A (ET-AR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ET-AR were designed and synthetized. The study was carried out using MTT method and binding assays. Results ET-AR-ODNs could move successfully across VSMC membranes. Photo-absorption in the MTT test was reduced significantly (P<0.05) in the antisense group at 5μmol/L; the reduction of CPM also occurred in the 125 I-ET-1 specific binding assay; and the sense and mismatched ODNs groups did not show this reduction. Conclusion Our study suggested that the antisense oligomers inhibited the proliferation of VSMCs by hindering the translation of target mRNA and by reducing the production of related protein.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

Semaphorin 7A promotes human vascular smooth muscle cell proliferation and migration through theβ-catenin signaling pathway

Background:Vascular smooth muscle cells(VSMCs)undergo a conversion from a contractile phenotype to a proliferative synthetic phenotype,contributing to the pathogenesis of cardiovascular diseases.Semaphorin 7A(SEMA7A)is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in vascular homeostasis by regulating endothelial cell behaviors.However,the expression and role of SEMA7A in VSMCs remain unclear.Methods:In this study,we screened for VSMC-regulating genes in publicly available datasets and analyzed the expression of SEMA7A in human coronary artery smooth muscle cells(hCASMCs)treated with platelet-derived growth factor-BB(PDGF-BB).The effects of SEMA7A overexpression and knockdown on hCASMC proliferation and migration were examined.The signaling pathways involved in the action of SEMA7A in hCASMCs were determined.Results:Bioinformatic analysis showed that SEMA7A was significantly dysregulated in VSMCs treated with oxidized low-density lipoprotein or overexpressing progerin,a pro-atherogenic gene.The PDGF-BB stimulation led to a concentration-and time-dependent induction of SEMA7A.Depletion of SEMA7A attenuated PDGF-BB-induced hCASMC proliferation and migration.Conversely,overexpression of SEMA7A enhanced hCASMC proliferation and migration.Mechanistically,SEMA7A stimulated the activation of theβ-catenin pathway and upregulated c-Myc,CCND1,and MMP7.Knockdown ofβ-catenin impaired SEMA7A-induced hCASMC proliferation and migration.Conclusions:SEMA7A triggers phenotype switching in VSMCs through theβ-catenin signaling pathway and may serve as a potential therapeutic target for cardiovascular diseases.

A bioinformatics-based study of the mechanism of JQ-1 on BET protein and atherosclerosis induced by vascular smooth muscle cell proliferation

Background:Based on previous theoretical studies,JQ-1 as a common inhibitor of bromodomain and extraterminal(BET)proteins was used to treat a variety of diseases.Therefore,we aimed to explore the mechanism of action of JQ-1 on BET proteins based on bioinformatics and build the novel hypothesis of JQ-1 in treating atherosclerosis(AS)caused by proliferation of vascular smooth muscle cells(VSMCs).Methods:We selected the chip GSE138323 which was searched with the key words“Vascular smooth muscle cell proliferation”in Gene Expression Omnibus(GEO)database,and differential gene analysis was performed between the GRO and JQ-1 groups.Then the top twenty significantly up-regulated genes and the top twenty significantly down-regulated genes were selected for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Thirdly,structured the PPI network of forty differential genes,and the core genes were screened by using the MCC algorithm which in“Cytohubba”plugin in the Cytoscapev3.9.1 software.After that,single gene Gene Set Enrichment Analysis(GSEA)enrichment analysis was performed on the selected core genes in R language.Finally molecular docking validation was performed.Results:Five core genes was selected:H3C2,H3C4,H3C7,H3C10 and AREG.The GO enrichment analysis results showed that there were twenty-five entries in biological process,eight entries in cellular components(CC),and twenty-five entries in molecular function.The KEGG enrichment analysis results showed that there were seven pathways,mainly including systemic lupus erythematosus and external neutrophil trap formation.The GSEA results showed that the five genes were mainly through the regulation of cytochrome P450 metabolism,PPAR signaling pathway and other pathways.The molecular docking results showed that JQ-1 had binding activity with these five genes.Conclusions:JQ-1 may regulate the expression of the genes that H3C2,H3C4,H3C7,H3C10 and AREG,to mainly regulate the genes in cytochrome P450 metabolism,PPAR singling pathway and other pathways,to make some influence in the proliferation of VSMCs,and improved atherosclerotic symptoms due to vascular smooth muscle proliferation,thus treating cardiovascular disease.

Urocortin, the neuropeptide, inhibits the viability of ECV304 cells and rat vascular smooth muscle cells

Objective: This study aims to investigate the effects of urocortin (Ucn) on the viability of endothelial cells (ECV304) and rat vascular muscle cells (VSMC). Methods: Rat aortic VSMC were isolated from the rats' thoracic aorta. We studied the effect of Ucn on the viability of ECV304 cells and VSMC by using a tetrazolium (MTT) assay.Results: Ucn (10 -7 mol/L) inhibited the viability of ECV304 cells and VSMC. Inhibition rates are 13% and 15%, respectively(P<0.05, compared with Control). This inhibition was not dependent on the affecting time and was not affected by the addition of ATP-sensitive potassium channel (KATP channel) blocker, glybenclamide (Gly, 10 mol/L). Conclusion: Ucn inhibits the viability of ECV304 and VSMC. Our results suggest that Ucn may be a new vasoactive agent and may have a beneficial effect in the process of vascular remodeling (VR).

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 pro- liferation, 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 recep- tor [~ （PDGFR~）-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 PDGFR^-ERK1/2 signaling cascade.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.

Inhibitory Effects of Saponins From Anemarrhena asphodeloides Bunge on the Growth of Vascular Smooth Muscle Cells

Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge （SAaB） （Botanical Name： Anemarrhena Asphodeloidis Rhizoma） on the growth of vascular smooth muscle cells （VSMCs）. Methods Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate （DAF-2, DA）. Cell aldose reductase （AR） activity, as well as the effect of Epalrestat and interleukin-1β were also explored. Results WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB （P〈0.01）. Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs （P〈0.01）. Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-Iβ Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat （P〈 0.01）. Conclusion SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.

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

Summary： 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） path- way 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 -T＋SiRNA-control group. The pYrAd-rSOCS3 and SiRNA-rSOCS3 were transfected into VSMCs in- duced 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 re- lated-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 be- fore 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.

Yangxueqingnao particles inhibit rat vascular smooth muscle cell proliferation induced by lysophosphatidic acid

Objective: To observe the effect of Yangxueqingnao particles on rat vascular smooth muscle cell (VSMC) prolif- eration induced by lysophosphatidic acid (LPA). Methods: The amount of 3H-TdR (3H-thymidine) admixed in cultured rat VSMC was measured and mitogen-activated protein kinase (MAPK) activity and lipid peroxidation end product malondialdehyde (MDA) content of the VSMC were assayed. Results: 1×10?9, 1×10?8, 1×10?7 mol/L LPA in a concentration dependent manner, induced the amount of 3H-TdR admixed, MAP kinase activity, and MDA content of the cultured rat VSMC to increase. However, 5%, 10%, and 15% Yangxueqingnao serum preincubation resulted in a decrease of 23.0%, 42.0%, and 52.0% (P<0.01) respectively in the amount of 3H-TdR admixed, a decline in VSMC MAP kinase activity of 13.9% (P<0.05), 29.6% (P<0.01), and 48.9% (P<0.01) respectively, and also, a decrease in MDA content of VSMC of 19.4%, 24.7%, and 43.2% (P<0.01) respectively, in the 1×10?7 mol/L LPA-treated VSMC. Conclusions: LPA activates the proliferation and lipid peroxidation of VSMC in a concentration dependent manner. The LPA-induced VSMC proliferation is related to the activity of MAP kinases, enzymes involved in an intracellular signalling pathway. The results of the present study showed that Yangxueqingnao particles can effectively inhibit LPA-induced VSMC proliferation, MAP kinase activation, and reduce lipid peroxidative lesion.

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine （Hcy）-induced tissue factor （TF） expression in human vascular smooth muscle cells （VSMCs） and the effect of Hcy on the activity of nuclear factor-kappaB （NF-кB） and the expression of inducible nitric oxide synthase （iNOS） were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC （NF-кB inhibitor）. Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

Valsartan Inhibits Angiotensin Ⅱ-induced Proliferation of Vascular Smooth Muscle Cells via Regulating the Expression of Mitofusin 2

Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

Different Responses of Cell Cycle between Rat Vascular Smooth Muscle Cells and Vascular Endothelial Cells to Paclitaxel

Summary： Although previous reports showed dmg-eluting stent （DES） could effectively inhibit neointima formation, in-stent restenosis （ISR） remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells （VSMCs） and vascular endothelial cells （VECs） of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, prolif- eration of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1 S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.

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

Background In 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 ex- amined the effects and the related mechanisms of glycation of HDL on the proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods ＆ Results Glycated 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 immunofiuorescence. 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-medi- ated 2＇,7＇-dichlorofluorescein （DCFH-DA） fluorescence. Compared to native HDL （N-HDL）, G-HDL remarkably promoted VSMC prolif- eration 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. Conclusions HDL 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.

Resveratrol inhibits angiotensin II-induced ERK1/2 activation by downregulating quinone reductase 2 in rat vascular smooth muscle cells

Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells （VSMCs） and repress mRNA and protein expression of quinone reductase 2 （NQO2）. This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs. Lentiviral vectors that incorporated NQO2 small interfering RNA （siRNA） were constructed and transduced into rat VSMCs. The cell proliferation was detected using the bromodeoxyuridine （BrdU） assay. Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species （ROS） was measured using a ROS assay kit. A realtime quantitative PCR was used to detect NQO2 mRNA levels. Extracellular signal-regulated kinase （ERK1/2） and NQO2 protein expression were determined by Western blotting analysis. The inhibitory effect of resveratrol （10 and 50 μmol/L） on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group （P 〈 0.01）. The ROS level in the NQO2 siRNA and resveratrol （50 μmol/L） treatment groups were lower than that in the normal and scrambled siRNA groups （P 〈 0.01 in both）. Compared with the normal and scrambled siRNA group, the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol （50 μmol/L） treatment group （P 〈 0.01 in both）. In conclusion, high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.

Effect of Amlodipine on the Growth of Vascular SmoothMuscle Cells of Spontaneously Hypertensive Rats

The effect of anti-hypertensive drug amlodipine on regression of cardiovascular hypertrophy due to hypertension was studied by using cultured smooth muscle cells derived from arteries of spontaneously hypertensive rats (SHR) and measuring [3H]-TdR and [3H]-Leucine binding. 48 h after adding amlodipine.[3H] -TdR binding in arterial smooth muscle cells from SHR in vitro was reduced by 50.5% and [3H]-Leucine binding was reduced by 56. 2% as compared with neuropeptide Y (NPY)-treated group. However, there was no significant change in cell number. The results showed that amlodipine could effectively inhibit increase of DNA and protein synthesis of vascular smooth muscle cell (VSMC) due to NPY. It indicates that amlodipine is of great significance on regression of genesis and development of cardiovascular hypertrophy due to hypertension.

Effects of high glucose on expression of OPG and RANKL in rat aortic vascular smooth muscle cells

Objective:To explore effect of high glucose on expression of osteoprotegerin(OPG) and receptor activator of NF- κB ligand(RANKL) in rat aortic vascular smooth muscle cells.Methods:SD rats were intraperitoneally injected with streptozotocin,OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining.In cultured vascular smooth muscle cells(VSMCs)(A7r5),qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL.Results:Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs.while RANKL expression was decreased.Besides,in vitro experiments high glucose induced OPG expression,but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5.Conclusions:Our findings suggested that high glucose could promote the expression of OPG,and inhibit the expression of RANKL in VSMCs,which may be partly be the molecular mechanism of diabetic vascular calcification.

Microscopic study of ultrasound-mediated microbubble destruction effects on vascular smooth muscle cells

Objective:To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy(AFAM).Methods:A7r5 rat aortic smooth muscle cells were divided into groups:control group(without ultrasonic irradiation,no micro bubbles)and US+MB group(45 kHz、0.4 W/cm^2 ultrasound irradiate for 20 seconds with a SonoVuc^(TM)concentration of[(56-140)×10~5/mL].Cell micromorphological changes(such as topographic and acoustic prognosis)were detected,before and after ultrasound destruction by AFAM.Results:In cell morphology,smooth muscle cells were spread o and connected to each another by fibers.At the center of the cell,the nuclear area had a rough surface and was significantly elevated from its surroundings.The cytoskeletal structure of the reticular nucleus and cytoplasm in the morphology of A7r5 cells(20μm×20μm)were clear before microbubble intervention.After acoustic exciting,the cell structure details of the acoustic image were improved with better resolution,showing the elasticity of different tissues.In the acoustic image,the nucleus was harder,more flexible and uneven compared with the cytoplasm.Many strong various-sized echo particles were stuck on the rough nuclear membrane's substrate surface.The nuclear membrane did not have a continuous smooth surface;there were many obstructions(pores).After ultrasound-intervention was combined with microbubbles,the dark areas of the A7r5 cell images was increased in various sizes and degrees.The dark areas showed the depth or low altitudes of the lower regions,suggesting regional depressions.However,the location and scope of the acoustic image dark areas were not similar to those found in the topographic images.Therefore,it was likely that the dark areas,both from the topographic and acoustic images,were sound-holes.In addition,some cell nuclei become round in different degrees after irradiation.Conclusions:Atomic force microscopy and acoustic excitation method can noninvasively and completely display a cell's structure,connections and elastic properties at a nano scale in just several minutes.The dark areas,both from the topographic and acoustic images,may be sound-holes;therefore,it would be helpful if these sound-holes were found.These findings provide a relationship between cell apoptosis after ultrasound and microbubble ultrasound irradiation,and the sound-hole effect.

Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells （VSMCs） in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-α- actin monoclonal antibody before serial subcuhivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 （CCK-8） in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Success- ful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells （F=40. 154, P〈0.01）. It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.