Influence of Angiotensin II on α1-Adrenergic Receptors Function in Rat Aorta and Expression in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT1 and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α1-adrenergic receptors (α1-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10−5 M), and in some experiments, 5-Methylurapidil (α1A-AR antagonist), AH11110A (α1B-AR antagonist), or BMY-7378 (α1D-AR antagonist), were used to identify the α1-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α1D-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α1-ARs proteins were evaluated. α1D-AR increased at 30 and 60 min post Ang II exposure, the α1A-AR diminished from 1 to 4 h, while α1B-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α1D-AR at short times, and BMY-7378 protected α1D-AR from desensitization.

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.

Inhibitory Effect of PPARδAgonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway

Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1αexpression

Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.

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.

Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

Effects of Tetrandrine on Cytosolic Free Calcium in Cultured Bovine Aortic Smooth Muscle Cells

利用Ｆｕｒａ２技术和ＡＲＣＭＭＩＣ阳离子系统研究了粉防己碱对传代培养牛主动脉血管平滑肌细胞内游离钙的影响。粉防己碱（１～１００μｍｏｌ·Ｌ－１）不影响血管平滑肌细胞的静息钙，但在有胞外钙存在时，对高钾，５羟色胺，ＡＴＰ，血管紧张素Ｉ，去甲肾上腺素引起的胞内钙升高有抑制作用；在无胞外钙存在时，粉防己碱对苯肾上腺素引起的胞内钙升高也有一定的抑制作用。粉防己碱对高钾升高内钙的抑制作用呈剂量依赖性，ＩＣ５０值为９．２（９５％可信限：５．７～１４．９）μｍｏｌ·Ｌ－１。上述结果提示粉防己碱对血管平滑肌细胞的电压依赖性钙通道和受体调控性钙通道均有阻滞作用，对受体调控性钙通道阻滞作用主要表现为阻滞钙离子内流。

Overexpression of heme oxygenasel protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-l) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation,we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector.The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-l, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism

This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.

Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis)

A new cell line, CSEC, has been successfully established from embryos at gastrula stage of a cultured marine fish, half smooth tongue sole （Cynoglossus sernilaevis）. CSEC cells grow actively and stably more than 50 passages for over 200 d in DMEM medium supplemented with 15% FBS （fetal bovine serum）, 2.5 ng/cm^3 bFGF （basic fibroblast growth factor）, 1 ng/cm^3 LIF （leukemia inhibitory factor） and 50 mmol/dm^3 2-ME （2-mecaptoethanol）. The cells grew well in the temperature range of 24-30 ℃ and the optimal growth temperature was 24 ℃. FBS and bFGF concentrations are the two key components for CSEC cells proliferation. Chromosome analysis reveals that CSEC cells have a normal diploid karyotype with 2n=42t. The significant fluorescent signals were observed in CSEC after transfection with the GFP reporter gene, suggesting that the CSEC cell line can be used as a useful tool for transgenic and genetic manipulation studies. CSEC cells showed the cytopathic effect （CPE） after infection with lymphosystis disease virus （LCDV） in 2 d. Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy. It suggests that CSEC could be potentially used for the study of aquatic virus.

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.

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 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.

Influence of Oxidized Low Density Lipoprotein on the Proliferation of Human Artery Smooth Muscle Cells in vitro

The effects of oxidized low density lipoprotein （ox-LDL） on the proliferation of cultured human vascular smooth muscle cells （vSMC） were investigated in vitro. By using NaBr density gradient centrifugation, LDL was isolated and purified from human plasma. Ox-LDL was produced from LDL by being incubated with CuSO4. ox-LDL was then added to the culture medium at different concentrations （35, 60, 85, 110, 135 and 160μg/mL） for 7 days. The influence of ox-LDL on vSMC proliferation was observed in growth curve, mitosis index, and in situ determination of apoptosis. The data were analyzed with SPSS 10.0 software. The results showed that the ox-LDL produced in vitro had a good purity and optimal oxidative degree, which was similar to the intrinsic ox-LDL in atherosclerotic plaque, ox-LDL at a concentration of 35 μg/mL demonstrated the strongest proliferation inducement, and at a concentration of 135 μg/mL, ox-LDL could inhibit the growth of vSMC. ox-LDL at concentrations of 35 and 50 μg/mL presented powerful mitotic trigger, and with the increase of ox-LDL concentration, the mitotic index of vSMC was decreased gradually, ox-LDL at higher concentrations promoted more apoptotic vSMCs, ox-LDL at lower concentrations triggered proliferation of vSMCs, and at higher concentrations induced apoptosis in vSMCs, ox-LDL played a promotional role in the pathogenesis and development of atherosclerosis by affecting vSMC proliferation and apoptosis.

Down-regulation of the gax gene in smooth muscle cells of the splenic vein of portal hypertension patients

BACKGROUND: The expression of gax, an anti-prolifera-tive homeobox gene, is rapidly down-regulated in vascular smooth muscle cells (VSMCs) following arterial injury. Whether the down-regulation of gax is involved in modulating the proliferation of smooth muscle cells of the splenic vein in patients with portal hypertension has not yet been elucidated. The aim of this study was to investigate the expression of the mRNA of the gax gene in smooth muscle cells of the splenic vein in patients with portal hypertension. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of gax mRNA and immunohistochemistry staining was performed to demonstrate the expression of PCNA protein in the splenic veins of 28 patients with portal hypertension and those of 12 normal controls. This study was approved by the Institutional Ethics Committee and informed consent was obtained from all participants. RESULTS: RT-PCR showed that the expression of gax mRNA was PCNA-positive and negative in the splenic vein of patients with portal hypertension (1.08 ± 0.04 and 1.39 ± 0.27, respectively). There was a significant difference in the 28 patients compared with the 12 normal controls ( P < 0.01). The relative expression of PCNA protein in the vascular tissues was significantly higher in the experimental group than in the control group. CONCLUSIONS: Down-regulation of gax mRNA and the overexpression of PCNA protein were seen in smooth muscle cells of the splenic vein in patients with portal hypertension, regulating the proliferation, migration and phenoty-pic change and resulting in remodelling of the splenic vein, which may play an important role in the pathogenesis and maintenance of portal hypertension.

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 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.

Iptakalim, a novel ATP-sensitive potassium channel opener, inhibits pulmonary arterial smooth muscle cell proliferation by downregulation of PKC-α

Iptakalim is a new ATP-sensitive potassium （KATp） channel opener, and it inhibits the proliferation of pulmonary arterial smooth muscle cells （PASMCs） and pulmonary vascular remodeling. However, the underlying mechanism remains unclear. In the present study, we found that iptakalim significantly decreased pulmonary artery pressure, inhibited pulmonary ariery remodeling and PKC-α overexpression in chronic hypoxia in a rat pulmonary hypertension model. Iptakalim reduced hypoxia-induced expression of PKC-α, and abolished the effect of hypoxia on PASMC proliferation significantly in a dose-dependent manner in vitro. Moreover, these effects were abol- ished by glibenclamide, a selective KArp channel antagonist. These results indicate that iptakalim inhibits PASMC proliferation and pulmonary vascular remodeling induced by hypoxia through downregulating the expression of PKC-α. Iptakalim can serve as a novel promising treatment for hypoxic pulmonary hypertension.

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.