Adv-shRNA抑制NRP2表达对胃癌SGC7901细胞增殖的影响

目的通过特异性抑制胃癌细胞株SGC7901中神经纤毛蛋白2(NRP2)基因的表达,检测RNA干扰片段对SGC7901细胞增殖能力的影响。方法将NRP2的特异性RNA干扰腺病毒载体转染至胃癌细胞SCG7901 72 h后荧光显微镜观察转染情况;采用RT-PCR法检测转染后NRP2基因mRNA表达情况;MTT法检测胃癌细胞增殖情况;Western印迹检测转染后NRP2蛋白表达情况。结果成功将NRP2-shRNA重组腺病毒载体转染至胃癌细胞SGC7901。各重组腺病毒转染组NRP2 mRNA水平均低于阴性对照组和空白组,其中以NRP2-shRNA-3抑制效应最强。阴性对照组与空白组细胞增殖迅速,两组间无显著差异(P>0.05),实验组经腺病毒载体转染后细胞增殖程度显著减少,与前两组相比有显著差异(P<0.05)。阴性对照组与空白组NRP2蛋白表达量明显高于实验组(P<0.01),而阴性对照组与空白组之间无显著差异(P>0.05)。结论重组腺病毒载体NRP2-shRNA可有效抑制胃癌细胞SCG7901中NRP2基因的mRNA和蛋白的表达,并在一定程度上抑制癌细胞增殖,可作为动物实验的理论基础和前期条件。     

RNA干扰真核表达载体对前列腺癌细胞CXCR4基因表达的抑制作用

目的 研究RNA干扰(RNAi)真核表达载体对前列腺癌细胞趋化因子CXC受体4(CXCR4)基因的抑制作用.方法 构建针对CXCR4基因的RNA干扰质粒表达载体,采用脂质体法转染前列腺癌PC-3m、LNCaP细胞系,半定量逆转录聚合酶链反应(RT-PCR)、Western blot法检测其对CXCR4 mRNA及蛋白表达的影响.结果 构建的重组真核表达载体在前列腺癌PC-3m、LNCaP细胞系中均抑制了CXCR4 mRNA及蛋白表达.与空白载体组细胞作对照,PC-3m细胞中小干扰RNA(siRNA)对CXCR4 mRNA的抑制率24、48 h分别为(87.8±10.2)%、(56.1±9.3)%,LNCaP细胞中siRNA对CXCR4 mRNA的抑制率分别为(56.9±8.8)%、(49.2±11.2)%,siRNA对PC-3m和LNCaP细胞蛋白抑制率,于24 h,前者为(64.7±6.7)%,后者为(58.7±11.6)%,与阴性载体转染组细胞比较,两组差异有统计学意义(P＜0.05).结论 RNA干扰真核表达载体可以有效地抑制两种前列腺癌细胞PC-3m、LNCaP细胞中CXCR4基因的表达.     

血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用

目的观察血管紧张素Ⅱ(AngⅡ)对小鼠主动脉血管平滑肌细胞(VSMC)自噬的影响以及自噬对细胞表型转换的调控作用。方法原代培养小鼠VSMC,用10~(-6)mol/L AngⅡ作用VSMC不同时间,采用Western blot检测微管相关蛋白轻链-3-Ⅱ(LC3-Ⅱ)的表达以观察AngⅡ对VSMC自噬的影响,透射电镜观察对照组及AngⅡ组的自噬小体。用Western blot检测自噬抑制剂3-MA和Baf-A1干预后对AngⅡ诱导自噬及细胞表型转换的影响。使用siRNA抑制自噬相关基因Atg7的表达,qRT-PCR检测转染后Atg7的表达变化,Western blot检测转染siRNA Atg7后对LC3-Ⅱ及细胞表型蛋白标志物的影响。结果 AngⅡ以时间依赖方式促进LC3-Ⅱ表达,自噬抑制剂3-MA抑制AngⅡ促LC3-Ⅱ的表达作用,而Baf-A1则增强AngⅡ促LC3-Ⅱ的表达作用,两种自噬抑制剂均可抑制AngⅡ促VSMC表型转换作用。转染siRNA Atg7后显著抑制AngⅡ促LC3-Ⅱ的表达作用,并可抑制AngⅡ促细胞表型转换作用。结论 AngⅡ促进VSMC从收缩表型转化为合成表型可能是自噬依赖性的,抑制自噬可以抑制AngⅡ诱导的VSMC表型转换。     

沉默脂肪细胞型脂肪酸结合蛋白基因对脂肪细胞合成甘油三酯及脂联素分泌的影响

目的应用RNA干扰(RNAi)技术,构建针对3T3-L1脂肪细胞型脂肪酸结合蛋白(A-FABP)的microRNA表达载体,研究沉默A-FABP基因的表达对脂肪细胞合成甘油三酯及脂联素分泌的影响。方法构建靶向A-FABP基因的microRNA表达载体转染3T3-L1脂肪细胞后,RT-PCR及Western blot法检测A FABP mRNA及蛋白表达。建立A-FABP基因沉默细胞模型,0.5 mmol/L游离脂肪酸和成熟脂肪细胞共孵育24 h,分为对照组、脂肪酸组、RNAi组、RNAi+脂肪酸组。ELISA法分别检测甘油三酯及脂联素水平,RT PCR法检测脂肪细胞A-FABP及脂联素mRNA的表达。结果 microRNA表达载体转染脂肪细胞后,能显著抑制A-FABP mRNA和蛋白表达。随着脂肪酸浓度的升高,脂肪细胞甘油三酯合成明显增加,脂联素分泌明显减少(P<0.05)。与对照组和脂肪酸组比较,RNAi组和RNAi+脂肪酸组甘油三酯水平明显降低,脂联素水平明显升高,脂联素mRNA表达明显上调(P<0.05)。结论阻断A-FABP基因有可能成为防治动脉粥样硬化及糖尿病的新靶点。     

干扰SET8调节血管平滑肌细胞增殖、凋亡促进血管钙化

背景与目的血管平滑肌细胞(VSMC)凋亡在许多血管钙化疾病的病理过程中起关键作用。近期研究表明,赖氨酸甲基转移酶SET8参与调节细胞增殖、凋亡过程。文章旨在探寻SET8是否通过调节VSMC增殖、凋亡而影响血管钙化。方法体外培养原代大鼠VSMC,将VSMC随机分为正常对照组、空质粒组和SET8-shRNA组。以脂质体Lipofectamine~(TM)2000为载体,转染VSMC。采用茜素红染色法和钙含量测定法判断细胞钙化程度;采用MTT法检测三组细胞的增殖能力;实时荧光定量PCR、Western blot法检测三组细胞中增殖基因Survivin和凋亡基因Caspase-3的表达水平,分析干扰SET8对VSMC增殖、凋亡及其相关基因和蛋白表达的影响。结果 (1)干扰SET8可成功抑制VSMC中SET8蛋白的表达(P0.05)。(2)转染VSMC后,与正常对照组和空质粒组比较,SET8-shRNA组钙含量明显升高(P0.05)。(3)MTT结果显示,在第12 h、24 h、36 h、48 h,SET8-shRNA组VSMC的增殖能力较正常对照组和空质粒组明显降低(P0.05)。(4)实时荧光定量PCR和Western blot结果显示SET8-shRNA组Survivin的mRNA和蛋白表达较正常对照组和空质粒组明显下降,而Caspase-3的mRNA和蛋白表达则相反(P0.05)。结论干扰SET8能够增加促凋亡基因表达,抑制增殖基因表达,SET8有可能参与调节大鼠血管钙化。     

27nt-miRNA通过靶向哺乳动物雷帕霉素靶蛋白调控血管平滑肌细胞凋亡及其分子机制研究

目的:探讨来源于一氧化氮合酶基因第四内含子的27碱基重复序列微小RNA(27nt-miRNA)对哺乳动物雷帕霉素靶蛋白(mTOR)介导的主动脉血管平滑肌细胞(VSMC)凋亡调控作用及潜在分子机制。方法:将大鼠主动脉VSMC分为空白对照组、雷帕霉素组、27nt-miRNA组、27nt-miRNA反义序列(anti-27ntmiRNA)组、阴性对照组。27nt-miRNA组:转染27nt-miRNA正义链慢病毒载体;anti-27nt-miRNA组:转染27ntmiRNA反义链慢病毒载体;阴性对照组:转染随机阴性序列慢病毒载体。除空白对照组外,雷帕霉素组与各转染组细胞给予100 ng/ml雷帕霉素诱导培养2 h诱导凋亡。荧光定量逆转录PCR(qRT-PCR)验证27nt-miRNA慢病毒稳转细胞株构建情况;CCK-8法、流式细胞术、qRT-PCR法与蛋白免疫印迹(Western blot)法分别检测各组细胞增殖能力、细胞凋亡率与周期分布以及mTOR、磷酸化-mTOR(p-mTOR)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(caspase-3)的信使RNA(mRNA)和(或)蛋白表达水平。结果:慢病毒转染VSMC后,27nt-miRNA稳转细胞株构建成功,雷帕霉素可抑制VSMC的mTOR mRNA与p-mTOR蛋白表达水平。与阴性对照组相比,27nt-miRNA组细胞的活力与增殖能力显著增强,且细胞凋亡率显著降低(P均<0.05),细胞周期G1期向S期分裂进程阻滞减弱(P<0.05);Bax和caspase-3蛋白表达量显著减少,Bcl-2蛋白表达量显著增加(P均<0.05);mTOR基因转录水平与p-mTOR蛋白表达水平均下降(P<0.05)。结论:27nt-miRNA通过负调控m TOR基因转录水平与蛋白磷酸化水平抑制VSMC凋亡,其机制可能与上调Bcl-2蛋白表达,下调Bax和caspase-3蛋白表达有关。     

RNAi选择性下调大鼠心肌细胞上Gsα蛋白的体外实验

目的:用RNA干扰技术(RNA interference, RNAi)选择性下调大鼠心肌细胞上乌苷酸结合蛋白(Gs)α亚基(Gs protein alphala subunit, Gsα)的表达,筛选出抑制Gsα蛋白效果最明显的特异短发夹RNA(short hairpin RNA, shRNA)表达质粒.方法:构建3条靶向gnas基因的特异与RNA质粒载体(shRNA1-3),以含非特异性shRNA编码序列的质粒载体为阴性对照(HK),用脂质体LipofectamineTM LTX & PLUS转染原代培养的大鼠心肌细胞,并设空白对照组,转染后通过半定量RT-PCR和Western blot法检测Gsα mRNA和蛋白的表达情况,以内参照三磷酸甘油醛脱氢酶(GAPDH)进行标化.结果:shRNA1-3均使Gsα的mRNA和蛋白质表达明显降低,差异有统计学意义(P<0.01),且shRNA3的抑制效果最显著,阴性对照质粒组与空白对照组Gsα表达差异无统计学意义.结论:利用RNAi技术成功下调了原代培养的心肌细胞中Gsα的表达,并筛选出沉默效果最明显的shRNA真核表达质粒,为心肌细胞的转染提供了可行的方法.     

Smoothened shRNA载体的构建及其对乳腺癌细胞增殖的影响

目的构建并筛选Smoothened（SMO）基因的RNAi表达质粒,了解Hedgehog信号通路影响乳腺癌细胞增殖的作用机制。方法以脂质体Lipofectamine^TM2000介导转染乳腺癌MCF-7细胞。转染后48 h,采用实时定量RT-PCR技术检测转染细胞中SMO基因mRNA的转录水平,筛选有效的SMO RNAi质粒,Western blot检测SMO蛋白在乳腺癌MCF-7细胞中的表达,CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测SMO短发夹状RNA（shRNA）对cyclinD1 mRNA表达的影响。结果 4个SMO shRNA表达载体SMO shRNA-1、SMO shRNA-2、SMO shRNA-3和SMO shRNA-4经测序鉴定证明插入正确。转染48 h后,与MOCK组相比,SMO shRNA-1、SMOshRNA-3、SMO shRNA-4组SMO mRNA表达量均下降（P=0.015、0.002、0.000）,其中转染SMO shRNA-4的MCF-7细胞中SMO mRNA表达水平低于SMO shRNA-1、SMO shRNA-2、SMO shRNA-3组（P=0.007、0.000、0.046）。SMOshRNA-4干扰抑制效率为87%。Western blot可检测到SMO在MCF-7中的表达,干扰片段SMO shRNA-4的抑制效果较好。转染SMO shRNA-4的MCF-7细胞增殖受到明显抑制（P〈0.01）,转染SMO shRNA后cyclinD1 mRNA表达下降（P=0.000）。结论已成功构建并筛选出SMO基因的RNAi表达质粒,SMO shRNA对乳腺癌细胞增殖有明显的抑制作用,Hedgehog信号通路可通过活化cyclinD1诱导细胞恶性增殖。     

Notch3表达下调的血管平滑肌细胞模型的建立

目的通过RNA干扰建立Notch3表达下调的血管平滑肌细胞(vascular smooth muscle cell,VSMC)模型,探讨Notch3在VSMC表型转化中的作用。方法根据Notch3序列设计siRNA,建立腺病毒表达载体;VSMC分为空白组、空载体组及试验组;将载体导入VSMC中,通过荧光实时定量PCR检测Notch3表达水平。结果在感染复数为300时,VSMC的感染效率最高。以空白组为基准,试验组Notch3的相对表达量为0.64,下降了36%,而空载体组为0.88,仅下降了12%。结论成功构建Notch3表达下调的VSMC模型,为后期研究奠定了良好的基础。     

大鼠microRNA-145慢病毒表达载体的构建及其对血管平滑肌细胞表型转化的影响

目的构建针对Rno-miR-145的慢病毒表达载体并探讨其在血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)表型转化中的作用。方法人工合成含有酶切位点粘端miR-145 shDNA双链模板序列,克隆于LV3 pGLV/H1/GFP+Puro-miRNA慢病毒穿梭载体中,转染293T细胞,收获并浓缩慢病毒颗粒,感染大鼠原代VSMC,倒置荧光显微镜下观察VSMC感染后的荧光表达情况,实时荧光定量PCR检测miR-145的表达情况;实验分为空白对照组、PDGF组、PDGF+miR-145组和细胞转染阴性慢病毒载体组(miR-NC组);采用实时荧光定量PCR测定miR-145对VSMC增殖相关基因PCNA、c-Jun及分化相关基因SM22αmRNA表达水平的影响。结果成功构建了microRNA-145慢病毒载体,测定病毒滴度为1×109TU/mL。倒置荧光显微镜下观察大鼠microRNA-145慢病毒表达载体感染成功,MOI值为50,感染72 h时感染率最高。实时荧光定量PCR结果显示PDGF可使PCNA、c-Jun表达增加,而使SM22α表达降低;miR-145可使PDGF诱导的去分化型VSMC增殖相关基因PCNA、c-Jun表达降低,分化相关基因SM22α表达增加。结论 miR-145慢病毒载体可高效感染大鼠原代VSMC。感染miR-145慢病毒后可抑制VSMC的表型转化。     

大鼠局灶性脑缺血再灌注小动脉血管平滑肌细胞表型转化的实验研究

目的　观察大脑中动脉缺血再灌注大鼠模型小动脉血管平滑肌细胞 (VSMC)中两种血管舒缩功能相关蛋白(α 平滑肌肌动蛋白和细丝蛋白 )的免疫组织化学表达变化 ,以期了解脑小动脉病变中VSMC的表型转化及细丝蛋白在表型转化中的意义 ,指导临床小动脉硬化型脑卒中的准确防治。方法　缺血再灌注组依照再灌注时间的不同分为 2、6、12、2 4h、3d及 7d共 6个亚组 ,另设正常对照组及大脑中动脉持续缺血组。按时相点取材 ,免疫组织化学染色 ,图像分析。结果　α 平滑肌肌动蛋白在再灌注 6h后表达明显下降 ,在 2 4h时表达最低 ,在 3d时表达有所升高 ,7d时表达最高 ;细丝蛋白缺血 2h后表达稍有下降 ,以 12h时的表达最低 ,2 4h时表达升高 ,在 3d时表达最高。结论　再灌注损伤可引起小动脉VSMC表型的变化 ;细丝蛋白也可作为VSMC表型变化的指标之一 ,与α 平滑肌肌动蛋白相比意义更大 ;细丝蛋白和α 平滑肌肌动蛋白可能在再灌注中起下调炎症反应及保护细胞的作用     

Novel mechanisms of the antiproliferative effects of amlodipine in vascular smooth muscle cells from spontaneously hypertensive rats.

The calcium channel blocker amlodipine continues to be of interest due to its potential proven ability to hinder the progression of atherosclerosis and reduce the number of clinical ischemic events. Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) are useful in the study of atherosclerosis because they show exaggerated growth with production of angiotensin II (Ang II) by conversion to the synthetic phenotype. To clarify mechanisms of the antiproliferative effects of amlodipine, we evaluated effects of the expression of growth factors, the changes in phenotype, and the proliferation of VSMC from SHR. Amlodipine significantly inhibited basal DNA synthesis and proliferation of VSMC from SHR. Amlodipine also inhibited expression of platelet-derived growth factor (PDGF) A-chain, transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) mRNAs in VSMC from SHR. Decreases in levels of PDGF A-chain and bFGF mRNAs in VSMC from SHR were greater with amlodipine than with nifedipine. Amlodipine significantly inhibited expression of the synthetic phenotype markers osteopontin and matrix Gla mRNAs, indicating that it inhibited the exaggerated growth of VSMC from SHR and suppressed the change from the contractile phenotype to the synthetic phenotype. Thus, amlodipine may be a beneficial therapeutic agent for patients with hypertensive vascular diseases.     

The epigenetic phenotypic switch of vascular smooth muscle cells involved in atherosclerosis

Atherosclerosis is a progressive and chronic inflammatory response to pathological fatty deposits in the arterial wall. The resulting disease spectrum ranges from angina pectoris, myocardial infarction, and sudden cardiac death to peripheral vascular disease and stroke. Vascular smooth muscle cells (VSMCs) are one of the major cell types contributing to neointimal formation of the affected arteries leading to atherosclerosis and in-stent restenosis. The severity of this disease has been linked to the cellular plasticity of VSMCs. At least two phenotypic states have been described: a contractile state, in which cells have increased cytoplasmic myofilaments and are involved in maintaining vascular tone, and a synthetic state, in which cells have relatively few contractile elements but in contrast upregulate the machinery required for protein synthesis and extracellular matrix secretion. In healthy vessels, VSMC can switch between these states, but regulation of this switch is disrupted in atherosclerosis and thought to contribute to the progression of disease.Our aim was to characterise the epigenetic signature of these VSMC phenotypes to better understand the pathophysiology of atherosclerosis. We used primary mouse VSMC from aorta and characterised the phenotypic changes occurring in response to transforming growth factor β, which promote the contractile phenotype, and platelet-derived growth factor that is involved in VSMC proliferation and migration. First we showed that VSMC develop into a contractile or synthetic phenotype in response to growth factor stimulation. We then characterised the gene expression profile and pattern of epigenetic histone modifications of VSMC selective genes. This identified novel histone modification proteins which could be involved in promoting the atherosclerotic phenotype. We have also shown that key VSMC selective genes are regulated through different epigenetic gene regulation mechanisms. This cell model will be used to investigate the molecular pathways involved in VSMC phenotypic regulation and identify novel candidates for future therapeutic intervention.FundingUK National Health Service and British Heart Foundation.     

Vascular smooth muscle cell-derived adiponectin: a paracrine regulator of contractile phenotype

Adiponectin is a cardioprotective adipokine derived predominantly from visceral fat. We recently demonstrated that exogenous adiponectin induces vascular smooth muscle cell (VSMC) differentiation via repression of mTORC1 and FoxO4. Here we report for the first time that VSMC express and secrete adiponectin, which acts in an autocrine and paracrine manner to regulate VSMC contractile phenotype. Adiponectin was found to be expressed in human coronary artery and mouse aortic VSMC. Importantly, siRNA knock-down of endogenous adiponectin in VSMC significantly reduced the expression of VSMC contractile proteins. Contractile protein deficiency was also observed in primary VSMC isolated from Adiponectin(-/-) mice. This deficiency could be rescued by culturing Adiponectin(-/-) VSMC in conditioned media from wild type (WT) VSMC. Moreover, the paracrine effect of VSMC-derived adiponectin was confirmed as adiponectin neutralizing antibody blocked the rescue. Overexpressed adiponectin also exerted paracrine effects on neighboring untransfected VSMC, which was also blocked by adiponectin neutralizing antibody. Interestingly, adiponectin expression was inducible by the PPARγ agonist rosiglitazone. Our data support an important role for VSMC-derived adiponectin in maintaining VSMC contractile phenotype, contributing to critical cardioprotective functions in the vascular wall. This article is part of a Special Issue entitled "Local Signaling in Myocytes".     

溶血磷脂酸受体3介导溶血磷脂酸诱导的血管平滑肌细胞表型转化

目的探讨溶血磷脂酸(LPA)受体在LPA诱导的血管平滑肌细胞(VSMC)表型转化中的作用及相关信号传导通路。方法培养SD大鼠分化表型VSMC,培养液加胰岛素样生长因子(IGF-1)为IGF-1组,加溶剂载体为空白组,以不同浓度水平LPA(0.1～10μmol/L)刺激,依次为LPA 0.1组、LPA 1组和LPA 10组,并在LPA(1μmol/L)条件下,以LPA受体1,3拮抗剂二辛烷甘油焦磷酸盐(DGPP 8:0)为DGPP 1组,RT-PCR法检测平滑肌肌动蛋白α(SMA-α)和骨桥蛋白mRNA表达,Western blot法检测p38分裂原活化蛋白激酶(p38MAPK),细胞外信号调节激酶(ERK)及其磷酸化蛋白水平。结果与空白组和IGF-1组比较,LPA 0.1组、LPA 1组和LPA 10组呈剂量依赖促进骨桥蛋白mRNA表达上升,SMA-αmR NA表达下降(P<0.01);与空白组比较,LPA 1组p38MAPK和ERK激活(P<0.01),DGPP 1组p38MAPK和ERK无明显变化(P>0.05)。结论与Gq蛋白偶联的LPA受体3介导了LPA诱导的VSMC表型转化,阻滞上述通路有可能成为控制与动脉粥样硬化和再狭窄等血管疾病相关的VSMC表型转化潜在的治疗干预靶点。     

Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation

The phenotype of vascular smooth muscle cells (VSMCs) is dynamically regulated in response to various stimuli. In a cellular process known as phenotype switching, VSMCs alternate between a contractile and synthetic phenotype state. Deregulation of phenotype switching is associated with vascular disorders such as atherosclerosis, restenosis after angioplasty, and pulmonary hypertension. An important role for microRNAs (miRNAs) in VSMC development and phenotype switching has recently been uncovered. Individual miRNAs are involved in promoting both contractile and synthetic VSMC phenotype. In this review, we summarize recent advances in the understanding of miRNA function in the regulation of VSMC phenotype regulation.     

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl(2), synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01-1.0 microM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-beta(1)-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.     

Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.