【作者】 颜凤；

【导师】 蒋凡；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究背景转化生长因子-β (TGF-β)在调节心血管生理和疾病中发挥多种作用,而其中重要的作用之一就是调节血管系统中的内皮细胞功能。TGF-β信号通路的失衡会导致胚胎血管发育的异常,而且TGF-β在出生后毛细血管新生中发挥重要的作用。除此之外,研究结果提示TGF-β能调节内皮细胞的增殖,凋亡,通透性变化和形态发生。有证据表明在有心血管疾病危险因素的患者中TGF-β血浆浓度是升高的,这些危险因素包括肥胖和糖尿病。而且,研究检测到患有先兆子痫的女性循环血中TGF-β的水平是升高的。在易患动脉粥样硬化的载脂蛋白E基因敲除小鼠中,TGF-β表达增加导致内皮细胞氧化应激和内皮功能紊乱。重要的是,TGF-β在内皮细胞中的生物学作用往往是矛盾的(或有保护作用或者是有害的),作用的不同取决于细胞的环境和细胞的类型。在内皮细胞中, TGF-β发挥作用主要由活化素受体样激酶1(ALK1)和ALK5两种膜受体介导。激活的ALK1受体磷酸化Smadl/5/8,然而ALK5的激活引起Smad2/3的磷酸化。这些活化的Smad蛋白与Smad4结合,作为一个转录激活复合物调节各种各样靶基因的表达,然而ALK1和ALK5的激活可能会引起不同的内皮细胞生物学效应。除这些经典信号途径外,最近研究提示NADPH氧化酶依赖的氧化还原机制或许介导了TGF-β生物学作用的调节。特别是,TGF-β特异性上调多种细胞中Nox4型NADPH氧化酶的表达。更重要的是,这些研究证实阻断Nox4的表达或功能抑制TGF-β对多种细胞功能的影响,提示Nox4在TGF-β信号通路中发挥重要的作用。多项证据表明TGF-β能引起不同类型内皮细胞的凋亡,然而TGF-β引起凋亡的信号机制还知之甚少。研究证实TGF-β引起肺毛细血管内皮细胞凋亡,这种作用依赖于ALK5-Smad2通路和下调抗凋亡基因Bcl-2和cFLIP。另一方面,多项研究指出p38丝裂原激活蛋白激酶(MAPK)介导了TGF-β引起的内皮细胞凋亡。然而,迄今为止鲜有研究表明TGF-β引起的内皮细胞凋亡涉及氧化还原依赖的信号途径。鉴于最近的结果显示活性氧簇(ROS)生成的增加或许在TGF-β引起的内皮细胞的凋亡中有重要作用,我们提出以下假设：Nox4依赖的氧化还原调节途径或许介导了TGF-β引起的内皮细胞的凋亡。研究目的1. TGF-β在内皮细胞凋亡中的作用2.Nox4氧化还原信号通路是否介导了TGF-β引起的内皮细胞凋亡3.MAPK信号通路在TGF-β引起内皮细胞凋亡中的作用及机制研究研究方法1.人内皮细胞培养人脐静脉内皮细胞HUVECs和永生化的人微细血管内皮细胞HMVECs购买自ATCC。细胞在含有5%胎牛血清的完全内皮细胞培养基(ECM)中培养,置于5%CO2,37℃条件的细胞培养箱中。待细胞长至融合时,用胰酶消化细胞进行传代培养,第3到6代之间的HUVECs用于实验。2.Caspase3/7活性测定培养在96孔板中的细胞经药物处理后,加入Caspase-Glo3/7试剂检测Caspase3/7活性。样品转移到白色96孔板中,用全波长扫描式多功能读数仪进行读板测定。3.蛋白质免疫印迹处理细胞后,用裂解液裂解细胞,提取细胞总蛋白,SDS-PAGE电泳分离蛋白样品,湿转法将蛋白转移到硝酸纤维素膜上,封闭后用特异性一抗4℃孵育过夜,然后二抗室温2小时。ECL化学发光液显影目的蛋白条带,LAS-4000化学发光仪检测。4. TUNEL法检测细胞凋亡末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定(TUNEL)法按照Millipore的步骤进行,其中标记的棕褐色为凋亡细胞的细胞核,绿色为正常细胞。随机取10个视野计数TUNEL阳性的细胞和总细胞数,TUNEL阳性细胞与总细胞的比值作为细胞凋亡率。5.ROS检测细胞内的ROS用DCFH-DA荧光染料检测,处理后的细胞加入DCFH-DA在37。C孵育20分钟并用激光共聚焦显微镜拍照留取图片,或者将DCFH-DA孵育的细胞悬液转移到96孔白板中检测荧光强度。6.线粒体膜电位(△ψm)的检测荧光探针JC-1用来检测线粒体膜电位的变化。药物处理后的细胞用JC-1进行染色,然后流式细胞术或者激光共聚焦显微镜检测红绿荧光的变化。7.Nox4干扰试验从备选的3条Nox4shRNA中挑选出干扰效率最高的一条进行实验,携带有Nox4shRNA和对照shRNA的慢病毒载体由上海吉玛公司构建。其中靶向Nox4的shRNA序列是5’GCTGTATATTGATGGTCCTTT3’,对照shRNA序列为5’GGTGTCTTCGAATTTATGTCT3’。将慢病毒转染细胞48小时后进行后续实验。8.实时荧光定量PCR检测mRNA水平的表达TRIzol试剂提取处理细胞的总RNA,逆转录成cDNA,再用Taqman探针-引物体系进行PCR,18S作为管家基因检测：mRNA表达的相对变化。9.细胞免疫荧光法培养于Lab-Tek腔室玻片上的细胞固定后,加一抗和荧光二抗孵育,用DAPI复染细胞核,激光共聚焦显微镜拍照。10.统计学分析数据用均数±标准误表示,用SPSS18.0软件进行统计分析,非配对t检验或单因素方差分析(ANOVA)分别分析两组和多组数据,p<0.05认为有统计学意义。研究结果1. TGF-β引起人内皮细胞的凋亡反应用不同浓度的TGF-β刺激HUVECs24小时,发现TGF-β在浓度范围1ng/ml到20ng/ml时引起明显的caspase3/7活性增加和caspase3裂解。用lOng/ml TGF-β刺激HUVECs不同时间发现TGF-β引起的凋亡作用是随时间变化的,24和48小时效果最强。我们还选用了另一种内皮细胞HMVECs进行研究。用不同浓度的TGF-β刺激HMVECs24小时和用10ng/ml TGF-β刺激不同的时间发现,TGF-β以剂量依赖和时间依赖的方式引起HMVECs相似的凋亡反应。2. TGF-β引起内皮细胞凋亡是由ALK5受体介导的通过测定caspase3/7活性和蛋白质免疫印迹法检测caspase3裂解,我们发现TGF-β引起的内皮细胞的凋亡作用被ALK5的抑制剂SB431542所阻断。而且,结果显示TGF-β能引起内皮细胞线粒体膜电位的下降,这种作用也被SB431542所抑制。通过检测caspase3/7的活性,发现staurosporine引起的内皮细胞凋亡不能被SB431542所阻断,证明了SB431542在TGF-β凋亡作用中的特异性。3.TGF-β通过ROS引起内皮细胞凋亡TGF-β引起内皮细胞ROS生成增加,而且TGF-β引起的ROS增加被SB431542所抑制,说明TGF-β通过ALK5受体使细胞内ROS水平增加。两种ROS清除剂NAC和EUK-134均能阻断TGF-β引起的内皮细胞凋亡,而ROS清除剂本身有轻微的抗凋亡作用(无统计学意义)。4. TGF-β上调内皮细胞中Nox4的表达首先,用TGF-β刺激HUVECs发现TGF-β增加Nox4mRNA的表达和上调Nox4的蛋白表达水平。而加入ALK5受体抑制剂后,TGF-β上调Nox4表达的作用被抑制,说明TGF-β是通过ALK5受体激活引起Nox4转录和翻译水平的变化的。同时,我们用细胞免疫荧光的方法证实了TGF-β及ALK5对Nox4表达及分布的影响。Nox4不仅在胞浆中存在,还分布于细胞核中,TGF-β刺激后,胞浆和胞核中的Nox4都增加了。5.Nox4在TGF-β引起内皮细胞凋亡中的作用研究发现TGF-β引起的内皮细胞凋亡作用被Nox4shRNA所阻断,而本身Nox4shRNA没有明显的抗凋亡作用。用Nox4的抑制剂VAS2870预处理细胞后,发现VAS2870阻断了TGF-β对caspase3的激活,caspase3的裂解和线粒体膜电位的下降。6.MAPKs在TGF-β引起内皮细胞凋亡中的作用TGF-β刺激内皮细胞,p38的磷酸化增加,从12小时开始一直持续到48小时。相反,JNK的磷酸化水平只是在12小时一过性增高。我们用p38和JNK特异的激酶抑制剂预处理细胞,证明抑制p38通路能阻断TGF-β的致凋亡作用,而JNK抑制剂没有作用。为了进一步验证,我们用p38和JNK特异的siRNA干扰p38和JNK通路,发现结果与用激酶抑制剂相一致。7. TGF-β通过氧化还原信号途径激活MAPKsNAC预处理细胞后发现,NAC完全阻断了TGF-β引起的p38的磷酸化。然而,NAC本身则增加了JNK的磷酸化,而有NAC存在时TGF-β引起的JNK的磷酸化不明显。这些结果提示p38和JNK的激活都是氧化还原敏感的。而且,用VAS2870抑制Nox4的功能削弱了TGF-β引起的p38磷酸化水平的增加。不同于MAPKs,无论在基础状态,还是有NAC或VAS2870存在的条件下,TGF-β对Akt的磷酸化水平没有影响。结论1. TGF-β通过ALK5受体引起内皮细胞凋亡反应2. TGF-β通过增加Nox4表达和ROS的生成增加促进细胞凋亡3.p38介导了TGF-β引起的内皮细胞凋亡,并且是氧化还原依赖的研究背景ROS是生物系统中普遍存在的信号分子,ROS的来源主要是NADPH氧化酶。NADPH氧化酶(Nox)家族由7个成员组成,包括Noxl-5和Duox1、2,其中4种类型的NADPH氧化酶是血管系统中ROS的重要来源：Nox1, Nox2,Nox4和Nox5。NADPH氧化酶将电子从NADPH传递到氧分子从而产生ROS, ROS包括局部有效的超氧化物和作用持久的具有膜扩散特性的过氧化氢(H2O2)。Nox4与其他类型的NADPH氧化酶不同的是其在心血管组织中的高表达,独特的固有酶活性和主要产生ROS的类型是H2O2。Nox4是一个多效性的信号分子,具有多种功能,包括细胞凋亡、细胞生长、分化、血管新生和细胞骨架重构等。研究表明Nox4在很多心血管疾病中表达增加,比如粥样硬化、高血压、再狭窄、血管老化和纤维化等。最近越来越多的研究结果显示Nox4具有正反两方面的作用,由于Nox4过表达小鼠模型的成功建立,研究者得出了惊人的结果,Nox4实际上具有血管保护作用而非血管损伤作用。我们之前的研究表明在内皮细胞中Nox4表达的增加或许会通过激活ERK1/2MAPK通路增加内皮细胞增殖(认为是一个促进存活的作用)。因此在这项研究中,我们试图解释Nox4在调节内皮细胞凋亡和存活方面看似相反的作用。蛋白酪氨酸磷酸酶(PTPs)是ROS敏感的酶类,可以被ROS氧化而失去其酶活性。PTPs负调控多种受体酪氨酸激酶(RTKs),这些RTK的配体包括EGF、 IGF-1、PDGF和VEGF。PTP1B是第一个被提纯分离的经典型PTP,是2型糖尿病、肥胖和肿瘤的一个新的治疗靶点。PTEN作为一个重要的肿瘤抑制物也是一种PTP,研究发现其在大部分人类的肿瘤中突变。之前研究表明,PTPs通过去磷酸化磷脂酰肌醇(3,4,5)-三磷酸(PIP3)抑制Akt信号通路。我们在此部分探究PTPs及Akt是否参与了过表达Nox4对细胞凋亡的作用。研究目的1.过表达外源性Nox4在内皮细胞凋亡中的作用2.Akt通路及ERK1/2通路在过表达外源性Nox4抗内皮细胞凋亡中的作用3.PTPs是否参与了过表达外源性Nox4对Akt通路的激活及作用机制研究方法1.慢病毒或腺病毒过表达Nox4人类野生型Nox4的cDNA克隆购买自Origene,亚克隆到pLV.EX3d.P/neo-EF1A。表达Nox4或eGFP的慢病毒转染细胞,48小时之后进行后续实验。感谢Goldstein教授赠与的表达人野生型Nox4的腺病毒。2. Caspase3/7活性测定细胞经药物处理后,加入Caspase-Glo3/7试剂检测Caspase3/7活性。加入检测试剂的样品转移到白色96孔板中,读板测定。3.蛋白质免疫印迹裂解液裂解细胞,提取细胞总蛋白,SDS-PAGE电泳分离蛋白样品,将蛋白转移到硝酸纤维素膜上,封闭后一抗孵育过夜,然后二抗孵育2小时。ECL化学发光液显影目的蛋白条带,LAS-4000化学发光仪检测。4.细胞免疫荧光法培养于Lab-Tek腔室玻片上的细胞固定后,加一抗和荧光二抗孵育,用DAPI复染细胞核,激光共聚焦显微镜拍照。5.蛋白质免疫沉淀细胞裂解液预清洗之后,加入一抗和蛋白A或G琼脂糖珠进行孵育,然后通过离心去除琼脂糖珠。将蛋白样品通过SDS-PAGE电泳分离,转移到硝酸纤维素膜上,封闭后加一抗二抗孵育,最后化学发光检测蛋白条带。6.PTP1B活性测定处理细胞后,免疫沉淀获取PTP1B蛋白,用购买自Millipore的PTP比色试剂盒,按照说明书的方法检测PTP1B的活性。7.统计学分析数据用均数±标准误表示,用SPSS18.0软件进行统计分析,非配对t检验或单因素方差分析(ANOVA)分别分析两组和多组数据,p<0.05认为有统计学意义。研究结果1.过表达外源性Nox4对抗内皮细胞凋亡反应研究结果证实慢病毒或腺病毒过表达Nox4(vNox4)不仅抑制了内皮细胞的基础凋亡反应而且抑制了血清饥饿引起的内皮细胞凋亡,还减少了staurosporine导致的内皮细胞凋亡。另外,我们发现在内皮细胞中过表达Nox4后,再给予TGF-β刺激,并不能引起明显的凋亡反应。2.过表达外源性Nox4通过激活Akt发挥抗凋亡作用我们检测了vNox4对促进存活因子Akt和ERK1/2的作用,结果显示vNox4剂量依赖性增加Akt的磷酸化水平。但是,vNox4组与对照病毒组相比,并没有引起明显的ERK1/2激活。用Akt通路抑制剂Ⅶ或wortmannin处理细胞能明显减弱vNox4的抑制凋亡作用,而ERK1/2抑制剂U0126没有这种作用。我们也证实用特异性siRNA干扰Akt表达后部分阻断了vNox4的抗凋亡作用。鉴于TGF-β不影响Akt的磷酸化水平,以上数据提示vNox4对Akt的激活或许是内源性和过表达Nox4对凋亡反应作用不同的原因。3.过表达外源性Nox4与内源性Nox4亚细胞分布相同为了明确过表达Nox4和内源性Nox4是否存在亚细胞分布的不同,免疫荧光法检测过表达Nox4的细胞中Nox4的表达,发现其表达在胞浆和胞核中均增加,与之前TGF-β刺激后的反应相似,由此看来细胞分布不是过表达Nox4作用不同的原因。4. PTPs抑制剂对Akt磷酸化的影响为了研究vNox4激活Akt的信号机制,我们首先验证了两个涉及细胞内氧化还原信号途径的ROS敏感的PTP1B和PTEN受抑制之后是否影响Akt的磷酸化。实验结果表明PTP1B的抑制剂而非PTEN的抑制剂处理内皮细胞之后,Akt的磷酸化水平增加了。抑制PTP1B的作用类似于VEGF引起的作用,然而VEGF并没有在存在PTP1B抑制剂的条件下进一步增加Akt的磷酸化。相反,VEGF在存在和缺少PTEN抑制剂的条件下,对Akt有相似的激活作用。5. vNox4抑制PTP1B活性和增加VEGFR的激活为了进一步证明Nox4过表达对PTPIB功能的作用,我们检测了对照组细胞和Nox4过表达细胞免疫沉淀的PTPIB的磷酸酶活性,发现Nox4过表达抑制了PTPIB的活性。为了确定PTP1B活性的抑制是氧化还原敏感的,在所有样品中加入还原剂二硫苏糖醇(DTT),结果显示Nox4对PTP1B活性的影响被DTT阻断了。因为PTP1B是VEGF受体(VEGFR)信号途径的负调节因子,我们检测了vNox4和PTP1B抑制剂是否调节VEGFR2的磷酸化水平。免疫沉淀检测结果提示抑制PTP1B或者过表达Nox4均能增加VEGFR2的磷酸化。结论1.过表达外源性Nox4具有抗内皮细胞凋亡的作用2.Akt信号通路而非ERK1/2参与了Nox4的抗凋亡作用3.PTPs抑制Akt通路的激活4.过表达外源性Nox4通过抑制PTP1B的活性进而激活VEGFR研究背景细胞分化不仅是心血管的胚胎发育中一个重要的过程,而且在成年期也同样具有维持血管系统稳态的重要作用。内皮细胞动脉和静脉表型的错误界定会对循环系统的发育和功能造成严重的后果,比如在胚胎发育中的缺陷和死亡,另外还有动静脉畸形(AVM)。 AVM是遗传性毛细血管扩张症(HTT)的一个重要特点,而且脑动静脉畸形是导致年轻人颅内出血性中风的主要原因。人类和小鼠基因研究表明TGF-β信号通路在毛细血管新生中发挥重要作用,TGF-β信号通路组成成分的缺失会导致小鼠因为血管缺陷而胚胎死亡。以往研究发现TGF-β受体endoglin (TGF-β的辅助受体)或ALK1受体的缺失会导致类似于动静脉畸形血管异常的血管病理学改变,与HHT有关,这种病理变化往往伴随内皮细胞动静脉特异性的紊乱。间接的研究证据提示TGF-β信号通路或许参与了内皮细胞表型的特异性分化。在发育中,内皮细胞动脉和静脉的特异性分化是受严格转录调控的。某些细胞的标志物,比如酪氨酸激酶受体EphB4和其配体EphrinB2在细胞中特异性表达,EphrinB2主要在动脉内皮细胞中表达而EphB4主要在静脉内皮细胞中表达,这一对配体-受体被认为分别是动脉型内皮细胞和静脉型内皮细胞的标志物。静脉表型被认为是内皮细胞分化中的缺省状态,而Notch信号通路的激活(在VEGFA和sonic hedgehog的下游)注定细胞向动脉表型分化。进化上高度保守的Notch信号通路在哺乳动物中己发现4种Notch受体(Notch1-4)和5种Notch配体(Jagged1、2, DLL1、3、4),研究显示Notch信号通路在决定动脉细胞表型中发挥重要作用。细胞膜上的Notch配体与相邻细胞的受体结合,引起Y-分泌酶剪切跨膜受体形成Notch细胞内结构域(NICD),然后NICD从原来的受体上释放转位进入细胞核。进入细胞核的NICD与DNA结合蛋白CSL (RBP-Jκ/CBF1)和共同激活因子Mastermind-like (MAML)结合,激活下游靶基因HES和HEY家族的转录。有意思的是,越来越多的证据提示ROS介导的信号途径在干细胞或祖细胞分化中是一个重要的调节因子。但是,ROS在内皮细胞动静脉分化中的作用还知之甚少,因此,我们的研究的目的是探究TGF-β的氧化还原调节作用在内皮细胞动脉和静脉表型特异性变化中的潜在作用。研究目的1. TGF-β对内皮细胞动静脉标志物的影响2. TGF-β是否对Notch信号通路有作用3.氧化还原信号通路是否参与了TGF-β引起的内皮细胞表型变化研究方法1.实时荧光定量PCR检测mRNA表达TRIzol试剂提取细胞总RNA,逆转录成cDNA,再用Taqman探针-引物体系或SYBR Green法进行PCR, GAPDH或18S作为管家基因检测mRNA表达的相对变化。2.蛋白质免疫印迹裂解液裂解细胞,提取细胞总蛋白,测定蛋白浓度,SDS-PAGE电泳分离蛋白样品,将蛋白转移到硝酸纤维素膜上,封闭后用一抗、二抗孵育。ECL化学发光液显影目的蛋白条带,LAS-4000化学发光仪检测。3.细胞免疫荧光法生长于Lab-Tek腔室玻片上的细胞处理后固定,加一抗和荧光二抗孵育,用DAPI复染细胞核,激光共聚焦显微镜拍照。4.细胞核蛋白和胞浆蛋白的分离提取收集处理的内皮细胞,提取细胞的细胞核和细胞浆蛋白,SDS-PAGE电泳分离蛋白,进行western blot的后续操作,检验NICD的核转位情况。5.统计学分析数据用均数±标准误表示,用SPSS18.0软件进行统计分析,非配对t检验或单因素方差分析(ANOVA)分别分析两组和多组数据,p<0.05认为有统计学意义。研究结果1. TGF-β刺激HUVECs表达动脉标志物TGF-β上调动脉标志物EphrinB2的表达,而对静脉标志物EphB4的mRNA水平没有显著作用。TGF-β没有显著影响EphrinB2的蛋白水平。2. TGF-β增加Notch受体和配体及靶基因的表达检测不同Notch信号通路基因表达谱的变化,包括Notch受体(Notch1和Notch4)、Notch配体(Jagged1和DLL4)和Notch的靶基因(HEY1),结果显示TGF-β增加上述Notch信号通路标志物的表达。我们又用western blot验证了TGF-β对DLL4、Notchl和Jagged1的蛋白表达量的影响,结果与基因表达相似。3. TGF-β增加NICD的表达和促进核转位TGF-β处理增加了NICD的蛋白表达,并且用免疫荧光和蛋白质免疫印迹的方法证明TGF-β引起NICD的核转位,提示TGF-β激活了Notch信号通路。4. TGF-β未影响HMVECs的动静脉标志物的表达研究结果显示TGF-β对动脉标志物表达的影响是细胞特异性的,因为我们并没有观察到TGF-β对HMVECs的EphrinB2、EphB4、Notch1、Notch4、Jagged1或HEY1的表达有明显影响。5. TGF-β增加动脉标志物的表达是氧化还原依赖的在NAC存在的条件下,TGF-β与对照组相比,对动脉标志物表达没有显著的作用。我们发现应用外源性H202模拟了TGF-β对动脉标志物表达的作用。重要的是,我们发现在转染了慢病毒Nox4shRNA的内皮细胞中,TGF-β对Notch1、 Jagged1、HEY1和EphrinB2表达的影响减弱了。结论1. TGF-β细胞特异性刺激HUVECs向动脉表型转化2. TGF-β激活Notch信号通路3. TGF-β对内皮细胞的动脉型分化作用是氧化还原依赖的更多还原

【Abstract】 BackgroundTransforming growth factor-β (TGF-β) has pleiotropic effects in regulating cardiovascular physiology and disease, while one of the major targets of TGF-P in the vasculature is endothelial cells. Disruption of TGF-P signaling in endothelial cells results in impaired vascular development in the embryo, and TGF-P also has pivotal roles in postnatal angiogenesis. In addition, TGF-β has been implicated in modulating endothelial cell proliferation, apoptosis, permeability and morphogenesis. There is evidence that TGF-P concentration in the plasma is elevated in patients with risk factors of cardiovascular disease, including obesity and diabetes. Moreover, an increased level of circulating TGF-P was found in women with preeclampsia. In atherosclerosis-prone apolipoprotein(E)-deficient mice, systemic elevation of TGF-β expression induced endothelial oxidative stress and dysfunction. Remarkably, the biological effects of TGF-P in endothelial cells are often dichotomous (either protective or detrimental), depending on the cellular context and the type of endothelial cells involved.In endothelial cells, TGF-β-induced effects are mainly mediated by the membrane receptors activin receptor-like kinase (ALK)1and ALK5. Stimulation of the ALK1receptor phosphorylates Smadl/5/8, while stimulation of ALK5triggers phosphorylation of Smad2/3. These activated Smad proteins then work together with Smad4as a transcriptional activating complex to regulate the expression of various target genes, while activation of ALK1and ALK5may induce distinct cellular effects in endothelium. In addition to these canonical signaling pathways, emerging evidence suggests that NADPH oxidase-dependent redox mechanisms may also be involved in mediating the biological actions of TGF-β. In particular, TGF-P specifically upregulates expression of the Nox4type of NADPH oxidase in a variety of cells (including vascular cells). More importantly, these studies have demonstrated that blockade of Nox4expression or function inhibits TGF-β-induced effects on multiple cell functions, indicating a pivotal role of Nox4in TGF-β signaling.Several lines of evidence have shown that TGF-β can trigger an apoptotic response in different types of endothelial cells. However, the signaling mechanisms underlying this action of TGF-β are poorly understood. Lu et al. demonstrated that TGF-β-induced apoptosis in pulmonary microvascular endothelial cells was dependent on the ALK5-Smad2pathway and subsequent downregulation of the antiapoptotic genes Bcl-2and cFLIP. On the other hand, several studies have pointed to a critical role of activation of the p38mitogen-activated protein kinase (MAPK) in mediating TGF-β-induced endothelial apoptosis. Nevertheless, so far there is little information about whether TGF-β-induced apoptosis in endothelial cells involves redox-dependent signaling mechanisms. In light of some recent results showing that increased reactive oxygen species (ROS) production may have a prominent role in promoting cell apoptosis in response to TGF-β, we hypothesize that Nox4-dependent redox regulation may mediate TGF-P-induced endothelial cell apoptosis.Objectives1. To investigate the effect of TGF-β on endothelial apoptosis2. To clarify whether Nox4and redox signaling mediate TGF-β-induced endothelial apoptosis3. To explore the mechanisms of TGF-β-induced endothelial apoptosis and role of MAPKsMaterials and Methods1. Cell cultureHUVECs and immortalized HMVECs were purchased from the American Type Culture Collection (ATCC). Cells were cultured in complete endothelial cell medium (ECM) containing5%fetal bovine serum in a humidified incubator with5%CO2at37℃. HUVECs used for the experiments were between passages3and6.2. Caspase3/7activity assayCells seeded in96-well plates were incubated with the Caspase-Glo3/7substrate reagent. The samples were transferred to a white-walled plate and the luminescence signal was measured in a Varioskan Flash plate reader.3. Western blot analysisCells were homogenized in lysis buffer after stimulation, then protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membranes. The membrane was blocked with5%nonfat milk and incubated with specific primary antibodies. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000luminescent image analyzer.4. TUNEL labelingTUNEL was performed using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore) following the manufacturer’s protocol. The number of TUNEL-positive cells were counted and averaged across10random fields from4independent experiments each.5. ROS measurementIntracellular ROS was measured with DCFH-DAnfluorescence. Cells were incubated with DCFH-DA at37℃for20min. After staining, cells were detected with a laser-scanning confocal microscope or transferred to a96-well white-walled plate to detect the fluorescent signal with a Varioskan Flash plate reader.6. Measurement of mitochondrial membrane potential (△Ψm)The fluorescent probe JC-1was used to determine changes in△Ψm. Cells were stained with JC-1and analyzed by either flow cytometry or confocal microscopy. Decreased△Ψm is reflected by the decrease in red fluorescence (dye accumulated in mitochondria).7. Nox4interferenceLentiviral vectors carrying a shRNA targeting human Nox4were purchased from GenePharma (Shanghai, China). The targeting sequence for Nox4was5’GCTGTATATTGATGGTCCTTT3’.A scrambled sequence of5’GGTGTCTTCGAATTTATGTCT3’was used as control. Experiments were performed48hr after transfection8. Real-time PCRTotal RNA was extracted with TRIzol reagent and reverse transcripted to cDNA. Real-time PCR was performed using predesigned Taqman probe-primer sets and18S was used as house keeping gene.9. ImmunofluorescenceCells cultured on Lab-Tek chamber slides were fixed and then incubated with primary antibodies and Alexa Fluor594-conjugated secondary antibodies. After counter staining with DAPI, cells were photographed using a confocal microscope.10. Statistical analysisData are presented as mean±standard error of the mean (SEM). Data analysis was performed with unpaired t-test or one-way analysis of variance (ANOVA) using SPSS18.0software. P<0.05was considered as statistically significant.Results1. TGF-P induces a moderate apoptotic response in human endothelial cellsWe found that TGF-β significantly increased caspase3/7activity and triggered caspase3cleavage from lng/ml to20ng/ml (Figure1B). We further demonstrated that the effects of TGF-β on apoptosis were time-dependent, with the strongest effects being observed at24and48hr. To confirm our findings in HUVECs, we treated HMVECs with TGF-β and showed that TGF-P induced similar apoptotic reactions in a dose-and time-dependent manner.2. TGF-β-induced apoptosis is dependent on ALK5Treatment with SB431542abolished the apoptotic response induced by TGF-p as indicated by caspase3/7activation and caspase3cleavage. Moreover, we showed that SB431542also prevented the collapse of mitochondrial membrane potential (△Ψm) in response to TGF-P as measured with JC-1staining. Moreover, we confirmed that SB431542had no effects on staurosporine-induced apoptosis.3. ROS mediates TGF-p-induced apoptosisTGF-P treatment enhanced intracellular ROS production in HUVECs, and this effect could be blocked by SB431542. We preincubated the cells with antioxidant compounds NAC (N-acetyl-L-cysteine) or EUK-134and showed that both NAC and EUK-134abrogated TGF-β-induced caspase3/7activation.4. TGF-P increases the expression of Nox4in mRNA and protein levelWe found that Nox4expression was increased by TGF-P in a time-and concentration-dependent manner as measured by real-time PCR and western blot, which was blocked by SB431542. Immunofluorescence experiments confirmed that TGF-β increased the abundance of Nox4protein, both in the nuclei and in the cytosol, and this effect was also blocked by SB431542.5. Nox4mediates TGF-β-induced endothelial apoptosisIt was revealed that Nox4gene silencing blunted TGF-β-induced apoptosis, as determined by caspase3/7activity. To further confirm the results, we treated the cells with the pharmacological Nox inhibitor VAS2870, and showed that VAS2870also blocked caspase3/7activation, caspase3cleavage and disruption of△Ψm induced by TGF-p.6. Role of MAPKs in TGF-β-induced endothelial apoptosis TGF-P treatment increased phosphorylation of p38from12hr to48hr as compared to untreated cells. In contrast, phosphorylation of JNK was only transiently increased around12hr. We treated the cells with specific p38or JNK kinase inhibitors and demonstrated that inhibition of the p38pathway with SB202190blocked the proapoptotic effects of TGF-β, whereas the JNK Inhibitor II had no significant effects. We further confirmed these results by knocking down p38and JNK expression with specific siRNAs.7. TGF-β activates MAPKs in a redox-sensitive mannerWe found that NAC completely blocked the effect of TGF-p on p38phosphorylation. However, NAC per se somehow increased the basal level of JNK phosphorylation, while the effect of TGF-P on JNK phosphorylation in the presence of NAC was not significant, indicating that activation of both p38and JNK were redox sensitive. Moreover, we showed that inhibition of the Nox4function with VAS2870diminished TGF-β-induced p38phosphorylation. In contrast to MAPKs, TGF-P had no effects on Akt phosphorylation either under the basal condition or in the presence of NAC or VAS2870.Conclusions1. TGF-P induces endothelial cell apoptosis through ALK5receptor2. Nox4and redox signaling mediate TGF-β-induced endothelial apoptosis3. TGF-P induces endothelial apoptosis through p38MAPK in a redox-dependent manner. BackgroundROS are molecules existing widely in biological systems, and accumulating evidence has identified that NADPH oxidase is the major source of ROS generation in vasculature. The catalytic subunit of the NADPH oxidase family consists of seven members, namely Nox1-5and Duox1and2. Noxl, Nox2, Nox4and Nox5are four main members in vasculature with Nox4and Nox2predominant in endothelial cells. The enzymes transfer electrons from NADPH to molecular oxygen, producing ROS consisting of locally effective superoxide and/or the relatively long-lasting and membrane-diffusible ROS hydrogen peroxide (H2O2). Nox4differs from other NADPH oxidase isoforms by its abundant expression in vascular tissues and constitutive activity.Nox4type NADPH oxidase has multiple regulatory functions including apoptosis, cell growth, differentiation, angiogenesis and cytoskeletal remodeling. Nox4expression is increased in many cardiovascular diseases, such as atherosclerosis, hypertension, restenosis, vascular aging and fibrosis. Recently, more and more studies have revealed its controversial functions, and many researches indicate Nox4as a protective factor in cardiovascular systems since the model of gene-modified mice with Nox4overexpression successfully established.Moreover, our previous studies in endothelial cells suggest that increased Nox4expression may enhance cell proliferation (presumably a prosurvival effect) via activating the ERK1/2MAPK pathway. In the present study, therefore, we also attempted to reconcile these seemingly paradoxical effects of Nox4in modulating endothelial cell apoptosis and survival.Protein tyrosine phosphatases (PTPs) are sensitive to ROS and can be oxidized to lose their activities. PTPs negatively regulate receptor tyrosine kinases (RTKs), including receptors of some growth factors such as EGF, IGF-1, PDGF and VEGF. PTP1B, belonging to the classical PTPs, is the first isolated PTP in its pure form and it is a treatment target for type2diabetes, obesity and cancer.PTEN is a hallmark of cancer suppressor which is commonly found mutant in most human tumor cells. PTPs negatively regulate Akt signaling by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3). In this part, we will explore whether PTPs and (or) Akt participate Nox4-induced anti-apoptotic effect.Objectives1. To explore role of Nox4overexpression in endothelial apoptosis2. To evaluate whether Akt or ERK1/2signaling pathway mediates the anti-apoptotic effect of Nox4in endothelial cells3. To investigate the mechanisms of Nox4-stimulated Akt activation and the role of PTPsMaterials and Methods1. Nox4overexpression by viral vectorsA cDNA clone of human wild type Nox4was purchased from Origene and subcloned into pLV.EX3d.P/neo-EF1A. Experiments were performed48hr after transfection with lentiviruses expressing human Nox4or eGFP (control). Adenoviruses expressing wild type human Nox4were gifts from Dr Goldstein.2. Caspase3/7activity assayCells seeded in96-well plates were incubated with the Caspase-Glo3/7substrate reagent. The samples were transferred to a white-walled plate and the luminescence signal was measured in a Varioskan Flash plate reader.3. Western blot analysisCells were homogenized in lysis buffer after stimulation, then protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membranes. The membrane was blocked with5%nonfat milk and incubated with specific primary antibodies. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000luminescent image analyzer.4. ImmunofluorescenceCells cultured on Lab-Tek chamber slides were fixed and then incubated with primary antibodies and Alexa Fluor594-conjugated secondary antibodies. After counter staining with DAPI, cells were photographed using a confocal microscope.5. ImmunoprecipitationCell lysates were precleared and incubated with antibody and protein A or G agarose bead slurry. The beads were washed and protein samples were separated by SDS-PAGE and the following procedures were taken as western blot.6. PTP1B activity assayPTP1B activity was measured in immunoprecipitated PTP1B protein samples using a PTP colorimetric assay kit from Millipore according to the manufacturer’s instruction.7. Statistical analysisData are presented as mean±SEM. Data analysis was performed with unpaired t-test or one-way ANOVA using SPSS18.0software. P<0.05was considered as statistically significant.Results1. Nox4overexpression is protective against endothelial apoptosisWe demonstrated that lentivirus-mediated Nox4overexpression (vNox4) suppressed both of the basal level of and serum starvation-induced apoptosis in HUVECs. In addition, we showed that overexpression of Nox4(using adenovirus) also reduced the apoptosis induced by staurosporine. TGF-β failed to induce endothelial apoptosis following Nox4overexpression.2. vNox4inhibits endothelial apoptosis through Akt activationWe showed that phosphorylation of Akt was dose-dependently increased by vNox4treatment. However, vNox4had no significant effects on ERK1/2phosphorylation. Treatment with Akt Inhibitor ⅧIor wortmannin significantly attenuated the antiapoptotic effects of vNox4, whereas treatment with U0126produced no effects. We also confirmed that knocking down of Akt with siRNA partially blocked the inhibitory effects of vNox4on apoptosis.3. Subcellular distribution of Nox4between ectopic and endogenous Nox4We performed immunofluorescence in vNox4-treated cells and found that vNox4increased the Nox4protein level in both of the cytoplasm and nuclei, a pattern that was similar to that following TGF-β treatment.4. PTP1B inhibitor stimulates Akt activationWe demonstrated that treatment with PTP1B inhibitor, but not PTEN inhibitor, increased Akt phosphorylation in resting endothelial cells. This effect of PTP1B inhibition was comparable to that induced by vascular endothelial growth factor (VEGF), while VEGF did not further increase Akt phosphorylation in the presence of PTP1B inhibitor. In contrast, VEGF similarly induced Akt phosphorylation in the absence and presence of the PTEN inhibitor.5. vNox4suppresses PTP1B activity and enhances VEGFR activationWe showed that Nox4overexpression suppressed PTP1B activity, an effect that was abolished by the reducing agent DTT. Immunoprecipitation with anti-VEGFR2or anti-phospho-tyrosine followed by western blot analysis revealed that inhibition of PTP1B or overexpression of Nox4enhanced VEGFR2phosphorylation. Similar to the effects on Akt phosphorylation, VEGF only increased the basal level of VEGFR2phosphorylation, but had smaller effects on VEGFR2phosphorylation in the presence of vNox4or PTP1B inhibitor.Conclusions1. Ectopic Nox4overexpression protects against endothelial apoptosis2. Akt signaling mediates ectopic Nox4inhibition of endothelial apoptosis3. The inhibitor of PTP1B stimulates Akt activation4. Ectopic Nox4enhances VEGFR activation through suppressing PTP1B activity BackgroundCellular differentiation is not only an important progress in cardiovascular embryonic development but also found in adulthood to maintain the homeostasis of vasculatures. Misidentification of arterial or venous endothelial cell fate would cause drastic consequences of the circulatory system in the development and function. Despite arterial and venous disturbing would bring about deficiency and lethality in embryonic development, arteriovenous malformation (AVM) is one characteristic of hereditary hemorrhagic telangiectasia (HHT) and AVM in the brain is the mainly leading cause of intracranial hemorrhagic stroke, particularly in younger people.TGF-β signaling pathway is reported to play important roles in angiogenesis and mice deficient for components of TGF-β signaling display embryonic lethalities due to vascular defects. People have shown that lack of endoglin (an accessory receptor of TGF-β) or ALK1results in vascular pathologies resembling AVM related to HHT, which are accompanied by disturbance of arterial and venous specificities.Indirect evidence indicates that TGF-P signaling may have impacts on the specification of endothelial phenotypes. Specification of arterial or venous type of endothelium during development is under strict transcriptional control. In the cardiovascular system, EphrinB2expression is restricted to the arteries and its receptor EphB4is predominantly expressed in venous endothelial cells. This pair of ligand-receptor has been used as markers of arterial and venous endothelial cells respectively.It is thought that venous phenotype is the default state during endothelial differentiation, while activation of the Notch signaling pathway (which is downstream of vascular endothelial growth factor A and sonic hedgehog) is obligatory for arterial differentiation. The evolutionarily conserved Notch signaling pathway consists of four Notch receptors (Notch1-4) and five Notch ligands (Jagged1,2and DLL1,3,4) in mammals. Cell-bound ligand initiates Notch signaling and triggers y-secretase to cleave the transmembrane receptor. The Notch intracellular domain (NICD) is then released from the receptor and translocates into the nucleus in association with the DNA-binding protein CSL (RBP-Jκ/CBF1) and the coactivator Mastermind-like (MAML) to activate target genes transcription such as HES and HEY families.Interestingly, mounting evidence indicates that ROS-mediated signaling is an important regulator of stem or progenitor cell differentiation. However, little is known about the effects of ROS on arterial-venous specification of endothelial cells. Therefore, the aim of this part is to explore the potential involvement of TGF-β-induced redox regulation in changes of the arterial and venous phenotype specification of endothelial cells.Objectives1. To investigate the effect of TGF-β on expression of arterial and venous markers2. To study whether TGF-β influences Notch signaling pathway3. To explore role of redox signaling in TGF-β-inducedarterial phenotypic switchMaterials and Methods1. Real-time PCRTotal RNA was extracted with TRIzol reagent and reverse transcripted to cDNA. Real-time PCR was performed using predesigned Taqman probe-primer sets or SYBR Green method and GAPDH or18S was used as house keeping gene.2. Western blot analysisCells were homogenized in lysis buffer after stimulation, then protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membranes. The membrane was blocked with5%nonfat milk and incubated with specific primary antibodies. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000luminescent image analyzer.3. ImmunofluorescenceCells cultured on Lab-Tek chamber slides were fixed and then incubated with primary antibodies and Alexa Fluor594-conjugated secondary antibodies. After counter staining with DAPI, cells were photographed using a confocal microscope.4. Nuclear and cytoplasmic protein extraction and western blotCells were harvested after treatment with TGF-β, nuclear and cytoplasmic protein was obtained using Nuclear and Cytoplasmic Protein Extraction Kit from Beyotime Company. Then western blot was performed to detect the nuclear translocation of NICD.5. Statistical analysisData are presented as mean±EM. Unpaired t-test or one-way ANOVA was performed to analyze data. p<0.05was considered as statistically significant.Results1. TGF-β stimulates expression of arterial markers in HUVECsTGF-β upregulated expression of the arterial marker EphrinB2, but had no effects on expression of the venous maker EphB4.We did not observe a significant change in the EphrinB2protein level.2. TGF-β activates Notch signaling pathwayTGF-β significantly increased the expression levels of Notch receptors (Notchl and Notch4) and ligands (Jaggedl and DLL4), and the Notch target gene (HEY1) in mRNA level. The increased expression of DLL4, Notchl and Jaggedl were also confirmed with western blot.3. TGF-β increases NICD production and nuclear translocationTGF-β treatment increased the amount of NICD, and stimulated nuclear translocation of NICD as detected with immunofluorescence and western blot of nuclear and cytoplasmic protein, indicating an enhanced Notch signaling.4. The effect of TGF-P on arterial switch is cell-specificIn HMVECs, we did not observe any significant effects of TGF-β on EphrinB2, EphB4, Notchl, Notch4, Jagged1or HEY1, indicating that the effects of TGF-β on expression of the arterial markers were cell type-specific.5. TGF-β-induced arterial marker expression is redox dependentWe demonstrated that in the presence of NAC, the effects of TGF-P on arterial marker expression were all diminished. Consistently, we found that application of exogenous H2O2mimicked the effects of TGF-β on expression of the arterial markers. Importantly, we demonstrated that TGF-β-induced effects on expression of Notchl, Jaggedl, HEY1and EphrinB2were diminished in cells pre-treated with the lentivirus expressing Nox4shRNA.Conclusions1. TGF-β induces endothelial cell arterial phenotypic switch in a cell-specific manner 2. TGF-P activates Notch signaling pathway in HUVECs3. Redox signaling mediates TGF-β-induced arterial phenotypic switch of HUVECs更多还原