【作者】 张澄；

【导师】 王兴利； 安丰双；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 论文一肿瘤坏死因子TNFα抑制胶原合酶限速亚单位P4Hα1的作用及其分子机制的实验研究1.背景:动脉粥样硬化斑块不稳定所导致的斑块破裂和血栓形成是引起急性心脑血管事件的主要原因。研究发现典型的易损斑块具有以下特点:较大的脂核、较薄的纤维帽、斑块胶原纤维和平滑肌细胞含量减少、巨噬细胞密度和活性增加、活化T细胞浸润增加等,其中斑块胶原的过度降解是导致斑块不稳定的主要原因,因此斑块内胶原的代谢平衡已成为近年来心血管病基础研究的热点之一。动脉粥样硬化斑块纤维帽的完整性和高强度主要有赖于细胞外基质的支持,其中最主要的成分是Ⅰ型、Ⅱ型和Ⅲ型胶原纤维。胶原代谢主要由胶原的合成与降解平衡来调节。此外,胶原纤维的三维结构也是影响其功能的因素之一。分布在动脉内膜的血管平滑肌细胞是粥样斑块内胶原的主要来源。斑块内胶原的代谢失调如胶原合成减少和/或降解增加均可导致斑块表面的纤维帽变薄和斑块的不稳定。因此,明确斑块的细胞外基质代谢及其调节机制对于稳定斑块具有重要的意义。胶原蛋白的生物学合成过程包括一系列的前骨胶原的翻译后修饰,其中细胞修饰过程需要5种酶的参与,包括3种胶原羟化酶和2种胶原糖基转移酶。在这些羟化酶中,4羟基-脯氨酸羟化酶（P4H）是一个具有2个α亚基（P4Hα1,P4Hα2）和2个β亚基（P4Hβ1,P4Hβ2）的四聚体。其中β亚基是二硫化异构酶,起催化作用的主要部位存在于β亚基,而α亚基的主要作用是决定酶的活性,是胶原合成的重要限速酶。P4H是在所有的已知21种类型胶原合成过程中起关键作用的酶。P4H的过表达会导致胶原的合成增多,而抑制P4H的产生则可导致胶原的不稳定和降解。炎症在许多心血管疾病的发生和发展过程中都起着重要的作用,如动脉粥样硬化、心力衰竭、心肌病和动脉瘤等。在急性冠状动脉综合征（ACS）的发病过程中,炎症是斑块易损和破裂的始动环节。炎症可以诱导多种细胞因子的分泌,如肿瘤坏死因子（TNFα）、转录生长因子（TGF-β）以及各种白细胞介素等,这些细胞因子可通过激活基质金属蛋白酶（MMP）和抑制胶原合成引起细胞外基质的降解。在这些因子中,TNFα由激活的巨噬细胞所分泌,在细胞外基质降解和ACS的发病过程中起着非常重要的作用。2.目的:（1）在体外试验中,研究肿瘤坏死因子（TNFα）对胶原酶限速亚单位P4Hα1的抑制作用。（2）在体外试验中,探讨TNFα对P4Hα1抑制作用的分子机制,为稳定易损斑块寻找新的治疗靶点。3.方法:3.1质粒构建pGL3-baisc多克隆载体被用来构建包含P4Hα1启动子片断的质粒。我们首先应用带有KpnⅠ和HindⅢ内切酶双酶切位点,并含有P4Hα1启动子片断的引物对P4Hα1启动子进行扩增。我们选取了-580bp至+76bp这一部分的启动子序列,采用逐段删除的方法,对引物进行设计,最后得到了包含9段P4Hα1启动子片断,顺序依次为:-580至+76bp、-480至+76bp、-417至+76bp、-320至+76bp、-271至+76bp、-184至+76bp、-145至+76bp、-97至+76bp、-32至+76bp、+18至+76bp的pGL3-P4Hα1载体。3.2对细胞的转染与刺激在剂量实验中,0 ng/ml,1 ng/ml,10 ng/ml和100 ng/ml TNFα纯品被分别加入到细胞培养基中,在时间实验中,我们在4小时,8小时,24小时和48小时,分别向培养基中加入100 ng/ml TNFα,与培养基充分混匀后进行检测。将1 ug含有P4Hα1启动子片断的pGL3-baisc质粒转染进平滑肌细胞中,进行下一步检测。为了检测细胞因子在TNFα对P4Hα1抑制过程中所起的作用,我们设计了因子NonO、hnRNP-K、BUB3、ILF2、DJ-1和HIF1等的RNA干扰片断,将siRNA转染入细胞进行检测。3.3 RT-PCR通过RNA提取,逆转录和RT-PCR的方法对平滑肌细胞内P4Hα1,β-actin,luciferase等的mRNA进行检测。3.4 EMSA将探针与细胞核蛋白混合,用抗体标记后进行凝胶电泳,观察探针与蛋白的结合能力。3.5 ChIP分析将抗目的蛋白的抗体与核蛋白结合后,用含有目的片段的引物进行PCR扩增,观察目的蛋白与目的DNA片段的结合能力。3.6 Western Blot经过提取蛋白,凝胶电泳,转膜,标记一抗和二抗,曝光等步骤观察目的蛋白的表达。4.结果:4.1.TNFα对P4Hα1的抑制作用我们用100ng/ml TNFα分别刺激平滑肌细胞4,8,24和48小时后,提取细胞RNA,检测P4Hα1mRNA的表达水平。发现经TNFα刺激8小时后,P4Hα1mRNA产量明显下降。用1ng/ml,10ng/ml和100ng/ml TNFα刺激平滑肌细胞8小时,观测P4Hα1mRNA的表达,发现随着TNFα浓度的升高,P4Hα1的mRNA水平呈递减趋势,在100ng/ml达到最大的抑制效应。4.2.P4Hα1启动子上TNFα反应元件（TaRE）的鉴定我们用脂质体转染的方式将含有P4Hα1启动子片断的PGL3 Basic载体转入平滑肌细胞中,经TNFα刺激后,用RT-PCR的方法检测PGL3 Basic载体上荧光素酶的表达。发现转染了含有+18至+76bp启动子片断的载体后,超过80%的TNFα的抑制作用消失,荧光素酶表达基本恢复正常,证明TNFα反应元件位于P4Hα1启动子-32至+18片断上,TNFα通过作用于这一片断抑制P4Hα1的mRNA表达。4.3.NonO与P4Hα1启动子的结合在EMSA中,我们将平滑肌细胞核蛋白与含有-32至+18片断的探针结合后,用抗NonO抗体标记,在经TNFα刺激后,Supershift条带显著增强,证明NonO在TNFα刺激后,与P4Hα1启动子的结合力增强。在平滑肌细胞核蛋白中,用抗NonO抗体免疫共沉淀下与NonO结合的DNA片段,用包含P4Hα1启动子-32至+18片断的引物进行PCR扩增后,发现只有经TNFα刺激后,NonO蛋白才能与P4Hα1启动子结合,从而扩增出清晰的PCR条带。将NonOsiRNA转入平滑肌细胞后,经TNFα刺激,用RT-PCR检测P4Hα1mRNA的表达。发现敲低NonO蛋白后,60%以上的TNFα对P4Hα1mRNA的抑制作用消失。4.4.TNFα通过下游通路ASK1和JNK实现对P4Hα1的抑制作用TNFα刺激平滑肌细胞前,先用JNK抑制剂-SP600125刺激平滑肌细胞1小时阻断JNK通路,观测P4Hα1mRNA的表达。发现加入JNK抑制剂后,TNFα对P4Hα1mRNA的抑制作用基本消失。ASK1抑制剂-Thioredoxin刺激平滑肌细胞1小时后,继续用TNFα刺激8小时,RT-PCR检测P4Hα1的mRNA水平变化,发现ASK1抑制剂几乎全部消除了TNFα对P4Hα1mRNA的抑制作用。4.5.DJ-1在TNFα对P4Hα1抑制过程中的作用我们将DJ-1siRNA转入平滑肌细胞中沉默DJ-1基因,经TNFα刺激后,观察P4Hα1mRNA的表达。结果显示DJ-1基因沉默消除了超过50%的TNFα对P4Hα1的抑制作用。将DJ-1siRNA转入平滑肌细胞中,并分别用TNFα和JNK1腺病毒刺激细胞,用抗NonO抗体免疫共沉淀后用含有P4Hα1启动子TaRE片段的引物PCR扩增,发现经TNFα或JNK1刺激都不能扩增出PCR片段,证明DJ-1RNA干扰后,NonO与TaRE不再结合。我们进一步将NonOsiRNA转入平滑肌细胞,用TNFα或JNK1腺病毒刺激后,用抗DJ-1抗体进行ChIP分析,发现不仅将NonO敲低后,DJ-1不能与TaRE结合,而且在没有转染NonOsiRNA的细胞中,TNFα或JNK1也都不能导致DJ-1与TaRE的结合。进一步用抗氧化DJ-1抗体进行ChIP分析,发现TNFα和JNK1均可使氧化DJ-1与TaRE结合,在将NonO基因沉默后,这种结合力明显下降。4.6.TNFα导致的组蛋白改变平滑肌细胞经TNFα或JNK1腺病毒刺激后,提取核蛋白,用抗组蛋白4第12个赖氨酸的抗体进行ChIP分析。结果显示,经TNFα刺激后,组蛋白4第12个赖氨酸的乙酰化明显增强。平滑肌细胞经TNFα或JNK1腺病毒刺激后,用抗组蛋白3第9个赖氨酸的抗体和包含P4Hα1启动子TaRE片段的引物进行ChIP分析。发现TNFα刺激后,组蛋白3第9个赖氨酸的去乙酰化明显增强。用20uMHATI刺激平滑肌细胞24小时,提取核蛋白,用抗NonO抗体进行ChIP分析后发现,不能扩增出PCR片段,证明经HATI刺激后,NonO不能再与P4Hα1启动子片段结合。经TSA刺激后,用抗NonO抗体免疫共沉淀NonO蛋白后,经ChIP分析,发现TSA对NonO与TaRE片段的结合并不能产生影响。用NonOsiRNA敲低NonO蛋白后,用抗组蛋白4第12个赖氨酸乙酰化抗体进行ChIP分析,结果显示组蛋白4第12个赖氨酸乙酰化没有变化,NonO的RNA干扰不能影响其乙酰化过程。经TNFα或JNK1刺激后,用抗组蛋白3第9个赖氨酸乙酰化抗体进行ChIP分析,发现扩增出了PCR片段,证明NonO的RNA干扰影响了组蛋白3第9个赖氨酸的构型,使其由去乙酰化变成乙酰化。5.结论:（1）TNF-α通过作用于P4Hα1启动子上的反应元件,激活ASK1-MKK4-JNK1-NonO信号通路,使转录因子DJ-1氧化和转录因子NonO磷酸化,实现对P4Hα1的抑制作用。（2）这些结果揭示了炎症因子引起胶原降解的关键分子,对于减少斑块纤维帽的胶原降解、稳定易损斑块、抑制主动脉瘤的形成,提供了具有重要学术意义和潜在应用价值的治疗新靶点。论文二动脉粥样硬化斑块中精氨酸酶Ⅱ表达水平和调控机制的实验研究1.背景近年来,由动脉粥样硬化（AS）导致的心脑血管疾病已成为世界许多国家第一位致死性疾病。研究证明,在AS的长期病程中,血管内皮功能异常是最早可检出的异常,在多项临床试验中,高频血管超声技术所测量的肱动脉内皮功能异常已被作为预测急性心脑血管事件的替代终点。由内皮型一氧化氮合酶（endothelial nitric-oxide synthase,eNOS）所介导的一氧化氮（NO）的产生是血管内皮功能的最重要调节因素。NO具有扩张血管的直接作用,并可通过激活下游通路,抑制血小板粘附和聚集、血管平滑肌细胞的增殖和迁移以及内皮细胞表达黏附分子,从而起到抗AS的作用。已有研究表明,NO合成的减少会导致血管内皮功能的失调,继而促进AS病变或诱发心脑血管事件。大量的基础和临床研究已证明由eNOS所介导的NO的产生对于AS的发生和发展起着关键的作用。目前,文献中已报告3种类型的一氧化氮合酶,第一种一氧化氮合酶首先在大脑中被分离出来,故称为神经元型一氧化氮合酶（neuronal nitric oxidesynthase,nNOS）,但其后在多种细胞包括血管内皮细胞中发现了nNOS的表达;第二种一氧化氮合酶首先从巨噬细胞中被分离出来,称为诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）;第三种一氧化氮合酶首先在主动脉内皮细胞中被发现,故称为内皮型一氧化氮合酶（eNOS）。在这三种一氧化氮合酶中,eNOS对于血管功能的调节最为重要。eNOS可被内皮细胞中的一些信号传导分子所激活,eNOS的构型变化尤其是磷酸化改变会直接影响eNOS的活性。左旋精氨酸是一个参与许多病理过程的半必需氨基酸,可被催化成为左旋脯氨酸、左旋鸟氨酸以及多聚氨酸,同时也是NO合酶的作用底物。在血管内皮细胞中,经eNOS作用后,左旋精氨酸转化为左旋瓜氨酸和NO,是NO的前体物质。由于NO在维持内皮功能中的关键作用和左旋精氨酸在NO合成中的重要地位,近年来一些学者对左旋精氨酸的血管保护作用进行了研究,结果表明左旋精氨酸-eNOS-NO通路参与AS的发生和发展过程。动物实验的结果证实,左旋精氨酸治疗可降低AS病变的发生率。精氨酸酶是一种尘物锰金属酶,可催化左旋精氨酸水解为左旋鸟氨酸和尿素。精氨酸酶有两个60%同源序列的同功异构体—精氨酸酶Ⅰ和精氨酸酶Ⅱ,它们在组织中的表达和细胞中的定位均不相同。精氨酸酶Ⅰ在肝脏内大量表达,并调节着体内大部分精氨酸酶的活性。精氨酸酶Ⅱ则在大部分组织中都有表达,尤其是肾脏和前列腺,但在肝脏中含量很少。最近的研究表明,在主动脉、肺动脉、颈动脉和冠状动脉等血管组织中,精氨酸酶Ⅰ和精氨酸酶Ⅱ的含量都很丰富。由于精氨酸酶有水解左旋精氨酸为左旋鸟氨酸和尿素的生物学功能,因此可与eNOS竞争分解左旋精氨酸,使左旋精氨酸向NO的转化减少。在巨噬细胞中对精氨酸的代谢过程的研究发现,左旋精氨酸向尿素的转化要多于向NO的转化,在动物体内抑制了精氨酸酶的表达后,NO的产量明显增高,在内皮细胞中的实验也证实抑制了精氨酸酶后,可刺激NO的合成。另外,精氨酸酶Ⅰ和精氨酸酶Ⅱ的过表达在降低左旋精氨酸水平的同时,也显著抑制了NO的含量。在精氨酸酶的两个同功异构体中,精氨酸酶Ⅱ与AS的关系更为密切。在人动脉内皮细胞中,针对精氨酸酶Ⅱ的RNA干扰降低了Ox-LDL对精氨酸酶的上调程度,增加了NO的表达。在ApoE基因敲除小鼠的主动脉早期AS病变中,精氨酸酶Ⅱ活性升高,NO水平降低。但这些研究主要局限于AS早期病变,随着斑块负荷的增大,精氨酸酶Ⅱ表达水平是否持续升高,eNOS表达水平是否持续下降,二者之间的关系如何,尚不清楚。因此,对于AS晚期病变,精氨酸酶Ⅱ能否作为敏感的生化标记物和有效的干预靶点是一个悬而未决的问题。此外,调节精氨酸酶Ⅱ的转录过程迄今不明,能否在启动子和转录因子水平抑制精氨酸酶Ⅱ的合成成为另一个悬而未决的问题。2.目的（1）在放置颈动脉狭窄套管的ApoE基因敲除小鼠中,建立斑块负荷较大的颈动脉AS模型,检测斑块内精氨酸酶Ⅱ和eNOS的表达水平。（2）寻找精氨酸酶Ⅱ的启动子活性片断以及与精氨酸酶Ⅱ启动子结合的转录因子,明确精氨酸酶Ⅱ的转录过程。3.方法3.1.质粒构建pGL3-baisc多克隆载体被用来构建包含精氨酸酶Ⅱ启动子片断的质粒。我们首先应用带有XhoⅠ和HindⅢ内切酶双酶切位点,并含有精氨酸酶Ⅱ启动子片断的引物对精氨酸酶Ⅱ启动子进行扩增。然后经PCR扩增、目的片断与载体的连接后构建pGL3-精氨酸酶Ⅱ启动子质粒。3.2.apoE基因敲除小鼠动脉粥样硬化模型的建立80只雄性apoE-/-小鼠均全程高脂饲料喂养（0.25%胆固醇+15%脂肪）,并行颈动脉套管术,以诱发颈总动脉粥样硬化病变,喂养8周后提取斑块组织进行检测。3.3.细胞培养人HELA细胞和内皮细胞经原代培养和细胞传代后进行实验研究。3.4.免疫组化经漂洗,封闭,一抗和二抗孵育后测量NO,eNOS和精氨酸酶Ⅱ等因子在斑块的含量3.5.RT-PCR经RNA提取,逆转录和RT-PCR等步骤检测精氨酸酶启动子片断的活性。4.结果4.1.组织学检测4.1.1.HE染色HE染色发现,小鼠右侧颈总动脉狭窄套管内出现较大的斑块,斑块内膜和中膜面积明显增大,左侧颈总动脉无斑块生成。4.1.2.Masson染色ApoE-/-小鼠右侧颈总动脉斑块中的胶原含量丰富。4.1.3.天狼猩红染色ApoE-/-小鼠右侧颈总动脉斑块中的Ⅰ型、Ⅱ型和Ⅲ型胶原纤维均明显增多。4.1.4.油红0染色ApoE-/-小鼠右侧颈总动脉斑块中脂质成分明显增多。4.2.eNOS在斑块组织中的表达用免疫组化的方法检测了eNOS在ApoE-/-小鼠双侧颈总动脉中的蛋白表达,发现apoE-/-小鼠右颈总动脉的eNOS蛋白表达水平显著低于左侧。4.3.精氨酸酶Ⅱ在斑块组织中的表达通过免疫组化观察精氨酸酶Ⅱ在斑块中的表达后,发现与左侧颈总动脉相比,右颈颈总动脉斑块内的精氨酸酶Ⅱ蛋白表达水平明显升高。4.4.精氨酸酶Ⅱ启动子片断活性的检测我们将包含精氨酸酶Ⅱ启动子片段的质粒转入HELA细胞中,24小时后,用RT-PCR的方法观察荧光素酶的表达。发现启动子-704bp至-644bp片断承担了大部分的精氨酸酶Ⅱ启动子活性。4.5.与精氨酸酶Ⅱ启动子结合的转录因子的鉴定我们设计了包含精氨酸酶Ⅱ启动子-704bp至-644bp片断的生物素标记探针。用免疫共沉淀的方法沉淀下与探针结合的蛋白,经凝胶电泳后和考马斯亮蓝染色,切下清晰的条带后,进行质谱分析,发现了与精氨酸酶Ⅱ启动子片断结合的蛋白PARP1、PSPC1和SFPQ。5.结论（1）在斑块负荷较大的AS病变中,精氨酸酶Ⅱ的蛋白表达水平明显升高而eNOS蛋白表达水平明显降低。PARP1、PSPC1和SFPQ等转录因子结合于精氨酸酶Ⅱ启动子的活性区域,参与调节精氨酸酶Ⅱ的转录过程。（2）这些结果提示精氨酸酶Ⅱ可作为AS的生化标记物和干预靶点,通过高效、特异地抑制精氨酸酶Ⅱ的合成,可增强eNOS的活性和改善血管内皮功能,具有重要的学术意义和潜在的应用价值。论文三ACE2基因转染抑制早期动脉粥样硬化病变及其分子机制的实验研究1.背景近年来的研究认为,AS发生的始动环节是血管内皮细胞（VEC）损伤,VEC损伤后可表达粘附分子,后者可使单核细胞与血管内皮细胞相互粘附,继而在趋化因子的作用下,使单核细胞与T-淋巴细胞迁移入内膜下组织,然后单核细胞转变为巨噬细胞并吞噬脂质形成单核源性泡沫细胞,同时产生一系列炎症和免疫反应,从而促进AS的形成。单核细胞趋化因子（MCP-1）是一种强有力的趋化因子,主要作用是促进单核细胞聚集于内膜下,在AS的形成中起重要作用。近年研究还发现,在人类内皮细胞上有一种能够结合Ox-LDL的受体,称之为植物血凝素样氧化低密度脂蛋白受体（Lectin like Ox-LDL Receptor-1,LOX-1）,LOX-1的激活进一步诱导了粘附分子的产生,导致内皮细胞功能的损伤。LOX-1的表达是VEC出现功能异常的早期标志,在AS的形成中起重要作用。因此,深入研究MCP-1、LOX-1等炎症因子和内皮细胞功能的变化,对明确AS早期病变的发生机制和防治措施至关重要。传统的观念认为血管紧张素Ⅱ（AngⅡ）是RAS系统的主要成分,最近发现了这一系统的许多新成员,包括ACE2、Ang-（1-7）、Ang-（1-9）等,这些物质的发现和对其病理生理学意义的研究使人们对RAS有了许多新的认识。ACE2是近年来发现的第一个ACE的同系化合物,与ACE一样,ACE2也是一种含锌的金属蛋白酶,完整的人类ACE2蛋白由805个氨基酸组成。目前发现的ACE2的生理功能主要有两点:能够高效催化AngⅡ转化为Ang-（1-7）,这一途径的催化活性较ACE水解AngⅠ的催化活性高400倍。ACE2亦可使AngⅠ转化为Ang-（1-9）,后者还可以进一步被ACE或其他的酶继续降解成为Ang-（1-7）。研究发现Ang-（1-7）是一种有重要生物学作用的血管紧张素家族的终末活性产物,是AngⅡ的内源性拮抗因子,具有扩张血管、降低血压、抑制平滑肌细胞增殖、利尿、利钠及抑制血管新生内膜增生等多种功能。近年来,ACE2及其催化产物Ang-（1-7）与心血管病的关系日益受重视。研究发现,新西兰大白兔AS斑块中有大量ACE2蛋白的表达,主要分布在血管内皮细胞和泡沫细胞,提示ACE2与AS斑块的病理过程密切相关。急性心肌梗死（AMI）的大鼠中ACE2及ACE基因的表达明显增多,免疫组化研究发现ACE2蛋白主要表达在血管内皮细胞、平滑肌细胞、巨噬细胞以及心肌细胞中。心力衰竭患者中心肌ACE2基因表达增多,ACE2过表达可抑制高血压大鼠的心肌纤维化,改善左室舒张功能,提示ACE2拮抗了ACE的功能。直接应用Ang-（1-7）还可改善急性心肌梗死的左室重构。这些研究提示ACE2过表达具有保护心血管的功能。通过增强ACE2的表达或直接应用Ang-（1-7）,可拮抗ACE的作用,对AS及心脑血管病的治疗可起到积极的作用,从而有望成为心脑血管病的一个治疗新靶点。然而,ACE2过表达对于早期AS病变的作用及其分子机制至今不明。2.目的（1）在体内研究中,观察ACE2过表达对AS早期病变的抑制作用。（2）在体内和体外研究中,探讨ACE2过表达抑制早期AS病变的分子机制。3.方法3.1.ACE2表达质粒—复制缺陷重组腺病毒质粒Ad5-ACE2的构建将质粒PMD18-T-ACE2进行酶切,然后构建穿梭质粒（pDC316—ACE2）。穿梭质粒与腺病毒骨架质粒进行同源重组,形成重组腺病毒质粒,重组腺病毒质粒在HEK293细胞内包装成为复制缺陷重组腺病毒质粒Ad5-ACE2。2.2.ACE2基因治疗100只新西兰大白兔随机分为Ad-ACE2组（20只）、Ad-ACE2+A779组（20只）、Ad-EGFP组（20只）、单纯高脂对照组（20只）及单纯高脂对照+A779组（20只）。在所有实验兔中行腹主动脉内膜球囊损伤术并行4周高脂喂养,于4周末时开始基因治疗,向转染部位的管腔中分别注入滴度为2.5×10⁹pfu/mL的Ad-ACE2或Ad-EGFP。在Ad-ACE2+A779组,除进行ACE2基因治疗外,加用Ang-（1-7）受体拮抗剂A779,用药具体用法为:经颈静脉泵入,浓度为200 ngkg^-1 min^-1,用药28天。3.3.内皮细胞的ACE2腺病毒转染首先用Ang-Ⅱ(终浓度为10^-6M/L)刺激细胞24小时,更换培养基后将ACE2腺病毒载体和EGFP腺病毒载体按1×10⁶ pfu的浓度分别加入细胞培养基中,在ACE2基因转染24小时,48小时和72小时后提取细胞蛋白进行检测。用1×10⁶mol Ang-（1-7）加入到内皮细胞培养基中,充分混匀后于37℃培养箱放置,于4小时,8小时,16小时和24小时提取蛋白进行检测。3.4.质谱分析用质谱分析的方法测量AS病变组织中Ang-（1-7）/Ang-Ⅱ比值和Ang-Ⅰ/Ang-Ⅱ比值,分别反映ACE2和ACE的活性。3.5.血脂水平检测在实验开始、4周末和8周末抽取兔的血液样本,留取血清,用酶学检测血清胆固醇（TC）和甘油三酯（TG）水平。3.6.免疫组化检测经漂洗、封闭、一抗和二抗孵育后测量LOX-1、MCP-1、ERK、p38等因子在AS组织中的含量3.7.RT-PCR经RNA提取,逆转录和RT-PCR检测AS病变组织中ACE2的表达。3.8.Western Blot经凝胶电泳、转膜、加入一抗和二抗后检测AS病变组织中ACE2、ACE、AT1R、MCP-1、LOX-1等因子的蛋白表达水平。4.结果4.1.基因转染后ACE2的表达提取兔的斑块组织,发现与Ad-EGFP组,单纯高脂对照组和单纯高脂对照+A779组相比,Ad-ACE2组和Ad-ACE2+A779组的ACE2基因表达明显增高。用Western Blot的方法观察Ad-ACE2在细胞内的转染效率,发现与EGFP腺病毒转染组和对照组相比,ACE2的蛋白水平在腺病毒转染24小时后显著增高。4.2.ACE2基因转染对AS病变的影响通过对兔腹主动脉斑块的HE染色观察到,Ad-EGFP组、单纯高脂对照组、单纯高脂对照+A779组与Ad-ACE2组相比,AS病变的内膜面积显著增加,内膜与中膜面积比值增大。在加入了Ang-（1-7）受体拮抗剂-A779后,Ad-ACE2+A779组的内膜面积和内膜与中膜面积比值比Ad-ACE2组又明显增大。4.3.ACE2基因转染对MCP-1和LOX-1表达的影响以免疫组化方法检测斑块内MCP-1和LOX-1的蛋白表达,与Ad-ACE2组相比,Ad-EGFP组、单纯高脂对照组、单纯高脂对照+A779组中MCP-1和LOX-1蛋白表达水平升高。以ACE2腺病毒和A779刺激细胞后,用Western Blot观测MCP-1和LOX-1的蛋白水平,发现在ACE2腺病毒转染48小时后,与EGFP腺病毒转染组和对照组相比,MCP-1和LOX-1蛋白表达水平明显下降,但在加入A779后,发现ACE2对MCP-1和LOX-1表达的抑制作用了大部分被逆转。4.4.ACE2过表达对Ang-（1-7）蛋白表达的影响通过对腹主动脉斑块内Ang-（1-7）蛋白水平的检测发现,Ad-ACE2组和Ad-ACE2+A779组的Ang-（1-7）表达高于Ad-EGFP组、对照组和A779组。ACE2基因转染24小时后,Ang-（1-7）蛋白水平与EGFP基因转染组和对照组相比明显升高。4.5.ACE2腺病毒转染对ACE表达的影响用免疫组化的方法对体内ACE蛋白表达水平的检测发现,Ad-ACE2组和Ad-ACE2+A779组的ACE表达明显低于Ad-EGFP组、对照组和A779组。Western Blot检测内皮细胞ACE表达后发现,在ACE2腺病毒转染细胞24小时后,与EGFP基因转染组和对照组相比,ACE蛋白表达水平明显降低。4.6.ACE2腺病毒转染对AT1R表达的影响对AS组织进行Western Blot分析,发现Ad-ACE2组和Ad-ACE2+A779组的AT1R蛋白表达水平明显低于Ad-EGFP组、对照组和A779组。提取细胞蛋白后,用Western Blot检测AT1R的表达水平,发现在ACE2腺病毒转染细胞48小时后,AT1R蛋白表达水平明显下降。4.7.ACE2腺病毒转染对ERK-p38表达的影响对AS组织进行免疫组化检测,发现Ad-ACE2组和Ad-ACE2+A779组的ERK和p38蛋白表达比Ad-EGFP组、对照组和A779组显著降低。ACE2基因转染内皮细胞24小时后,与EGFP基因转染组和对照组相比,ERK和p38的蛋白表达水平明显下降。4.8.ACE2腺病毒转染PI3K-Akt表达的影响用Western Blot的方法观察了AS组织内PI3K和Akt通路的表达情况,结果显示,Ad-ACE2组和Ad-ACE2+A779组的PI3K和Akt蛋白表达比Ad-EGFP组、对照组和A779组明显升高。ACE2腺病毒转染细胞24小时后,与Ad-EGFP组和对照组相比,PI3K和Akt蛋白表达水平明显升高。4.9.ACE2腺病毒转染对ROS表达的影响我们在细胞中对Ang-Ⅱ下游通路ROS的表达进行了观测,发现ACE2基因转染48小时后,与Ad-EGFP组和对照组相比,ROS蛋白表达水平显著下降。加入A779后,与Ad-ACE2组相比,Ad-ACE2+A779组ROS蛋白表达水平明显回升。5.结论（1）ACE2通过多条信号通路的交互对话实现对AS早期病变的显著抑制作用,这些通路包括:Ang-Ⅱ-AT1R-ERK-p38-ACE,Ang-Ⅱ-AT1R-ROS-MCP-1/LOX-1,Ang-Ⅱ-AT1R-PI3K-Akt-LOX-1/MCP-1,Ang-（1-7）-ERK-p38-ACE,Ang-（1-7）-PI3K-Akt-LOX-1/MCP-1和Ang-（1-7）-ROS-MCP-1/LOX-1等。（2）通过选择性增强或抑制Ang-Ⅱ和Ang-（1-7）信号通路的交互作用,有可能起到抑制AS早期病变的作用,这为AS的治疗提供了多个具有重要理论意义和潜在应用价者的新靶点。更多还原

【Abstract】 Paper One An Experimental Study on the Suppressive Effects of Tumor Necrosis Factorαon the Rate-limiting Subunit of Collagen Synthase（P4Hα1） and the Underlying Molecular Mechanisms1.IntriductionMany pathologic changes in the cardiovascular system can be triggered by inflammation,which contributes to the disease process.Metabolic imbalance in the extracellular matrix（ECM） in the myocardium and arterial wall represents one of the key structural changes that mark the development and progression of most cardiovascular diseases.ECM components,especially collagen—the main constituent of the fibrous cap in atheroma—determines plaque stability and vulnerability to rupture.Furthermore,dysregulated ECM metabolism in the aortic wall,such as inadequate collagen degradation or elastin disruption,leads to aortic aneurysm and rupture.ECM is the structural framework of all tissues including the arterial wall,in which fibrillar proteins（collagen and elastin） and adhesive proteins（eg, laminin and fibronectin） form the structural backbone of the tissue.In the arterial wall,various cells including endothelial cells,smooth muscle cells,and fibroblasts,contribute to ECM metabolism.Collagen is one of the most metabolically active ECM components,with at least 39 subtypes;typesⅠandⅢare the ones most commonly found in the arterial wall.The collagen molecule consists of 3 identical polypeptide chains,calledαchains.This molecule has at least 1 triple-helical collagenous domain with repeating （Gly-X-Y） n sequences,ie,a glycine residue at every third amino acid and, frequently,proline and 4-hydroxyproline in the X and Y positions.Collagen biosynthesis involves a number of posttranslational modifications of procollagens and proteolytic conversion to collagens.The intracellular modifications require 5 specific enzymes,including 3 collagen hydroxylases and 2 collagen glycosyltransferases.Prolyl-4-hydroxylase（P4H） is one of the key intracellular enzymes required for the synthesis of all known types of collagens.It catalyzes the formation of hydroxyproline from proline residues located in repeating X-Pro-Gly triplets in the procollagens during posttranslational processing.It is essential for folding the procollagen polypeptide chains into stable triple helical molecules.Inhibition of P4H produces unstable collagen associated with collagen decrease.P4H is composed ofαandβsubunits in whichαsubunit is rate-limiting and essential for collagen maturation and secretion.Cytokines,including TNF-α,transforming growth factor（TGF）-α,and various interleukins,are dysregulated in inflammation and may participate in ECM metabolism by increasing ECM degradation through activation of matrix metalloproteinases（MMPs） and inhibition of collagen synthesis.TNF-α,which is released by activated macrophages,is one of the most potent cytokines involved in cardiovascular pathogenesis1 and actively regulates ECM metabolism.2.Objectives（1） To investigate whether TNF-αexerts an inhibitive effect on P4Hα1 in vitro;（2） To elucidate the underlying molecular mechanisms of TNF-α-mediated inhibition of P4Hα1 in vitro and explore a novel therapeutic target of plaque stabilization.3.Methods3.1 Plasmid ConstructionTo define promoter regions that are responsive to TNF-α-mediated P4Hα1 suppression,we generated P4Hα1 promoter-pGL3 constructs with serial deletions of the P4Hα1 promoter.The pGL3-basic vector（Cat#:E1751, Promega,Madison,Wi） was used for the plasmid construction.We produced plasmids containing P4Hα1 promoter regions from -580,-480,-417,-320,-271, -184,-145,-97,-32,and -18 to +76bp between the multiple cloning sites Kpnl and HindⅢfor the purpose of directional cloning.3.2 Cell Transfection and StimulationFor dose-dependent effects,HASMCs were treated with 0,1,10,and 100 ng/mL human recombinant TNF-α（Cat#T6674,Sigma-Aldrich） for 8 hours before cells were harvested for measurements of target gene mRNA levels. For the time-course study,we treated cells for 4,8,24,and 48 hours,1ug recombinant P4Hα-pGL3 plasmids were transfected to HASMCs with Lipofectamine 2000.we used NonO,hnRNP-K,BUB3,and ILF2 specific siRNAs（100 nmol/L） to inhibit the expression of these proteins in cells。3.3 RT-PCRP4Hα1,β-actin and luciferase mRNA levles were measured by RT-PCR after RNA extraction and reverse transcription.3.4 EMSAWe carried out electrophoretic mobility shift assay（EMSA） to ascertain whether the TaRE in the P4Hα1 promoter was binding with any transcription factors.For the antibody-based supershift assay,antibodies against transcription factors were added to a mixture of biotin-labeled oligonucleotide probes and nuclear proteins.3.5 Chromatin Immunoprecipitation Assay The chromatin immunoprecipitation（ChIP） assay was performed with the histone ChIP assay.We also used anti-transcription factor antibodies instead of anti-H3 histone antibody for the immunoprecipitation process.3.6 Western BlotProteins were extracted form HASMC,separated with 10%SDS-PAGE, and transferred to nitrocellulose membranes.After incubation with the primary and secondary antibodies and exposure,protein expression was determined.4.Results4.1.TNF-α-mediated Suppression of P4Hα1 ExpressionWe treated HASMCs with TNF-α100 ng/mL for 4,8,24,and 48 hours.At the end of each of these time points,cells were harvested and P4Hα1 mRNA levels were measured.We found that P4Hα1 mRNA levels were significantly decreased up to 8 hours after treatment.In examining dose-response effects, we observed a linear reduction in P4Hα1 mRNA levels in HASMCs treated with 1 to 100 ng/mL TNF-αfor 8 hours in culture.4.2.Identification of TNF-αRegulatory Element（TaRE） in the P4Hα1 5’-Flanking SequenceWe transfected HASMCs with several pGL3 reporter constructs containing the progressively deleted 5’-flanking regions of the P4Hα1 gene and measured mRNA levels of the luciferase gene after treatment with TNF-α.Compared with no treatment,TNF-αdramatically reduced promoter activity in all P4Hα1 promoter vectors except when the region of -32bp to +18bp was deleted in the promoter region（Figure 2）,which abolished more than 80%of the TNF-α-induced inhibition.4.3.Direct Binding of NonO and hnRNP-K to the P4Hα1 PromoterThe density of the supershift band with the anti-NonO antibody was significantly increased when nuclear proteins extracted from TNF-α-treated HASMCs were used in the EMSA.Only TNF-α-treated HASMCs showed a NonO-TaRE interaction in the ChIP assay.NonO silencing abolished more than 60%of TNF-α-mediated P4Hα1 suppression.4.4.Involvement of the ASK1-JNK PathwayJNK inhibitor recovered almost 100%of the TNF-α-mediated P4Hα1 suppression,after treatment with the ASK1 inhibitor,TNF-α-mediated P4Hα1 suppression recovered to a degree similar to that of JNK inhibitor.4.5.Role of DJ-1 in TNFα-MKK4-JNK1-NonO-mediated P4Hα1 SuppressionAfter DJ-1 expression was silenced by DJ-1-specific siRNA in HASMCS,we observed a 50%recovery of P4Hα1 expression in HASMCs treated with TNFα. Using the ChIP assay with the anti-NonO antibody for immunoprecipitation,we showed that TNF-αinduced NonO-TaRE binding was abolished when the expression of DJ-1 was suppressed by gene specific siRNA.The ChIP assay （with anti-oxidized DJ-1 antibody） showed that the binding of DJ-1 to TaRE was abrogated by NonO siRNA.4.6.Role of histone acetylation in TNFα-MKK4-JNK1-NonO-mediated P4Hα1 suppressionIn contrast,although TNFαor JNK1 induced H4 lysine 12（H4L12） acetylation,the treatments induced deacetylation at H3 lysine.HATI abolished the TNFα-induced H4L12 acetylation,and the HDAC inhibitor abrogated TNFα-induced H3L9 deacetylation.These results demonstrated that silencing NonO by gene specific siRNA had no effect on TNFα-induced H4L12 acetylation.However,NonO suppression abolished TNFα-induced H3L9 deacetylation.5.Conclusions（1） Through direct binding to TaRE in the TNF-αpromoter,TNFαactivates the ASK1-MKK4-JNK1-NonO pathway,oxidizes DJ-1 and phosphorylates NonO,thereby suppressing P4Hα1 transcription.（2） These results revealed the key molecules involving inflammation-related collegen degradation and provided novel therapeutic targets for reducing collegen degradation in the fibrous cap of atherosclerotic plaques and in the aortic wall,thereby stabilizing vulnerable plaques and inhibiting the formation of aortic aneurysm. Paper Two An Experimental Study on the Expression Level and Regulatory Mechanisms of ArginaseⅡin Atherosclerotic Plaques1.IntroductionIn recent years,atherosclerosis（AS）-induced cardiovascular disease has become the number one killer in many countries of the world.Many studies have demonstrated that vascular endothelial dysfunction is the earliest detectable abnormalities in the long time course of AS and brachial arterial endothelial dysfunction measure by high frequency ultrasound has been used as a surrogate endpoint for acute cardiovascular events in multiple clinical trials.Nitric oxide（NO） produced by endothelial nitric oxide synthase（eNOS） is the most important factor for regulating endothelial function.NO directly dilates blood vessels and inhibits adhesion and aggregation of platelets, proliferation and migration of smooth muscle cells and expression of adhesion molecules by endothelial cells,thereby exerting an anti-atherosclerosis effect. Previous studies have confirmed that the reduction in NO synthesis may lead to endothelial dysfunction,accelerate AS lesions and induce cardiovascular events.Accumulating evidence now exists that NO plays a key role in the development and progression of AS.At present,three types of NO synthase have been reported in the literature.The first type of NO synthase was found in the brain and thus named neuronal nitric oxide synthase（nNOS）,although it was detected later in many other cells including vascular endothelial cells.The second type was discovered in macrophages and named inducible nitric oxide synthase（iNOS）. The third type was found in the aortic endothelial cells and therefore named eNOS.Among the three types,eNOS is the most important in mediating vascular function,eNOS can be activated by some signals from the endothelial cells and the configuration changes,especially phosphorylation,of eNOS may affect the activity of this particular enzyme.L-arginine is a semi-essential amino acid engaging many pathological processes,which can be catalyzed into L-arginine,L-ornithine or polyamines and serve as substrate of NO synthase.Converted by eNOS in endothelial cells,L-arginine becomes L-citrulline and NO,and hence is the precursor of NO.Because of the key role of NO in vascular protection and the important function of L-arginine in the synthesis of NO,some authors have stepped forward to investigate the function of L-arginine for vessel protection and they are able to show that L-arginine can activate eNOS-NO pathway and treatment with L-arginine can even reduce the incidence of AS-related diseases in animals.Arginase is a manganese metalloprotease which can catalyze L-arginine into L-ornithine and urea.Arginase has two isozymes-arginineⅠand arginineⅡ, with 60%homologous sequence.ArginaseⅠexpresses mainly in the liver and undertakes the most activities of arginase in the body.ArginaseⅡexpresses in most tissues,in particular,kidney and prostate.Recent studies have reported that vascular tissues including the aortic,pulmonary,carotid and coronary arteries are rich in both arginaseⅠand arginaseⅡ.Since arginase is capable of catalyzing arginine into L-ornithine and urea, it may compete with eNOS in catalyzing L-arginine and impede the conversion of L-arginine to NO.Metabolic Studies of L-arginine in macrophages found that there was more conversion of L-arginine to urea than to NO.The production of NO was increased after inhibition of arginase both in vitro and in vivo.On the other hand,overexpression of arginaseⅠand arginaseⅡreduced the expression levels of L-arginine and NO at the same time.ArginaseⅡhas been found to be more closely related to AS than arginaseⅠas RNA interference of arginineⅡattenuated arginine upregulation by Ox-LDL and enhanced NO expression in human aortic endothelial cells.In the early aortic atherosclerotic lesions in Apo-/- mice,the activity of arginaseⅡwas increased while the level of NO decreased.These studies,however,were limited to early atherosclerotic lesions and it remains unclear whether the expression level of arginaseⅡcontinues to increase and that of eNOS continues to decrease with increased plaque burden.Therefore,it is still an open question whether arginaseⅡcan serve as a sensitive biomarker and an effective therapeutic target in the late stage of atherosclerosis with a big plaque burden.Besides,as the transcription process of arginaseⅡis unknown,whether arginase can be inhibited at the levels of promoter or transcription factors constitutes another open question.2.Objectives:（1） To establish a carotid atherosclerotic model with a large plaque burden in ApoE-/- mice with placement of a stenotic carotid collar and to measure the expression levels of arginaseⅡand eNOS in these plaques;（2） To detect the positive region of arginaseⅡpromoter and the transcription factors binding with this promoter and thereby clarify arginiaseⅡtranscription process.3.Methods3.1.Plasmid ConstructionpGL3-basic vector was used to define positive promoter regions of arginaseⅡ.We generated arginaseⅡpromoter-pGL3 constructs with serial deletions of the arginaseⅡpromoter.After the PCR amplification and plasmid construction,we produced plasmids containing arginaseⅡpromoter regions between the multiple cloning sites XhoⅠand HindⅢfor the purpose of directional cloning. 3.2.Animal Model80 male apoE-/- mouse were fed with an atherogenic chow and underwent right carotid collar placement to induce atherosclerotic lesions with a big plaque burden.The left carotid artery without collar placement was used as a internal control.3.3.Cell CultureHuman Hela cells and endothelial cells were cultured for experimental studies.3.4.Immunohistochemical AnalysisExpression levels of eNOS and arginaseⅡproteins in atherosclerotic lesions were measured using appropriate primary antibodies.3.5 Real-time RT-PCRThe gene expression levels of luciferase were quantitatively analyzed using RT-PCR.4.Results4.1.Histological Measurement4.1.1.Hematoxylin and Eosin StainingIncreased intima and media area and significant plaques were detected in the right carotid atherosclerotic lesions.No plaques were found in the left carotid artery.4.1.2.Masson StainingCollagen expression was abundant in the right common carotid artery of ApoE^-/- mice.4.1.3.Picrosirius Red StainingThe relative content of typeⅠ,ⅡandⅢcollagen fibers was increased significantly in atherosclerotic plaques of the right carotid artery4.1.4.Oil Red O StainingThe relative content of lipids was significantly increased in atherosclerotic plaques of the right carotid artery of ApoE^-/- mice. 4.2.Expression of eNOS in PlaquesImmunohistochemical staining revealed that the expression level of eNOS proteins was significantly lower in the right carotid artery than that in the left carotid artery.4.3.Expression of ArginaseⅡin PlaquesImmunohistochemical staining showed that the expression level of arginaseⅡproteins was significantly higher in the right carotid artery than that in the left carotid artery.4.4.Promoter Region Responsible for ArginaseⅡTranscription ActivitypGL3-basic vectors combined with arginaseⅡpromoter regions were transfected into HELA cells for 24h and the expression of luciferase was detected by RT-PCR.The result showed that the region of-704bp to-644bp was responsible for most activities of arginaseⅡpromoter.4.5.Identification of Transcription Factors Biding with ArginaseⅡPromoterWe designed biotin-labeled probe containing the -704bp to -644bp region of arginaseⅡpromoter.Proteins binding with the probe were pulled-down using immunoprecipitation and stained with Coomassie blue after gel electrophoresis.A clear band was cut for mass spectrometry and proteins PARP1、PSPC1 and SFPQ binding with the positive region of arginaseⅡpromoter were finally indentified as the transcription factors.5.Conclusions（1） The expression level of arginaseⅡwas increased whereas that of eNOS decreased in atherosclerotic lesions with a big plaque burden.Transcription factors PARP1、PSPC1and SFPQ bind with the positive region of arginaseⅡpromoter and mediate the transcription process of arginaseⅡ.（3） These results suggest that arginaseⅡcan serve as a biomarker and a therapeutic target in atherosclerosis.By effectively inhibiting the synthesis of arginaseⅡ,the activity of eNOS can be enhanced and endothelial function improved.Thus,our finding has important theoretical significance as well as application potentials.Ⅰ Paper Three An Experimental Study on the Therapeutic Effects of ACE2 Gene Transfection on Early Atheroscleriotic Lesions and the Underlying Molecular Mechanisms1.IntriductionAccumulating evidence indicates that the renin-angiotensin system（RAS） plays an important role in the pathogenesis of atherosclerosis.Angll increases mRNA and protein expression of monocyte chemoattractant protein-1（MCP-1） and lectin-like oxidized low-density lipoprotein receptor-1（LOX-1） and enhances the uptake of low-density lipoprotein（LDL） through LOX-1 by endothelial cells,macrophages and smooth muscle cells.MCP-1 is the major chemotactic factor contributing to monocyte adhesion to endothelial cells,one of the earliest events in the pathogenesis of atherosclerosis.Likewise,LOX-1 plays an essential role in endothelial injury and dysfunction.Therefore, inhibition of ACE by ACE inhibitors or AT1 receptor blockers is effective against atherosclerosis.Angiotensin-converting enzyme 2（ACE2）,a zinc-bearing metalloproteinase,is the first angiotensin-converting enzyme（ACE） homologue identified in 2000.The human ACE2 protein consists of 805 amino acids.The expression of ACE2 is highly tissue specific and is found to be distributed mainly in heart,kidney and didymus tissue.Recent studies have demonstrated that in physiological and pathological conditions,ACE2 competes with ACE by converting vasoconstrictive angiotensinⅡ（AngⅡ） into vasodilative Ang-（1-7）.ACE2 appears to play a protective role in cardiovascular system.Both ACE and ACE2 mRNA and protein expression is increased in rats with acute myocardial infarction.Many studies have found ACE2 protein expressed in atherosclerotic plaques in New Zealand white rabbits,mainly in endothelial cells and foam cells.Cardiac function was shown to decline in ACE2 gene knock-out mice,but overexpression of ACE2 by use of an adenovirus（Ad） vector significantly inhibited the development of myocardial fibrosis.These studies suggest that ACE2 may protect myocardium and vessels by counteracting ACE and relieving the harmful effects of RAS and hence is a potential therapeutic target in cardiovascular diseases.2.Objectives:（1）To attest the therapeutic effects of ACE2 overexpression on early atherosclerotic lesions;（2） To elucidate the molecular mechanisms of ACE2-mediated therapeutic effects on atherosclerotic lesions.3.Methods:3.1.Preparation of ACE2 Ad VectorThe murine ACE2 cDNA was amplified by RT-PCR from RNA of mouse kidney.Recombinant adenoviruses（Ad） carrying the murine ACE2（AdACE2） or a control transgene EGFP（Ad-EGFP） were prepared as previously described with the AdMax system.3.2.Animal Model and Gene TransferOne hundred of New Zealand white rabbits were fed an atherogenic chow and underwent balloon induced arterial endothelial injury after anesthesia using a previously described method.Rabbits were randomly divided to 5 groups（n=20 in each group） and gene therapy was initiated at the end of week 4.Rabbits in group Ad-ACE2 or Ad-EGFP or Ad-ACE2+A779 or A779 or control received a suspension of Ad-ACE2（2.5×10⁹pfu） or Ad-EGFP （2.5×10⁹pfu） or Ad-ACE2+A779 or A779 or no injection,respectively.A779 is an antagonist of Ang-（1-7） receptor and was administered at a dose of 200 ng kg^-1 min^-1 for 28 days.Rabbits were maintained on the high-cholesterol diet for additional 4 weeks.The rabbits were anesthetized and their aortas were collected for pathological and biochemical analysis.3.3.Cell Culture and Gene TransferThe HUVECs were incubated with Ang-Ⅱfor 24 hours before they were divided into 10 groups that received Ad-ACE2,Ad-EGFP,Ad-ACE2+A779 （1μmol/l）,A779,Ad-ACE2+ERK inhibitor,ERK inhibitor,Ad-ACE2+Akt inhibitor,Akt inhibitor,Ang-（1-7） and no treatment（control group）,respectively. Ad-ACE2（1×10⁶ pfu） or Ad-EGFP（1×10⁶ pfu） was transfected into cells per well in a 6-well plate and cells were harvested 24,48 and 72 hours after gene transfection for western blot analysis.Cells in the Ang-（1-7） group were collected 4,8,16 and 24 hours after treatment with Ang-（1-7） at a dose of 1×10⁶ mol.3.4.Measurement of ACE2 and ACE ActivityThe enzymatic activities of ACE2 for converting Ang-Ⅱto Ang-（1-7） and those of ACE for converting Ang-Ⅰto Ang-Ⅱwere evaluated by surface-enhanced laser desorption/ionization time of flight mass spectrometry.3.5.Serum Lipid MeasurementBlood samples were collected at baseline and at the end of week 4 and week 8,respectively,and serum concentrations of total cholesterol（TC） and triglyceride（TG） were determined by enzymatic assays.3.6.Immunohistochemical AnalysisMacrophages,LOX-1,MCP-1,Ang-（1-7）,ERK and p38 in atherosclerotic lesions were identified using appropriate primary antibodies.3.7.Real-time RT-PCR The gene expression levels of ACE2 were quantitatively analyzed using RT-PCR.3.8.Western Blot AnalysisThe protein expression of ACE2,ACE,Ang-Ⅱ,AT₁R,MCP-1,LOX-1, Ang-（1-7）,ERK,p38,PI3K,AKT and ROS were assayed by Western blot both in Vivo and in Vitro,respectively.4.Results4.1.ACE2 Expression and Activity after Gene TransferACE2 expression was high in atherosclerotic lesions of Ad-ACE2 and Ad-ACE2+A779 groups but low in lesions of Ad-EGFP,A779 and control groups.Similarly,western blot analysis showed that ACE2.protein expression in human endothelial cells was significantly increased 24 hours after Ad-ACE2 transfection in comparison with Ad-EGFP and control groups.4.2.Effects of ACE2 Gene Transfer on Atherosclerotic LesionsMarked intimal thickening was present in the Ad-EGFP,control and A779 groups.However,the intima area and the ratio of intimal area/media area（I/M area） were significantly lower in the Ad-ACE2 group than in the Ad-EGFP, control or A779 groups.Conversely,with the administration of A779 in the Ad-ACE2+A779 group,the intimal area and the ratio of I/M area were significantly increased compared with those in the Ad-ACE2 group.4.3.Effects of ACE2 Gene Transfer on MCP-1 and LOX-1 ExpressionImmunohistochemistry was used to examine the expression of MCP-1 and LOX-1.The abdominal aorta in Ad-EGFP,control and A779 groups were heavily stained,as compared with those in the Ad-ACE2 group.Western blot analysis in cultured endothelial cells demonstrated a lower expression of MCP-1 in the Ad-ACE2 group than that in the Ad-EGFP and the control groups 48 hours after ACE2 transfection.Similarly,LOX-1 was significantly suppressed by ACE2 gene transfer but this effect was not observed until 72 hours after gene transfer. 4.4.Effects of ACE2 Transfection on Ang-（1-7） ExpressionThe Ang-（1-7） protein expression level was higher in the Ad-ACE2 and Ad-ACE2+A779 groups than that in the Ad-EGFP,control and A779 groups in the aortic lesions.Ang-（1-7） protein expression level in the Ad-ACE2 group was significantly increased in comparison with that in the Ad-EGFP and control groups in HECs.4.5.Effects of ACE2 Transfection on ACE ExpressionThe ACE protein expression determined by immuohistochemistry were significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.Western blot analysis indicated that the ACE protein expression level was significantly reduced 24 hours after gene transfection in the Ad-ACE2 group compared with that in the Ad-EGFP and control groups.4.6.Effects of ACE2 Transfection on AT₁R ExpressionAT₁R protein expression level detected by western blot were significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.Reduction of the AT₁R protein expression level was also observed 48 hours after Ad-ACE2 transfection and 16 hours after Ang-（1-7） treatment in HECs.4.7.Effects of ACE2 on ERK-p38-ACE ExpressionERK and p38 protein expression level was significantly lower in the Ad-ACE2,Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups according to immunohistochemical analysis.The ERK and p38 protein expression level was significantly reduced in the Ad-ACE2 group 24 hours after gene transfection in comparison with that in the control and Ad-EGFP groups.4.8.Effects of ACE2 on PI3K-Akt-MCP1/LOX1 ExpressionThe PI3K and Akt protein expression in atherosclerotic lesions were detected by western blot.The results indicated that protein expression levels of PI3K and Akt were significantly higher in the Ad-ACE2 and Ad-ACE2+A779 group than that in the Ad-EGFP,A779 and control groups.The PI3K and Akt protein expression level in the Ad-ACE2 group 24 hours after gene transfection was significantly higher than that in the control and Ad-EGFP groups.4.9.Effects of AdACE2 on ROS Expression in vitroThe ROS protein expression level was significantly reduced in the Ad-ACE2 group in comparison with that in the control and Ad-EGFP groups and this effect was not enhanced by increasing gene transfection time to 48 and 72 hours5.Conclusions（1） ACE2 overexpression significantly attenuated early atherosclerotic lesions via cross-talk among multiple signaling pathways including Ang-Ⅱ-AT1R-ERK-p38-ACE,Ang-Ⅱ-AT1R-ROS-MCP-1/LOX-1, Ang-Ⅱ-AT1R-PI3K-Akt-LOX-1/MCP-1,Ang-（1-7）-ERK-p38-ACE, Ang-（1-7）-PI3K-Akt-LOX-1/MCP-1 and Ang-（1-7）-ROS-MCP-1/LOX-1 pathways.（2） Through selective enhancement or inhibition of the cross-talk among Ang-Ⅱ和Ang-（1-7） signaling pathways,early AS lesions may be suppressed.Thus, our results provide multiple novel targets for the treatment of early AS lesions.更多还原