血管紧张素-（1-7）抑制动脉粥样硬化形成和进展的实验研究
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摘要
背景
动脉粥样硬化(atherosclerosis, AS)斑块形成是心脑血管疾病的共同病理学基础。大量研究表明,血管平滑肌细胞(vascular smooth muscle cells, VSMCs)在AS斑块形成过程中发挥着关键作用。正常血管中膜VSMCs呈“静止状态”,但促AS因素的作用下,VSMCs的增殖和迁移能力大大增强,向内膜下迁移增多,从而促进AS的形成。近年来,一些学者认为,在AS发生过程中VSMCs的增殖和迁移改变可能早于内皮细胞的功能异常。尽管在这方面仍有争议,但越来越多的证据支持VSMCs在AS早期病变中具有重要地位。
研究表明,肾素-血管紧张素系统(renin-angiotensin system, RAS)在VSMCs增殖和迁移及AS斑块形成和进展过程中起着重要的作用,血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是这一作用的关键分子。血管紧张素-(1-7)[angiotensin-(1-7), Ang-(1-7)]是近年来发现的R-AS系统新成员,主要由血管紧张素转化酶-2(angiotensin converting enzyme-2, ACE2)分解血管紧张素Ⅱ所产生。血管紧张素-(1-7)在多种病理生理过程中拮抗血管紧张素Ⅱ,对于RAS系统过度激活所导致的病变起保护作用,如高血压、心力衰竭和动脉粥样硬化等。既往的体内研究曾发现,Ang-(1-7)可抑制内膜损伤模型的中膜平滑肌细胞的增殖,但其抑制’VSMCs增殖和迁移活性的分子机制至今未明。
新近的研究发现,促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPKs)在AngⅡ诱导的VSMCs增殖中起有重要作用。SM22a是VSMCs重要的表型蛋白,在AS病变、主动脉瘤和肿瘤血管中SM22a表达明显下降。SM22α基因敲除可促进VSMCs增殖和迁移活性,促进AS的形成。
综上所述,虽然Ang-(1-7)与VSMCs增殖关系的研究取得了一些进展,仍存一些重大的科学问题未能解决：(1)Ang-(1-7)调节VSMCs增殖活性的分子机制是什么?(2)MAPKs家族蛋白ERK、P38和JNK与SM22α表型蛋白在Ang-(1-7)调节VSMCs增殖活性的过程中是否发挥独立或联合发挥作用?因此,我们提出如下假设：Ang-(1-7)可有效抑制AngⅡ诱导的VSMCs增殖,这一作用是通过调节MAPKs与SM22α信号转导通路的交互对话而实现的。
目的
1.检测Ang-(1-7)对血管紧张素Ⅱ诱导的VSMCs增殖和迁移的影响；
2.检测血管紧张素-(1-7)对VSMCs增殖和迁移相关信号蛋白(ERK1／2,P38,JNK1／2和SM22α)的影响；
3.明确上述信号蛋白在VSMCs增殖和迁移过程中的内在联系。
方法
1.细胞培养：人主动脉平滑肌原代细胞购自Sciencell公司,采用常规方法培养,当达到80-90%融合时传代,实验采用第4～8代细胞。
2.实验分组：为了观察AngⅡ和Ang-(1-7)对VSMCs功能和相应信号转导通路蛋白的影响,分为6个组：对照组、AngⅡ组、Ang-(1-7)组、AngⅡ+Ang-(1-7)组、AngⅡ+Ang-(1-7)+A779组、A779组、PD98059组、SB203580组、PD98059组+SB203580组、SM22α干扰组。AngⅡ在培养基的终浓度为0.1μM；Ang-(1-7)和A779在培养基的终浓度为1μM。
3.MTT和BrdU掺入实验：根据实验目的的不同,分别观察AngⅡ、Ang-(1-7)、AngⅡ+Ang-(1-7)、AngⅡ+Ang-(1-7)+A779、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰后VSCMs的增殖能力。
4.Transwell平板实验：根据实验目的的不同,分别观察AngⅡ、Ang-(1-7)、AngⅡ+Ang-(1-7)、AngⅡ+Ang-(1-7)+A779、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰后VSCMs的增殖能力。
5.RT-PCR：观察AngⅡ和Ang-(1-7)干预后,VSMCs SM22αmRNA的表达。
6.Western blot:观察AngⅡ、Ang-(1-7)、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰等不同干预后,VSMCs有关因子的表达。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
结果
1.Ang-(1-7)对AngⅡ诱导的VSMCs增殖活性的影响：MTT和BrdU实验证实,与对照相比,AngⅡ组的VSMCs增殖活性显著增强,Ang-(1-7)能有效抑制AngⅡ诱导的VSMCs增殖,而A779可有效逆转Ang-(1-7)的作用。
2..Ang-(1-7)对AngⅡ诱导的VSMCs迁移的影响：Transwell平板实验证实,与对照相比,AngⅡ组的VSMCs迁移能力显著增强,Ang-(1-7)能有效抑制AngⅡ诱导的VSMCs迁移,而A779可部分逆转Ang-(1-7)的作用。
3. VSMCs增生信号转导蛋白的研究：与对照相比,AngⅡ组的VSMCsERK1／2、P38的活性增强,Ang-(1-7)能有效抑制AngⅡ诱导的ERK1／2、P38激活,而A779可有效逆转Ang-(1-7)抑制ERK1／2、P38的作用。与对照组相比,AngⅡ组的VSMCs活性增强,Ang-(1-7)对JNK1／2活性无显著影响。与对照组相比,AngⅡ组的VSMCs SM22a表达显著减少,Ang-(1-7)组SM22α表达显著增加,而A779可有效逆转Ang-(1-7)的作用。
4.与对照组相比,PD98059、SB203580组、PD98059+SB203580组VSMCs增殖和迁移能力显著下降；SM22αsiRNA组VSMCs增生和迁移能力显著升高；PD98059、SB203580组、PD98059+SB203580组VSMCs SM22α表达升高。
结论
1.Ang-(1-7)能有效抑制AngⅡ对VSMCs增殖和迁移的促进作用。
2.AngⅡ能有效激活ERK1／2、P38、JNK1／2,抑制SM22α表达。Ang-(1-7)能够下调AngⅡ引起的ERK1／2、P38激活,增加SM22α表达,A779可大部分逆转Ang-(1-7)的作用。Ang-(1-7)对JNK1／2的激活无显著作用。
3. Ang-(1-7)-MAS-ERK/P38-SM22α可能是影响VSMCs生物活性的主要信号通路。
1.背景
动脉粥样硬化(atherosclerosis, AS)是导致心脑血管疾病的病理学基础。肾素-血管紧张素系统(renin-angiotensin system, RAS)在血管平滑肌细胞(vascular somooth muscle cells,VSMCs)增殖、迁移及AS斑块形成和进展过程中起着重要的作用。血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是RAS系统的主要活性成分。大量的研究证实,AngⅡ可显著促进VSMCs的增殖和迁移活性,促进AS形成和进展。
血管紧张素-(1-7)[Angiotensin-(1-7), Ang-(1-7)]是近年来发现的RAS家族新成员,在体内主要由血管紧张素转化酶-2(angiotensin converting enzyme-2,ACE2)分解AngⅡ所产生。近年研究发现,Ang-(1-7)是AngⅡ重要的内源性拮抗剂,具有抑制炎症和氧化应激、激活磷酸酯酶、促进肾脏排钠、促进血管舒张等作用,介导了心肌重构和纤维化、高血压血管重构、血管平滑肌细胞增殖、炎症、血管内皮细胞功能调节等多个病理生理过程。
以往研究发现,Ang-(1-7)可抑制球囊内皮损伤模型中主动脉VSMCs增殖和迁移以及血管内膜增厚。本实验室的研究也发现,ACE2过表达可抑制早期AS病变的形成。这些研究提示,Ang-(1-7)在AS的进程中可能发挥着重要的保护作用。因此,通过直接应用Ang-(1-7),拮抗体内AS进程中过度激活的AngⅡ,对AS病变得预防和治疗可能起到有益的作用。然而,Ang-(1-7)对于AS病变形成的量效关系至今不明。此外,我们的体外研究证实,Ang-(1-7)可调节VSMCs的ERK1／2、P38、SM22α等信号蛋白的表达,抑制VSMCs的增殖和迁移,但这些体外实验的结果能否在体内得到验证尚不明了。
因此,本研究提出如下假设：在ApoE-／-小鼠AS病变模型中,Ang-(1-7)可通过调节体内MAS-ERK/P38-SM22α信号转导通路,抑制VSMCs增殖和迁移,进而抑制AS的发展。
2.目的
2.1在体内试验中,观察不同剂量的Ang-(1-7)对ApoE-／-小鼠AS病变的抑制作用；
2.2在体内实验中,验证Ang-(1-7)抑制ApoE-／-小鼠AS病变形成的机制。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
8周龄的雄性ApoE-／-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养8周。
3.2 Ang-(1-7)体内干预实验
高脂喂养4周后,随机分为PBS对照组(12只)、Ang-(1-7)干预组(36只)和A779干预组(36只)。Ang-(1-7)和A779组又分别分为3个剂量组(100ng／kg.min、200ng／kg.min、400ng／kg.min)。用药具体方法为：经植入式胶囊渗透压泵皮下持续泵入,用药28天。
分别于药物干预4周后处死动物,进行斑块的病理学和免疫组织化学检测。
3.3血液生化指标检测
分别检测血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
将主动脉纵剖后行大体油红O染色,对主动脉根部斑块进行H&E染色,心肌组织进行Masson染色；并通过免疫组织化学方法检测主动脉SM22α和肾脏内MOMA-2的表达。
3.5 Western blot检测
取新鲜主动脉根部组织,提取蛋白质,Western blot检测AS病变组织p-ERK1／2和p-P38的表达。
4.结果
4.1血液生化指标检测：
对照组、Ang-(1-7)各亚组和A779各亚组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.2 Ang-(1-7)的抗AS作用
大体油红O染色显示,与对照组相比,各Ang-(1-7)干预组均能有效抑制主动脉表面AS病灶范围。Ang-(1-7)抑制主动脉表面AS病变的作用呈剂量依赖性；随Ang-(1-7)浓度的增加,主动脉表面AS病灶范围逐渐减少。Ang-(1-7)最大剂量组与对照组主动脉表面AS病灶范围有显著统计学差异(2.58% vs.4.14%,P<0.05)。
HE染色显示,与对照组相比,各Ang-(1-7)干预组均能有效抑制主动脉根部AS斑块的形成。Ang-(1-7)抑制主动脉根部AS面积的作用呈剂量依赖性；随Ang-(1-7)浓度的增加,主动脉根部AS斑块横截面积逐渐减少。Ang-(1-7)最大剂量组与对照组主动脉根部AS斑块面积有显著统计学差异(12.08% vs.20.28%,P<0.05)。
4.3 A779对AS的影响
大剂量A779干预组主动脉表面AS病变范围(4.79% vs.4.14%,P<0.05)和主动脉根部(23.22%vs.20.28%,P<0.05)AS病变横截面积显著高于PBS对照组,低、中剂量A779干预对主动脉表面和主动脉根部AS病变范围无显著影响。
4.4 Ang-(1-7)抗AS的机制
血清学分析显示,各组间ApoE-／-小鼠的血清TC、TG、HDL-C、LDL-C的差异无统计学差异。上述结果提示Ang-(1-7)抗AS的作用可能独立于对血脂水平的影响。
研究发现,RAS的激活可促进血管中膜平滑肌细胞的激活,促进VSMCs迁移和增殖活性,启动AS的形成和进展。我们的体外实验证实,促分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)、ERK1/2、P38MAPK和SM22a在Ang-(1-7)介导的平滑肌细胞的迁移和增殖活性改变中发挥着重要的作用。因此我们在体内观察了上述信号蛋白的改变。SM22a的免疫组织化学染色发现,大剂量Ang-(1-7)干预组主动脉根部血管中膜SM22a含量显著高于对照组(59.57% vs.8.80%,P<0.01)和大剂量A779干预组(59.57% vs.7.75%,P<0.01),提示Ang-(1-7)干预可显著提高VSMCs SM22α含量。Western blot检测结果发现,Ang-(1-7)干预显著降低血管内ERK1／2和P38MAPK活性,A779显著升高血管内ERK1／2和P38MAPK活性。
4.5 Ang-(1-7)对ApoE-／-小鼠肾脏巨噬细胞浸润的影响
既往研究显示,Ang-(1-7)可增加肾脏巨噬细胞浸润,促进肾脏炎症反应。因此,本研究观察了大剂量组Ang-(1-7)对ApoE-／-小鼠肾脏巨噬细胞浸润的影响。免疫荧光检测发现,各组肾脏无明显阳性染色,Ang-(1-7)组和PBS对照组之间无显著差异。
4.6 Ang-(1-7)对ApoE-／-小鼠心肌胶原含量的影响
Masson染色显示,各组心肌胶原染色无显著差异,无明显的纤维化改变。
5.结论
1.Ang-(1-7)对高脂喂养的ApoE-／-小鼠AS病变有显著的抑制作用。
2.Ang-(1-7)抗AS的作用可能与调节MAS-ERK/P38-SM22α信号转导通路,抑制VSMCs增殖和迁移活性有关。
1.背景
大量临床研究显示,急性冠状动脉综合征(acute coronary syndrome, ACS)主要是由于AS易损斑块(vulnerable plaque)破裂和血栓形成造成急性冠脉完全或部分闭塞所致。因此,斑块易损性已成为国内外研究的热点。斑块是否破裂取决于斑块内在因素和外在因素的共同作用,而内在因素是斑块不稳定性的决定因素。因此,深入研究斑块易损性的发生机制,增强易损斑块的稳定性,是预防临床急性心血管事件的重要方法。
大量研究证实,炎症是AS发生和发展的重要机制。炎症反应贯穿于AS的整个过程,而炎性细胞在AS病变的进程中发挥了重要的作用。在外界因素的刺激下,血管内皮细胞、平滑肌细胞、巨噬细胞及外膜的成纤维细胞等均可产生炎性因子,促进血管炎症反应和AS病变的形成。斑块内炎症反应的活跃程度与斑块易损性密切相关,因此斑块内炎症反应是斑块易损机制中最重要的因素之一。
肾素-血管紧张素系统(renin-angiotensin system, RAS)在炎症反应和AS斑块的发生和发展中起着重要的作用。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)能够通过多种信号传导通路介导炎症反应,而AngⅡ与AS的关系已经得到了证实。传统的观念认为,AngⅡ是RAS系统的主要成分,近年来随着angiotensin converting enzyme 2 (ACE2、angiotensin-(1-7) [Ang-(1-7)]等RAS家族新成员的发现,人们对RAS家族各成员的病理生理学意义有了许多新的认识。Ang-(1-7)在体内主要由ACE2高效分解AngⅡ所生成。新近的研究发现血管紧张素-(1-7)是一种有重要生物学作用的AngⅡ内源性拮抗剂,能有效抑制炎症反应和氧化应激。
我们既往的研究也发现,ACE2过表达可以稳定AS易损斑块。本研究第Ⅱ部分的结果也发现,Ang-(1-7)能有效抑制ApoE-／-小鼠AS早期病变的形成。这些研究提示我们是否可以通过体内直接应用Ang-(1-7),抑制体内过度激活的RAS,从而促进AS斑块的稳定性呢?然而,直接应用Ang-(1-7)稳定AS易损斑块的研究甚少,其确切作用和分子机制有待阐明。
2.目的
2.1在体内研究中,观察大剂量的Ang-(1-7)对ApoE-／-小鼠AS斑块稳定性的影响；
2.2在体内研究中,观察大剂量的Ang-(1-7)对ApoE-／-小鼠AS斑块炎症的影响机制；
2.3在体外实验中,观察Ang-(1-7)对巨噬细胞炎症因子表达的影响。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
8周龄的雄性ApoE-／-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养,分为两组：单纯高脂喂养组(高脂喂养12周+药物干预4周)和颈动脉套管组(高脂喂养2周+颈动脉套管8周+药物干预4周)。
3.2 Ang-(1-7)体内干预实验
单纯高脂喂养组和颈动脉套管组分别于高脂喂养12周和颈动脉套管8周后,随机分为PBS对照组(12只)、Ang-(1-7)干预组(12只,400ng／kg.min)和A779干预组(12只,400ng／kg.min)。用药具体方法为：经植入式胶囊渗透压泵皮下持续泵入,用药28天。
分别于药物干预4周后处死动物,检测斑块的病理学形态结构。
3.3血液生化指标检测
分别检测血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
分别对主动脉根部和颈动脉套管近心端斑块分别进行油红O染色、HE染色和天狼猩红染色,并通过免疫组织化学方法检测斑块内平滑肌肌动蛋白α-actin、巨噬细胞MOMA-2、肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)、单核细胞趋化因子(MCP-1)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)和金属蛋白酶组织抑制剂-1(TIMP-1)的表达。检测斑块内脂质、巨噬细胞、胶原和VSMCs的含量,然后计算易损指数：易损指数=(巨噬细胞+脂质)阳性面积百分比／(平滑肌细胞+胶原)阳性面积百分比。
3.5 RT-PCR采用RT-PCR检测巨噬细胞TNF-α、IL-1β、IL-6和MCP-1 mRNA的表达水平。
4.结果
3.1血液生化指标检测：
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组血清TC、TG、HDL-C、LDL-C未见统计学差异。
高脂喂养+颈动脉套管组：对照组、Ang-(1-7)组和A779组血清TC、TG、HDL-C、LDL-C未见统计学差异。
3.2 Ang-(1-7)对主动脉根部AS斑块易损指标的影响
对照组、Ang-(1-7)组和A779组主动脉根部斑块脂质含量百分比分别为20.65%、15.76%和24.45%；巨噬细胞含量百分比分别为17.58%、12.32%和27.84%；平滑肌细胞含量百分比分别为8.59%、23.34%和11.45%；胶原含量百分比分别为14.56%、17.25%和9.58%。易损指数分别为1.65±0.15；0.89±0.24和2.38±0.26。
3.3 Ang-(1-7)对主动脉根部AS斑块炎症的作用
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组主动脉根部斑块IL-6含量百分比分别为20.25%、12.17%和25.87%；IL-1β含量百分比分别为17.07%、16.39%和25.98%；TNF-α含量百分比分别为20.36%、23.65%和35.68%；MCP-1含量百分比分别为20.36%、12.65%和19.68%。
3.4 Ang-(1-7)对巨噬细胞炎症因子表达的影响
RT-PCR结果显示,尽管Ang-(1-7)对巨噬细胞炎症因子的表达无明显影响,但AngⅡ可显著增加巨噬细胞TNF-α、IL-6、IL-1β和MCP-1的表达,而Ang-(1-7)显著降低AngⅡ诱导的上述细胞因子的表达上调。相反的,A779可大部分逆转Ang-(1-7)的作用。
3.5 Ang-(1-7)对主动脉根部AS斑块基质降解酶表达的影响
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组主动脉根部斑块TIMP-1含量百分比分别为5.79%、30.74%和5.42%；MMP-2含量百分比分别为28.45%、11.23%和37.92%；MMP-9含量百分比分别为17.16%、11.23%和15.43%。
3.6 Ang-(1-7)对颈动脉AS斑块易损指标的影响
对照组、Ang-(1-7)组和A779组颈动脉斑块脂质含量百分比分别为20.43%、14.76%和25.37%；巨噬细胞含量百分比分别为12.46%、7.21%和19.95%；平滑肌细胞含量百分比分别为5.15%、8.24%和4.20%；胶原含量百分比分别为15.21%、17.98%和10.08%。易损指数分别为1.62、0.84和3.17。
5.结论
1.大剂量Ang-(1-7)能有效地抑制斑块局部巨噬细胞浸润和脂质沉积,增强斑块的稳定性。
2.大剂量Ang-(1-7)稳定易损斑块的作用可能与抑制斑块局部炎症因子和基质金属降解酶的表达,从而抑制炎症反应和基质降解有关。
1.背景
动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病共同的病理学基础。肾素-血管紧张素系统(renin-angiotensin system, RAS)在炎症反应和AS发生和发展过程中起着重要的作用,在AS患者和动物模型中都发现有RAS的激活。RAS主要通过促进血管炎症和氧化应激反应诱发AS病变。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)是RAS发挥生物学活性的主要成员,AngⅡ促进AS的机制包括促进内皮细胞粘附分子的表达、促进单核细胞趋化和向巨噬细胞分化、促进巨噬细胞氧化应激反应,促进血管平滑机细胞增殖和迁移等。
越来越多的研究证实,抑制RAS能减轻AS的发生和发展。应用ACE抑制剂(ACEI)和血管紧张素受体拮抗剂(ARB)可显著抑制高脂喂养的兔和ApoE-／-小鼠主动脉AS病变的进展。然而,临床研究发现,充分剂量的ACEI和／或ARB治疗并不能完全预防心血管事件的发生。血管紧张素-(1-7)[angiotensin-(1-7), Ang-(1-7)]是近年来发现的AngⅡ的内源性拮抗剂,可在多种疾病如心肌重构和纤维化、高血压血管重构、平滑肌细胞增殖、血管炎症、血管内皮细胞功能异常及肾脏疾病等进程中发挥拮抗AngⅡ的作用,减轻体内过度激活的RAS带来的损害。因此本研究的第Ⅱ、Ⅲ部分着重观察了Ang-(1-7)对AS病变形成和进展的影响,结果发现Ang-(1-7)可以有效的抑制早期AS病变形成,稳定晚期AS斑块。然而,ACEI和Ang-(1-7)对于AS早期病变的发生和晚期斑块的稳定是否具有相同的作用尚不明了。
为了对比ACEI及Ang-(1-7)对AS发生和发展的影响,本实验在高脂喂养的ApoE-／-小鼠AS病变模型中,采用组织病理学和免疫组织化学技术观察不同阶段AS病变的形成和斑块的稳定性,对比研究了雷米普利和Ang-(1-7)对AS发生和发展的影响,为AS的预防和治疗提供新的途径。
2.目的
2.1在体内实验中,对比观察雷米普利和Ang-(1-7)对AS病变形成的抑制作用；
2.2在体内实验中,对比观察雷米普利和Ang-(1-7)对AS晚期斑块的稳定作用。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
根据AS模型的不同,分为单纯高脂喂养组和高脂喂养+颈动脉套管组。
单纯高脂喂养组：8周龄的雄性ApoE-／-小鼠,实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养8周。高脂喂养4周后,进行分组和药物干预。
高脂喂养+颈动脉套管组：8周龄的雄性ApoE-／-小鼠,实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。高脂喂养2周后,行颈动脉套管术,放置套管8周后,进行分组和药物干预。
3.2 Ang-(1-7)体内干预实验
单纯高脂喂养组和高脂喂养+颈动脉套管组分别于高脂喂养4周和10周后,随机分为PBS对照组(12只)、雷米普利组(12只,5mg／kg.d)和Ang-(1-7)组(12只,400ng/kg.min)。分别于药物干预4周后处死动物,检测斑块的病理学形态结构。
3.3血液生化指标检测
分别检测血清总胆固醇、甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
单纯高脂喂养组：将主动脉纵剖后行大体油红O染色,对主动脉根部斑块进行H&E染色。
高脂喂养+颈动脉套管组：对颈动脉斑块分别进行HE染色、油红O染色和天狼猩红染色,并通过免疫组织化学染色检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)的表达。检测斑块内脂质、巨噬细胞、胶原、VSMCs的含量,计算斑块易损指数。
4.结果
4.1血液生化指标检测：
对照组、雷米普利组和Ang-(1-7)组之间血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.2雷米普利和Ang-(1-7)的抗AS作用
4.2.1雷米普利和Ang-(1-7)对主动脉表面AS形成抑制作用
大体油红O染色显示,与对照组相比,雷米普利和Ang-(1-7)均能显著减少主动脉表面AS病灶范围。对照组、雷米普利组和Ang-(1-7)组主动脉表面的病灶范围分别为：4.14%、2.52%和2.58%。雷米普利组和Ang-(1-7)组主动脉表面的病灶范围无显著差异(P>0.05),但均显著低于对照组(P值均<0.05)。
4.2.2雷米普利和Ang-(1-7)对主动脉根部AS形成的影响
H&E染色显示,与对照组相比,雷米普利和Ang-(1-7)均能显著减少主动脉根部AS病灶横截面积。对照组、雷米普利组和Ang-(1-7)组主动脉根部的斑块面积分别为：20.28%、15.32%、12.08%。Ang-(1-7)组主动脉根部的AS斑块面积显著低于雷米普利组(P>0.05),且均显著低于对照组(P值均<0.05)。
4.3雷米普利和Ang-(1-7)对颈动脉AS斑块稳定性的影响
对照组、雷米普利组和Ang-(1-7)组颈动脉斑块脂质含量百分比分别为20.43%、15.48%和14.76%；巨噬细胞含量百分比分别为12.46%、6.68%和7.21%；平滑肌细胞含量百分比分别为5.15%、7.72%和8.24%：胶原含量百分比分别为15.21%、16.49%和17.98%。易损指数分别为1.62±0.38、0.91±0.26和0.84±0.24。与对照组相比,雷米普利和Ang-(1-7)均可显著降低颈动脉AS斑块的易损指数(P值均<0.05),但雷米普利和Ang-(1-7)对斑块稳定性的影响无显著差异。
5.结论
1.体内实验结果表明,雷米普利和Ang-(1-7)均能显著抑制高脂喂养的ApoE-／-小鼠AS病变形成。
2.雷米普利和Ang-(1-7)均可有效稳定高脂喂养的ApoE-／-小鼠AS斑块。
Background
Atherosclerosis formation and plaque rupture are the common pathological basis of most of cardio-cerebrovascular diseases. Understanding the molecular and cellular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit disease progression before it leads to clinical consequences. Studies have revealed that many different cell types, including macrophages, lymphocytes, endothelial cells and smooth muscle cells (SMCs), are involved in atherosclerotic lesion formation. Most reviews on atherosclerosis focus on the role of endothelial and inflammatory cells in the initiation of atherosclerosis and discuss SMCs largely in the context of late atherosclerosis when they migrate into the neointima and secrete matrixproteins to stabilize the plaque. Histological studies of autopsy specimens of human coronary arteries ranging from infants to adults provide evidence that regions prone to the development of atherosclerosis contain abundant SMCs whereas regions that are more resistant to atherosclerosis contain few, raising the interesting question: Do SMCs in areas of intimal thickening play a pathogenic role in the increased development of atherosclerosis at these sites? Recent studies, coupled with significant in vitro mechanistic data and in vivo correlative data reviewed herein, provide strong evidence that SMCs proliferation and migration are indeed important in early atherogenesis.
Present evidence suggests that intimal SMCs differ significantly from medial SMCs and as such may have unique atherogenic properties that make them fertile ground for the initiation of plaques. Arterial medial SMCs predominantly express proteins involved in the contractile function and quiet, and could swith to the "synthetic" phenotypic states in response to a variety of atherogenic stimuli, while the latter migrate and proliferate more readily than "contractile" SMCs, leading to the atherogenesis.
Renin-angiotensin system (RAS) plays a critical role in VSMCs proliferation, migration and atherogenesis and atherosclerosis acceleration. In particular, confirmed data have shown that angiotensinⅡ(AngⅡ) contributed a lot to VSMCs proliferative activity and aterial remodeling. Angiotensin-(1-7) [Ang-(1-7)], a new member of RAS family, was mostly produced by angiotensin converting enzyme-2 (ACE2) activity. Recent studies have found Ang-(1-7) plays an important protective role in various pathophysiological processes, including cardiac remodeling and fibrosis, hypertensive vacular remodeling, inflammation and regulation of endothelial cells function, through antagonizing AngⅡ. Previous studies have shown that Ang-(1-7) inhibited VSMCs proliferation in a balloon-injured model, however, the mechanism of VSMCs proliferation and migration is still to be elucidated.
Mitogen-activated protein kinases (MAPKs) play a pivotal role in AngⅡ-mediated VSMCs proliferative activity, and recent studies found that SM22a-knockout promoted VSMCs proliferation and migration, and atherosclerotic process.
To sum up, we hypothesize that Ang-(1-7) can effectively inhibit AngⅡ-induced VSMCs proliferation and migration, through blocking the MAPKs and SM22a signaling pathway crosstalk, and to regulate VSMCs bioactivities.
According to the questions mentioned above, our present study was designed to test our hypothesis in vitro.
Aims
1. To test the effect of Ang-(1-7) on AngⅡ-induced VSMCs proliferation and migration.
2. To test the effect of Ang-(1-7) on AngⅡ-induced VSMCs proliferation and migration-related signaling proteins, such as ERK1/2, P38, JNK1/2 and SM22a.
3. To clarify the signaling pathway connection and cross-talk in the VSMCs proliferation and migration.
Methods
1. Cell culture
The human VSMCs were purchased from Sciencell Company and cultured by the routine methods. When cells confluence reached 80-90%, the cells were removed again and those at passage 4-8 were selected for the study.
2. Experimental grouping
In order to inspect the effects of AngⅡand Ang-(1-7) on VSMCs poliferative activity and the related signaling proteins, VSMCs were divided into six groups: control group, AngⅡgroup, Ang-(1-7) group, AngⅡ+Ang-(1-7) group, AngⅡ+Ang-(1-7)+A779 group, A779 group, PD98059 group, SB203580 group, PD98059+SB203580 group and SM22 a knockdown group. In VSMCs culture medium, the concentration of AngⅡ, Ang-(1-7) and A779 were 0.1μM, 1μM and 1μM, respectively.
3. MTT and BrdU incorporation assay
These experiments were undertaken to compare VSMCs reproductive activities after AngⅡ, Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention for 24h.
4. Transwell plate assay
The transwell plate assay was undertaken to detect VSMCs migration acivity after AngⅡ,Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention for 12h.
5. RT-PCR
RT-PCR was taken to explore some cytokines mRNA expression in VSMCs after AngⅡand Ang-(1-7) intervention.
6. Western blot
To observe the expression of some cytokines in VSMCs after AngⅡ,Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention, Western blot was undertaken. The relative expression of protein was demonstrated by the ratio of integral optical density (IOD) value between target protein andβ-actin.
Results
1. Ang-(1-7) regulating AngⅡ-mediated VSMCs proliferative activity:Both MTT assay and BrdU incorporation assay confirmed that Ang II could effectively increase the proliferative activity, while Ang-(1-7) could remarkably abolish the efficacy of AngⅡ. Meanwhile, A779 could effectively reverse the effect of Ang-(1-7).
2. Effect of Ang-(1-7) on AngⅡ-induced VSMCs migration:Transwell plate assay showed that, compared with the control group, Ang II could effectively increase the migration of VSMCs. Similar with the effect of Ang-(1-7) on VSMCs proliferation, Ang-(1-7) could remarkably reverse the effect of AngⅡ, while A779 effectivley abolish the effect of Ang-(1-7).
3. The study of VSMCs proliferation signal transduction pathway:The activities of ERK1/2 and P38 were significantly higher in Ang II group than control group, and Ang-(1-7) treatment effectively decrease the efficacy of AngⅡ, while A779 could effectively abolish the effect of Ang-(1-7). The activity of JNK1/2 significantly increased after AngⅡtreatment, however, no significant change was found with Ang-(1-7) incubation. There were less mRNA and protein expression of SM22a in AngⅡgroup than control group. There were more mRNA and protein expression of SM22a in Ang-(1-7) group than control group, while A779 could effectively abolish the effect of Ang-(1-7).
4. Crosstalk between MAPKs and SM22a pathway:Both the proliferative and migrative activities significantly decreased in PD98059 group, SB203580 group and PD98059+SB203580 group, and remarkably increased in SM22a knockdown group. On the other hand, the mRNA and protein expression of SM22a in PD98059 group, SB203580 group and PD98059+SB203580 group were significantly higher than the control group.
Conclusion:
1. AngⅡcan enhance the VSMCs activities of proliferation and migration, while Ang-(1-7) can effectively inhibit these functions.
2.AngⅡcan activate ERK1/2, P38, and JNK1/2 and inhibit SM22a, however, its effects on ERK1/2, P38 and SM22a were significantly abolished by Ang-(1-7).
3. The Ang-(1-7)-MAS-ERK/P38-SM22a signaling pathway might play an important role in regulating the VSMCs bioactivities.
1. Background
Atherosclerosis formation and progression is the common pathological basis of the most cardio-cerebrovascular diseases. Accumulating evidence indicates that the rennin-angiotensin system (RAS) plays an important role in vascular smooth muscle cell (VSMCs) proliferation, migration and atherosclerosis formation and progression. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is a key cytokine that regulates cell growth, migration, inflammation and oxidative stress and therefore contributed to VSMCs proliferation, migration and atherosclerosis formation and progression.. Angiotensin-(1-7), a new member of RAS, is mainly produced by angiotension converting enzyme (ACE2), through degradating AngⅡ. A large mount of rencent studies demonstrated that Ang-(1-7) was an important endogenous antagonist of AngⅡ, via anti-inflammation and oxidative stress, vascular vasodiation, reduction of fibrosis and phosphatases activation, and thus played an important role in cardiac reconstruction and fibrosis, hypertensive vascular reconstruction, VSMCs proliferation and migration, and endothelial cells function regulation.
Previous studies demonstrated that Ang-(1-7) significantly inhibited VSMCs proliferation and migration in a balloon-injured arterty model, and our rencent study showed that overexpression ACE2 inhibted early AS formation. All the above studies indicated that Ang-(1-7) might play a critical role in AS progression. So, chronic injection of Ang-(1-7) in ApoE-/-mice might attenuate over-activited AngⅡeffectively, and thus inhibit AS formation.
The effect of Ang-(1-7) on AS formation and the possible mechanism is unclear. Our in vitro study demonstrated that Ang-(1-7) regulated VSMCs ERK1/2, P38 and SM22 a activities and expression, which play a critical role in AS formation and progression. However, whether Ang-(1-7) could regulate the above signaling protein and AS progression in vivo is unknown.
So, we raise the following hypothesis:Ang-(1-7) might regulate the MAS-ERK/P38-SM22 a signaling pathway and inhibit AS formation in ApoE-/-mice.
2. Objectives
2.1 To assess the therapeutic effects of Ang-(1-7) on atherosclerotic lesions formation in ApoE-/-mice.
2.2 To elucidate the molecular mechanisms of Ang-(1-7)-mediated therapeutic effects on atherosclerotic lesions.
3. Methods
3.1 Atherosclerosis animal model
ApoE-/-mice aged 8 weeks were fed an atherogenic chow (0.25% cholesterol and 15% cocoa butter). After 8 weeks for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Ang-(1-7) treatment
At the end of week 4 for atherogenic diet, the mice were randomly allocated to phosphate buffered saline (PBS) control group, Ang-(1-7) group and A779 group. All the animals were implanted with a subcutaneous osmotic pump for the duration of 4 weeks. In order to demonstrate the dose-response in mice, the Ang-(1-7) and A779 groups were divided three subgroups(100ng.kg-1.min-1,200ng.kg-1.min-1 and 400ng.kg-1.min-1), respectively.
The mice were anesthetized and their aortas were collected for pathological and biochemical analysis at the end of experiment.
3.3 Serum lipid measurement
Serum samples were collected after 12 hours of fasting and stored at-80℃. Total cholesterol, Triglyceride, high density lipoprotein cholesterol and low density lipoprotein cholesterol were measured.
3.4 Histopathological and immunohistochemical measurement
The whole aortas were stained with Oil Red O, and the cardiac muscles were stained by Masson. SM22ain aortas and MOMA-2 in kidney were identified using appropriate primary antibodies.
3.5 Western blot analysis
The protein expression of p-ERK1/2 and p-P38 were analyzed by Western blot measurement.
4. Results
4.1 Ang-(1-7) protects against atherosclerosis
After 8 weeks of high fat diet, we found that, with the dose of Ang-(1-7) increasing, the inhibitory action of Ang-(1-7) on atherosclerotic lesions formation was increased. The mice treated with high-dose Ang-(1-7) showed significantly smaller atherosclerotic lesions in their aortas, as shown by both en face (2.58% vs.4.14%, P<0.05) and aortic root section analysis (12.08% vs.20.28%, P<0.05), comparing with the control mice.
4.2 The effect of A779 on atherosclerosis
After 8 weeks of high fat diet, we found that high dose A779 significantly increased both the en face (4.79% vs.4.14%, P<0.05) and aortic root section (23.22% vs.20.28%, P<0.05) atherosclerotic lesions formation, comparing with the control mice.
4.3 Ang-(1-7) has no influence on the serum lipid profiles
Serum analysis demonstrates that no significant differences were found among the various treatment groups, which indicating the protective effect of Ang-(1-7) on atherosclerosis might independent of lipid profiles.
4.4 Ang-(1-7) inhibits the activities of ERK1/2 and P38MAPK in vivo
Western blot analysis showed that the phosphorylations of ERK1/2 and P38MAPK were significantly attenuated by Ang-(1-7) in vivo. Conversely, with the administration of A779, the phosphorylations of ERK1/2 and P38 were significantly increased in the A779 treatment group.
4.5 Ang-(1-7) increases the protein expression of SM22a
Immunohistochemical staining demonstrated that the protein expression of SM22ain aortas of Ang-(1-7)-treated mice were significantly higher than the PBS control mice (59.57% vs.8.80%, P<0.01). However, no significant difference of protein expression of SM22a between PBS and A779 groups (8.80% vs.7.75%).
4.6 Ang-(1-7) has no effect on macrophages infiltration in kidney
Immunofluorescence analysis did not found evident macrophages infiltration in kidney of Ang-(1-7)-treated mice, as well as PBS and A779-treated mice.
4.7 Ang-(1-7) has no effect on cardiac muscle collagen content
Masson staining demonstrates no remarkable cardiac fibrosis, and no significant differences of collagen contents among various groups.
5. Conclusions
1. Chronic Ang-(1-7) treatment significantly attenuated atherosclerotic lesions formation in ApoE-/-mice.
2. The anti-atherosclerosis effect of Ang-(1-7) were partly mediated by the effect of anti-proliferation and migration activity on VSMCs through cross-talk of MAS-ERK/P38-SM22a signaling pathways
1. Background
Recent studies have shown that plaque rupture and subsequent intraluminal thrombosis is the most common cause of acute coronary syndrome (ACS). Early prevention of plaque rupture might be an effective approach to reduce the risk of ACS. Ruptured plaques have a large necrotic core, a thin fibrous cap depleted of extracellular matrix (collagen and proteoglycans) and smooth muscle cells (SMCs), and infiltration by macrophages and T-cells with outward positive remodeling and increased plaque vascularity. In most patients with ACS, more than one vulnerable plaque are present in their coronary arteries, and thus, systemic drug therapy should be the appropriate treatment. Unfortunately, there is still not an ideal drug for stabilizing atherosclerotic plaques. Although clinical and basic trials have demonstrated the capability of angiotensin converting enzyme inhibitors (ACEI) and angiotensin type I receptor blockers (ARB) in stabilizing vulnerable plaques, a large part of patients experienced an acute coronary event despite an intensive ACEI and ARB therapy. Therefore, exploration of new drugs with high efficacy and low side effects for stabilizing vulnerable plaques is clearly warranted.
Atherosclerosis is a chronic inflammatory disease. Although the precise mechanisms of plaque rupture are poorly understood, most researchers agree that the disruption of a fibrous cap rich in macrophages and T-lymphocytes in addition to the necrotic core coming in contact with circulating blood are events that lead to the development of thrombi in fatal plaques.
Accumulating evidence demonstates that the rennin-angiotensin system (RAS) contributes a lot to atherogenesis and atherosclerotic progress. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is well proved in atherosclerotic formation and progression. Inhibition of the RAS with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARB) is one of the current therapeutic strategies with proven beneficial effects in the treatment of atherosclerosis. However, a large part of patients experienced an acute coronary event despite an intensive ACEI and ARB therapy. Recent studies have shown that, angiotensin converting enzyme 2 (ACE2), Ang-(1-7), new components of the RAS, play important roles in atherosclerosis, cardiac and vascular remodeling. Ang-(1-7) may exert actions that oppose the atherogenic effects of AngⅡ, which plays an important role in cardiovascular pathophysiology. Our previous study also demonstrated that overexpression of ACE2 stabilized vulnerable plaques. However, whether Ang-(1-7) has protective effect against vulnerable atherosclerotic plaques has not been elucidated. In this study, we observed the effect of Ang-(1-7) on atherosclerotic plaque stability in ApoE-/-mice by systemic application.
2. Objectives
2.1 To assess the therapeutic effects of Ang-(1-7) on atherosclerotic lesions stability in ApoE-/-mice.
2.2 To explore the mechanisms of Ang-(1-7)-mediated therapeutic effects on atherosclerotic lesions inflammation infiltration.
3. Methods
3.1 Atherosclerosis animal model
ApoE-/-mice (aged 8 weeks), which were fed an atherogenic chow (0.25% cholesterol and 15%cocoa butter) throughout the experimental procedure, were divided into two group:group 1:atherogenic chow; group 2:atherogenic chow+ Carotid collar placement. After 16 (group 1) or 14 (group 2) weeks for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Ang-(1-7) treatment
Group 1:At the end of 12 weeks for atherogenic diet, the mice were randomly allocated to phosphate buffered saline (PBS) group, Ang-(1-7) (400ng.kg-1.min-1) group and A779 (400ng.kg-1.min-1)group. All the animals were implanted with a subcutaneous osmotic pump for the duration of 4 weeks.
Group 2:At the end of 2 weeks for atherogenic diet, a 2-mm long constricting silastic tube was placed on the right common carotid artery. Eight weeks later, the collars were removed and a subcutaneous osmotic pump was implanted for the duration of 4 weeks. The mice were randomly allocated to phosphate buffered saline (PBS) group, Ang-(1-7) (400ng.kg-1.min-1) group and A779 (400ng.kg-1.min-1) group.
3.3 Histopathological and immunohistochemical measurement
Mice were perfused through the left ventricle with PBS, followed by 4% paraformaldehyde under 100 mmHg pressure. The aortic root and right common carotid artery was excised and immersed in 4%formaldehyde overnight (4℃), embedded in OCT, and stored at-80℃until use. In all of the mice, OCT-embedded aortic root and carotid artery were cross-sectioned into pieces 6μm thick at 50-μm intervals. Sections were stained with hematoxylin and eosin (H&E), and further stained for lipids and collagen using Oil-red O and picrosirius red, respectively. Smooth muscle cells (SMCs, anti-a-actin) and macrophages (MOMA-2), tumor necrosis factor-a(TNF-α), Interleukin-6 (IL-6), Interleukin-1β(IL-1β), macrophage chemoattractant protein-1 (MCP-1), matrix matalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were immunostained with corresponding antibodies. The vulnerability index was calculated by the following formula:(macrophage staining %+lipid staining %)/(smooth muscle cell%+fiber%).
3.4 RT-PCR
The mRNA expression levels of TNF-α、IL-6、IL-1βand MCP-1 in macrophages were tested by RT-PCR.
4. Results
4.1 The effect of Ang-(1-7) on atherosclerotic plaque stability
Group 1:Immunohistochemistry analysis indicated that plaques from Ang-(1-7)-injected ApoE-knockout mice showed increased percentages of collagen (18.5%) and smooth muscle cells (66.9%), and reduced percentages of macrophages (29.9%) and lipid area (23.7%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased 46.1% by Ang-(1-7) treatment.
Group 2:Immunohistochemistry analysis indicated that plaques from carotid arteries in Ang-(1-7)-injected ApoE-knockout mice showed increased percentages of collagen (18.2%) and smooth muscle cells (60.0%), and reduced percentages of macrophages (42.1%) and lipid area (27.8%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased 48.1%by Ang-(1-7) treatment.
4.2 The effect of Ang-(1-7) on inflammatory cytokines in plaques
Immunohistochemical analysis indicated the protein expression levels of IL-6, IL-1β, TNF-αand MCP-1 in atherosclerotic plaques of aortic roots were significantly decreased by Ang-(1-7) injection in ApoE-/-mice.
4.3 The effect of Ang-(1-7) on MMPs expression in atherosclerotic plaques
Immunohistochemical analysis demonstrated that the differences of protein expression from aortic root plaques between PBS and Ang-(1-7) subgroups were significant for both MMP-2 (25.1%versus 20.2%, p<0.05) and MMP-9 (17.2% versus 11.3%,p<0.05) staining.
4.4 The effect of Ang-(1-7) on AngⅡ-mediated proinflammatory cytokines expression in macrophages
The results in vitro demonstrated that the mRNA expression levels of IL-6, IL-1β, TNF-αand MCP-1 in RAW264.7 mouse macrophage were significantly increased after incubation with AngⅡcompared to control group, and this up-regulation was significantly reversed by Ang-(1-7), while A779 counteracted the effect of Ang-(1-7).
5. Conclusions
1. Chronic Ang-(1-7) treatment significantly attenuated lipids and macrophages infiltration, and increased the contents of smooth muscle cells and collagen in atherosclerotic plaques in ApoE-/-mice.
2. The effect of Ang-(1-7) on plaque stablity was partly mediated by the the inhibitory effect on inflammation reaction and MMPs activities in atherosclerotic plaques.
1. Background
A large amount of evidence demonstates that the rennin-angiotensin system (RAS) plays a critical role in the atherogenesis and atherosclerosis progression. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is an important cytokine that regulates cell growth, migration, inflammation and oxidative stress and therefore contributed to atherosclerotic progression. Inhibition of the RAS with angiotensin-converting enzyme (ACE) inhibitors is one of the current therapeutic strategies with proven beneficial effects in the treatment of atherosclerosis. Blockade of RAS with ACE inhibitors significantly attenuate the development of aortic atherosclerosis in the hyperlipidemic rabbit and ApoE-/-mice. The link between RAS and human atherosclerosis has also been proved by the results of the Heart Outcomes Prevention Evaluation (HOPE) trial, which showed a 22% reduction in the incidence of myocardial infarction, stroke, cardiac arrest and revascularization procedures in patients taking the ACE inhibitor ramipril compared with those on placebo. In addition, in the Perindopril pROtection aGainst REcurrent Stroke Study (PROGRESS), treatment with the ACE inhibitor perindopril reduced the incidence of major vascular events by 26% and myocardial infarction by 38%. Thus, ACE inhibitor treatment may have protective effects on vascular cells via the antagonism of AngⅡactions.
Recent studies have shown that, angiotensin converting enzyme 2 (ACE2) and angiotensin-(1-7) [Ang-(1-7)], new components of the RAS, play important roles in atherosclerosis, cardiac and vascular remodeling and kidney function. Ang-(1-7) may exert actions that oppose the proliferative, hypertrophic and proinflammatory effects of AngⅡ, which may play an important role in cardiovascular pathophysiology. Our foregoing studies have demonstrated the protective effect of Ang-(1-7) on atherogenesis and plaques stability. This study was carried out to test the hypothesis that Ang-(1-7) enhances stability of atherosclerotic plaque, similar to a high-dose ramipril therapy. Since recent studies revealed a better outcome in patients with vascular disease by ramipril treatment, a high-dose ramipril was chosen as a therapeutic standard to compare with a high dose of Ang-(1-7) in a ApoE-/-mice atherosclerosis model.
2. Objectives
2.1 To compare the protective effects of ramipril and Ang-(1-7) on atherosclerotic lesions formation in ApoE-/-mice.
2.2 To assess the therapeutic effects of ramipril and Ang-(1-7) on atherosclerotic plaque stability in ApoE-/-mice.
3. Methods
3.1 Atherosclerosis animal model
All the animals were fed an atherogenic chow(0.25% cholesterol and 15% cocoa butter) during all the experiment period.
Group 1:ApoE-/-mice aged 8 weeks (n=36) were fed an athrogenic chow for 4 weeks.Then the mice were divided into three groups randomly:control group (n=12), ramipril group (n=12) and Ang-(1-7) group (n=12).
Group 2:ApoE-/-mice aged 8 weeks (n=36) were fed an athro genic chow for 2 weeks. Then, a 2-mm long constricting silastic tube was placed on the right common carotid artery near its bifurcation. Eight weeks later, the collars were removed, and the mice were divided into three groups randomly:control group (n=12), ramipril group (n=12) and Ang-(1-7) group (n=12).
After a further 4-week treatment for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Serum lipid measurement
Serum samples were collected after overnight fasting and stored at-80℃. Total Triglyceride, cholesterol, low density lipoprotein and high density lipoprotein cholesterol cholesterol were measured.
3.3 Histopathological and immunohistochemical measurement
Group 1:The whole aortas were stained with Oil Red O, and the aortic root lesions were stained with hematoxylin and eosin (H&E).
Group 2:Sections of carotid plaques were stained with H&E for histological analysis. The collagen and lipids in plaques were stained by using picrosirius red and Oil-red O, respectively. Smooth muscle cells (SMCs) and macrophages were immunostained with anti-a-actin antibodies and MOMA-2, respectively.
4. Results
4.1 The effect of Ramipril and Ang-(1-7) on serum lipid levels
Serum analysis demonstrates that no significant differences were found among the various treatment groups.
4.2 Ramipril and Ang-(1-7) protects against atherogenesis in the whole surface of aortas
After 8 weeks of high fat diet, we found that, both the two groups treated with ramipril and Ang-(1-7) showed significantly smaller atherosclerotic lesions in their aortas, as shown by both en face (ramipril group:2.52%; Ang-(1-7) group:2.58% and PBS group:4.14%). However, the atherosclerotic lesion area in ramipril group was not different from the Ang-(1-7) (P>0.05), while the lesion areas in both ramipril and Ang-(1-7) groups were larger than control group (both.P<0.05).
4.3 Ramipril and Ang-(1-7) protects against atherosclerosis in the aortic roots
After 8 weeks of high fat diet, we found that, all the three groups treated with ramipril and Ang-(1-7) showed significantly smaller atherosclerotic lesions by aortic root section analysis (ramipril group:15.32%; Ang-(1-7) group:12.08% and PBS group:20.28%). The atherosclerotic lesion area in ramipril group was significantly larger than the Ang-(1-7) group (P<0.05), while the lesion areas in both ramipril and Ang-(1-7) groups were larger than control group (both P<0.05).
4.4 The effect of Ramipril and Ang-(1-7) on carotid atherosclerotic plaque stability
Histopathological and Immunohistochemical analysis indicated that plaques from carotid arteries in ramipril and Ang-(1-7)-treated ApoE-/-mice showed increased percentages of collagen (ramipril group:16.49%; Ang-(1-7) group:17.98% and PBS group:15.21%) and smooth muscle cells (ramipril group:7.72%; Ang-(1-7) group:8.24%and PBS group:5.15%), and reduced percentages of macrophages (ramipril group:6.68%; Ang-(1-7) group:7.21% and PBS group:12.46%) and lipid area (ramipril group:15.48%; Ang-(1-7) group:14.76% and PBS group:20.43%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased (ramipril group:0.91±0.26; Ang-(1-7) group: 0.84±0.24 and PBS group:1.62±0.38).
5. Conclusions
1. Ramipril and Ang-(1-7) offered similar protection in terms of atherosclerotic lesions formation in ApoE-/-mice.
2. Ramipril and Ang-(1-7) showed similar protective effect on the atherosclerotic plaques stability in ApoE-/-mice.
动脉粥样硬化(atherosclerosis, AS)斑块形成是心脑血管疾病的共同病理学基础。大量研究表明,血管平滑肌细胞(vascular smooth muscle cells, VSMCs)在AS斑块形成过程中发挥着关键作用。正常血管中膜VSMCs呈“静止状态”,但促AS因素的作用下,VSMCs的增殖和迁移能力大大增强,向内膜下迁移增多,从而促进AS的形成。近年来,一些学者认为,在AS发生过程中VSMCs的增殖和迁移改变可能早于内皮细胞的功能异常。尽管在这方面仍有争议,但越来越多的证据支持VSMCs在AS早期病变中具有重要地位。
研究表明,肾素-血管紧张素系统(renin-angiotensin system, RAS)在VSMCs增殖和迁移及AS斑块形成和进展过程中起着重要的作用,血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是这一作用的关键分子。血管紧张素-(1-7)[angiotensin-(1-7), Ang-(1-7)]是近年来发现的R-AS系统新成员,主要由血管紧张素转化酶-2(angiotensin converting enzyme-2, ACE2)分解血管紧张素Ⅱ所产生。血管紧张素-(1-7)在多种病理生理过程中拮抗血管紧张素Ⅱ,对于RAS系统过度激活所导致的病变起保护作用,如高血压、心力衰竭和动脉粥样硬化等。既往的体内研究曾发现,Ang-(1-7)可抑制内膜损伤模型的中膜平滑肌细胞的增殖,但其抑制’VSMCs增殖和迁移活性的分子机制至今未明。
新近的研究发现,促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPKs)在AngⅡ诱导的VSMCs增殖中起有重要作用。SM22a是VSMCs重要的表型蛋白,在AS病变、主动脉瘤和肿瘤血管中SM22a表达明显下降。SM22α基因敲除可促进VSMCs增殖和迁移活性,促进AS的形成。
综上所述,虽然Ang-(1-7)与VSMCs增殖关系的研究取得了一些进展,仍存一些重大的科学问题未能解决：(1)Ang-(1-7)调节VSMCs增殖活性的分子机制是什么?(2)MAPKs家族蛋白ERK、P38和JNK与SM22α表型蛋白在Ang-(1-7)调节VSMCs增殖活性的过程中是否发挥独立或联合发挥作用?因此,我们提出如下假设：Ang-(1-7)可有效抑制AngⅡ诱导的VSMCs增殖,这一作用是通过调节MAPKs与SM22α信号转导通路的交互对话而实现的。
目的
1.检测Ang-(1-7)对血管紧张素Ⅱ诱导的VSMCs增殖和迁移的影响；
2.检测血管紧张素-(1-7)对VSMCs增殖和迁移相关信号蛋白(ERK1／2,P38,JNK1／2和SM22α)的影响；
3.明确上述信号蛋白在VSMCs增殖和迁移过程中的内在联系。
方法
1.细胞培养：人主动脉平滑肌原代细胞购自Sciencell公司,采用常规方法培养,当达到80-90%融合时传代,实验采用第4～8代细胞。
2.实验分组：为了观察AngⅡ和Ang-(1-7)对VSMCs功能和相应信号转导通路蛋白的影响,分为6个组：对照组、AngⅡ组、Ang-(1-7)组、AngⅡ+Ang-(1-7)组、AngⅡ+Ang-(1-7)+A779组、A779组、PD98059组、SB203580组、PD98059组+SB203580组、SM22α干扰组。AngⅡ在培养基的终浓度为0.1μM；Ang-(1-7)和A779在培养基的终浓度为1μM。
3.MTT和BrdU掺入实验：根据实验目的的不同,分别观察AngⅡ、Ang-(1-7)、AngⅡ+Ang-(1-7)、AngⅡ+Ang-(1-7)+A779、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰后VSCMs的增殖能力。
4.Transwell平板实验：根据实验目的的不同,分别观察AngⅡ、Ang-(1-7)、AngⅡ+Ang-(1-7)、AngⅡ+Ang-(1-7)+A779、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰后VSCMs的增殖能力。
5.RT-PCR：观察AngⅡ和Ang-(1-7)干预后,VSMCs SM22αmRNA的表达。
6.Western blot:观察AngⅡ、Ang-(1-7)、A779、PD98059、SB203580、PD98059+SB203580和SM22α干扰等不同干预后,VSMCs有关因子的表达。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。
结果
1.Ang-(1-7)对AngⅡ诱导的VSMCs增殖活性的影响：MTT和BrdU实验证实,与对照相比,AngⅡ组的VSMCs增殖活性显著增强,Ang-(1-7)能有效抑制AngⅡ诱导的VSMCs增殖,而A779可有效逆转Ang-(1-7)的作用。
2..Ang-(1-7)对AngⅡ诱导的VSMCs迁移的影响：Transwell平板实验证实,与对照相比,AngⅡ组的VSMCs迁移能力显著增强,Ang-(1-7)能有效抑制AngⅡ诱导的VSMCs迁移,而A779可部分逆转Ang-(1-7)的作用。
3. VSMCs增生信号转导蛋白的研究：与对照相比,AngⅡ组的VSMCsERK1／2、P38的活性增强,Ang-(1-7)能有效抑制AngⅡ诱导的ERK1／2、P38激活,而A779可有效逆转Ang-(1-7)抑制ERK1／2、P38的作用。与对照组相比,AngⅡ组的VSMCs活性增强,Ang-(1-7)对JNK1／2活性无显著影响。与对照组相比,AngⅡ组的VSMCs SM22a表达显著减少,Ang-(1-7)组SM22α表达显著增加,而A779可有效逆转Ang-(1-7)的作用。
4.与对照组相比,PD98059、SB203580组、PD98059+SB203580组VSMCs增殖和迁移能力显著下降；SM22αsiRNA组VSMCs增生和迁移能力显著升高；PD98059、SB203580组、PD98059+SB203580组VSMCs SM22α表达升高。
结论
1.Ang-(1-7)能有效抑制AngⅡ对VSMCs增殖和迁移的促进作用。
2.AngⅡ能有效激活ERK1／2、P38、JNK1／2,抑制SM22α表达。Ang-(1-7)能够下调AngⅡ引起的ERK1／2、P38激活,增加SM22α表达,A779可大部分逆转Ang-(1-7)的作用。Ang-(1-7)对JNK1／2的激活无显著作用。
3. Ang-(1-7)-MAS-ERK/P38-SM22α可能是影响VSMCs生物活性的主要信号通路。
1.背景
动脉粥样硬化(atherosclerosis, AS)是导致心脑血管疾病的病理学基础。肾素-血管紧张素系统(renin-angiotensin system, RAS)在血管平滑肌细胞(vascular somooth muscle cells,VSMCs)增殖、迁移及AS斑块形成和进展过程中起着重要的作用。血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是RAS系统的主要活性成分。大量的研究证实,AngⅡ可显著促进VSMCs的增殖和迁移活性,促进AS形成和进展。
血管紧张素-(1-7)[Angiotensin-(1-7), Ang-(1-7)]是近年来发现的RAS家族新成员,在体内主要由血管紧张素转化酶-2(angiotensin converting enzyme-2,ACE2)分解AngⅡ所产生。近年研究发现,Ang-(1-7)是AngⅡ重要的内源性拮抗剂,具有抑制炎症和氧化应激、激活磷酸酯酶、促进肾脏排钠、促进血管舒张等作用,介导了心肌重构和纤维化、高血压血管重构、血管平滑肌细胞增殖、炎症、血管内皮细胞功能调节等多个病理生理过程。
以往研究发现,Ang-(1-7)可抑制球囊内皮损伤模型中主动脉VSMCs增殖和迁移以及血管内膜增厚。本实验室的研究也发现,ACE2过表达可抑制早期AS病变的形成。这些研究提示,Ang-(1-7)在AS的进程中可能发挥着重要的保护作用。因此,通过直接应用Ang-(1-7),拮抗体内AS进程中过度激活的AngⅡ,对AS病变得预防和治疗可能起到有益的作用。然而,Ang-(1-7)对于AS病变形成的量效关系至今不明。此外,我们的体外研究证实,Ang-(1-7)可调节VSMCs的ERK1／2、P38、SM22α等信号蛋白的表达,抑制VSMCs的增殖和迁移,但这些体外实验的结果能否在体内得到验证尚不明了。
因此,本研究提出如下假设：在ApoE-／-小鼠AS病变模型中,Ang-(1-7)可通过调节体内MAS-ERK/P38-SM22α信号转导通路,抑制VSMCs增殖和迁移,进而抑制AS的发展。
2.目的
2.1在体内试验中,观察不同剂量的Ang-(1-7)对ApoE-／-小鼠AS病变的抑制作用；
2.2在体内实验中,验证Ang-(1-7)抑制ApoE-／-小鼠AS病变形成的机制。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
8周龄的雄性ApoE-／-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养8周。
3.2 Ang-(1-7)体内干预实验
高脂喂养4周后,随机分为PBS对照组(12只)、Ang-(1-7)干预组(36只)和A779干预组(36只)。Ang-(1-7)和A779组又分别分为3个剂量组(100ng／kg.min、200ng／kg.min、400ng／kg.min)。用药具体方法为：经植入式胶囊渗透压泵皮下持续泵入,用药28天。
分别于药物干预4周后处死动物,进行斑块的病理学和免疫组织化学检测。
3.3血液生化指标检测
分别检测血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
将主动脉纵剖后行大体油红O染色,对主动脉根部斑块进行H&E染色,心肌组织进行Masson染色；并通过免疫组织化学方法检测主动脉SM22α和肾脏内MOMA-2的表达。
3.5 Western blot检测
取新鲜主动脉根部组织,提取蛋白质,Western blot检测AS病变组织p-ERK1／2和p-P38的表达。
4.结果
4.1血液生化指标检测：
对照组、Ang-(1-7)各亚组和A779各亚组血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.2 Ang-(1-7)的抗AS作用
大体油红O染色显示,与对照组相比,各Ang-(1-7)干预组均能有效抑制主动脉表面AS病灶范围。Ang-(1-7)抑制主动脉表面AS病变的作用呈剂量依赖性；随Ang-(1-7)浓度的增加,主动脉表面AS病灶范围逐渐减少。Ang-(1-7)最大剂量组与对照组主动脉表面AS病灶范围有显著统计学差异(2.58% vs.4.14%,P<0.05)。
HE染色显示,与对照组相比,各Ang-(1-7)干预组均能有效抑制主动脉根部AS斑块的形成。Ang-(1-7)抑制主动脉根部AS面积的作用呈剂量依赖性；随Ang-(1-7)浓度的增加,主动脉根部AS斑块横截面积逐渐减少。Ang-(1-7)最大剂量组与对照组主动脉根部AS斑块面积有显著统计学差异(12.08% vs.20.28%,P<0.05)。
4.3 A779对AS的影响
大剂量A779干预组主动脉表面AS病变范围(4.79% vs.4.14%,P<0.05)和主动脉根部(23.22%vs.20.28%,P<0.05)AS病变横截面积显著高于PBS对照组,低、中剂量A779干预对主动脉表面和主动脉根部AS病变范围无显著影响。
4.4 Ang-(1-7)抗AS的机制
血清学分析显示,各组间ApoE-／-小鼠的血清TC、TG、HDL-C、LDL-C的差异无统计学差异。上述结果提示Ang-(1-7)抗AS的作用可能独立于对血脂水平的影响。
研究发现,RAS的激活可促进血管中膜平滑肌细胞的激活,促进VSMCs迁移和增殖活性,启动AS的形成和进展。我们的体外实验证实,促分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)、ERK1/2、P38MAPK和SM22a在Ang-(1-7)介导的平滑肌细胞的迁移和增殖活性改变中发挥着重要的作用。因此我们在体内观察了上述信号蛋白的改变。SM22a的免疫组织化学染色发现,大剂量Ang-(1-7)干预组主动脉根部血管中膜SM22a含量显著高于对照组(59.57% vs.8.80%,P<0.01)和大剂量A779干预组(59.57% vs.7.75%,P<0.01),提示Ang-(1-7)干预可显著提高VSMCs SM22α含量。Western blot检测结果发现,Ang-(1-7)干预显著降低血管内ERK1／2和P38MAPK活性,A779显著升高血管内ERK1／2和P38MAPK活性。
4.5 Ang-(1-7)对ApoE-／-小鼠肾脏巨噬细胞浸润的影响
既往研究显示,Ang-(1-7)可增加肾脏巨噬细胞浸润,促进肾脏炎症反应。因此,本研究观察了大剂量组Ang-(1-7)对ApoE-／-小鼠肾脏巨噬细胞浸润的影响。免疫荧光检测发现,各组肾脏无明显阳性染色,Ang-(1-7)组和PBS对照组之间无显著差异。
4.6 Ang-(1-7)对ApoE-／-小鼠心肌胶原含量的影响
Masson染色显示,各组心肌胶原染色无显著差异,无明显的纤维化改变。
5.结论
1.Ang-(1-7)对高脂喂养的ApoE-／-小鼠AS病变有显著的抑制作用。
2.Ang-(1-7)抗AS的作用可能与调节MAS-ERK/P38-SM22α信号转导通路,抑制VSMCs增殖和迁移活性有关。
1.背景
大量临床研究显示,急性冠状动脉综合征(acute coronary syndrome, ACS)主要是由于AS易损斑块(vulnerable plaque)破裂和血栓形成造成急性冠脉完全或部分闭塞所致。因此,斑块易损性已成为国内外研究的热点。斑块是否破裂取决于斑块内在因素和外在因素的共同作用,而内在因素是斑块不稳定性的决定因素。因此,深入研究斑块易损性的发生机制,增强易损斑块的稳定性,是预防临床急性心血管事件的重要方法。
大量研究证实,炎症是AS发生和发展的重要机制。炎症反应贯穿于AS的整个过程,而炎性细胞在AS病变的进程中发挥了重要的作用。在外界因素的刺激下,血管内皮细胞、平滑肌细胞、巨噬细胞及外膜的成纤维细胞等均可产生炎性因子,促进血管炎症反应和AS病变的形成。斑块内炎症反应的活跃程度与斑块易损性密切相关,因此斑块内炎症反应是斑块易损机制中最重要的因素之一。
肾素-血管紧张素系统(renin-angiotensin system, RAS)在炎症反应和AS斑块的发生和发展中起着重要的作用。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)能够通过多种信号传导通路介导炎症反应,而AngⅡ与AS的关系已经得到了证实。传统的观念认为,AngⅡ是RAS系统的主要成分,近年来随着angiotensin converting enzyme 2 (ACE2、angiotensin-(1-7) [Ang-(1-7)]等RAS家族新成员的发现,人们对RAS家族各成员的病理生理学意义有了许多新的认识。Ang-(1-7)在体内主要由ACE2高效分解AngⅡ所生成。新近的研究发现血管紧张素-(1-7)是一种有重要生物学作用的AngⅡ内源性拮抗剂,能有效抑制炎症反应和氧化应激。
我们既往的研究也发现,ACE2过表达可以稳定AS易损斑块。本研究第Ⅱ部分的结果也发现,Ang-(1-7)能有效抑制ApoE-／-小鼠AS早期病变的形成。这些研究提示我们是否可以通过体内直接应用Ang-(1-7),抑制体内过度激活的RAS,从而促进AS斑块的稳定性呢?然而,直接应用Ang-(1-7)稳定AS易损斑块的研究甚少,其确切作用和分子机制有待阐明。
2.目的
2.1在体内研究中,观察大剂量的Ang-(1-7)对ApoE-／-小鼠AS斑块稳定性的影响；
2.2在体内研究中,观察大剂量的Ang-(1-7)对ApoE-／-小鼠AS斑块炎症的影响机制；
2.3在体外实验中,观察Ang-(1-7)对巨噬细胞炎症因子表达的影响。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
8周龄的雄性ApoE-／-小鼠实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养,分为两组：单纯高脂喂养组(高脂喂养12周+药物干预4周)和颈动脉套管组(高脂喂养2周+颈动脉套管8周+药物干预4周)。
3.2 Ang-(1-7)体内干预实验
单纯高脂喂养组和颈动脉套管组分别于高脂喂养12周和颈动脉套管8周后,随机分为PBS对照组(12只)、Ang-(1-7)干预组(12只,400ng／kg.min)和A779干预组(12只,400ng／kg.min)。用药具体方法为：经植入式胶囊渗透压泵皮下持续泵入,用药28天。
分别于药物干预4周后处死动物,检测斑块的病理学形态结构。
3.3血液生化指标检测
分别检测血清甘油三酯、总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
分别对主动脉根部和颈动脉套管近心端斑块分别进行油红O染色、HE染色和天狼猩红染色,并通过免疫组织化学方法检测斑块内平滑肌肌动蛋白α-actin、巨噬细胞MOMA-2、肿瘤坏死因子(TNF-α)、白介素-6(IL-6)、白介素-1β(IL-1β)、单核细胞趋化因子(MCP-1)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)和金属蛋白酶组织抑制剂-1(TIMP-1)的表达。检测斑块内脂质、巨噬细胞、胶原和VSMCs的含量,然后计算易损指数：易损指数=(巨噬细胞+脂质)阳性面积百分比／(平滑肌细胞+胶原)阳性面积百分比。
3.5 RT-PCR采用RT-PCR检测巨噬细胞TNF-α、IL-1β、IL-6和MCP-1 mRNA的表达水平。
4.结果
3.1血液生化指标检测：
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组血清TC、TG、HDL-C、LDL-C未见统计学差异。
高脂喂养+颈动脉套管组：对照组、Ang-(1-7)组和A779组血清TC、TG、HDL-C、LDL-C未见统计学差异。
3.2 Ang-(1-7)对主动脉根部AS斑块易损指标的影响
对照组、Ang-(1-7)组和A779组主动脉根部斑块脂质含量百分比分别为20.65%、15.76%和24.45%；巨噬细胞含量百分比分别为17.58%、12.32%和27.84%；平滑肌细胞含量百分比分别为8.59%、23.34%和11.45%；胶原含量百分比分别为14.56%、17.25%和9.58%。易损指数分别为1.65±0.15；0.89±0.24和2.38±0.26。
3.3 Ang-(1-7)对主动脉根部AS斑块炎症的作用
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组主动脉根部斑块IL-6含量百分比分别为20.25%、12.17%和25.87%；IL-1β含量百分比分别为17.07%、16.39%和25.98%；TNF-α含量百分比分别为20.36%、23.65%和35.68%；MCP-1含量百分比分别为20.36%、12.65%和19.68%。
3.4 Ang-(1-7)对巨噬细胞炎症因子表达的影响
RT-PCR结果显示,尽管Ang-(1-7)对巨噬细胞炎症因子的表达无明显影响,但AngⅡ可显著增加巨噬细胞TNF-α、IL-6、IL-1β和MCP-1的表达,而Ang-(1-7)显著降低AngⅡ诱导的上述细胞因子的表达上调。相反的,A779可大部分逆转Ang-(1-7)的作用。
3.5 Ang-(1-7)对主动脉根部AS斑块基质降解酶表达的影响
单纯高脂喂养组：对照组、Ang-(1-7)组和A779组主动脉根部斑块TIMP-1含量百分比分别为5.79%、30.74%和5.42%；MMP-2含量百分比分别为28.45%、11.23%和37.92%；MMP-9含量百分比分别为17.16%、11.23%和15.43%。
3.6 Ang-(1-7)对颈动脉AS斑块易损指标的影响
对照组、Ang-(1-7)组和A779组颈动脉斑块脂质含量百分比分别为20.43%、14.76%和25.37%；巨噬细胞含量百分比分别为12.46%、7.21%和19.95%；平滑肌细胞含量百分比分别为5.15%、8.24%和4.20%；胶原含量百分比分别为15.21%、17.98%和10.08%。易损指数分别为1.62、0.84和3.17。
5.结论
1.大剂量Ang-(1-7)能有效地抑制斑块局部巨噬细胞浸润和脂质沉积,增强斑块的稳定性。
2.大剂量Ang-(1-7)稳定易损斑块的作用可能与抑制斑块局部炎症因子和基质金属降解酶的表达,从而抑制炎症反应和基质降解有关。
1.背景
动脉粥样硬化(atherosclerosis, AS)是心脑血管疾病共同的病理学基础。肾素-血管紧张素系统(renin-angiotensin system, RAS)在炎症反应和AS发生和发展过程中起着重要的作用,在AS患者和动物模型中都发现有RAS的激活。RAS主要通过促进血管炎症和氧化应激反应诱发AS病变。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)是RAS发挥生物学活性的主要成员,AngⅡ促进AS的机制包括促进内皮细胞粘附分子的表达、促进单核细胞趋化和向巨噬细胞分化、促进巨噬细胞氧化应激反应,促进血管平滑机细胞增殖和迁移等。
越来越多的研究证实,抑制RAS能减轻AS的发生和发展。应用ACE抑制剂(ACEI)和血管紧张素受体拮抗剂(ARB)可显著抑制高脂喂养的兔和ApoE-／-小鼠主动脉AS病变的进展。然而,临床研究发现,充分剂量的ACEI和／或ARB治疗并不能完全预防心血管事件的发生。血管紧张素-(1-7)[angiotensin-(1-7), Ang-(1-7)]是近年来发现的AngⅡ的内源性拮抗剂,可在多种疾病如心肌重构和纤维化、高血压血管重构、平滑肌细胞增殖、血管炎症、血管内皮细胞功能异常及肾脏疾病等进程中发挥拮抗AngⅡ的作用,减轻体内过度激活的RAS带来的损害。因此本研究的第Ⅱ、Ⅲ部分着重观察了Ang-(1-7)对AS病变形成和进展的影响,结果发现Ang-(1-7)可以有效的抑制早期AS病变形成,稳定晚期AS斑块。然而,ACEI和Ang-(1-7)对于AS早期病变的发生和晚期斑块的稳定是否具有相同的作用尚不明了。
为了对比ACEI及Ang-(1-7)对AS发生和发展的影响,本实验在高脂喂养的ApoE-／-小鼠AS病变模型中,采用组织病理学和免疫组织化学技术观察不同阶段AS病变的形成和斑块的稳定性,对比研究了雷米普利和Ang-(1-7)对AS发生和发展的影响,为AS的预防和治疗提供新的途径。
2.目的
2.1在体内实验中,对比观察雷米普利和Ang-(1-7)对AS病变形成的抑制作用；
2.2在体内实验中,对比观察雷米普利和Ang-(1-7)对AS晚期斑块的稳定作用。
3.方法
3.1 ApoE-／-小鼠AS模型的建立
根据AS模型的不同,分为单纯高脂喂养组和高脂喂养+颈动脉套管组。
单纯高脂喂养组：8周龄的雄性ApoE-／-小鼠,实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养8周。高脂喂养4周后,进行分组和药物干预。
高脂喂养+颈动脉套管组：8周龄的雄性ApoE-／-小鼠,实验全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。高脂喂养2周后,行颈动脉套管术,放置套管8周后,进行分组和药物干预。
3.2 Ang-(1-7)体内干预实验
单纯高脂喂养组和高脂喂养+颈动脉套管组分别于高脂喂养4周和10周后,随机分为PBS对照组(12只)、雷米普利组(12只,5mg／kg.d)和Ang-(1-7)组(12只,400ng/kg.min)。分别于药物干预4周后处死动物,检测斑块的病理学形态结构。
3.3血液生化指标检测
分别检测血清总胆固醇、甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇的水平。
3.4组织病理学检测
单纯高脂喂养组：将主动脉纵剖后行大体油红O染色,对主动脉根部斑块进行H&E染色。
高脂喂养+颈动脉套管组：对颈动脉斑块分别进行HE染色、油红O染色和天狼猩红染色,并通过免疫组织化学染色检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)的表达。检测斑块内脂质、巨噬细胞、胶原、VSMCs的含量,计算斑块易损指数。
4.结果
4.1血液生化指标检测：
对照组、雷米普利组和Ang-(1-7)组之间血清TC、TG、HDL-C、LDL-C未见统计学差异。
4.2雷米普利和Ang-(1-7)的抗AS作用
4.2.1雷米普利和Ang-(1-7)对主动脉表面AS形成抑制作用
大体油红O染色显示,与对照组相比,雷米普利和Ang-(1-7)均能显著减少主动脉表面AS病灶范围。对照组、雷米普利组和Ang-(1-7)组主动脉表面的病灶范围分别为：4.14%、2.52%和2.58%。雷米普利组和Ang-(1-7)组主动脉表面的病灶范围无显著差异(P>0.05),但均显著低于对照组(P值均<0.05)。
4.2.2雷米普利和Ang-(1-7)对主动脉根部AS形成的影响
H&E染色显示,与对照组相比,雷米普利和Ang-(1-7)均能显著减少主动脉根部AS病灶横截面积。对照组、雷米普利组和Ang-(1-7)组主动脉根部的斑块面积分别为：20.28%、15.32%、12.08%。Ang-(1-7)组主动脉根部的AS斑块面积显著低于雷米普利组(P>0.05),且均显著低于对照组(P值均<0.05)。
4.3雷米普利和Ang-(1-7)对颈动脉AS斑块稳定性的影响
对照组、雷米普利组和Ang-(1-7)组颈动脉斑块脂质含量百分比分别为20.43%、15.48%和14.76%；巨噬细胞含量百分比分别为12.46%、6.68%和7.21%；平滑肌细胞含量百分比分别为5.15%、7.72%和8.24%：胶原含量百分比分别为15.21%、16.49%和17.98%。易损指数分别为1.62±0.38、0.91±0.26和0.84±0.24。与对照组相比,雷米普利和Ang-(1-7)均可显著降低颈动脉AS斑块的易损指数(P值均<0.05),但雷米普利和Ang-(1-7)对斑块稳定性的影响无显著差异。
5.结论
1.体内实验结果表明,雷米普利和Ang-(1-7)均能显著抑制高脂喂养的ApoE-／-小鼠AS病变形成。
2.雷米普利和Ang-(1-7)均可有效稳定高脂喂养的ApoE-／-小鼠AS斑块。
Background
Atherosclerosis formation and plaque rupture are the common pathological basis of most of cardio-cerebrovascular diseases. Understanding the molecular and cellular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit disease progression before it leads to clinical consequences. Studies have revealed that many different cell types, including macrophages, lymphocytes, endothelial cells and smooth muscle cells (SMCs), are involved in atherosclerotic lesion formation. Most reviews on atherosclerosis focus on the role of endothelial and inflammatory cells in the initiation of atherosclerosis and discuss SMCs largely in the context of late atherosclerosis when they migrate into the neointima and secrete matrixproteins to stabilize the plaque. Histological studies of autopsy specimens of human coronary arteries ranging from infants to adults provide evidence that regions prone to the development of atherosclerosis contain abundant SMCs whereas regions that are more resistant to atherosclerosis contain few, raising the interesting question: Do SMCs in areas of intimal thickening play a pathogenic role in the increased development of atherosclerosis at these sites? Recent studies, coupled with significant in vitro mechanistic data and in vivo correlative data reviewed herein, provide strong evidence that SMCs proliferation and migration are indeed important in early atherogenesis.
Present evidence suggests that intimal SMCs differ significantly from medial SMCs and as such may have unique atherogenic properties that make them fertile ground for the initiation of plaques. Arterial medial SMCs predominantly express proteins involved in the contractile function and quiet, and could swith to the "synthetic" phenotypic states in response to a variety of atherogenic stimuli, while the latter migrate and proliferate more readily than "contractile" SMCs, leading to the atherogenesis.
Renin-angiotensin system (RAS) plays a critical role in VSMCs proliferation, migration and atherogenesis and atherosclerosis acceleration. In particular, confirmed data have shown that angiotensinⅡ(AngⅡ) contributed a lot to VSMCs proliferative activity and aterial remodeling. Angiotensin-(1-7) [Ang-(1-7)], a new member of RAS family, was mostly produced by angiotensin converting enzyme-2 (ACE2) activity. Recent studies have found Ang-(1-7) plays an important protective role in various pathophysiological processes, including cardiac remodeling and fibrosis, hypertensive vacular remodeling, inflammation and regulation of endothelial cells function, through antagonizing AngⅡ. Previous studies have shown that Ang-(1-7) inhibited VSMCs proliferation in a balloon-injured model, however, the mechanism of VSMCs proliferation and migration is still to be elucidated.
Mitogen-activated protein kinases (MAPKs) play a pivotal role in AngⅡ-mediated VSMCs proliferative activity, and recent studies found that SM22a-knockout promoted VSMCs proliferation and migration, and atherosclerotic process.
To sum up, we hypothesize that Ang-(1-7) can effectively inhibit AngⅡ-induced VSMCs proliferation and migration, through blocking the MAPKs and SM22a signaling pathway crosstalk, and to regulate VSMCs bioactivities.
According to the questions mentioned above, our present study was designed to test our hypothesis in vitro.
Aims
1. To test the effect of Ang-(1-7) on AngⅡ-induced VSMCs proliferation and migration.
2. To test the effect of Ang-(1-7) on AngⅡ-induced VSMCs proliferation and migration-related signaling proteins, such as ERK1/2, P38, JNK1/2 and SM22a.
3. To clarify the signaling pathway connection and cross-talk in the VSMCs proliferation and migration.
Methods
1. Cell culture
The human VSMCs were purchased from Sciencell Company and cultured by the routine methods. When cells confluence reached 80-90%, the cells were removed again and those at passage 4-8 were selected for the study.
2. Experimental grouping
In order to inspect the effects of AngⅡand Ang-(1-7) on VSMCs poliferative activity and the related signaling proteins, VSMCs were divided into six groups: control group, AngⅡgroup, Ang-(1-7) group, AngⅡ+Ang-(1-7) group, AngⅡ+Ang-(1-7)+A779 group, A779 group, PD98059 group, SB203580 group, PD98059+SB203580 group and SM22 a knockdown group. In VSMCs culture medium, the concentration of AngⅡ, Ang-(1-7) and A779 were 0.1μM, 1μM and 1μM, respectively.
3. MTT and BrdU incorporation assay
These experiments were undertaken to compare VSMCs reproductive activities after AngⅡ, Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention for 24h.
4. Transwell plate assay
The transwell plate assay was undertaken to detect VSMCs migration acivity after AngⅡ,Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention for 12h.
5. RT-PCR
RT-PCR was taken to explore some cytokines mRNA expression in VSMCs after AngⅡand Ang-(1-7) intervention.
6. Western blot
To observe the expression of some cytokines in VSMCs after AngⅡ,Ang-(1-7), A779, PD98059, SB203580, PD98059+SB203580 and SM22 a knockdown intervention, Western blot was undertaken. The relative expression of protein was demonstrated by the ratio of integral optical density (IOD) value between target protein andβ-actin.
Results
1. Ang-(1-7) regulating AngⅡ-mediated VSMCs proliferative activity:Both MTT assay and BrdU incorporation assay confirmed that Ang II could effectively increase the proliferative activity, while Ang-(1-7) could remarkably abolish the efficacy of AngⅡ. Meanwhile, A779 could effectively reverse the effect of Ang-(1-7).
2. Effect of Ang-(1-7) on AngⅡ-induced VSMCs migration:Transwell plate assay showed that, compared with the control group, Ang II could effectively increase the migration of VSMCs. Similar with the effect of Ang-(1-7) on VSMCs proliferation, Ang-(1-7) could remarkably reverse the effect of AngⅡ, while A779 effectivley abolish the effect of Ang-(1-7).
3. The study of VSMCs proliferation signal transduction pathway:The activities of ERK1/2 and P38 were significantly higher in Ang II group than control group, and Ang-(1-7) treatment effectively decrease the efficacy of AngⅡ, while A779 could effectively abolish the effect of Ang-(1-7). The activity of JNK1/2 significantly increased after AngⅡtreatment, however, no significant change was found with Ang-(1-7) incubation. There were less mRNA and protein expression of SM22a in AngⅡgroup than control group. There were more mRNA and protein expression of SM22a in Ang-(1-7) group than control group, while A779 could effectively abolish the effect of Ang-(1-7).
4. Crosstalk between MAPKs and SM22a pathway:Both the proliferative and migrative activities significantly decreased in PD98059 group, SB203580 group and PD98059+SB203580 group, and remarkably increased in SM22a knockdown group. On the other hand, the mRNA and protein expression of SM22a in PD98059 group, SB203580 group and PD98059+SB203580 group were significantly higher than the control group.
Conclusion:
1. AngⅡcan enhance the VSMCs activities of proliferation and migration, while Ang-(1-7) can effectively inhibit these functions.
2.AngⅡcan activate ERK1/2, P38, and JNK1/2 and inhibit SM22a, however, its effects on ERK1/2, P38 and SM22a were significantly abolished by Ang-(1-7).
3. The Ang-(1-7)-MAS-ERK/P38-SM22a signaling pathway might play an important role in regulating the VSMCs bioactivities.
1. Background
Atherosclerosis formation and progression is the common pathological basis of the most cardio-cerebrovascular diseases. Accumulating evidence indicates that the rennin-angiotensin system (RAS) plays an important role in vascular smooth muscle cell (VSMCs) proliferation, migration and atherosclerosis formation and progression. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is a key cytokine that regulates cell growth, migration, inflammation and oxidative stress and therefore contributed to VSMCs proliferation, migration and atherosclerosis formation and progression.. Angiotensin-(1-7), a new member of RAS, is mainly produced by angiotension converting enzyme (ACE2), through degradating AngⅡ. A large mount of rencent studies demonstrated that Ang-(1-7) was an important endogenous antagonist of AngⅡ, via anti-inflammation and oxidative stress, vascular vasodiation, reduction of fibrosis and phosphatases activation, and thus played an important role in cardiac reconstruction and fibrosis, hypertensive vascular reconstruction, VSMCs proliferation and migration, and endothelial cells function regulation.
Previous studies demonstrated that Ang-(1-7) significantly inhibited VSMCs proliferation and migration in a balloon-injured arterty model, and our rencent study showed that overexpression ACE2 inhibted early AS formation. All the above studies indicated that Ang-(1-7) might play a critical role in AS progression. So, chronic injection of Ang-(1-7) in ApoE-/-mice might attenuate over-activited AngⅡeffectively, and thus inhibit AS formation.
The effect of Ang-(1-7) on AS formation and the possible mechanism is unclear. Our in vitro study demonstrated that Ang-(1-7) regulated VSMCs ERK1/2, P38 and SM22 a activities and expression, which play a critical role in AS formation and progression. However, whether Ang-(1-7) could regulate the above signaling protein and AS progression in vivo is unknown.
So, we raise the following hypothesis:Ang-(1-7) might regulate the MAS-ERK/P38-SM22 a signaling pathway and inhibit AS formation in ApoE-/-mice.
2. Objectives
2.1 To assess the therapeutic effects of Ang-(1-7) on atherosclerotic lesions formation in ApoE-/-mice.
2.2 To elucidate the molecular mechanisms of Ang-(1-7)-mediated therapeutic effects on atherosclerotic lesions.
3. Methods
3.1 Atherosclerosis animal model
ApoE-/-mice aged 8 weeks were fed an atherogenic chow (0.25% cholesterol and 15% cocoa butter). After 8 weeks for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Ang-(1-7) treatment
At the end of week 4 for atherogenic diet, the mice were randomly allocated to phosphate buffered saline (PBS) control group, Ang-(1-7) group and A779 group. All the animals were implanted with a subcutaneous osmotic pump for the duration of 4 weeks. In order to demonstrate the dose-response in mice, the Ang-(1-7) and A779 groups were divided three subgroups(100ng.kg-1.min-1,200ng.kg-1.min-1 and 400ng.kg-1.min-1), respectively.
The mice were anesthetized and their aortas were collected for pathological and biochemical analysis at the end of experiment.
3.3 Serum lipid measurement
Serum samples were collected after 12 hours of fasting and stored at-80℃. Total cholesterol, Triglyceride, high density lipoprotein cholesterol and low density lipoprotein cholesterol were measured.
3.4 Histopathological and immunohistochemical measurement
The whole aortas were stained with Oil Red O, and the cardiac muscles were stained by Masson. SM22ain aortas and MOMA-2 in kidney were identified using appropriate primary antibodies.
3.5 Western blot analysis
The protein expression of p-ERK1/2 and p-P38 were analyzed by Western blot measurement.
4. Results
4.1 Ang-(1-7) protects against atherosclerosis
After 8 weeks of high fat diet, we found that, with the dose of Ang-(1-7) increasing, the inhibitory action of Ang-(1-7) on atherosclerotic lesions formation was increased. The mice treated with high-dose Ang-(1-7) showed significantly smaller atherosclerotic lesions in their aortas, as shown by both en face (2.58% vs.4.14%, P<0.05) and aortic root section analysis (12.08% vs.20.28%, P<0.05), comparing with the control mice.
4.2 The effect of A779 on atherosclerosis
After 8 weeks of high fat diet, we found that high dose A779 significantly increased both the en face (4.79% vs.4.14%, P<0.05) and aortic root section (23.22% vs.20.28%, P<0.05) atherosclerotic lesions formation, comparing with the control mice.
4.3 Ang-(1-7) has no influence on the serum lipid profiles
Serum analysis demonstrates that no significant differences were found among the various treatment groups, which indicating the protective effect of Ang-(1-7) on atherosclerosis might independent of lipid profiles.
4.4 Ang-(1-7) inhibits the activities of ERK1/2 and P38MAPK in vivo
Western blot analysis showed that the phosphorylations of ERK1/2 and P38MAPK were significantly attenuated by Ang-(1-7) in vivo. Conversely, with the administration of A779, the phosphorylations of ERK1/2 and P38 were significantly increased in the A779 treatment group.
4.5 Ang-(1-7) increases the protein expression of SM22a
Immunohistochemical staining demonstrated that the protein expression of SM22ain aortas of Ang-(1-7)-treated mice were significantly higher than the PBS control mice (59.57% vs.8.80%, P<0.01). However, no significant difference of protein expression of SM22a between PBS and A779 groups (8.80% vs.7.75%).
4.6 Ang-(1-7) has no effect on macrophages infiltration in kidney
Immunofluorescence analysis did not found evident macrophages infiltration in kidney of Ang-(1-7)-treated mice, as well as PBS and A779-treated mice.
4.7 Ang-(1-7) has no effect on cardiac muscle collagen content
Masson staining demonstrates no remarkable cardiac fibrosis, and no significant differences of collagen contents among various groups.
5. Conclusions
1. Chronic Ang-(1-7) treatment significantly attenuated atherosclerotic lesions formation in ApoE-/-mice.
2. The anti-atherosclerosis effect of Ang-(1-7) were partly mediated by the effect of anti-proliferation and migration activity on VSMCs through cross-talk of MAS-ERK/P38-SM22a signaling pathways
1. Background
Recent studies have shown that plaque rupture and subsequent intraluminal thrombosis is the most common cause of acute coronary syndrome (ACS). Early prevention of plaque rupture might be an effective approach to reduce the risk of ACS. Ruptured plaques have a large necrotic core, a thin fibrous cap depleted of extracellular matrix (collagen and proteoglycans) and smooth muscle cells (SMCs), and infiltration by macrophages and T-cells with outward positive remodeling and increased plaque vascularity. In most patients with ACS, more than one vulnerable plaque are present in their coronary arteries, and thus, systemic drug therapy should be the appropriate treatment. Unfortunately, there is still not an ideal drug for stabilizing atherosclerotic plaques. Although clinical and basic trials have demonstrated the capability of angiotensin converting enzyme inhibitors (ACEI) and angiotensin type I receptor blockers (ARB) in stabilizing vulnerable plaques, a large part of patients experienced an acute coronary event despite an intensive ACEI and ARB therapy. Therefore, exploration of new drugs with high efficacy and low side effects for stabilizing vulnerable plaques is clearly warranted.
Atherosclerosis is a chronic inflammatory disease. Although the precise mechanisms of plaque rupture are poorly understood, most researchers agree that the disruption of a fibrous cap rich in macrophages and T-lymphocytes in addition to the necrotic core coming in contact with circulating blood are events that lead to the development of thrombi in fatal plaques.
Accumulating evidence demonstates that the rennin-angiotensin system (RAS) contributes a lot to atherogenesis and atherosclerotic progress. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is well proved in atherosclerotic formation and progression. Inhibition of the RAS with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARB) is one of the current therapeutic strategies with proven beneficial effects in the treatment of atherosclerosis. However, a large part of patients experienced an acute coronary event despite an intensive ACEI and ARB therapy. Recent studies have shown that, angiotensin converting enzyme 2 (ACE2), Ang-(1-7), new components of the RAS, play important roles in atherosclerosis, cardiac and vascular remodeling. Ang-(1-7) may exert actions that oppose the atherogenic effects of AngⅡ, which plays an important role in cardiovascular pathophysiology. Our previous study also demonstrated that overexpression of ACE2 stabilized vulnerable plaques. However, whether Ang-(1-7) has protective effect against vulnerable atherosclerotic plaques has not been elucidated. In this study, we observed the effect of Ang-(1-7) on atherosclerotic plaque stability in ApoE-/-mice by systemic application.
2. Objectives
2.1 To assess the therapeutic effects of Ang-(1-7) on atherosclerotic lesions stability in ApoE-/-mice.
2.2 To explore the mechanisms of Ang-(1-7)-mediated therapeutic effects on atherosclerotic lesions inflammation infiltration.
3. Methods
3.1 Atherosclerosis animal model
ApoE-/-mice (aged 8 weeks), which were fed an atherogenic chow (0.25% cholesterol and 15%cocoa butter) throughout the experimental procedure, were divided into two group:group 1:atherogenic chow; group 2:atherogenic chow+ Carotid collar placement. After 16 (group 1) or 14 (group 2) weeks for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Ang-(1-7) treatment
Group 1:At the end of 12 weeks for atherogenic diet, the mice were randomly allocated to phosphate buffered saline (PBS) group, Ang-(1-7) (400ng.kg-1.min-1) group and A779 (400ng.kg-1.min-1)group. All the animals were implanted with a subcutaneous osmotic pump for the duration of 4 weeks.
Group 2:At the end of 2 weeks for atherogenic diet, a 2-mm long constricting silastic tube was placed on the right common carotid artery. Eight weeks later, the collars were removed and a subcutaneous osmotic pump was implanted for the duration of 4 weeks. The mice were randomly allocated to phosphate buffered saline (PBS) group, Ang-(1-7) (400ng.kg-1.min-1) group and A779 (400ng.kg-1.min-1) group.
3.3 Histopathological and immunohistochemical measurement
Mice were perfused through the left ventricle with PBS, followed by 4% paraformaldehyde under 100 mmHg pressure. The aortic root and right common carotid artery was excised and immersed in 4%formaldehyde overnight (4℃), embedded in OCT, and stored at-80℃until use. In all of the mice, OCT-embedded aortic root and carotid artery were cross-sectioned into pieces 6μm thick at 50-μm intervals. Sections were stained with hematoxylin and eosin (H&E), and further stained for lipids and collagen using Oil-red O and picrosirius red, respectively. Smooth muscle cells (SMCs, anti-a-actin) and macrophages (MOMA-2), tumor necrosis factor-a(TNF-α), Interleukin-6 (IL-6), Interleukin-1β(IL-1β), macrophage chemoattractant protein-1 (MCP-1), matrix matalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) were immunostained with corresponding antibodies. The vulnerability index was calculated by the following formula:(macrophage staining %+lipid staining %)/(smooth muscle cell%+fiber%).
3.4 RT-PCR
The mRNA expression levels of TNF-α、IL-6、IL-1βand MCP-1 in macrophages were tested by RT-PCR.
4. Results
4.1 The effect of Ang-(1-7) on atherosclerotic plaque stability
Group 1:Immunohistochemistry analysis indicated that plaques from Ang-(1-7)-injected ApoE-knockout mice showed increased percentages of collagen (18.5%) and smooth muscle cells (66.9%), and reduced percentages of macrophages (29.9%) and lipid area (23.7%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased 46.1% by Ang-(1-7) treatment.
Group 2:Immunohistochemistry analysis indicated that plaques from carotid arteries in Ang-(1-7)-injected ApoE-knockout mice showed increased percentages of collagen (18.2%) and smooth muscle cells (60.0%), and reduced percentages of macrophages (42.1%) and lipid area (27.8%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased 48.1%by Ang-(1-7) treatment.
4.2 The effect of Ang-(1-7) on inflammatory cytokines in plaques
Immunohistochemical analysis indicated the protein expression levels of IL-6, IL-1β, TNF-αand MCP-1 in atherosclerotic plaques of aortic roots were significantly decreased by Ang-(1-7) injection in ApoE-/-mice.
4.3 The effect of Ang-(1-7) on MMPs expression in atherosclerotic plaques
Immunohistochemical analysis demonstrated that the differences of protein expression from aortic root plaques between PBS and Ang-(1-7) subgroups were significant for both MMP-2 (25.1%versus 20.2%, p<0.05) and MMP-9 (17.2% versus 11.3%,p<0.05) staining.
4.4 The effect of Ang-(1-7) on AngⅡ-mediated proinflammatory cytokines expression in macrophages
The results in vitro demonstrated that the mRNA expression levels of IL-6, IL-1β, TNF-αand MCP-1 in RAW264.7 mouse macrophage were significantly increased after incubation with AngⅡcompared to control group, and this up-regulation was significantly reversed by Ang-(1-7), while A779 counteracted the effect of Ang-(1-7).
5. Conclusions
1. Chronic Ang-(1-7) treatment significantly attenuated lipids and macrophages infiltration, and increased the contents of smooth muscle cells and collagen in atherosclerotic plaques in ApoE-/-mice.
2. The effect of Ang-(1-7) on plaque stablity was partly mediated by the the inhibitory effect on inflammation reaction and MMPs activities in atherosclerotic plaques.
1. Background
A large amount of evidence demonstates that the rennin-angiotensin system (RAS) plays a critical role in the atherogenesis and atherosclerosis progression. AngiotensinⅡ(AngⅡ), the main peptide of RAS, is an important cytokine that regulates cell growth, migration, inflammation and oxidative stress and therefore contributed to atherosclerotic progression. Inhibition of the RAS with angiotensin-converting enzyme (ACE) inhibitors is one of the current therapeutic strategies with proven beneficial effects in the treatment of atherosclerosis. Blockade of RAS with ACE inhibitors significantly attenuate the development of aortic atherosclerosis in the hyperlipidemic rabbit and ApoE-/-mice. The link between RAS and human atherosclerosis has also been proved by the results of the Heart Outcomes Prevention Evaluation (HOPE) trial, which showed a 22% reduction in the incidence of myocardial infarction, stroke, cardiac arrest and revascularization procedures in patients taking the ACE inhibitor ramipril compared with those on placebo. In addition, in the Perindopril pROtection aGainst REcurrent Stroke Study (PROGRESS), treatment with the ACE inhibitor perindopril reduced the incidence of major vascular events by 26% and myocardial infarction by 38%. Thus, ACE inhibitor treatment may have protective effects on vascular cells via the antagonism of AngⅡactions.
Recent studies have shown that, angiotensin converting enzyme 2 (ACE2) and angiotensin-(1-7) [Ang-(1-7)], new components of the RAS, play important roles in atherosclerosis, cardiac and vascular remodeling and kidney function. Ang-(1-7) may exert actions that oppose the proliferative, hypertrophic and proinflammatory effects of AngⅡ, which may play an important role in cardiovascular pathophysiology. Our foregoing studies have demonstrated the protective effect of Ang-(1-7) on atherogenesis and plaques stability. This study was carried out to test the hypothesis that Ang-(1-7) enhances stability of atherosclerotic plaque, similar to a high-dose ramipril therapy. Since recent studies revealed a better outcome in patients with vascular disease by ramipril treatment, a high-dose ramipril was chosen as a therapeutic standard to compare with a high dose of Ang-(1-7) in a ApoE-/-mice atherosclerosis model.
2. Objectives
2.1 To compare the protective effects of ramipril and Ang-(1-7) on atherosclerotic lesions formation in ApoE-/-mice.
2.2 To assess the therapeutic effects of ramipril and Ang-(1-7) on atherosclerotic plaque stability in ApoE-/-mice.
3. Methods
3.1 Atherosclerosis animal model
All the animals were fed an atherogenic chow(0.25% cholesterol and 15% cocoa butter) during all the experiment period.
Group 1:ApoE-/-mice aged 8 weeks (n=36) were fed an athrogenic chow for 4 weeks.Then the mice were divided into three groups randomly:control group (n=12), ramipril group (n=12) and Ang-(1-7) group (n=12).
Group 2:ApoE-/-mice aged 8 weeks (n=36) were fed an athro genic chow for 2 weeks. Then, a 2-mm long constricting silastic tube was placed on the right common carotid artery near its bifurcation. Eight weeks later, the collars were removed, and the mice were divided into three groups randomly:control group (n=12), ramipril group (n=12) and Ang-(1-7) group (n=12).
After a further 4-week treatment for atherogenic chow, they were fasted overnight and killed by euthanasia.
3.2 Serum lipid measurement
Serum samples were collected after overnight fasting and stored at-80℃. Total Triglyceride, cholesterol, low density lipoprotein and high density lipoprotein cholesterol cholesterol were measured.
3.3 Histopathological and immunohistochemical measurement
Group 1:The whole aortas were stained with Oil Red O, and the aortic root lesions were stained with hematoxylin and eosin (H&E).
Group 2:Sections of carotid plaques were stained with H&E for histological analysis. The collagen and lipids in plaques were stained by using picrosirius red and Oil-red O, respectively. Smooth muscle cells (SMCs) and macrophages were immunostained with anti-a-actin antibodies and MOMA-2, respectively.
4. Results
4.1 The effect of Ramipril and Ang-(1-7) on serum lipid levels
Serum analysis demonstrates that no significant differences were found among the various treatment groups.
4.2 Ramipril and Ang-(1-7) protects against atherogenesis in the whole surface of aortas
After 8 weeks of high fat diet, we found that, both the two groups treated with ramipril and Ang-(1-7) showed significantly smaller atherosclerotic lesions in their aortas, as shown by both en face (ramipril group:2.52%; Ang-(1-7) group:2.58% and PBS group:4.14%). However, the atherosclerotic lesion area in ramipril group was not different from the Ang-(1-7) (P>0.05), while the lesion areas in both ramipril and Ang-(1-7) groups were larger than control group (both.P<0.05).
4.3 Ramipril and Ang-(1-7) protects against atherosclerosis in the aortic roots
After 8 weeks of high fat diet, we found that, all the three groups treated with ramipril and Ang-(1-7) showed significantly smaller atherosclerotic lesions by aortic root section analysis (ramipril group:15.32%; Ang-(1-7) group:12.08% and PBS group:20.28%). The atherosclerotic lesion area in ramipril group was significantly larger than the Ang-(1-7) group (P<0.05), while the lesion areas in both ramipril and Ang-(1-7) groups were larger than control group (both P<0.05).
4.4 The effect of Ramipril and Ang-(1-7) on carotid atherosclerotic plaque stability
Histopathological and Immunohistochemical analysis indicated that plaques from carotid arteries in ramipril and Ang-(1-7)-treated ApoE-/-mice showed increased percentages of collagen (ramipril group:16.49%; Ang-(1-7) group:17.98% and PBS group:15.21%) and smooth muscle cells (ramipril group:7.72%; Ang-(1-7) group:8.24%and PBS group:5.15%), and reduced percentages of macrophages (ramipril group:6.68%; Ang-(1-7) group:7.21% and PBS group:12.46%) and lipid area (ramipril group:15.48%; Ang-(1-7) group:14.76% and PBS group:20.43%) compared with those treated with PBS. Accordingly, the histological plaque vulnerability index was also decreased (ramipril group:0.91±0.26; Ang-(1-7) group: 0.84±0.24 and PBS group:1.62±0.38).
5. Conclusions
1. Ramipril and Ang-(1-7) offered similar protection in terms of atherosclerotic lesions formation in ApoE-/-mice.
2. Ramipril and Ang-(1-7) showed similar protective effect on the atherosclerotic plaques stability in ApoE-/-mice.
引文
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