血管紧张素AT1受体介导动脉内皮PP2A激活的机制研究

Endothelium exerts a number of vasoprotective effects. Many of these effects are largely mediated by endothelium-derived nitric oxide, the most potent endogenous vasodilator. Accumulating evidence suggests that endothelial dysfunction is an early marker for many cardiovascular diseases. The impairment of endothelium-dependent vasodilation caused by a loss of NO. A decline in NO availability may be caused by decreased expression of the endothelial nitric oxide synthase (eNOS). Angiontensin Ⅱ is the major bioactive peptide of the renin/angiotensin system, plays a fundamental role in controlling cardiovascular homeostasis. One of its important receptors, AT1 receptor, is G protein-coupled receptor. AT1 receptor signaling can be mediated by G protein-dependent pathway and by G-independent pathway. An increasing body of evidance sugguests that angionten Ⅱ/AT1 receptor signaling pathway which activated by multiple mechanisms contributes to the occurrence of endothelial dyfunction. Also, endothelial NADPH-oxidase-derived reactive oxygen specises (ROS) induced by angioten Ⅱ/AT1 receptor was reported to uncoupling of eNOS.It has been shown that AT1 receptor mediated-pathway activated PP2A in cardiomyocyte. Our previous experimental results also suggested that angiotensin Ⅱ/AT1 receptor pathway increased PP2A activity in two-kidney-one-clip hypertensive rats mesenteric artery, leading to eNOS dephosphorylation and decreased NO production. However, molecular mechanisms underlying AT1 receptor mediated-PP2A activation remains unclear. This project intends to explore molecular mechanism underlying PP2A regulation at transcriptional level, translational level, post-translational modulation and protein-protein interaction, and the signaling pathways may be involved in. The objective of this study is to further clarify the pathogenesis of endothelial dyfuntion induced by angioten Ⅱ through AT1 receptor.

血管内皮功能障碍（endothelial dysfunction，ED）的重要表现之一是内皮依赖的血管舒张功能降低。已知内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）功能障碍是ED发生的一个关键因素。血管紧张素Ⅱ（AngⅡ）是导致ED的重要原因，AngⅡ/AT1R信号可经G蛋白依赖或非依赖的途径介导，AT1R通路激活可通过多个机制引起eNOS/NO功能障碍而导致ED发生。已表明AngⅡ/AT1R通路可使PP2A活化。本课题组前期实验结果也发现双肾一夹高血压大鼠肠系膜动脉AngⅡ/AT1R通路激活可引起PP2A活性增强、eNOS去磷酸化、eNOS/NO功能障碍。但迄今AngⅡ/AT1R通路使PP2A活性增强的机制尚不清楚，本项目拟从转录水平、翻译水平、翻译后水平研究其发生机制，并探索所涉及的信号通路，以进一步阐明AngⅡ导致ED的机制。

内皮功能障碍（endothelial dysfunction，ED）是心血管疾病发病的一个重要始动环节。血管紧张素Ⅱ（Angiotensin II, AngⅡ）作为参与疾病发生发展的一个非常重要的体液因子，是导致ED的重要因素。本项目以PP2A降低eNOS磷酸化水平为切入点，研究AngII激活PP2A的分子机制，以进一步阐明AngⅡ导致ED，促进心血管疾病发生发展的机制。本项目以AngⅡ处理人脐静脉内皮HUVECs、AngⅡ孵育离体大鼠肠系膜动脉组织和微量泵持续灌注AngⅡ的SD大鼠为研究对象，从细胞、离体组织、动物整体水平开展研究。. AngⅡ处理HUVECs的研究结果表明，AngⅡ降低eNOS Ser1177磷酸化水平及NO产量，且效应呈时间和浓度依赖性。eNOS Ser1177磷酸化水平降低是1型受体（AT1R）介导PP2A激活所致。PP2A的激活与AngⅡ降低PP2A内源性抑制蛋白I2（I2PP2A）的表达及PP2A C亚基酪氨酸307位点（PP2Ac Tyr307）磷酸化水平有关。在用AngⅡ孵育离体大鼠肠系膜动脉组织，以及微量泵持续灌注AngⅡ的SD大鼠的肠系膜动脉组织的实验中，取得了一致性结果。. 利用HUVECs进一步研究I2PP2A的作用及PP2Ac Tyr307水平降低的分子机制。结果表明，siRNA转染敲低或过表达I2PP2A蛋白可以降低或提高eNOS Ser1177水平；免疫共沉淀实验表明AngⅡ使I2PP2A蛋白与PP2A结合减少，PP2A活性增加，同时AngⅡ促进PP2A与eNOS结合，使eNOS Ser1177去磷酸化。AngⅡ通过AT1R通路使p22phox增加，Nox激活，ROS产生增多，PP2Ac Tyr307水平降低。用Nox抑制剂APO预处理或siRNA转染敲低p22phox后，PP2Ac Tyr307水平提高，表明AngⅡ降低PP2Ac Tyr307水平、激活PP2A是依赖于Nox/ROS通路的。用Src激酶抑制剂PP2预处理的结果表明AngⅡ降低PP2Ac Tyr307水平是Src Tyr418水平降低、Src激酶活性降低的结果。. 本项目研究了AngⅡ激活血管内皮细胞PP2A的分子机制，进一步阐明AngⅡ引起ED的机制，为研发防治AngⅡ参与发病的心血管疾病药物提供详实可靠的实验依据。