血管紧张素Ⅱ-1型受体内吞障碍在其自身抗体致血管持续收缩中的作用及机制

Sustained vasoconstriction is reported as the vital pathogenesis for hypertension development. Amounts of evidences have demonstrated that angiotensin II type 1 receptor autoantibody (AT1-AA) may play an important role on sustained vasoconstriction; however, the mechanism was still unclear. Compared with endogenous Ang II, AT1-AA can induce less AT1R internalization on membrane and longer-term vasoconstriction. This study aim to confirm the reduced endocytosis of AT1R is the main cause of decreased AT1R internalization and sustained vasoconstriction, by using over-expression/knock down the protein associated endocytosis, cell surface biotinylation, vascular ring, etc. From two aspects both the phosphorylation of AT1R and formation of endocytic vesicles, we attempt to explore the key point of reduced AT1R endocytosis by AT1-AA via mass spectrometry, total internal reflection fluorescence microscope and immunofluorescence co-localization. In the aspect of conformation change, we purpose to certify that AT1-AA molecular weight and its combining site on AT1R will be the upstream mechanism on endocytosis inhibition and sustained activation of AT1R, by FRET, Fab fragement separation and locus mutation of amino acid. This study is designed to investigate the scientific hypothesis: AT1-AA can lead to sustained vasoconstriction by inhibiting the AT1R endocytosis.

血管持续收缩是高血压发生发展的重要病理生理学基础。研究证实，血管紧张素II（Ang II）1型受体（AT1R）自身抗体（AT1-AA）可引起血管持续收缩，但机理不明。本课题组前期发现，同AT1R的内源性配体Ang II相比，AT1-AA引起膜AT1R内化减少可能与血管持续收缩有关。本研究拟利用过表达/敲减内吞相关蛋白、生物素示踪、血管环等方法，从不同水平证实AT1R内吞障碍是AT1-AA致膜AT1R内化减少、血管持续收缩的重要原因；利用质谱、多色全内反射荧光显微镜成像、荧光共定位等技术，从受体磷酸化和内吞囊泡形成方面寻找AT1-AA致AT1R内吞障碍的关键环节；利用氨基酸位点突变、FERT、抗体Fab片段分离等方法，从构象改变的角度证实AT1-AA分子量和受体结合位点是其引起AT1R内吞障碍和受体信号持续激活的上游机制，以证明科学假说：AT1-AA通过抑制AT1R的内吞导致血管持续收缩。

血管持续收缩是高血压等多种心血管疾病发生发展的重要病理生理学基础。研究证实，血管紧张素II（Ang II）1型受体（AT1R）自身抗体（AT1-AA）存在于多种血管张力异常相关心血管疾病患者体内，可直接引起血管持续收缩，但潜在机制不清。本课题组前期原创性发现，同AT1R的内源性配体Ang II相比，AT1-AA引起膜AT1R内化减少可能与血管持续收缩有关。（1）本项目利用多色全内反射荧光显微镜成像、scFv构建、血管环、ELISA等方法，证实AT1R内吞障碍是AT1-AA致膜AT1R内化减少、血管持续收缩的重要原因，且AT1-AA的上述效应与分子量大小无关；（2）利用免疫荧光共定位、生物发光共振能量转移（BRET）、Confocal、Western blot等技术，证实AT1-AA通过限制AT1R招募β-arrestin1/2，导致内吞过程难以启动，AT1R的内吞减少，AT1R下游信号持续存在；（3）利用致质粒构建、siRNA干扰/过表达、基因测序、分子对接等方法，证实AT1-AA与Ang II结合在AT1R的不同位点，二者可引起显著不同的AT1R构象变化和磷酸化编码模式，这可能是造成AT1-AA限制AT1R内吞，导致血管持续收缩的关键上游机制。本项目从受体信号传导的不同环节，阐明了AT1-AA特殊的致AT1R持续活化的分子机制，可为针对性干预AT1-AA造成的血管损伤效应提供新的潜在靶点和策略。