内皮细胞TRPV4-Nox2复合体在细胞ROS稳态调控和血管通透性调节中的作用及机制研究

Unbalance of homeostasis in endothelial ROS plays a key role on vascular permeability and this process was found with chronic disease such as obesity and diabetes. Previous studies suggest Nox2 was mainly involved in vascular dysfunction, but the mechanism of regulating its activity has not yet been elucidated. On the basis of founding TRPV4-Nox2 coupling, we proposed the hypothesis"TRPV4-Nox2 coupling regulates the endothelial ROS homeostasis and vascular permeability". We took high glucose/high fat treated cells, high fat diet induced obesity mice and clinical samples as targets, tested by multiple technical such as co-IP, Proximity Ligation Assay, immuno-FRET, in vitro endothelial cell monolayer permeability assay, Miles vascular permeability assay, measurement of intracellular ROS generation, visualization of actin filaments, immunofluorescence, to clarify the role of TRPV4-Nox2 coupling on ROS induced vascular permeability. Our study will uncover the mechanism of regulating ROS homeostasis and provide new ideas and potential effective molecular targets for ROS induced vascular dysfunction treatment.

内皮细胞活性氧（ROS）稳态失衡在血管高通透性等血管功能稳态失衡中起关键作用，肥胖和糖尿病等慢性疾病均可引起ROS稳态失衡。既往研究发现Nox2主要参与血管功能稳态失衡，但对Nox2活性的调控机制尚未阐明。本课题组在发现TRPV4-Nox2耦联复合体的基础上，提出“内皮细胞ROS稳态和血管通透性的调节是由TRPV4-Nox2耦联复合体所介导”的假说，本项目以高糖/高脂细胞模型，高脂饮食诱导的肥胖小鼠模型和临床标本为研究对象，应用co-IP，PLA，immuno-FRET，单层内皮细胞通透检测，Miles 血管通透检测，ROS检测和荧光免疫等技术，探索TRPV4-Nox2耦联稳态失衡在ROS稳态失衡所介导的血管高通透性中的病理机制。研究结果有助于揭示ROS稳态失衡的调控机制,为ROS稳态失衡导致的血管功能障碍的预防和治疗提供新思路，新靶点。

血管内皮细胞在维持血管功能稳态平衡的过程中发挥重要作用。正常的血管通透性是维持组织液生成和回流的关键因素。肥胖等慢性疾病，均存在血管内皮细胞活性氧(reactive oxygen species，ROS）稳态失衡，进而导致血管通透性增高等血管功能稳态失衡。我们前期发现激动TRPV4可介导ROS生成，并且和Nox相关。TRPV4和Nox2存在物理耦联，并且该耦联在肥胖小鼠模型中增高。在此基础上，本项目对TRPV4-Nox2功能复合体参与动脉内皮细胞ROS的稳态形成，以及TRPV4-Nox2功能复合体在高脂饮食诱导的肥胖小鼠模型中调控ROS稳态的失衡，进而促进内皮通透性增高等血管功能稳态紊乱进行了研究。本项目应用基因敲减，线粒体膜电位荧光标记，化学抑制剂，co-IP，免疫荧光共振能量转移(immuno-FRET)进一步确定TRPV4-Nox2的物理以及功能耦联，应用ROS荧光标记检测其功能耦联变化，并应用对TRPV4-Nox2功能复合体的干预策略，检测其对单层细胞通透性的影响（FITC-葡聚糖通透实验，细胞骨架蛋白重构，紧密连接蛋白和粘附连接蛋白形态以及表达检测）确该功能复合体在维持ROS稳态中的作用机制。应用野生型小鼠，TRPV4基因敲除小鼠构建高脂饮食肥胖小鼠模型，检测TRPV4-Nox2功能复合体物理以及功能耦联的变化，干预该功能复合体后对ROS稳态失衡，以及血管通透性增高等血管功能稳态紊乱的影响。针对TRPV4-Nox2功能复合体靶向小分子药物的筛选以及生物学验证。本研究从血管内皮细胞ROS稳态调控的角度认识血管稳态的分子机制、解析血管稳态失衡引起心脑血管疾病的动态过程及其规律，对于有效干预策略的发现具有重要的现实意义。