锚定蛋白ENH调控eNOS磷酸化在血管重构中的作用及机制研究

Gas NO in vascularature is dominantly synthesized by eNOS, which suppresses the proliferation of vascular smooth muscle cells. NO is essential for maintaining vascular hemostasis. The pathogenesis of proliferative vascular disease including atherosclerosis is associated with NO inefficiency; therefore the activity of eNOS to synthesize NO is tightly regulated in vivo. Phosphoryaltion of eNOS, regulated by multiple protein kinases and phosphatases is the main regulation of eNOS activity. Our preliminary data showed that ENH is a new interacting protein of eNOS. ENH binds to PKA and facilitates PKA to phosphorylate eNOS. Carotid artery ligation induced significantly less neointima in mice with ENH endothelium-specific knockout. NO is decreased in ligated vessels, which is blocked in ENH knockout endothelium. All these data strongly lead to the hypothesis that ENH regulates phosphorylation of eNOS and its activity through anchoring certain protein kinases or phosphatases together with eNOS. To validate, first we will answer whether eNOS inhibitor will induce neointima formation in ENH knockout mice, which makes eNOS and NO as key events in ENH regulating vascular remodeling. Then we will investigate the role of ENH in regulating phosphorylation of eNOS caused by cessation of blood flow and involved protein kinases/phosphatases. At last, we study how ENH regulates phosphorylation of eNOS through the interaction between ENH and eNOS. Our research will bring new light in vascular proliferative diseases.

气体分子一氧化氮NO在血管中主要由内皮eNOS合成，通过抑制平滑肌细胞增殖维持心血管稳态。NO不足与动脉粥样硬化等多种堵塞性血管病的发生发展有关，因此体内合成NO的eNOS活性受到严密调控。多种蛋白激酶和磷酸酶参与的磷酸化修饰是调节eNOS活性的主要方式。我们前期研究发现新的eNOS结合蛋白ENH，ENH结合并协助PKA磷酸化eNOS。内皮条件敲除ENH显著抑制小鼠血管结扎诱导的NO减少和血管重构。由此推测：ENH通过调节eNOS磷酸化下调血管结扎后eNOS活性，从而促进血管重构。为验证假说，首先利用ENH内皮敲除小鼠和eNOS抑制剂研究eNOS活性在ENH调控血管重构中的重要作用，接着探究血管结扎后与ENH调节eNOS磷酸化相关的蛋白激酶，最后明确激酶-ENH-eNOS复合体对eNOS磷酸化的作用方式。本项目将阐明内皮ENH在血管重构中的作用机制，有望为理解堵塞性血管病提供新的实验基础。

ENH是一个含有PDZ和LIM结构域的蛋白激酶锚定蛋白。含有LIM结构域的ENH mRNA长剪接体在血管内皮和平滑肌中均有表达。eNOS以及其产生的气体分子NO是维持血管稳态的重要分子。eNOS活性受AKT为主的磷酸化调控。血管损伤后，血管内皮AKT活性降低， eNOS磷酸化（Ser1177）水平下降，NO生成减少，血管稳态破坏，平滑肌细胞发生表型转换后增殖迁移形成新生内膜。我们研究发现在人动脉粥样硬化斑块内皮和小鼠的重构血管中，ENH的表达升高。ENH敲除，包括血管内皮选择性敲除，抑制结扎以及钢丝损伤诱导的小鼠血管重构。此结果明确血管内皮ENH在血管重构中的重要作用。eENH与AKT1以及AKT磷酸酶PHLPP2相互作用，ENH作为一个锚定蛋白协助PHLPP2对AKT1去磷酸化，负调控AKT-eNOS。由此我们提出一条血管重构重要的调控通路。血管损伤诱导内皮细胞ENH与PHLPP2表达增加，使得AKT1的磷酸化和活性下降，进而eNOS的磷酸化水平和活性下降，NO产生减少。维持稳态所需要的NO介导的血管内皮与平滑肌之间的通讯破坏，血管发生堵塞性重构。抑制PHLPP2，或者破坏ENH与PHLPP2的结合都可以阻断其对AKT-eNOS的负调控，阻止血管发生重构。因此我们明确了血管内皮ENH在血管重构中的关键作用，并对其作用机制进行了详细阐述。此外，我们还发现ENH与 Smad3的E3连接酶Stub1竞争结合Smad3，阻止其泛素化降解。因此ENH通过稳定Smad3促进TGFβ1信号。这些研究丰富了ENH在血管中的作用。