BMX调控的PAR1磷酸化和内化在血管内皮中的保护机制研究

Vascular endothelial cells play a crucial role in vascular homeostasis. The increased permeability of vascular endothelial barrier and vascular leakage become the maker of lung injury during sepsis. However, the mechanism of pulmonary capillary vascular leakage is still unclear. BMX, a non-tyrosine kinase receptor, which expresses mainly in vascular endothelial cells plays a crucial role in vascular diseases. We have previously found that BMX participates in TNF alpha- and VEGF-induced angiogenesis and vascular remodeling. Our preliminary data show that BMX suppresses cecal ligation and puncture (CLP)-induced pulmonary capillary leakage and lung injury in mice. PAR1 is the receptor of thrombin on endothelial membrane and mediates thrombin-induced vascular permeability increase during acute sepsis. We found that BMX suppresses thrombin-induced PAR1 signal. Furthermore, we found that BMX directly binds to PAR1 and phosphorylates PAR1 without affecting its protein level. With these pilot studies, our guiding hypothesis is that BMX promotes PAR1 internalization and PAR1 signal inactivation through phosphorylation and BMX/PAR1 internalization axis attenuates vascular permeability dysfunction and vascular leakage during sepsis. To test our hypothesis, we will use multi-approaches including CRISPAR/Cas9 knockdown, BMX activity site and PAR1 phosphorylation site mutation, lentivirus package and transfection et al. to explore the mechanism of BMX-regulated PAR1 phosphorylation and internalization. To investigate the protective function of BMX/PAR1 internalization in endothelial permeability and vascular leakage, we will generate CLP-induced lung injury and sepsis model in BMX-KO mice, use ECIS instrument to detect trans-endothelial electrical resistance and inject Evans blue dye in mice tail vein. Also, we will use PAR1 inhibitor SCH79797 in animal model to explore the protective function of BMX/PAR1 axis in vascular barrier permeability and vascular leakage. Aiming at the mechanism of BMX-suppressed PAR1 signal and its protective function in vascular permeability and leakage, we will provide a new target and a new treatment method for vascular barrier dysfunction related diseases including sepsis.

血管内皮细胞在维持血管稳态中发挥重要作用。内皮高通透性和血管渗漏是脓毒症过程中肺损伤的标志。我们团队以往研究证明BMX在炎性因子TNFalpha和VEGF诱导的血管生成和重塑中发挥关键作用。预实验发现BMX抑制脓毒症诱导的小鼠肺血管渗漏和损伤；BMX磷酸化PAR1并抑制PAR1信号却不改变其蛋白水平。因此我们推测BMX通过磷酸化PAR1促进其内化和信号失活从而抑制内皮高通透性和血管渗漏。本项目拟首先在BMX敲除小鼠中利用盲肠结扎穿孔建立脓毒症诱导肺损伤模型，研究BMX在内皮高通透性和血管渗漏中的作用。然后利用CRISPAR/Cas9敲除、位点突变和慢病毒感染等多种手段来研究BMX调控PAR1信号的分子机制及其在内皮中的作用。最后利用动物模型探讨BMX介导的PAR1磷酸化和内化在脓毒症诱导肺损伤中的内皮保护作用。本项目将为血管内皮屏障紊乱相关疾病（如脓毒症）提供新治疗靶点和策略。

脓毒症是一种由微生物入侵引发的全身炎症、免疫失调和多器官损伤的高致死疾病，是继心血管疾病和癌症之后的第三大常见致死疾病。肺内皮的高通透性和微血管泄漏是脓毒症诱导肺损伤的标志。脓毒症导致凝血酶（thrombin）在肺微血管中沉积，thrombin通过内皮细胞膜上的蛋白酶激活受体1（PAR1）来促进血管泄漏从而导致肺损伤。BMX在血管中的作用表明BMX可能是治疗血管疾病的新靶点。然而，BMX在内皮通透性、血管泄漏和脓毒症中的作用尚不清楚。本项目证明了在早期脓毒症的病理过程中BMX调控thrombin/PAR1介导的内皮通透性改变和血管泄露从而在脓毒症进程中发挥保护作用。在机制方面，我们利用位点突变、生物化学和细胞分析等方法证明了BMX直接同PAR1结合，磷酸化PAR1并确定磷酸化位点（Y381和Y383）；进一步证明了BMX通过PAR1磷酸化促进其内化、信号失活并导致thrombin/PAR1信号通路关闭。在细胞水平，利用CRISPAR/Cas9敲除和慢病毒感染构建BMX稳定敲除细胞株，证明了BMX调控的thrombin/PAR1信号失活参与内皮细胞高通透性改变和炎症反应。在动物模型方面，在野生型（WT）和BMX敲除（BMX-KO）小鼠中用盲肠结扎和穿刺（Cecal ligation and puncture, CLP）手术诱导脓毒症；在体内利用Evans Blue染色检测血管泄露情况，确立了BMX在脓毒症中的保护作用；证明BMX通过抑制thrombin/PAR1通路发挥保护作用。在临床指导方面，我们利用PAR1阻断剂SCH79797预处理，证明了在早期脓毒症中阻断PAR1信号通路缓解BMX缺失诱导的内皮通透性增加和肺血管泄露；证明BMX调控的PAR1磷酸化、内化和信号失活在脓毒症诱导的内皮高通透性中的作用；提示BMX磷酸化PAR1可作为治疗血管屏障功能紊乱疾病的新靶点。总之，本项目证明了BMX通过直接磷酸化PAR1促进其内化和信号通路关闭从而防止内皮细胞通透性增强和血管泄露；利用CLP诱导的小鼠模型进一步证明了BMX在早期脓毒症发病过程中的保护作用机制，为脓毒症的治疗提供新的理论依据和作用靶点。项目资助期间，课题组共发表SCI论文3篇；培养了1名博士后出站和1名青年教师，培养了2名博士研究生和3名硕士研究生；课题组顺利完成了所有预定的研究计划。