内皮细胞SH3GL1在血管通透性调节中的作用和机制

Increasing of vascular permeability is involved in various diseases,there is no effective therapeutic methods clinically,the main reason is its mechanism is not clear.Recently our data shows that SH3GL1 abundance decreased followed by vascular permeability increasing induced by LPS and also global knockout of SH3GL1 caused vascular endothelium injury.Further more the increasing vascular permeability induced by LPS was restrained in the SH3GL1 global transgenic mice.Overexpressing SH3GL1 inhibits the apoptosis of EC through decreasing the stability of p53,on the other hand increases the total expression of VE-cadherin and β-catenin strengthening cell-cell junction.So we infer that SH3GL1 plays a key role in maintaining normal vascular permeability.On the basis of previous study,the present study established SH3GL1 endothelia special kockout and transgenetic mice is to investigate the effect of endothelia SH3GL1 to vascular permeability,meanwhile to reveal SH3GL1 through which ubiquitin ligase to mediate p53 degradation and the key domain that SH3GL1 interacts with p53;state the molecular mechanism that SH3GL1 upregulates the expression of adhension junction protein VE-cadherin and β-catenin in detail.Above findings will recognize the protencial new function of SH3GL1 in maintaining endothelial barrier homeostasis,and provide evidence of SH3GL1 as a more effective and innotive target of preventing vascular permeability increasing.

血管通透性增加可导致多种疾病，临床尚无有效治疗方法，主要原因是其发生机制尚不清楚。我们预实验发现LPS诱导血管通透性增加模型中，SH3GL1蛋白表达降低。全身性敲除SH3GL1诱导血管内皮损伤，而全身性高表达SH3GL1则对抗LPS诱导的血管通透性增加。上调SH3GL1降低p53稳定性，减少内皮细胞凋亡，同时增加VE-钙粘蛋白和β-环状蛋白表达，加强内皮细胞间连接。据此，我们推测：SH3GL1是维持正常血管通透性的关键分子。本研究拟采用SH3GL1 内皮特异性转基因和敲除小鼠，明确内皮细胞SH3GL1在血管通透性调控中的作用，同时揭示SH3GL1介导p53降解的泛素连接酶，以及二者相互作用的关键结构域；阐明SH3GL1上调VE-钙粘蛋白和β-环状蛋白表达的详细分子机理。该工作将揭示SH3GL1维持内皮屏障稳态的新功能，为评估SH3GL1是否可作为防治血管通透性增加的有效新靶点提供科学依据。

内皮通透性的稳定是维持血液与组织液间各种物质交换平衡的关键。血管内皮屏障的破坏，引起血管通透性异常升高，组织水肿，导致多种疾病的发生发展，如急性呼吸窘迫综合症、脓毒症、动脉粥样硬化、中风、心肌梗塞等；同时血管内皮通透性升高会促进肿瘤转移，增加癌症患者的死亡风险。血管内皮细胞完整、内皮细胞间连接功能正常是维持内皮通透性稳定的重要表现。SH3GL1是一种含有SH3结构域并参与调控突触囊泡以及膜上受体蛋白内吞的蛋白。本项目研究工作发现，与对照小鼠相比，内皮特异性敲除SH3GL1，小鼠肺部B16F10黑色素瘤细胞、LLC Lewis 肺癌细胞累积、转移灶数量以及转移面积显著增多；同时加剧LPS诱导肺血管通透性升高。而全身性高表达SH3GL1则显著减少肿瘤细胞在肺部累积及转移面积，降低LPS诱导肺血管通透性升高。免疫荧光及TUNEL染色显示，内皮特异性敲除SH3GL1，肺血管内皮细胞凋亡数量显著增多，肺血管内皮层黏着连接蛋白VE-cadherin 表达水平显著降低。机制研究发现沉默SH3GL1显著增加内皮细胞凋亡数量，增加凋亡小体形成；同时，黏着连接功能蛋白VE-cadherin和β-catenin，紧密连接蛋白ZO-1和Occludin在膜上的表达分布减少，内皮细胞间连接松散，缝隙加大。过表达SH3GL1, 上述作用反之。免疫共沉淀结果显示，SH3GL1通过其SH3结构域与凋亡调控蛋白p53相互作用，过表达SH3GL1加强p53与E3泛素连接酶HERC2相互作用，促进p53泛素化降解；而下调SH3GL1则稳定p53蛋白。以上研究结果得出结论：SH3GL1通过促进p53蛋白泛素化降解，抑制内皮细胞凋亡，以及维持黏着连接功能蛋白VE-cadherin和β-catenin和紧密连接蛋白ZO-1和Occludin在内皮细胞膜的稳定分布两方面发挥稳定内皮通透性作用。SH3GL1是维持内皮通透性稳定的关键分子，调控SH3GL1表达是改善内皮通透性异常疾病的新策略。