低密度脂蛋白(LDL)在内皮细胞(ECs)中穿胞过多,是触发动脉粥样硬化(AS)的早期事件。TNF-a、CRP等炎症因子可促进这一过程,但机制尚未阐明。本项目通过研究TNF-a、CRP等激活ECs NLRP3炎症小体的分子机制,探讨ECs NLRP3炎症小体对LDL在ECs中穿胞的调节作用和对与ECs共培养的血管平滑肌细胞增殖和表型转化等AS改变的影响和机制。在整体水平, 一方面利用ECs特异性表达重组酶小鼠(Tie2-Cre)与LoxP标记的突变NLRP3转基因小鼠(Nlrp3A350VneoR)杂交,实现小鼠NLRP3条件性激活; 另一方面,以血管内皮粘附分子1 (VCAM-1)抗体连接并包封NLRP3小干扰RNA的阳离子脂质体转染对小鼠ECs NLRP3进行特异性分子干预,分别揭示ECs NLRP3炎症小体对LDL穿胞和AS发生发展的作用机制,以期找到防治AS的关键分子和药物靶点。

Increased transcytosis of LDL across endothelial cells and deposition in the subendothelial space is the early event to initiate atherosclerosis. Inflammatory factors, TNF-a, CRP etc, can accelerate this process. This project is designed to in-depth study the molecular mechanisms underlying how TNF-a, CRP and other inflammatory cytokines activate NLRP3 inflammasome in endothelial cells and explore how endothelial NLRP3 inflammasome regulates LDL transcytosis across endothelial cells, as well as the proliferation or phenotypic transformation, migration or invasion of co-cultured vascular smooth muscle cells, ultimately contributing to the vascular remodeling process. In the in-vivo animal level , on one side, by crossbreeding the endothelial cell-specific expression of recombinant enzyme in mice (Tie2-Cre) with LoxP flanked neomycin resistance gene inserted in a dominated mutant NLRP3 mice (Nlrp3A350VneoR ) to achieve conditional knock-in of an endothelial specific dominated activated NLRP3 mutant mice; on the other side, mouse endothelial cell-specific knock-down of NLRP3 was achieved through injection of vascular cell adhesion molecule-1 (VCAM-1) antibody-connected and NLRP3 small interfering RNA-encapsulated cationic liposomes. On the basis of these two sets of mice experiments, the roles of endothelial NLRP3 inflammasome in the transcytosis of lipoprotein and the development of vascular remodeling and progression of atherosclerosis will be revealed through both endothelial specific NLRP3 inflammasome activation and deletion. These studies will clarify the essential roles of NLRP3 inflammasome in lipoprotein transcytosis and vascular remodeling, which will help to elucidate the molecular mechanisms of atherosclerosis, as well as find the key molecular target for early intervention of atherosclerosis.