内皮细胞NLRP3炎症小体加速LDL穿胞促进动脉粥样硬化发生的作用与分子干预

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.

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。

NLRP3炎症小体是一种多聚体蛋白复合物，在动脉粥样硬化（AS）的发生发展中起重要作用。低密度脂蛋白(LDL)在内皮细胞(ECs)中穿胞过多,是触发AS的早期事件，TNF-a、CRP等炎症因子可促进这一过程，但NLRP3炎症小体在内皮细胞脂蛋白穿胞中的作用与机制尚不明确。为研究内皮细胞NLRP3炎症小体对LDL穿胞的调节作用及其分子机制，我们使用一系列抑制剂处理人血管内皮细胞，首次发现TNF-α、CRP可通过结合细胞膜上相关受体CD32、CD64，激活细胞内的ROS/ TXNIP/NF-kB信号通路，活化pro-IL-1b的表达，上调溶酶体内组织蛋白酶活性，从而激活NLRP3炎症小体；通过内皮细胞体外穿胞模型发现，NLRP3炎症小体可促进LDL在内皮细胞中的穿胞作用。我们首次制备合成一种包封有NLRP3 siRNA的带VCAM-1结合肽的阳离子脂质体（PCL），通过体外细胞实验和体内ApoE-/-小鼠模型实验发现，NLRP3 siRNA-PCL可以有效抑制TNF-α诱导的NLRP3炎症小体激活和内皮细胞的LDL穿胞及在血管内皮下的沉积和动脉粥样硬化的发生发展。主要研究结果已发表SCI论文10篇，申请中国发明专利1项，为动脉粥样硬化相关疾病的防治提供了新的分子靶点和治疗选择。