抗动脉粥样硬化的新机制：Wnt5a/Ror2信号通路促进胆固醇逆向转运

Imbalance of intracellular cholesterol transport is an important factor in the development of atherosclerosis (As), and the capacity of reverse cholesterol transport is the key to determining the process and outcome in atherosclerosis.It has been shown that Wnt5a plays an important role in atherosclerosis, but the mechanism remains unclear. We previously confirmed that Wnt5a could dramatically reduce the cholesterol accumulation in the lipid-loaded cells, which was related to the upregulation of ABCA1 expression. In the present proposal, our central hypothesis is Wnt5a promotes cellular cholesterol efflux to exert the role of anti-atherosclerosis through its non-canonical signaling pathway; the binding of Wnt5a with its specific receptor Ror2 can accelerate cell reverse cholesterol transportation, hence increase cholesterol efflux via PKC/AP-1/ABCA1 signaling pathway. Established experimental methods and technologies will be used, including retroviral vector infected systems to establish of Wnt5a overexpression or knock-down stable cell lines, overexpression or knockdown Wnt5a in apoE-/- mice, Dil fluorescence labeling, high-performance liquid chromatography(HPLC), immunofluorescence staining, Western-blot, chromatin immunoprecipitation (CHIP), co-immunoprecipitation , luciferase assays and others. The aim of the proposal is to explore the effect and potential mechanism of Wnt5a/Ror2 pathway on PKC, AP-1 and ABCA1 signaling molecules, as well as to obtain reliable in vitro and in vivo evidence of Wnt5a in regulating cholesterol transport in lipid-loaded cells. By accomplishing this proposal, we expect to clarify the new molecular mechanism underlying pathogenesis of atherosclerosis and reveal several new targets for anti- atherosclerosis drug development.

胆固醇逆向转运能力是决定动脉粥样硬化（As）进程与转归的关键因素。而Wnt5a在As发生中具有重要作用，但机制未明。我们前期研究已证实Wnt5a显著减少荷脂细胞内胆固醇蓄积，并与上调ABCA1有关。因此，提出"Wnt5a通过非经典Wnt途径，即与受体Ror2的结合后经由PKC/AP-1/ABCA1通路促进细胞胆固醇逆向转运，增加胆固醇流出发挥抗As作用"的科研假说。本项目拟采用逆转录病毒载体感染系统建立Wnt5a敲低和过表达的稳定细胞株及apoE-/-小鼠模型，通过Dil荧光标记、HPLC、免疫印迹、荧光素酶报告基因检测、CHIP等方法，探讨Wnt5a/Ror2通路对PKC、AP-1和ABCA1等信号分子的影响和潜在机制，旨在获得Wnt5a在体内和体外模型中通过非经典途径调控荷脂细胞胆固醇逆向转运的可靠证据。本项目的研究结果将为As的发病机制研究提供新的理论依据，并为抗As提供新的分子靶点

动脉粥样硬化（atherosclerosis，AS）所致冠心病、中风等心脑血管疾病是严重危害人类健康的“头号杀手”，已成为世界首位人口死亡原因之一。AS是一个多因素参与、多基因异常调控的复杂病理过程，涉及多种学说，包括脂质紊乱学说、、炎症学说、血管平滑肌细胞克隆学说、血栓形成学说、氧化应激学说等。但其发病机制并不明确。本项目在国家自然基金的资助下，通过收集临床血样本、建立Wnt5a敲低和过表达稳定细胞株及动脉粥样硬化小鼠模型，主要开展了Wnt5a及其信号通路与动脉粥样硬化的研究。在体内外实验中，初步阐明了Wnt5a及其信号通路通过影响ABCA1和Caveolin-1的表达，减少荷脂细胞内脂质蓄积，启动胆固醇逆向转运；经由Wnt5a/Ror2/PKC信号通路抑制平滑肌细胞增殖，减少血管内膜的增生，进而发挥抗动脉粥样硬化的作用。同时，我们对上一项国家自然基金项目进行了补充，发现Wnt5a通过PKC非经典通路诱导上皮间质转化，促进鼻咽癌细胞的迁移侵袭和转移。提示，Wnt5a及其非经典Wnt信号分子介导的脂质代谢作用可能是心血管疾病和肿瘤发生的共同生物学机制，为疾病的药物防治提供了新的靶点和实验依据。此外，我们还拓展了抗动脉粥样硬化药物Ezetimibe的新机制，揭示了Ezetimibe可通过调节胆固醇代谢与转运、抑制细胞内脂质蓄积、抑制炎症应答、抗平滑肌细胞增殖等发挥抗动脉粥样硬化的作用。该项目的成果以科研论文和科研总结的形式提供，已经在《Clinical and Experimental Pharmacology and Physiology》、《Clin Chim Acta》、《International Journal of Molecular Sciences》、《Oncotarget》等国内外核心期刊发表论文十余篇，参编专著4部。