巨噬细胞ABCG1致动脉粥样硬化作用的机制研究

Reverse cholesterol transport (RCT) is an important defence process which protects against the development of atherosclerosis. Macrophage cholesterol efflux is the first step of RCT, in which transmembrane lipid transporter ABCA1 and ABCG1 play important roles in transferring cellular cholesterol into extracellular acceptor. Although numerous studies have demonstrated the anti-atherosclerotic effect of ABCA1, functional roles of another transporter-ABCG1 remain controversial, mostly because of inconsistent results from animal studies. Recent publication from our lab,which focused on the association between ABCG1 promoter region single nucleotide polymorphism (ABCG1-PromR-SNP) and coronary artery disease (CAD) in humans,found that human ABCG1-PromR-SNP -367G>A attenuated macrophage ABCG1 mRNA and protein expression and was significantly associated with deceased risk for CAD and myocardial infarction. For the first time, our findings suggested that ABCG1 in macrophages may promote the development of atherosclerosis in humans. Based on these findings, we hypothesize that genetic variant may alter ABCG1 function and contribute to atherosclerosis and CAD in patients. In this proposal, we will extend our in vitro studies in macrophages with ABCG1-PromR-SNP, and test our hypothesis in transgenic mice that lack endogenous ABCG1 but express the genetic variant. We will also enroll CAD patients and perform computed tomographic coronary angiography to analyze atherosclerotic lesions quantitatively based on ABCG1-PromR-SNP genotype. We will study macrophage cholesterol efflux, macrophage cholesterol uptake and deposition, and macrophage apoptosis as well. Our proposed studies should help us define the functional significance of ABCG1 in the development of atherosclerosis and discover the underlying mechanism, which may shed new light in the pathogenesis of atherosclerosis and eventually help people develop new measures in preventing and treating this life-threatening disease.

胆固醇逆转运是机体抗动脉粥样硬化（AS）的防御机制。巨噬细胞内胆固醇外流是胆固醇逆转运的第一步，跨膜转运蛋白ABCA1或ABCG1在胞内胆固醇外流中发挥重要作用。近期本实验室对ABCG1启动子区基因单核苷酸多态性（ABCG1-PromR-SNP）与冠心病发病相关性的研究显示，由ABCG1-PromR-SNP决定的ABCG1低表达显著降低冠心病发病的危险性，首次在人类发现ABCG1具有致AS作用，在国际上受到关注。本项目将深入探讨ABCG1-PromR-SNP决定的ABCG1表达差异影响AS进展的机制，在观察ABCG1影响其自身介导的胆固醇外流的同时，研究其对巨噬细胞吞噬功能、细胞凋亡、胞内胆固醇沉积、以及对ABCA1功能的影响；拟通过体外实验、转基因动物和对不同基因型冠心病患者冠状动脉病变范围和程度的定量分析，揭示由ABCG1-PromR-SNP决定的ABCG1表达影响AS发病的分子机制。

胆固醇逆转运（RCT）是机体对抗动脉粥样硬化（AS）的重要机制。ABC转运体介导的细胞胆固醇外流是RCT的第一步。ABCG1介导胞内胆固醇转运至成熟的HDL颗粒，本课题组以往对ABCG1启动子区基因单核苷酸多态性与冠心病发病进行的相关性分析显示，ABCG1启动子区rs57137919位点基因多态性-367G>A显著降低冠心病发病的危险性，提示巨噬细胞ABCG1可能具有致AS作用。然而其生物学机制尚未明确。.本项目对不同基因型人群外周血巨噬细胞的研究表明，ABCG1-367G>A位点改变削弱转录因子与ABCG1启动子结合作用，降低启动子转录活性，进而降低巨噬细胞ABCG1表达水平及其介导的胆固醇外流功能。ABCG1-367G>A位点改变上调巨噬细胞内凋亡相关基因表达，后者可能与细胞凋亡增加及AS发生降低相关。在此基础上构建稳定敲低或过表达ABCG1的THP-1细胞系，建立LC-MS/MS测定巨噬细胞氧化固醇外流的方法。研究发现ABCG1的主要作用是特异性地介导7-酮基胆固醇和7β-羟基胆固醇外流至HDL。ABCG1表达下调会导致ABCG1介导的胆固醇及氧化固醇外流减少，巨噬细胞内胆固醇和氧化固醇沉积，细胞炎症反应增强，细胞凋亡加速，同时对凋亡细胞的清除能力增加，可能是ABCG1表达下调发挥抗AS作用的原因所在。本项目进一步构建了ABCG1敲除小鼠和人ABCG1转基因小鼠模型，通过高脂饮食诱导建立AS模型，进行组织学与病理学分析；并筛选ABCG1启动子rs57137919位点不同基因型的冠心病患者，采用PCI定量分析冠脉AS斑块病变程度，明确巨噬细胞ABCG1表达变化对冠心病患者冠状动脉病变程度的影响。结果表明ABCG1-367AA型患者Gensini评分较GA、GG及(GA+GG)型患者均显著降低。AA型患者Syntax评分较GG和(GA+GG)型患者均显著降低。.综上所述，本项目揭示了ABCG1启动子区基因多态性影响巨噬细胞ABCG1表达和功能的分子生物学机制。明确了ABCG1介导外流的氧化固醇类型和能力，结合ABCG1表达差异对巨噬细胞泡沫化、吞噬能力和凋亡的影响，对ABCG1下调的抗AS机制提出了合理解释。最后从临床角度确立了ABCG1表达的差异对冠状动脉病变进展的影响，进一步阐明了ABCG1低表达时、巨噬细胞抗AS进程的分子机制，为AS的发病机制提供了新思路。