胆固醇酯转运蛋白在息肉状脉络膜血管病变发病中的作用机制研究

Polypoidal choroidal vasculopathy (PCV) is the leading cause of irreversible vision impairment among Chinese elderly. Our previous study first indentified that cholesteryl ester transfer protein (CETP) gene was significantly associated with the risk of PCV. We also observed that the risk allele of CETP decreased the serum level of high-density lipoprotein (HDL) in patients with PCV. Our preliminary experiment demonstrated that α1-antitrypsin, an elastase inhibitor carried by HDL, was also influenced by CETP risk variant. Overexpression of HTRA1, which has elastase activity, could induce PCV. Furthermore, α1-antitrypsin can bind to and form a stable complex with HTRA1. These findings indicate the function of CETP in regulating elastase inhibitor carried by HDL, which involve in the pathogenesis of PCV. This study was designed to investigate the biological plausibility of the association between CETP risk variant and PCV. The regulation function of CETP variant will be studied in serum CETP mass and activity, HDL level and particle size, α1-antitrypsin level, formation of α1-antitrypsin-HTRA1 complex, and downstream extracellular matrix-related factor regulated by HTRA1. The remodeling of Bruch membrane and choroidal vessels and the expression of each member in this pathway will be observed in a mice model with short hairpin RNA targeting α1-antitrypsin. This study will connect the pathways of CETP and HTRA1 by α1-antitrypsin. The approval of this study will give a functional answer for our previous findings and a scientific basis for the intervention, prevention and control of PCV.

息肉状脉络膜血管病变（PCV）是我国老年人群高发的不可逆致盲性眼病。申请人前期研究中首次鉴定出胆固醇酯转运蛋白（CETP）基因与PCV发病强相关，并发现该基因变异下调PCV患者血清高密度脂蛋白（HDL）；预实验表明HDL所携带的α1抗胰蛋白酶也同时下调。鉴于小鼠过度表达弹性蛋白酶HTRA1可诱发PCV，而α1抗胰蛋白酶可结合HTRA1抑制其活性，提示CETP通过HDL相关的细胞外基质代谢参与PCV发病。本项目在功能遗传学层面调查CETP基因致人群易感PCV的作用机制，分析CETP基因变异对PCV患者CETP活性、HDL颗粒及所附α1抗胰蛋白酶水平、α1抗胰蛋白酶-HTRA1复合物形成以及下游细胞外基质代谢通路的影响，并在小鼠体内观察沉默α1抗胰蛋白酶后脉络膜组织病理学以及通路上下游分子的表达变化。本项目联汇CETP脂代谢通路和HTRA1细胞外基质代谢通路，有望为PCV防治提供新的干预路径。

息肉状脉络膜血管病变（PCV）是我国老年人群高发的不可逆致盲性眼病。本项目大样本的病例对照关联分析发现和验证了脂质代谢相关的胆固醇酯转运蛋白（CETP）基因（rs3764261，P=4×10E-7；rs5882，P=5×10E-3）和FER1L4基因（rs57545942，P=2×10E-7）是国人PCV发病独立于CFH和HTRA1基因的危险因素；PCV患者存在血清高密度脂蛋白（HDL）胆固醇和血小板活化因子等脂质组学异常，且受CETP变异位点基因型影响；同时存在血清中性粒细胞弹性蛋白酶显著上调、α1 抗胰蛋白酶（AAT）显著下调的基质代谢失衡。临床观察发现PCV分支血管网是一种突破Bruch膜的1型脉络膜新生血管；该病理过程中Bruch膜弹性纤维层降解断裂是PCV发生基础，过表达后可诱导PCV表型的HTRA1具有弱且缓慢的弹性蛋白酶活性，但体外单独的HTRA1不足以降解Bruch膜弹性纤维层。我们发现体外Bruch膜弹性蛋白酶解可被HDL抑制，且呈剂量效应，其抗弹性蛋白酶活性来自于HDL自身结合的AAT；但HTRA1存在时，可裂解失活AAT，从而剥夺HDL抵抗Bruch膜降解的功能，同时数倍地增强中性粒细胞弹性蛋白酶活性，促成PCV发生。本项目在PCV发病机制中联汇了脂质代谢和基质代谢通路，表明CETP基因变异可能通过调控HDL在Bruch膜层面影响AAT-HTRA1相关的弹性蛋白降解，参与PCV发病；HTRA1应该作为PCV防治的重要靶标。