GPR55介导溶血磷脂酰肌醇通过mtROS通路活化内皮细胞促进动脉粥样硬化机制研究

The prevalence and mortality of cardiovascular disease (CVD) caused by atherosclerosis (AS) rise steadily in China. Hyperlipidemia promote AS via several mechanisms, which include elevation of plasma concentration of lysophospholipids (LPLs). LPLs can play a major role in pathogenesis of AS by activating G-protein coupled receptors (GPCRs). However, the molecular mechanisms underlying promotion of AS by LPLs-GPCRs remain poorly defined. Our previous studies demonstrated that: 1) lysophosphatidylinositol (LPI), a species of LPLs, was significantly increased in the early stage of AS; 2) LPI can induce endothelial cell (EC) activation, which was inhibited by G-protein coupled receptor 55 (GPR55) antagonist; 3) vascular inflammation was decreased by knocking out GPR55 in atherogenic apolipoprotein E (ApoE)-/- mice; and 4) mitochondrial reactive oxygen species (mtROS) signaling pathway plays a major role in hyperlipidemia-induced EC activation. Therefore, we hypothesized that LPI-GPR55 pathway induces EC activation, monocyte recruitment, and promotes AS via mtROS-mediated mechanisms. In the present proposal, we will use EC as the in vitro model with either GPR55 overexpression or GPR55 knock-down by small interference RNA; and high fat diet-fed ApoE-/- mice with GPR55 EC-specific knockout as the in vivo AS model to determine the roles of LPI-GPR55 in AS. To achieve this goal, we will use various experimental techniques to carry out the proposed studies in vitro and in vivo. We will also examine the preventive and therapeutic effects of GPR55 inhibitor on AS, which will provide novel targets for development of new drugs.

动脉粥样硬化AS引起的心血管疾病CVD在中国的患病率及死亡率仍处于持续上升阶段。高脂血症可通过多种机制参与AS，比如，其引起溶血磷脂LPLs增高，LPLs被相应的G蛋白偶联受体识别，参与AS形成，但具体机制尚不清楚。课题组前期发现溶血磷脂酰肌醇LPI，为LPLs的一种，是AS重要的脂代谢异常分子；其引起的内皮细胞EC活化可被GPR55拮抗剂所抑制；ApoE-/-小鼠敲除GPR55后，抑制血管炎症；mtROS通路是脂质代谢异常引起EC活化的重要通路。我们提出假设：LPI-GPR55激活mtROS，活化EC，促进单核细胞黏附与移行，参与AS形成发展。本研究拟利用GPR55过表达、siRNA干扰和敲除的血管EC以及结合CRISPR/Cas9技术构建的EC特异性GPR55敲除AS模型小鼠，通过各种实验技术在细胞和活体水平解析LPI-GPR55参与AS的分子网络，探索GPR55抑制剂防治AS新靶点。

动脉粥样硬化AS引起的心血管疾病CVD在中国的患病率及死亡率仍处于持续上升阶段。高脂血症可通过多种机制参与AS，比如，其引起溶血磷脂LPLs增高，LPLs被相应的G蛋白偶联受体识别，参与AS形成，但具体机制尚不清楚。课题组前期发现溶血磷脂酰肌醇LP为LPLs的一种，是AS重要的脂代谢异常分子；其引起的内皮细胞EC活化可被GPR55拮抗剂所抑制；ApoE-/-小鼠敲除GPR55后，抑制血管炎症；mtROS通路是脂质代谢异常引起EC活化的重要通路。我们提出假设：LPI-GPR55激活mtROS，活化EC，促进单核细胞黏附与移行，参与AS形成发展。本研究中，我们体外实验中采用18：0 LPI处理HAECs细胞，利用RT-PCR、Western Blot、细胞增殖实验以及单核细胞黏附实验阐明了在HAECs中，LPI能诱导GPR55活化，激活下游信号途径，使 HAECs活化产生自噬现象，从而导致HAECs损伤从而诱导AS的发生。而 GPR55抑制剂CID16020046可以抑制这一现象，阻断 HAECs的活化自噬，从而延缓AS的发生、发展。体内实验主要通过鉴定出ApoE-/- Cre+/+ GPR55flox/flox小鼠与ApoE-/-小鼠后，高脂喂养造模AS小鼠模型。后续我们将在体内验证AS可通过 LPI-GPR55信号通路促进EC自噬，同时敲除GPR55基因能够抑制这一现象。生物信息学主要通过机器学习，分子对接等方式筛选出新的GPR55抑制剂，探索GPR55抑制剂防治AS新靶点。