Apoa-II通过调控AMPK信号通路抑制动脉粥样硬化形成的机理研究

Extreme hyperlipidemia is the major causes in atherosclerosis (As). In this research, we fed the zebrafish with high-cholesterol diet (HCD) and found that the early phase of As formation, like lipid accumulation and disturbance of intima. Impressively, giving Ezetimibe, a cholesterol absorbing inhibitor, to the zebrafish, could antagonize the formation of As caused by CHD. By performing iTRAQ analysis, we identified apolipoprotein A-II (apoA-II) is the key protein which was specially increased during Eze treatment. But until now, the mechanism of how ApoA-II inhibits the As still unknow. By knocking down the expression of Apoa-II gene in zebrafish, we found that the activity of phospho-AMPK was inhibited. Thus, the ApoA-II protein may have the function of activating AMPK activity. AMPK can associate with Vasp, which is known as the leading signal of migration in the protrusion. Through the bridging of Pdlim5, activated AMPK can phosphorylate Vasp instantaneously by this quick path and regulate cell migration firmly. At last, we will establish As model by mice knocking out Apoa-II or specially expressing human Apoa-II gene. On this model, we will try to prove that ApoA-II inhibits cell migration by regulating AMPK-Pdlim5-Vasp signaling pathway. It could be the mechanism of As inhibition caused by up regulation of ApoA-II. This work may contribute to the theory of metastasis regulating on cell behaviors.

血脂异常是动脉粥样硬化（As）的发病基础。 本研究发现胆固醇吸收抑制剂依泽替米贝（Eze）可以减轻斑马鱼高胆固醇饮食（HCD）所导致的内膜增厚伴结构紊乱等As早期表现。经iTRAQ分析载脂蛋白A2（ApoA-II）是Eze起作用的关键蛋白。但ApoA-II抑制As形成的分子机制仍不十分清楚。我们通过敲低Apoa-II基因发现斑马鱼AMPK磷酸化减弱，从而初步证实了ApoA-II对AMPK可能有激活作用。AMPK可通过底物蛋白Pdlim5的桥连作用与突促内细胞迁移的引领分子Vasp相靠近，AMPK活化后迅速磷酸化Vasp并抑制平滑肌细胞的迁移。最后，我们将利用敲除Apoa-II基因或特异性表达人Apoa-II基因的小鼠建立As模型，在动物层面验证ApoA-II通过调控AMPK-Pdlim5-Vasp信号通路抑制细胞迁移，从而减轻As的形成，进一步完善物质代谢调控细胞行为这个新兴学说。

细胞的异常迁移与增殖是动脉粥样硬化（As）早期的病理生理学基础。预实验发现，高胆固醇饮食导致斑马鱼血管早期As形成。为此，我们联想到胆固醇代谢与细胞迁移之间或许存在重要的桥梁。高密度脂蛋白（HDL）是负责胆固醇转运的主要脂蛋白, 它有很多不同的成分，其中ApoA-II在抗As形成中的作用仍存争议。既往关于ApoA-II的研究多集中在物质代谢领域，而它对细胞行为学特别是细胞迁移力的影响一直没有明确解答。 我们发现，降脂新药依泽替米贝（ezetimibe，Eze）可诱导Apoa-II基因表达上调，敲低斑马鱼体内Apoa-II基因将阻断Eze所带来的血管保护作用。AMPK是调控细胞迁移运动的重要激酶。有报道发现ApoA-I可以激活AMPK，Eze也能使AMPK磷酸化增强。预实验发现通过Eze上调表达的Apoa-II蛋白同样可以活化激活AMPK。AMPK可以近距离的接触到运动调节蛋白Vasp，从而迅速高效的调控细胞运动。既往有报道Vasp可被AMPK磷酸化。血管损伤后的重塑涉及到多种细胞的迁移和表型转变，在这个过程中AMPK起到的作用不容忽视。AMPK对细胞命运转变的调控，是我们关注的更本质的问题。.本研究发现Apoa-II介导高脂条件下EZE对斑马鱼血管发育异常的拮抗作用，高脂对血管的损伤是从血管新生与发育阶段就开始的。EZE通过提升Apoa-II表达水平，调控AMPKa1活性，进而抑制细胞的迁移运动与血管新生。我们建立了内皮细胞条件特异性敲除AMPKa1小鼠模型，利用该小鼠模型，构建成年鼠和乳鼠视网膜染色模型，并创新性的发现了AMPKa1在乳鼠视网膜血管发育过程中的重要作用。还对AMPK的底物vasp在血管发育与新生中的功能与机制进行了初步探索，明确了vasp作为AMPK经典底物，磷酸化位点在S317.创新性的构建了平滑肌细胞条件敲除AMPKa1的小鼠模型，并利用As早期损伤模型进行单细胞测序，初步明确了内皮细胞、成纤维细胞、平滑肌细胞在这一过程中表型的转换；筛选出一群Scar1+细胞在损伤前后有较大变化，并贡献于血管内膜的异常增生。这些探索为进一步研究AMPKa1在As中、特别是在细胞命运转变中的作用机制提供理论和实验证据，为As治疗提供潜在靶点。