内皮细胞代谢紊乱调控线粒体自噬在低氧性肺动脉高压发生中的作用研究

Initial endothelial cells (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC is the crucial cause of hypoxic pulmonary arterial hypertension(HPAH),however, the pathogenesis remains unclear. Metabolic dysfunction of cells causes mitochondrial dysfunction and mitophagy to maintain cell homeostasis.But over-stimulation of mitophagy may give rise to apoptosis. We observed that there was metabolic dysfunction in pulmonary microvascular endothelial cells (PMVECs) when exposed to hypoxia. Glycolysis and expression of PDK1 which shifts metabolism away from oxidative phosphorylation toward glycolysis were both increased and then decreased. It was indicated that PDK1 might regulate metabolic dysfunction of PMVECs. We also observed that mitophagy related protein PINK1/Parkin expression was significantly increased at the early stage of hypoxia and after then decreased, which was similar to that of cells metabolism in PMVECs. We hypothesized that when exposed to hypoxia, metabolic dysfunction of cells caused over-stimulation of mitophagy and induced cell apoptosis at the early stage of hypoxia. Then the moderate of mitophagy was beneficial to the followed proliferation of EC, which contributed to HPAH. Therefore, the aim of our study is to investigate the effect of PDK1 which regulating metabolic dysfunction on mitophagy mediated by PINK1/Parkin, and influence apoptosis and proliferation of EC in vivo and in vitro. This study will provide a new insight for curing HPAH.

血管内皮细胞先凋亡后增殖是导致低氧性肺动脉高压（HPAH）的重要致病因素，但其发生机制仍未明确。代谢紊乱可致线粒体功能异常进而发生线粒体自噬，以维持细胞自身稳态。但线粒体自噬过度激活则可能引发凋亡。我们前期研究发现低氧可引起肺微血管内皮细胞(PMVECs)代谢紊乱，糖酵解先增强后减弱，而糖酵解关键调控酶PDK1可能对其进行调节。同时，PMVECs中线粒体自噬蛋白PINK1和Parkin在低氧初期显著增强而后期减弱。因此我们推测，低氧早期内皮细胞代谢紊乱致线粒体自噬过度激活进而引起内皮细胞凋亡；而后期适度的线粒体自噬使细胞死亡减少，并有利于细胞增殖，从而参与HPAH的发生。综上所述，本课题拟在整体和细胞水平研究低氧时PDK1调节的代谢紊乱通过影响PINK1/Parkin介导的线粒体自噬而对内皮细胞表型变化的调控作用，不仅有助于加深对HPAH发病机制的认识，而且为探索新的治疗靶点提供理论依据。

低氧引起的肺血管重塑是低氧性肺动脉高压（HPH）持续且难以逆转的主要原因。血管内皮细胞早期凋亡的增加和随后的增殖旺盛和凋亡抑制，是引发血管结构重塑并导致肺动脉高压的重要起始和致病因素。然而，为什么低氧过程中内皮细胞会表现为先凋亡后增殖，其发生机制尚未明确。.本课题主要研究以下内容：①HPH发生不同阶段中肺血管内皮细胞凋亡和增殖的表型变化。②线粒体自噬分子PINK1对肺血管内皮细胞的凋亡和增殖及肺血管结构重塑的调控。③PDK1对PINK1的调控作用，及其对内皮细胞的凋亡和增殖及肺血管结构重塑的影响。.研究结果发现，低氧促进血管内皮细胞线粒体自噬增多，线粒体自噬蛋白分子PINK1和Parkin在基因水平和蛋白水平表达都显著增强。PINK1参与低氧促进内皮细胞增殖以及低氧对内皮细胞促凋亡的作用。PINK1表达与低氧时内皮细胞ROS生成，炎症小体蛋白NLRP3表达，以及IL-1β水平呈正相关。我们研究还发现，低氧可使内皮细胞丙酮酸脱氢酶激酶PDK1明显增多。而且PDK1参与调控低氧时内皮细胞增殖和凋亡，以及氧化应激和炎症反应。我们用体内外研究证明PDK1可以调控PINK1的表达。.本研究结果表明低氧时PDK1可通过PINK1信号通路对线粒体的自噬进行调节，从而影响肺血管内皮细胞增殖和凋亡，参与肺血管结构重塑。研究不仅有助于加深对HPH发病机制的理解认识，而且为探索新的HPH治疗靶点提供理论依据。