FUNDC1介导的线粒体自噬功能障碍在糖尿病血管钙化中作用和机制研究

Vascular smooth muscle cell (VSMC) transformation to an osteoblast-like phenotype, which is often associated with oxidative stress, is a major factor contributing to diabetic vascular calcification. Mitochondrial damage is an important factor in oxidative stress, whereas mitophagy is the main path to eliminate damaged mitochondria. FUNDC1 is a newly found mitochondrial outer membrane protein that mediates mitochondrial autophagy through its characteristic LIR to specifically interact with LC3.Our previous studies have demonstrated that FUNDC1-mediated mitophagy is inhibited in VSMC treated with advanced glycation end products (AGEs), which causing the up-regulation of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4). In addition, the up-regulation of PDK4 promotes the phenotype transformation of VSMC. Therefore, we hypothesize that: 1) Inhibition of mitochondrial autophagy promotes damaged mitochondria aggregation and mitochondrial ROS formation, resulting in increased oxidative stress; 2) mtROS accelerates the vascular calcification of diabetes by increasing the stability of HIF1α as well as PDK4 gene transcription activated by HIF1α. Thus, gene knockout techniques will be applied to dissect the role of FUNDC1-mediated mitochondrial autophagy in acceleration of diabetic vascular calcification both in vitro and in vivo, revealing the detailed mechanisms underlying the mitochondrial autophagy. This study will provide a new target for the prevention and treatment of diabetic vascular calcification.

氧化应激导致平滑肌细胞（VSMC）向成骨样细胞转换是糖尿病血管钙化发生重要机制。线粒体损伤是导致氧化应激关键因素，线粒体自噬是清除损伤线粒体主要途径。FUNDC1是新发现的线粒体外膜蛋白，可与自噬泡结合介导线粒体自噬。我们预实验结果显示晚期糖基化终产物（AGEs）可抑制VSMC的FUNDC1蛋白表达，同时显著上调丙酮酸脱氢酶激酶4（PDK4）。已有研究表明PDK4可促进VSMC表型转换。因此，申请人提出以下假设：1）糖尿病条件下FUNDC1介导的线粒体自噬抑制导致线粒体来源ROS（mtROS）增加。2）mtROS可增加低氧诱导因子-1α（HIF1α）稳定性，进而促进PDK4转录，促使VSMC向成骨细胞表型转换，加速糖尿病血管钙化。本研究利用药理学手段和基因敲除技术在离体和整体水平探讨FUNDC1介导的线粒体自噬功能障碍对糖尿病血管钙化的作用及其机制，为防治糖尿病血管钙化提供新的干预靶点。

血管钙化与糖尿病患者心血管风险事件的发生密切相关，而线粒体功能障碍是血管钙化发生的重要机制之一。因此，探寻线粒体生物发生、线粒体融合-分裂及线粒体自噬之间的交互作用机制对于维持线粒体质量控制平衡、改善血管平滑肌细胞 (VSMCs)功能及减轻血管钙化具有重要意义。本项目在线粒体微环境层面，以线粒体自噬在糖尿病血管钙化中的作用机制为中心，围绕线粒体膜蛋白FUNDC1和糖代谢关键酶丙酮酸脱氢酶激酶4 (PDK4)与线粒体质量控制系统的相关性，并探索两者对糖尿病血管钙化的影响及机制。通过维生素D3、尼古丁构建大鼠主动脉钙化模型，体外通过β-GP诱导VSMCs钙化并添加晚期糖基化终产物(AGEs)模拟糖尿病状态；单独血管钙化过程中VSMCs总的自噬通量受阻，并伴随FUNDC1低表达，而PDK4的抑制剂DCA能够拮抗β-GP所致的FUNDC1表达下降，并与线粒体相关内质网膜相关。使用AGEs干预VSMCs模拟糖尿病钙化环境，通过GFP-mRFP-LC3双标腺病毒观察自噬通量，虽然在细胞水平短时间AGEs干预能够上调VSMC的自噬通量而减轻VSMCs内钙盐沉积；但长时间AGEs的干预会阻断胞内自噬流，从而加重VSMCs钙化；进一步机制探索发现AGEs能通过低氧诱导因子-1α (HIF-1α)/PDK4信号通路上调VSMCs中成骨分化基因表达，导致钙盐沉积；在巨噬细胞中我们也发现AGEs可通过HIF-1α/PDK4信号通路诱导巨噬细胞M1极化，再次证实PDK4在糖尿病血管并发症发生中的核心地位。我们继续发现钙化过程中上调的PDK4能够增加胞内氧化应激及凋亡，但二甲双胍可激活线粒体自噬/线粒体生物发生途径而抑制PDK4诱导的细胞凋亡，减轻VSMCs的成骨样分化。此外，基于PDK4作为糖代谢的关键调控酶，我们发现PDK4能够调节血管钙化过程中糖代谢重编程，促进VSMCs钙化。作为该项目的延伸，我们发现无氧糖酵解的终末代谢产物乳酸可过度激活线粒体分裂，抑制保护性线粒体自噬，加重血管钙化。我们也发现骨髓间充质干细胞来源的外泌体能通过调节氧化应激而拮抗AGEs诱导的血管钙化。本项目的实施为糖尿病血管并发症的机制研究提供了新的思路和方向，同时为课题组后续研究奠定了扎实的基础。