Endothelial function in human diabetes: role of mitochondrial fission proteins

 描述(申请人提供)：全球糖尿病(DM)患病率已飙升至3.8亿人以上。这些患者的发病率和死亡率的主要原因是糖尿病相关的大血管和微血管疾病。糖尿病血管疾病与非糖尿病患者的血管疾病有重要的病理生理差异。目前，与非糖尿病患者相比，糖尿病患者对抗血管疾病的治疗效果明显较差。针对糖尿病血管疾病中特别重要的病理生理途径的新疗法可能会为减少糖尿病的不良血管事件提供显著的益处。糖尿病血管疾病始于血管内皮功能障碍的发展--一种以血管炎症增加、血管收缩和血栓形成倾向增加为特征的状态。在糖尿病中，我们和其他人发现内皮功能障碍可以由内皮线粒体功能的严重变化引发，继而发生过度的线粒体分裂。这些变化既出现在急性血糖异常暴露之后，也表现在糖尿病患者慢性异常血糖暴露期间。我们的初步数据表明，无论是高糖还是低糖暴露引起的急性内皮功能损害和慢性DM内皮功能障碍都是通过一个共同的机制发生的--动力蛋白相关蛋白-1(Drp1)，一种基于胞浆的GTPase酶，与位于线粒体膜外膜上的对接蛋白结合。这种结合会引发线粒体过度分裂，并引发线粒体和内皮功能障碍。此外，我们的初步数据强烈表明，Fis1是这一过程中关键的Drp1对接蛋白。这一应用采用了一种创新的翻译方法，独特地将针对完整的人类血管和人体内皮细胞中Drp1-Fis1相互作用的关键药理学和分子研究与基于结构的药物设计和在相关患者衍生组织中产生的化合物测试相结合。我们的方法很有希望直接导致确定一种一流的药物，可以显著减少全球近4亿糖尿病患者的心脏病发作、中风、外周血管疾病、肾脏疾病、失明和神经病变。在目标1中，我们将确定在体内急性暴露于高或低葡萄糖水平是否会导致线粒体分裂和过量的线粒体活性氧产生。此外，我们将确定糖尿病患者完整小动脉的内皮依赖性血管扩张和一氧化氮(NO)生物利用度的损害是否是DRp1和/或FIS1依赖的方式。在目标2中，我们将确定是否可以通过抑制Fis1和/或Drp1的表达来逆转糖尿病患者完整小动脉的内皮依赖性血管扩张和NO生物利用度的慢性损害。在目标3中，我们将识别专门破坏Drp1-Fis1相互作用的小分子，验证这些发现，测试这些小分子对糖尿病患者体外人小动脉的疗效。

 DESCRIPTION (provided by applicant): The prevalence of diabetes (DM) worldwide has soared above 380 million individuals. The primary causes of morbidity and mortality in these patients are diabetes-related macrovascular and microvascular disease. DM vascular disease has critical pathophysiological differences from vascular disease seen in non-DM patients. Currently therapies to combat vascular disease are significantly less effective in DM patients compared to non-DM patients. Novel therapies targeted at disrupting pathophysiological pathways of particular importance in DM vascular disease may offer significant benefits for the reduction of adverse vascular events in DM. DM vascular disease begins with the development of vascular endothelial dysfunction-a state characterized by increased vascular inflammation and increased vasoconstrictive and pro-thrombotic tendencies. In DM, we and others have discovered endothelial dysfunction can be initiated by critical changes in endothelial mitochondrial function occurring secondary to excessive mitochondrial fission. These changes appear both following acute exposure to abnormal glucose as well as being evident during the chronic abnormal glucose exposures of DM. Our preliminary data suggest both acute impairment of endothelial function by high or low glucose exposure and chronic DM endothelial dysfunction occur through a common mechanism-the activation and binding of dynamin-related protein-1 (Drp1), a cytosolic-based GTPase enzyme, to docking proteins located on the outer mitochondrial membrane. This binding initiates excessive mitochondrial fission and triggers mitochondrial and endothelial dysfunction. Further, our preliminary data strongly suggest Fis1 is the critical Drp1 docking protein in this process. This application employs an innovative translational approach that uniquely combines critical pharmacological and molecular studies targeting the Drp1-Fis1 interaction in intact human vessels and endothelial cells from human subjects with structure-based drug design and testing of resulting compounds in relevant patient-derived tissues. Our approach holds great promise to lead directly to identifying a first-i-class pharmacological agent that could significantly reduce heart attacks, strokes, peripheral vascular disease, renal disease, blindness, and neuropathy in the world's nearly 400 million cases of diabetes. In Aim 1, we will determine whether acute in vivo exposure to high or low glucose levels induces mitochondrial fission and excess mitochondrial reactive oxygen species production. Further, we will determine whether impairment of endothelium-dependent vasodilation and nitric oxide (NO) bioavailability in intact arterioles from DM patients is Drp1 and/or Fis1-dependent manner. In Aim 2, we will determine whether the chronic impairment of endothelium-dependent vasodilation and NO bioavailability in intact arterioles from human with DM can be reversed by suppression of Fis1 and/or Drp1 expression. In Aim 3, we will identify small molecules to that specifically disrupt the Drp1-Fis1 interaction, validate these findings, an test the efficacy these small molecules on ex vivo human arterioles from DM subjects.