Mechanical regulation of vascular metabolism

该提案概述了David Wu博士在Gokhan Mutlu博士和Yun Fang博士的实验室进行为期两年的博士后培训的研究计划。这个研究项目的最终目标是在血管生物学方面做出有助于促进人类健康的发现；另一个目标是熟练掌握细胞和分子生物学的基本技术，并最终启动一个研究项目，最终申请血管生物学的k级奖。在为期两年的指导期内，吴博士将从芝加哥大学的导师和咨询委员会获得额外的学术和科学指导。研究的总体目标是确定剪切应力对内皮细胞（EC）代谢的影响，而EC在内皮细胞活化中起着基础性的作用。内皮细胞活化是内皮细胞屏障功能降低和炎症增加的过程。内皮细胞的激活与糖酵解的显著上调是最近发现的现象。“动脉粥样硬化倾向”血流产生的低剪切应力（与正常“动脉粥样硬化保护”血流产生的高剪切应力相反）在决定内皮细胞活化方面也很重要。然而，剪切应力的变化如何引起ECs代谢的变化尚不清楚。吴博士现在有确凿的数据表明，暴露于动脉粥样硬化酮流动的人主动脉内皮细胞（HAECs）上调糖酵解、血管生成和炎症中的关键酶。吴博士也有初步的数据表明，在动脉粥样硬化蛋白流动下，haec不能利用线粒体产生能量。令人兴奋的是，他最近发现，动脉粥样硬化性血流导致转录因子缺氧诱导因子-1α (HIF-1α)的常氧稳定，而HIF-1α在许多其他情况下都是上调糖酵解的。这是违反直觉的，因为HIF-1α活性被认为在高氧压环境下被抑制，例如在动脉血流中。此外，吴博士发现溶血磷脂酸（LPA）信号，在动脉粥样硬化流动中增加（通过RhoA和Rac1，调节细胞骨架组织的小gtpase），也是已知的动脉粥样硬化的关键介质，也诱导HIF-1α。总的来说，这些结果导致了他的中心假设：动脉粥样硬化酮流通过RhoA或Rac1介导的LPA信号调节以HIF-1α依赖的方式导致代谢变化。目的1将验证HIF-1α稳定是糖酵解上调、线粒体功能不全和动脉粥样硬化酮血流下EC激活所必需的假设。目的2将验证动脉粥样硬化酮流动通过LPA信号通过RhoA和Rac1稳定HIF-1α的假设。目标是实现对剪切应力相关代谢变化的机制理解。内皮细胞激活在动脉粥样硬化的流动状态（瓣膜和动脉分支点附近）自然发生，这导致慢性炎症和最终动脉粥样硬化，这是美国发病率和死亡率的主要原因。因此，有必要更好地了解剪切应力如何诱导代谢变化并因此激活EC。

The proposal outlines a research plan for David Wu, MD PhD, to perform postdoctoral training in the laboratories of Gokhan Mutlu, MD, and Yun Fang, PhD, for two years. The ultimate goal of this research project is to make discoveries in vascular biology that could be useful in promoting human health; another goal is to be- come fluent in the basic techniques in cellular and molecular biology, and ultimately to jump start a research program for the eventual application of a K-level award in vascular biology. During the 2 year mentored period, Dr. Wu will receive additional academic and scientific guidance from the mentors and an advisory committee at the University of Chicago. The overall research goal is to determine the role that shear stress plays on endothelial cell (EC) metabolism, which plays a fundamental role in endothelial activation. Endothelial activation is the process by which ECs have reduced barrier function and increased inflammation. That endothelial activation occurs with a significant upregulation of glycolysis is a recently discovered phenomenon. That low shear stress generated by “atheroprone” blood flow (as opposed to high shear stress during normal, “atheroprotective” blood flow) is important in determining endothelial activation is also well-described. However, how changes in shear stress produce changes in the metabolism of ECs is unknown. Dr. Wu now has confirmatory data that human aortic endothelial cells (HAECs) exposed to atheroprone flow upregulate key enzymes in glycolysis, angiogenesis, and inflammation. Dr. Wu also has preliminary data that suggests HAECs are unable to use their mitochondria to generate energy under atheroprone flow. Excitingly, he recently found that atheroprone flow led to normoxic stabilization of transcription factor hypoxia inducible factor-1α (HIF-1α), which is known to upregulate glycolysis in many other contexts. This is counterintuitive, as HIF-1α activity is thought to be suppressed in high oxygen tension settings, such as in arterial blood flow. Furthermore, Dr. Wu found that lysophosphatidic acid (LPA) signaling, increased in atheroprone flow (and working through RhoA and Rac1, small GTPases which modulate cytoskeletal organization), and also known to be a key mediator of atherosclerosis, also induces HIF-1α. Collectively, these results led to his central hypothesis: atheroprone flow-mediated modulation of LPA signaling through RhoA or Rac1 leads to metabolic changes in a HIF-1α dependent manner. Aim 1 will test the hypothesis that HIF-1α stabilization is required for upregulation of glycolysis, mitochondrial insufficiency, and EC activation under atheroprone flow. Aim 2 will test the hypothesis that atheroprone flow stabilizes HIF-1α through LPA signaling via RhoA and Rac1. The goal is to achieve a mechanistic understanding of shear-stress related changes in metabolism. Endothelial activation occurs naturally in atheroprone flow states (near valves and arterial branch points) – this results in chronic inflammation and ultimately atherosclerosis, a leading cause of morbidity and mortality in the United States. Thus, there is a significant need to better understand how shear stress induces metabolic changes and hence EC activation.