Flow-Mediated Dilation of Human Coronary Arterioles

描述（由申请人提供）：作用于内皮细胞的剪切应力产生血管舒张。这可以说是生理学上最重要的内皮扩张机制，几乎发生在每个血管床。我们实验室的最新数据表明，冠状动脉疾病患者的冠状动脉中发生了血流介导的扩张（FMD），但它是通过一种新的机制发挥作用的，该机制涉及内皮细胞产生活性氧（ROS），包括过氧化氢（H2O2）。令人惊讶的是，线粒体呼吸链在人类心脏的FMD中起着必要的作用，然而，尚不清楚线粒体如何参与内皮表面上的机械剪切应力的转导以引起扩张。我们的总体假设是，剪切作用于内皮细胞，通过连接的细胞骨架元素刺激释放线粒体的H2O2，内皮衍生的超极化因子（EDHF）。我们将从三个方面来检验这个假设。首先，将使用抗霉素A和TNF α来确定线粒体ROS释放的药理学刺激是否可以引起人冠状动脉扩张。在单独的研究中，我们将使用针对线粒体内膜的新型抗氧化剂来确定线粒体内产生的H2O2是否是FMD所必需的。生物测定系统将确认H2O2是否确实是人体冠状动脉循环中EDHF介导的FMD。其次，我们将使用免疫组织化学和特定的药理学和分子方法，包括siRNA，以确定是否内皮细胞骨架元素发挥必要的作用，在FMD和线粒体ROS的产生。第三，我们将测试一氧化氮通过其对线粒体呼吸的抑制作用减少人心脏FMD的假设。这将使用一氧化氮供体，测量线粒体复合物活性和ROS生成来完成。这些实验可以确定一种以前没有描述过的途径，通过这种途径一氧化氮可以抑制EDHF介导的扩张，即通过阻断线粒体产生ROS。总的来说，这些目标解决了一个新的机制，内皮依赖性血管舒张涉及线粒体产生的ROS，迄今为止只在人类心脏。这些研究应该确定细胞过程之间的新联系，包括机械转导，呼吸和氧化还原信号，调节生理事件，如血管舒张。

DESCRIPTION (provided by applicant): Shear stress acting on endothelial cells produces vasodilation. This is arguably the most physiologically important endothelial mechanism of dilation and occurs in virtually every vascular bed. Recent data from our laboratory indicate that flow-mediated dilation (FMD) occurs in coronary arterioles from patients with coronary disease however it operates through a novel mechanism involving endothelial production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2). Surprisingly the mitochondrial respiratory chain plays a necessary role in FMD in the human heart, however it is not known how mitochondria are involved in the transduction of mechanical shear stress on the surface of the endothelium to elicit dilation. Our overall hypothesis is that shear acting on endothelial cells through attached cytoskeletal elements stimulates release from the mitochondria of H2O2, an endothelial derived hyperpolarizing factor (EDHF). We shall test this hypothesis in three ways. First antimycin A and TNFalpha will be used to determine if pharmacological stimulation of mitochondrial ROS release can elicit dilation of human coronary arterioles. In separate studies, we shall use novel antioxidants targeted to the mitochondrial inner membrane to determine whether H2O2 generated from within the mitochondria is necessary for FMD. A bioassay system will confirm whether H2O2 is indeed an EDHF mediating FMD in the human coronary circulation. Second we will use immunohistochemistry and specific pharmacological and molecular approaches including siRNA to determine whether endothelial cytoskeletal elements play a necessary role in FMD and mitochondrial ROS generation. Third, we shall test the hypothesis that nitric oxide through its inhibitory effect on mitochondrial respiration reduces FMD in the human heart. This will be done using nitric oxide donors, measuring mitochondrial complex activity and ROS generation. These experiments may identify a pathway not previously described by which nitric oxide can inhibit EDHF-mediated dilation; namely, by blocking mitochondrial production of ROS. Collectively these aims address a novel mechanism of endothelium-dependent vasodilation involving mitochondrial generation of ROS, thus far reported only in human hearts. These studies should identify new links among cell processes including mechanotransduction, respiration, and redox signaling that regulate physiological events such as vasodilation.