Hydrogen Peroxide and Flow-Induced Dilation of Human Coronary Microcirculation

描述(申请人提供)：作用于内皮细胞的剪切力产生血管扩张。这可以说是最重要的内皮扩张生理机制，几乎发生在每一个血管床上。我们最近的数据表明，冠心病患者的冠状动脉微动脉存在血流介导的扩张(FMD)，但FMD通过一种新的机制起作用，包括内皮细胞产生包括过氧化氢(H_2O_2)在内的活性氧物种(ROS)。令人惊讶的是，线粒体呼吸链在人类心脏的口蹄疫中扮演着必要的角色。这项应用的总体目标是研究从内皮到平滑肌的FMD信号序列，研究3个目标。1)我们将研究内皮细胞产生过氧化氢的机制。使用来自冠心病患者的新鲜人冠状动脉小动脉和来自微血管组织和导管动脉的培养的人内皮细胞进行比较。我们将寻求令人兴奋的初步数据，表明线粒体和NADPH氧化酶都参与其中，可能是通过ROS诱导的ROS释放机制和rac1的激活。2)使用一种新的生物测定技术来评估血管扩张和平滑肌钾通道开放，我们将确定与血管扩张有关的内皮衍生超极化因子(EDHF)。花生四烯酸代谢产物和过氧化氢都是FMD所必需的，但初步研究表明，过氧化氢是可转移的扩张剂。3)我们将确定H_2O_2诱导扩张的机制，验证新的假设，即H_2O_2通过半胱氨酸氧化直接作用于PKG11，产生激活的二硫键二聚体形式的酶。这些目标跨越了一个广泛的、临床相关的氧化还原信号通路，从内皮过氧化氢的形成，到过氧化氢作为一种可转移的血管舒缩物质的释放，再到其对潜在的平滑肌细胞的作用机制。总而言之，这些目标解决了一种新的内皮依赖性扩张机制，涉及线粒体产生ROS，到目前为止只在人类心脏中报道过。结果应该确定细胞机械转导、呼吸和氧化还原信号之间的新联系，这些信号调节重要的生理事件，如小动脉血管扩张，负责组织灌流。与人类慢性冠状动脉疾病的直接相关性为这种理解微血管反应性的机械性方法提供了坚实的基础。公共卫生相关性：作用于内皮细胞的剪切力引起的扩张可以说是最重要的内皮细胞扩张的生理机制，几乎发生在每一个血管床上。虽然我们在动物身上进行了广泛的研究，但我们直接检查了患有心脏病的人类的血管，以阐明其中涉及的机制。我们的发现显示了一种独特的扩张机制，涉及线粒体产生ROS，到目前为止只在人类心脏中报道过。这一建议的结果应该确定细胞机械转导、呼吸和氧化还原信号之间的新联系，这些信号调节重要的生理事件，如小动脉血管扩张，负责组织灌流。与人类慢性冠状动脉疾病的直接相关性为这种理解微血管反应性的机械性方法提供了坚实的基础。

DESCRIPTION (provided by applicant): Shear stress acting on endothelial cells produces vasodilation. This is arguably the most important physiological endothelial mechanism of dilation and occurs in virtually every vascular bed. Our recent data indicate that flow-mediated dilation (FMD) occurs in coronary arterioles from patients with coronary disease but 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. The overall goal of this application is to examine the FMD signaling sequence from endothelium to smooth muscle studying 3 aims. 1) We will examine the mechanism of endothelial production H2O2. Using fresh human coronary arterioles from subjects with coronary disease and cultured human endothelial cells from both microvascular tissue and conduit arteries for comparison. We shall pursue exciting preliminary data that indicate both mitochondria and NADPH oxidase are involved, possibly through a ROS- induced ROS release mechanism and activation of Rac1. 2) Using a novel bioassay technique to assess vasodilation and smooth muscle potassium channel opening, we shall identify the endothelial derived hyperpolarizing factor (EDHF) responsible for dilation. Both arachidonic acid metabolites and H2O2 are necessary for FMD, but preliminary studies point to H2O2 as the transferable dilator agent. 3) We shall determine the mechanism of H2O2 -induced dilation, examining the novel hypothesis that H2O2 directly acts on PKG11 by cysteine oxidation, yielding an activated disulfide dimeric form of the enzyme. These goals span a broad, clinically relevant redox signaling pathway from endothelial H2O2 formation, to H2O2 release as a transferable vasomotor substance, to its mechanism of action on underlying smooth muscle cells. Collectively these aims address a novel mechanism of endothelium-dependent dilation involving mitochondrial generation of ROS, thus far reported only in human hearts. Results should identify new links among cellular mechanotransduction, respiration, and redox signaling that regulates important physiological events such as arteriolar vasodilation, responsible for tissue perfusion. The direct relevance to humans with chronic coronary disease provides a strong foundation for this mechanistic approach to understanding microvascular reactivity. PUBLIC HEALTH RELEVANCE: Dilation resulting from shear stress acting on endothelial cells is arguably the most important physiological endothelial mechanism of dilation, and occurs in virtually every vascular bed. Although examined extensively in animals, we examine blood vessels directly from humans with heart disease to clarify the mechanisms involved. Our findings show a unique mechanism of dilation involving mitochondrial generation of ROS, thus far reported only in human hearts. Results of this proposal should identify new links among cellular mechanotransduction, respiration, and redox signaling that regulates important physiological events such as arteriolar vasodilation, responsible for tissue perfusion. The direct relevance to humans with chronic coronary disease provides a strong foundation for this mechanistic approach to understanding microvascular reactivity.