Role of Hydrogen Peroxide in the Mechanism of Flow-Induced Dilation of the Human

描述（由申请人提供）：剪切应力作用于内皮细胞产生血管舒张。这可以说是最重要的生理内皮机制的扩张，发生在几乎每一个血管床。我们最近的数据表明，血流介导的扩张（FMD）发生在冠心病患者的冠状动脉中，但其作用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FMD中起着必要的作用。本应用程序的总体目标是研究从内皮到平滑肌的FMD信号序列。1)我们将探讨内皮细胞产生H2O2的机制。用冠心病患者的新鲜人冠状动脉与培养的人微血管组织和导管动脉内皮细胞进行比较。我们将追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舒张和平滑肌钾通道开放，我们将确定负责舒张的内皮源性超极化因子（EDHF）。花生四烯酸代谢物和H2O2对口蹄疫都是必需的，但初步研究表明H2O2是可转移的扩张剂。3)我们将确定H2O2诱导扩张的机制，检验H2O2通过半胱氨酸氧化直接作用于PKG11的新假设，产生活化的二硫二聚体形式的酶。这些目标跨越了广泛的、临床相关的氧化还原信号通路，从内皮H2O2的形成，到H2O2作为一种可转移的血管舒缩物质的释放，再到其对平滑肌细胞的作用机制。总的来说，这些目标解决了涉及线粒体生成ROS的内皮依赖性扩张的新机制，迄今为止仅在人类心脏中报道。结果应确定细胞机械转导、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，如负责组织灌注的小动脉血管扩张。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制方法提供了坚实的基础。公共卫生相关性：由作用于内皮细胞的剪切应力引起的扩张可以说是最重要的生理内皮扩张机制，并且几乎发生在每个血管床上。虽然在动物中进行了广泛的研究，但我们直接检查了患有心脏病的人的血管，以阐明所涉及的机制。我们的研究结果显示了一种独特的涉及线粒体生成ROS的扩张机制，迄今为止仅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，如负责组织灌注的小动脉血管扩张。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制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.