TRP channels in the regulation of vascular tone

血管动态平衡严重依赖于血管内皮释放的血管扩张因子。最多的 这些因素中最突出的是一氧化氮(NO)，它是内皮功能的主要晴雨表，并成为 在包括冠状动脉疾病(CAD)在内的多种疾病中受损。在人类的冠状动脉和心脏 脂肪微循环，我们已经证明了一种新的过程，其中NO依赖的流动介导的损失 冠心病受试者的扩张(FMD)是通过产生过氧化氢(H_2O_2)来补偿的 内皮线粒体和随后的过氧化氢依赖的扩张。虽然两者都是血管扩张剂，但在 对NO的反对通常会促进细胞活化、炎症和动脉粥样硬化，从而理解 负责从NO向H_2O_2转变的机制可能是制定新的战略以改善 冠心病患者血管内皮功能的研究这个项目的总体目标是阐明信号传递机制。 在CAD过程中将血管扩张剂开关从NO调节为H_2O_2。基于上一个周期的发现，这一次 该提案旨在确定导致以前未被评估的收益的细胞内途径 内皮细胞瞬时受体电位香草素4通道的功能及其对血管扩张剂的作用 切换到CAD。我们将检验一个中心假设，即剪切敏感的磷脂酶A2的协同作用源于 花生四烯酸和NADPH氧化酶信号促进TRPV4激活及随后的过氧化氢依赖 冠心病小动脉扩张的同时交叉抑制NO依赖的扩张。此外，NADPH氧化酶作为一种新的衰老- 而与CAD相关的上游调节器在启动切换过程中起着关键作用。这个应用程序带来了 结合血管运动调节、人类微循环和离子通道结构生物学的专业知识来识别 CAD过程中调节血管扩张剂开关的新分子机制和相互作用。具体目标：(1)我们 将确定TRPV4激活和微动脉血流扩张的分子机制；以及(2)我们将 确定NADPH氧化酶如何调节TRPV4的激活和从NO到H2O2的转化 冠状动脉小动脉的FMD。将对新鲜分离的人小动脉和内皮细胞进行研究 以及活体动物模型，使用多方面的方法结合分离的血管反应性，钙 成像、膜片钳电生理学、质谱学、RNA序列、突变和离子通道 分子建模。意义：我们提议的研究将提供对基本机制的洞察 在健康和疾病中调节人类微血管功能，并潜在地影响我们对冠状动脉的方法 微血管功能障碍与冠心病和其他各种血管病变相关。

Vascular homeostasis is critically dependent upon vasodilator factors released from the endothelium. The most prominent of these factors is nitric oxide (NO), which is the main barometer of endothelial function and becomes impaired in a broad range of diseases including coronary artery disease (CAD). In the human coronary and adipose microcirculation, we have demonstrated a novel process where loss of NO-dependent flow-mediated dilation (FMD) in subjects with CAD is compensated by the production of hydrogen peroxide (H2O2) from endothelial mitochondria and subsequent H2O2-dependent dilation. Although both are vasodilators, H2O2, in opposition to NO, generally promotes cell activation, inflammation, and atherosclerosis, and thus understanding mechanisms responsible for this transition from NO to H2O2 may be key to developing novel strategies to improve endothelial function in patients with CAD. The overall goal of this project is to elucidate the signaling mechanisms that regulate the vasodilator switch from NO to H2O2 during CAD. Building on findings from the last cycle, this proposal is designed to determine intracellular pathways responsible for a previously unappreciated gain of function of endothelial transient receptor potential vanilloid 4 (TRPV4) channels and its contribution to vasodilator switch in CAD. We will test the central hypothesis that a synergy of shear-sensitive phospholipase A2-derived arachidonic acid and NADPH oxidase signaling promotes TRPV4 activation and subsequent H2O2-dependent dilation while cross-inhibiting NO-dependent dilation in CAD arterioles. Further, NADPH oxidases as novel aging- and CAD-associated upstream regulators play a critical role in initiating the switch. This application brings together expertise in vasomotion regulation, human microcirculation, and ion channel structural biology to identify novel molecular mechanisms and interactions that regulate vasodilator switch during CAD. Specific Aims: (1) we will determine the molecular mechanism of TRPV4 activation and arteriolar dilation by flow; and (2) we will determine how NADPH oxidases regulate TRPV4 activation and conversion from NO to H2O2 as mediator of FMD in CAD arterioles. Studies will be conducted on freshly isolated human arterioles and endothelial cells as well as in vivo animal models, using a multifaceted approach incorporating isolated vessel reactivity, Ca2+ imaging, patch-clamping electrophysiology, mass spectrometry, RNA-Seq, mutagenesis, and ion channel molecular modeling. Significance: our proposed studies will provide insight into fundamental mechanisms regulating human microvascular function in health and disease and potentially impact our approach to coronary microvascular dysfunction associated with CAD and a variety of other vascular pathologies.