Regulations of Myoendothelial Function By Signaling Microdomains in Hypertension

高血压中信号微域对肌内皮功能的调节

基本信息

批准号：
8894077
负责人：
MARK T NELSON
金额：
$ 37.63万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-17 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8894077
关键词：
A kinase anchoring protein Adrenergic Receptor Affect Agonist Allosteric Regulation Angiotensin II Architecture Arteries Binding Proteins Biosensor Blood Vessels Blood flow Calcineurin Cardiovascular Diseases Cell Line Cell physiology Cells Cellular biology Cerebrum Coupled Coupling Cultured Cells Cyclic AMP-Dependent Protein Kinases Data Defect Elements Employee Strikes Endothelial Cells Endothelium Feedback Functional disorder G alpha q Protein Gap Junctions Genetic Glycerol Health Hypertension ITPR1 gene Inositol Ion Channel Mechanics Mediating Mediator of activation protein Mesenteric Arteries Mesentery Molecular Nature Nerve New Territories Phosphoric Monoester Hydrolases Phosphotransferases Production Protein Kinase C Proteins Receptor Activation Receptor Signaling Regulation Relaxation Research Resistance Role Scheme Signal Pathway Signal Transduction Site Smooth Muscle Smooth Muscle Myocytes Structure Techniques Testing Tissues Vanilloid Vascular Diseases Vascular Endothelium Vasodilation Work base caged IP3 caveolin 1 citrate carrier density endothelial dysfunction insight intercellular communication mouse model novel operation optogenetics photolysis receptor receptor coupling receptors for activated C kinase research study shear stress signal processing vasoconstriction

项目摘要

DESCRIPTION (provided by applicant): Endothelial cells (ECs) lining blood vessels are pivotal regulators of vascular tone. Their function is disrupted in cardiovascular diseases, including hypertension. Although some of the molecular players involved in mediating endothelial-dependent vascular regulation have been identified, key aspects of their signaling linkages remain poorly understood. Importantly, how these molecular circuits are spatially organized to enable efficient signaling is largely unknown. In this proposal, we test the novel hypothesis that EC A-kinase anchoring protein (AKAP150) and transient receptor potential vanilloid 4 (TRPV4) channels form the core of a dynamic integrator of endothelial and smooth muscle cell (SMC) signaling that is localized at myendothelial projections (MEPs)-specialized projections through the internal elastic lamina that connect ECs with adjacent SMCs through gap junctions. In support of this, we provide novel data that AKAP150, which binds protein kinase C (PKC), protein kinase A (PKA) and calcineurin (PP2B), is required for Gq-protein coupled receptor (GqPCR) activation of TRPV4 channels exclusively at MEPs. In contrast, shear stress preferentially stimulates non-MEP TRPV4 channels. Moreover, AKAP150 promotes cooperative gating of TRPV4 channels in a 4- channel metastructure but, surprisingly, is not a determining factor of TRPV4 channel agonist sensitivity, which is dramatically different between cerebral and mesenteric resistance arteries. Importantly, our data demonstrate that this signaling network is disrupted in hypertension through changes in local coupling caused by the loss of MEP AKAP150. In Aim 1, we investigate the roles of AKAP150-bound PKC, PKA and PP2B as well as caveolin-1 in the regulation of MEP TRPV4 activity and cooperativity using a genetically encoded, EC- specific Ca2+ biosensor (GCaMP2), an optogenetic technique for controlling spatial production of IP3/diacyl glycerol, and genetic mouse models of major network elements. We also explore the basis for the striking difference in TRPV4 agonist sensitivity between cerebral and systemic (mesenteric) arteries. In Aim 2, we use a variety of approaches, including multi-photolysis of caged IP3 and Ca2+, to define mechanisms of myoendothelial feedback to MEPs and shear stress-induced vasodilation via activation of non-MEP TRPV4 channels. In Aim 3, we use insights gained from Aims 1 and 2 to unravel the nature of the dysfunction of the MEP signaling network in hypertension using two mouse models. Taken together, these experiments will provide an unparalleled view of the bidirectional signaling network in MEPs and represent the first detailed exploration of the defects in local connections that likely contribute to endothelial dysfunction in hypertension.

描述（由申请人提供）：血管内壁的内皮细胞（EC）是血管张力的关键调节因子。它们的功能在心血管疾病（包括高血压）中受到破坏。尽管已经确定了一些参与介导内皮依赖性血管调节的分子参与者，但其信号传导联系的关键方面仍然知之甚少。重要的是，这些分子电路如何在空间上组织以实现有效的信号传导在很大程度上尚不清楚。在本提案中，我们测试了新的假设，即 EC A 激酶锚定蛋白 (AKAP150) 和瞬时受体电位香草酸 4 (TRPV4) 通道形成内皮细胞和平滑肌细胞 (SMC) 信号传导动态整合器的核心，该整合器位于肌内皮投射 (MEP) - 通过内部弹性层的特殊投射，通过间隙连接将 EC 与相邻的 SMC 连接起来。为了支持这一点，我们提供了新的数据，即结合蛋白激酶 C (PKC)、蛋白激酶 A (PKA) 和钙调神经磷酸酶 (PP2B) 的 AKAP150 是 TRPV4 通道的 Gq 蛋白偶联受体 (GqPCR) 激活所必需的。欧洲议会议员。相反，剪切应力优先刺激非 MEP TRPV4 通道。此外，AKAP150 促进 4 通道元结构中 TRPV4 通道的协同门控，但令人惊讶的是，它并不是 TRPV4 通道激动剂敏感性的决定因素，这在脑动脉和肠系膜阻力动脉之间存在显着差异。重要的是，我们的数据表明，在高血压中，由于 MEP AKAP150 缺失导致局部耦合变化，该信号网络被破坏。在目标 1 中，我们使用基因编码的 EC 特异性 Ca2+ 生物传感器 (GCaMP2)（一种光遗传学技术）研究了 AKAP150 结合的 PKC、PKA 和 PP2B 以及 Caveolin-1 在 MEP TRPV4 活性和协同性调节中的作用。控制IP3/二酰基甘油的空间产生，以及主要网络元件的遗传小鼠模型。我们还探讨了脑动脉和全身（肠系膜）动脉之间 TRPV4 激动剂敏感性显着差异的基础。在目标 2 中，我们使用多种方法，包括笼养 IP3 和 Ca2+ 的多重光解，来定义肌内皮对 MEP 的反馈机制，以及通过激活非 MEP TRPV4 通道来剪切应力诱导的血管舒张。在目标 3 中，我们利用从目标 1 和 2 中获得的见解，使用两种小鼠模型揭示了高血压中 MEP 信号网络功能障碍的本质。总而言之，这些实验将为 MEP 中的双向信号网络提供无与伦比的视图，并首次详细探索可能导致高血压内皮功能障碍的局部连接缺陷。