Regulations of myoendothelial function by signaling microdomains in hypertension

高血压中信号微结构域对肌内皮功能的调节

• 批准号: 8761552
• 负责人: MARK T NELSON
• 金额: $ 39.52万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 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/8761552
• 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; 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; public health relevance; receptor; receptor coupling; receptors for activated C kinase; research study; shear stress; signal processing; vasoconstriction

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(申请人提供)：血管内皮细胞(ECs)是血管张力的关键调节器。在包括高血压在内的心血管疾病中，它们的功能会被破坏。虽然一些参与介导内皮依赖性血管调节的分子已经被确定，但它们的信号联系的关键方面仍然知之甚少。重要的是，这些分子电路是如何在空间上组织起来以实现有效的信号传递的，这在很大程度上是未知的。在这个方案中，我们测试了一种新的假设，即EC A-激酶锚定蛋白(AKAP150)和瞬时受体潜在香草样蛋白4(TRPV4)通道构成了内皮和平滑肌细胞(SMC)信号的动态整合的核心，该整合信号定位于肌内皮细胞投射(MEPs)-通过内部弹性膜通过缝隙连接连接内皮细胞和相邻SMC的专门化投射。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Gq蛋白偶联受体(GqPCR)仅在MEP激活TRPV4通道所必需的。相反，切应力优先刺激非MEP TRPV4通道。此外，AKAP150在4通道元结构中促进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著差异。重要的是，我们的数据表明，这一信号网络在高血压中是通过MEP AKAP150缺失导致的局部偶联改变而被破坏的。在目标1中，我们使用遗传编码的EC特异的钙离子生物传感器(GCaMP2)和主要网络元件的遗传小鼠模型，研究了AKAP150结合的PKC、PKA和PP2B以及小窝蛋白-1在MEP TRPV4活性和协同性调节中的作用。GCaMP2是一种控制IP3/二酰甘油空间生产的光遗传技术。我们还探讨了大脑动脉和系统(肠系膜)动脉对TRPV4激动剂敏感性显著差异的基础。在目标2中，我们使用多种方法，包括笼状IP3和钙离子的多重光解，来确定肌内皮细胞对MEP的反馈机制，以及通过激活非MEP TRPV4通道而剪切力诱导的血管扩张。在目标3中，我们使用从目标1和目标2中获得的见解，通过两个小鼠模型来揭示高血压中MEP信号网络功能障碍的本质。综上所述，这些实验将提供MEP中双向信号网络的无与伦比的视角，并首次详细探索可能导致高血压患者内皮功能障碍的局部连接缺陷。