Regulations of Myoendothelial Function By Signaling Microdomains 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因子。它们的功能在包括高血压在内的心血管疾病中被破坏。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，这些分子电路如何在空间上组织以实现有效的信号传导在很大程度上是未知的。在本研究中，我们验证了一个新的假设，即EC a激酶锚定蛋白（AKAP150）和瞬时受体电位vanilloid 4 （TRPV4）通道构成了内皮和平滑肌细胞（SMC）信号动态整合器的核心，这些信号定位于my内皮突起（MEPs）——通过间隙连接连接EC和相邻SMCs的内部弹性层的特化突起。为了支持这一点，我们提供了新的数据，AKAP150结合蛋白激酶C (PKC)，蛋白激酶A （PKA）和钙调神经磷酸酶（PP2B），是gq蛋白偶联受体（GqPCR）激活TRPV4通道所必需的。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通道的协同门通，但令人惊讶的是，它不是TRPV4通道激动剂敏感性的决定因素，这在脑动脉和肠系膜抵抗动脉之间存在显著差异。重要的是，我们的数据表明，高血压患者通过MEP AKAP150缺失引起的局部偶联改变，该信号网络被破坏。在Aim 1中，我们利用基因编码的EC特异性Ca2+生物传感器（GCaMP2）、控制IP3/二酰基甘油空间生成的光遗传学技术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caveolin-1在MEP TRPV4活性和协同性调控中的作用。我们还探讨了脑动脉和全身动脉（肠系膜）之间TRPV4激动剂敏感性显著差异的基础。在Aim 2中，我们使用多种方法，包括笼中的IP3和Ca2+的多重光解，来定义肌内皮对mep的反馈机制和剪切应力通过激活非mep TRPV4通道诱导的血管舒张。在Aims 3中，我们利用Aims 1和Aims 2中获得的见解，利用两种小鼠模型揭示高血压MEP信号网络功能障碍的本质。综上所述，这些实验将为mep的双向信号网络提供一个无与伦比的视角，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。

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.