Regulations of Myoendothelial Function By Signaling Microdomains in Hypertension

描述（由申请人提供）：血管内皮细胞（EC）是血管张力的关键调节因子。它们的功能在心血管疾病，包括高血压中被破坏。虽然一些参与介导内皮依赖性血管调节的分子参与者已经被确定，但其信号联系的关键方面仍然知之甚少。重要的是，这些分子回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。在这个提议中，我们测试了新的假设，即EC A-激酶锚定蛋白（AKAP 150）和瞬时受体电位香草素4（TRPV 4）通道形成了内皮细胞和平滑肌细胞（SMC）信号传导的动态整合器的核心，该信号传导定位于myendothelial projections（MEPs）-通过内弹性膜将EC与相邻SMC通过间隙连接的专门投射。为了支持这一点，我们提供了新的数据，AKAP 150，结合蛋白激酶C（PKC），蛋白激酶A（PKA）和钙调磷酸酶（PP 2B），是所需的Gq-蛋白偶联受体（GqPCR）激活TRPV 4通道只在MEP。相反，剪切应力优先刺激非MEP TRPV 4通道。此外，AKAP 150促进TRPV 4通道在4通道亚结构中的协同门控，但令人惊讶的是，不是TRPV 4通道激动剂敏感性的决定因素，这在脑和肠系膜阻力动脉之间是显著不同的。重要的是，我们的数据表明，这种信号网络在高血压中通过MEP AKAP 150丢失引起的局部偶联变化而被破坏。在目的1中，我们研究AKAP 150结合的PKC，PKA和PP 2B以及小窝蛋白-1在MEP TRPV 4活性和协同性的调节中的作用，使用遗传编码的EC特异性Ca 2+生物传感器（GCaMP 2），用于控制IP 3/二酰基甘油的空间产生的光遗传学技术，以及主要网络元件的遗传小鼠模型。我们还探讨了TRPV 4激动剂敏感性脑动脉和全身（肠系膜）动脉之间的显着差异的基础。在目标2中，我们使用了多种方法，包括笼IP 3和Ca 2+的多重光解，以确定肌内皮反馈MEP和剪切应力诱导的血管舒张通过激活非MEP TRPV 4通道的机制。在目标3中，我们使用从目标1和2中获得的见解，使用两种小鼠模型来阐明高血压中MEP信号网络功能障碍的性质。总之，这些实验将提供一个无与伦比的双向信号网络的MEP视图，并代表了第一次详细探索的缺陷，在本地连接，可能有助于高血压内皮功能障碍。

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.