A novel TRPV4-eNOS signaling pathway in pulmonary endothelium

摘要 肺动脉高压（PAH）是一种肺血管疾病， 确诊后三年45%。PAH通常与内皮血管舒张功能丧失相关， 长期以来被认为是PAH发展的主要贡献者。PAH中的内皮功能障碍导致 减少肺血管系统中主要血管舒张分子-一氧化氮（NO）的释放;但 潜在的病理机制仍然未知。对这些途径的详细的机械理解 调节肺血管内皮一氧化氮合酶（eNOS）活性是破译 肺动脉高压患者内皮功能障碍的发生机制。在本申请中，我们专注于本地，单一的Ca 2 + 通过内皮TRPV 4（瞬时受体电位香草酸4）通道的流入事件-TRPV 4火花- 调节天然肺动脉内皮细胞eNOS活性和NO释放。我们的初步结果 揭示TRPV 4是肺内皮中的主要Ca 2+内流途径，其通过以下途径引起血管舒张： eNOS激活。此外，内皮TRPV 4通道作为一种控制机制，整合 来自不同生理刺激的信息，包括三磷酸腺苷（ATP）、流动/剪切应力，以及 平滑肌β 1肾上腺素能刺激，通过不同的信号机制。TRPV 4诱导的 慢性缺氧诱导的PAH中血管舒张和NO释放受损。我们假设 TRPV 4 Ca 2+信号转导是PAH中内皮血管舒张功能丧失的原因。具体目标1： 确定内皮TRPV 4通道作为整合来自以下信息的关键控制机制的作用： 不同的生理刺激通过不同的信号通路引起小鼠和人的血管舒张 肺动脉我们还将证实内皮TRPV 4通道在内皮细胞中的生理作用。 TRPV 4-/-小鼠。在具体目标2中，我们将定义TRPV 4-eNOS连接，其导致血管舒张， 对生理刺激的反应。我们还将确定NO本身如何调节TRPV 4通道活性 通过内皮鸟苷酸环化酶-蛋白激酶G（PKG）机制。在具体目标3中，我们将描述 在PAH小鼠模型中TRPV 4-eNOS功能障碍，并检验内皮PKG 上调是PAH中TRPV 4-eNOS轴功能受损的原因。这些研究将导致 新的发现，包括eNOS通过局部Ca 2+信号调节的第一个证据及其在 呸。这些研究的结果将为治疗PAH提供急需的新治疗靶点。

Abstract Pulmonary arterial hypertension (PAH) is a disease of pulmonary vasculature with a high mortality rate of up to 45% three years after diagnosis. PAH is often associated with the loss of endothelial vasodilation, which has long been thought to be a major contributor to development of PAH. Endothelial dysfunction in PAH results in reduced release of the predominant vasodilatory molecule in pulmonary vasculature– nitric oxide (NO); but the underlying pathological mechanisms remain unknown. A detailed mechanistic understanding of the pathways that regulate endothelial NO synthase (eNOS) activity in pulmonary vasculature is necessary for deciphering the mechanisms that cause endothelial dysfunction in PAH. In this application we focus on local, unitary Ca2+ influx events through endothelial TRPV4 (transient receptor potential vanilloid 4) channels–TRPV4 sparklets– that regulate eNOS activity and NO release in the native pulmonary artery endothelium. Our preliminary results reveal that TRPV4 is a major Ca2+ influx pathway in pulmonary endothelium that causes vasodilation through eNOS activation. Moreover, endothelial TRPV4 channels serve as a control mechanism that integrates information from different physiological stimuli, including adenosine triphosphate (ATP), flow/shear stress, and smooth muscle 1 adrenergic stimulation, via distinct signaling mechanisms. The TRPV4-induced vasodilations and NO release are impaired in chronic hypoxia-induced PAH. We hypothesize that impaired TRPV4 Ca2+ signaling is responsible for the loss of endothelial vasodilations in PAH. In Specific Aim 1, we will determine the role of endothelial TRPV4 channels as a key control mechanism that integrates information from different physiological stimuli via distinct signaling pathways to cause vasodilations in mouse and human pulmonary arteries. We will also confirm the physiological roles of endothelial TRPV4 channels in endothelium- specific TRPV4-/- mice. In Specific Aim 2, we will define the TRPV4-eNOS linkage that causes vasodilation in response to physiological stimuli. We will also determine how NO itself regulates TRPV4 channel activity through endothelial guanylyl cyclase-protein kinase G (PKG) mechanism. In Specific Aim 3, we will delineate the TRPV4-eNOS dysfunction in mouse models of PAH, and test the hypothesis that endothelial PKG upregulation is responsible for impaired function of TRPV4-eNOS axis in PAH. These studies will result in novel discoveries including first evidence of eNOS regulation by localized Ca2+ signals and its impairment in PAH. Results from these studies will provide much needed novel therapeutic targets for treating PAH.