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，死亡率高达 确诊三年后45%。PAH通常与内皮血管扩张的丧失有关，这有 长期以来，人们一直被认为是PAH发展的主要贡献者。PAH中的内皮功能障碍导致 肺血管系统中主要的血管扩张分子-一氧化氮(NO)的释放减少；但 潜在的病理机制仍不清楚。对路径的详细机械理解 调节肺血管内皮型一氧化氮合酶(ENOS)活性对于破译是必要的 PAH中导致内皮功能障碍的机制。在本应用程序中，我们将重点放在局部、单一的钙离子上 通过内皮TRPV4(瞬时受体电位香草素4)通道的内流事件-TRPV4小火花- 调节肺血管内皮细胞eNOS活性和NO释放。我们的初步结果 揭示TRPV4是肺内皮细胞钙离子内流的主要途径，通过以下途径引起血管扩张 ENOS激活。此外，内皮TRPV4通道作为一种控制机制，整合了 来自不同生理刺激的信息，包括三磷酸腺苷(ATP)、流动/剪切应力和 平滑肌-1肾上腺素能刺激，通过不同的信号机制。TRPV4诱导的 慢性低氧诱导的PAH血管扩张和NO释放受损。我们假设损害了 TRPV4Ca~(2+)信号转导参与了PAH内皮血管扩张功能的丧失。在具体目标1中，我们将 确定内皮细胞TRPV4通道作为整合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扩张 肺动脉。我们还将确认内皮TRPV4通道在内皮中的生理作用- 特异性TRPV4-/-小鼠。在特定的目标2中，我们将定义引起血管扩张的TRPV4-eNOS连接。 对生理刺激的反应。我们还将确定NO本身是如何调节TRPV4通道活动的 通过内皮细胞鸟苷酸环化酶-蛋白激酶G(PKG)机制。在具体目标3中，我们将描绘 在PAH小鼠模型中检测TRPV4-eNOS功能障碍，并验证内皮细胞PKG 上调是PAH中TRPV4-eNOS轴功能受损的原因之一。这些研究将导致 新发现包括内皮型一氧化氮合酶受局部钙信号调控及其损伤的首次证据 啊哈。这些研究的结果将为治疗PAH提供急需的新的治疗靶点。

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.