TRPV4参与调控缺氧性肺动脉收缩及其机制的研究

Hypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological mechanism which optimizes ventilation/perfusion (V/Q) matching by redirecting blood flow from poorly to better ventilated areas.Yet chronic HPV contributes to pulmonary hypertension and cor pulmonale.The underlying machanisms remain obscure. Recent studies have demonstated a critical role of Canonical transient receptor potential 6 (TRPC6)channel in HPV. We have lately reported that hypoxia increases the epoxyeicosatrienoic acids (EETs) production in pulmonary endothelial cells, which serve as the second messenger between the oxygen sensor (endothelium) and the HPV effector (pulmonay artery smooth muscle cells, PASMC). More importantly, EETs have been found to activate the transient receptor potenial vanilloid 4 (TRPV4)channel, which is widely expressed in the lung, including PASMCs. In preliminary study, we tested the role of TRPV4 channel in HPV.In both ex vivo and in vivo experiments, we found HPV response was largely attenuated in either TRPV4-/- mouse lungs or by TRPV4 inhibitor. In cultured mouse PASMCs, we further noticed that the translocation of TRPC6 to the caveolae, a prerequisite for HPV, is completely blocked by the TRPV4 inhibitor. In this study, combining both animal and cell models, we aim to test our hypothesis that TRPV4 in PASMCs plays a critical role in HPV. Under hypoxia, the interaction between TRPV4 and TRPC6 is required for the translocation of TRPC6 to the caveolae, which subsequently allows/facilitates Ca2+ influx into PASMCs through TRPC6 and causes PASMCs contraction. Our study may help to better understand the mechanism of HPV and thus provide a new therapeutic target for pulmonary hypertention and cor pulmonale.

缺氧性肺动脉收缩（HPV）是肺血管特有的保护性生理反应，但是持续的肺动脉收缩会导致肺动脉高压，甚至右心衰竭。HPV确切的机制尚未阐明。瞬时受体电位通道阳离子亚型6（TRPC6）被认为是参与缺氧性平滑肌细胞收缩的重要的Ca2+通道之一。本课题组发现缺氧增加了内皮细胞EETs的产量，而EETs是瞬时电位受体通道香草酸亚型4（TRPV4）的激动剂，在预实验中我们观察到抑制TRPV4能够减弱HPV反应，抑制TRPV4同时也阻断了TRPC6定位于细胞膜穴样内陷（caveolae）的过程，因此我们推测缺氧时内皮细胞释放的EETs激活平滑肌细胞TRPV4,与TRPC6形成复合物使之定位于caveolae,引起Ca2+内流。为证实这一假设，我们将复制小鼠急性和慢性HPV模型，从器官-细胞水平分别观察TRPV4对HPV的作用及其信号传递通路。本课题将从全新角度阐述HPV发生机制，为防治肺动脉高压提供新靶点。

缺氧性肺动脉收缩(HPV)是肺血管特有的保护性生理反应，但是持续的肺动脉收缩会导致肺动脉高压，甚至右心衰竭。HPV确切的机制尚未阐明。.我们通过小鼠原位肺灌注模型，观察到HPV反应在瞬时受体电势香草素亚型4 (TRPV4) 缺失小鼠和正常小鼠使用TRPV4 抑制剂GSK219组中明显减弱。通过运用活体显微镜技术，我们直接观察到正常小鼠肺小动脉在低氧情况下发生了收缩，而TRPV4缺失小鼠肺小动脉低氧下血管收缩反应减弱。我们通过造成小鼠局部缺氧的状态，监测动脉血气分析，发现在TRPV4缺失小鼠中发生了更严重的低氧血症，证明TRPV4缺失小鼠低氧下肺血流重新分配的能力下降。我们进一步研究发现在小鼠原位肺灌注模型中，caveolin-1 抑制剂、PI3K 抑制剂能够明显抑制HPV反应。通过Western blot技术，我们证明caveolin-1在人肺动脉平滑肌细胞中有表达。然后通过caveolae提取技术和Western blot技术，我们发现低氧刺激caveolae上瞬时受体电势阳离子亚型6 (TRPC6)表达明显上升，通过免疫共沉淀技术观察到低氧条件下TRPV4和TRPC6在caveolae上形成复合物增加。TRPV4 抑制剂、Cavolin-1 抑制剂和PI3K 抑制剂均明显减弱了低氧引起的TRPC6表达增加，而11-12EET能够增加TRPC6表达。我们还建立小鼠慢性缺氧模型，发现注射了caveolin-1抗体小鼠能够拮抗低氧引起的肺动脉高压，肺动脉肌化和右心室肥厚。综上结果，我们推测缺氧时内皮细胞释放的 EETs 激活平滑肌细胞 TRPV4，与TRPC6 形成复合物使之定位于 caveolae,引起 Ca2+内流，继而引起平滑肌细胞收缩，引起HPV反应。我们从器官-细胞水平研究了TRPV4 对 HPV 的作用及其信号传递通路，提出了新的HPV 发生机制，有望为防治肺动脉高压提供新的治疗靶点。