TRPV4 in Regulation of Lung Endothelial Permeability

描述(由申请人提供)：我们认为急性肺损伤的范例很可能以独特的内皮损伤“指纹”为特征，基于它们所针对的钙离子进入途径和这些通道蛋白的表达模式。研究结果表明，瞬时受体电位(TRP)蛋白的典型亚家族成员包括钙库操作的钙通道亚基，参与肺泡外血管内皮细胞通透性的钙离子依赖性调节。我们观察到，心力衰竭导致对储备耗竭的通透性反应丧失，但对14，15-环氧二十碳三烯酸(14，15-EET)的反应不会，14，15-环氧二十碳三烯酸(14，15-EET)是一种仅在肺泡间隔毛细血管促进钙离子进入依赖性急性肺损伤的脂质，表明14，15-EET靶向于间隔内皮细胞表达的一个独特的通道。我们的初步数据提示了一个新的候选基因--TRPV4--Trp蛋白香草子家族的成员，它受到内源性大麻素、花生四烯酸、ETS、热和机械扰动的不同调节。我们的假设是，肺泡间隔内皮细胞表达的TRPV4通道的调节整合了依赖钙离子进入的通透性对各种刺激的反应，包括内源性大麻素、EETs、高血管压、低张和热。为了解决我们的假设，我们设计了两个具体的目标。目的1将确定TRPV4是否是内源性大麻素、EETs、机械扰动、低张和热促进肺泡间隔内皮细胞通透性增加所需的钙内流的共同靶点。目的2揭示急性慢性心力衰竭是否由于保留了对TRPV4通道激活的反应性而失去了对储备耗竭的反应性而导致对急性肺损伤的选择性易感性。我们将通过对完整的肺内皮细胞的通透性和钙离子进入的特定测量，肺和血管腐蚀管型的显微镜来绘制通透性反应的空间异质性，并使用药物工具来操纵与TRPV4的门控有关的信号通路来解决这些目标。SiRNA下调大鼠肺中TRPV4的影响将与TRPV4-/小鼠的结果以及慢性心力衰竭中的结果进行比较，在慢性心力衰竭中，仅商店操作的Trp通道似乎下调。这项工作将首次对与完整肺内皮细胞通透性的分区调节有关的分区TRPV4通道表达进行严格的分析。

DESCRIPTION (provided by applicant): We propose that acute lung injury paradigms are likely to be characterized by unique endothelial injury "finger prints" based on the Ca2+ entry pathways they target and the expression pattern of these channel proteins. The consensus of data suggests that members of the canonical subfamily of transient receptor potential (TRP) proteins comprise subunits of store-operated Ca2+ channels and participate in Ca2+ entry-dependent regulation of lung endothelial permeability in extra-alveolar vessels. Our observation that heart failure leads to loss of the permeability response to store depletion but not that to 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid that promotes Ca2+ entry-dependent acute lung injury only in alveolar septal capillaries, suggests that 14,15-EET targets a distinct channel expressed in septal endothelium. Our preliminary data suggests a novel candidate -TRPV4-a member of the vanilloid subfamily of TRP proteins that is diversely regulated by endocannabinoids, arachidonic acid, EETs, heat, and mechanical perturbation. Our hypothesis is that regulation of TRPV4 channels expressed in alveolar septal endothelium integrates the Ca2+- entry-dependent permeability response to diverse stimuli, including endocannabinoids, EETs, high vascular pressure, hypotonicity, and heat. To address our hypothesis, we have devised 2 specific aims. AIM 1 will determine whether TRPV4 is a common target by which endocannabinoids, EETs, mechanical perturbation, hypotonicity and heat promote Ca2+ influx required for increased endothelial permeability in the alveolar septal compartment of the lung. AIM 2 will reveal whether acute chronic heart failure leads to a selective susceptibility to acute lung injury due to retention of responsiveness to activation of TRPV4 channels but loss of responsiveness to store depletion. We will address these aims using specific measures of permeability and Ca2 + entry in the intact lung endothelium, microscopy of lung and vascular corrosion casts to map spatial heterogeneity in the permeability response, and pharmacological tools to manipulate signaling pathways implicated in gating of TRPV4. The impact of TRPV4 down-regulation in rat lung by siRNA will be compared to outcomes in TRPV4-/ mice, and to that in chronic heart failure where store-operated TRP channels alone appear to be down regulated. This work will provide the first rigorous analysis of compartmentalized TRPV4 channel expression linked to compartmentalized regulation of endothelial permeability in the intact lung.