TRPV4 in Regulation of Lung Endothelial Permeability

描述（由申请人提供）：我们提出急性肺损伤范式可能具有独特的内皮损伤“指纹”特征，这是基于它们靶向的Ca2+进入途径和这些通道蛋白的表达模式。数据一致表明，瞬时受体电位（TRP）蛋白的典型亚家族成员包括储存操作的Ca2+通道亚基，并参与Ca2+进入依赖肺泡外血管肺内皮通透性的调节。我们观察到心力衰竭导致对储存库耗竭的通透性反应丧失，而不是对14,15-环氧二碳三烯酸（14,15- eet）的通透性反应丧失，14,15- eet是一种脂质，仅在肺泡间隔毛细血管中促进Ca2+进入依赖性急性肺损伤，这表明14,15- eet靶向间隔内皮中表达的独特通道。我们的初步数据表明了一种新的候选者- trpv4 -TRP蛋白的香草亚家族成员，受内源性大麻素、花生四烯酸、EETs、热量和机械扰动的不同调节。我们的假设是，肺泡间隔内皮中表达的TRPV4通道的调节整合了Ca2+进入依赖性通透性对各种刺激的反应，包括内源性大麻素、EETs、高血管压力、低张力和高温。为了证明我们的假设，我们设计了两个具体的目标。AIM 1将确定TRPV4是否是内源性大麻素、EETs、机械扰动、低张力和热促进Ca2+内流的共同靶标，而Ca2+内流是增加肺泡间隔室内皮通透性所必需的。AIM 2将揭示急性慢性心力衰竭是否由于对TRPV4通道激活的反应性保留而对储存耗尽的反应性丧失而导致对急性肺损伤的选择性易感性。我们将使用完整肺内皮通透性和Ca2 +进入的具体测量，肺和血管腐蚀铸型显微镜来绘制通透性反应的空间异质性，以及药理学工具来操纵与TRPV4门控有关的信号通路来解决这些目标。通过siRNA下调大鼠肺TRPV4的影响将与TRPV4-/小鼠的结果进行比较，并与慢性心力衰竭的结果进行比较，后者单独的储存操作TRP通道似乎下调。这项工作将首次提供与完整肺内皮通透性的区隔调节相关的区隔化TRPV4通道表达的严格分析。

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.