cGMP Compartmentalization in Pulmonary Endothelial Barrier Dysfunction

描述（由申请人提供）：肺内皮屏障失效导致肺水肿和急性肺损伤（All）。机械通气通过使肺过度通气伸展（VS），引起呼吸机性肺损伤（VILI），从而导致所有死亡。在VILI动物模型中，肺内皮屏障功能障碍与内源性一氧化氮（NO）生成增加有关。NO激活内皮可溶性鸟苷环化酶（sGC），产生环鸟苷单磷酸（cGMP）。内皮cGMP对vs诱导的内皮屏障功能的影响尚不清楚。我们在小鼠VILI模型中的初步实验表明，VS激活内皮细胞sGC以增加cGMP。VS发作后，sGC抑制或刺激分别减轻或加重屏障功能障碍，提示cGMP具有损伤作用。相反，VS之前的sGC激活具有屏障保护作用。通过非sGC机制（颗粒guanyyl环化酶，pGC）增加内皮细胞cGMP的产生对内皮屏障具有保护作用，与VS的发病时间无关。这些数据和其他数据表明，sGC介导的cGMP合成的细胞内区隔化被VS改变，从而改变cGMP信号传导对屏障功能的影响，可能是通过sGC从细胞质溶胶转位到腔泡膜结构域。本应用的目的是确定cGMP对VILI的影响，并特别关注cGMP区隔化。在Aim 1中，sgc产生的cGMP对内皮屏障功能的影响将在离体小鼠肺灌注制剂中进一步表征。将确定sGC激活对cGMP产生和内皮屏障功能障碍的剂量反应和特异性。此外，vs诱导的从cGMP保护到危害转换的时间将被确定。在Aim 2中，肺VS对内皮细胞sGC亚细胞区隔化的影响将通过共聚焦显微镜和亚细胞分离在完整的小鼠肺和循环拉伸的内皮细胞中确定。sGC-小泡相互作用的屏障作用将在离体灌注小泡蛋白-1 -/-小鼠肺中测定。vs诱导的sGC与磷酸二酯酶2 （cgmp介导的内皮屏障功能障碍的关键效应物）的相互作用将通过特异性抑制剂、免疫沉淀和cAMP测量来探索。相关性：感染或外伤引起的急性肺损伤导致肺衰竭，因为血管内皮细胞允许液体渗漏到空气中。机械通气支持是至关重要的，但肺组织的过度扩张可通过内皮分子（如cGMP）的改变加重损伤。了解cGMP如何恶化内皮细胞渗漏有助于更好地治疗急性肺损伤。

DESCRIPTION (provided by applicant): Failure of the lung endothelial barrier leads to pulmonary edema and acute lung injury (All). Mechanical ventilation contributes to all mortality by subjecting the lung to excessive ventilatory stretch (VS), causing ventilator induced lung injury (VILI). In animal models of VILI, pulmonary endothelial barrier dysfunction is associated with increased endogenous nitric oxide (NO) production. NO activates endothelial soluble guanyl cyclase (sGC), producing cyclic guanosine monophosphate (cGMP). The effect of endothelial cGMP on VS-induced endothelial barrier function is unclear. Our preliminary experiments in a mouse model of VILI suggest that VS activates endothelial sGC to increase cGMP. After VS onset, sGC inhibition or stimulation attenuates or exacerbates barrier dysfunction, respectively, suggesting an injurious role for cGMP. In contrast, sGC activation before VS is barrier protective. Increasing endothelial cGMP production by non- sGC mechanisms (particulate guanylyl cyclase, pGC) is protective of the endothelial barrier, regardless of timing with the onset of VS. These and other data suggest an intracellular compartmentalization of sGC- mediated cGMP synthesis that is altered by VS to change the effect of cGMP signaling on barrier function, possibly by translocation of sGC from the cytosol to a caveolar membrane domain. The goal of this application is to determine the effect of cGMP on VILI with a specific focus on cGMP compartmentalization. In Aim 1, the effect of sGC-produced cGMP on endothelial barrier function will be further characterized in an isolated perfused mouse lung preparation. The dose-response and specificity of sGC activation on cGMP production and endothelial barrier dysfunction will be determined. In addition, the timing of the VS-induced switch from cGMP protection to harm will be determined. In Aim 2, the effect of lung VS on the subcellular compartmentalization of endothelial sGC will be determined in both intact mouse lungs and cyclically- stretched endothelial cells by confocal microscopy and subcellular fractionation. The barrier effect of sGC- caveolae interaction will be determined in isolated, perfused caveolin-1 -/-mouse lungs. VS-induced interactions of sGC with phosphodiesterase 2, a key effector of cGMP-mediated endothelial barrier dysfunction, will be explored using specific inhibitors, immunoprecipitation, and cAMP measuremnts. Relevance: Acute lung injury from infection or trauma causes lung failure because the endothelial cells lining blood vessels allow fluid to leak into the air spaces. Mechanical ventilatory support is vital, but over- distension of lung tissue can worsen injury through changes in endothelial molecules such as cGMP. Understanding how cGMP worsens endothelial leakiness could lead to better therapies of acute lung injury.