The Effect of cGMP on Pulmonary Endothelial Apoptosis

描述(申请人提供)：急性肺损伤(ALI)的特征是肺内皮细胞通透性增加和由活性氧(ROS)和损伤性细胞因子引发的肺水肿。内皮功能障碍包括细胞旁对水和蛋白质的通透性增加，以及内皮细胞因坏死和凋亡而死亡的增加。细胞凋亡是细胞程序性死亡的一种形式，具有明显的生化和形态特征。越来越多的证据表明，内皮细胞凋亡是急性肺损伤的主要组成部分，但对肺内皮细胞凋亡的调控仍知之甚少。在一些系统内皮细胞中的研究发现，信号分子环鸟苷一磷酸(CGMP)是一种促生存、抗凋亡的因子，而在其他细胞中，cGMP则触发细胞凋亡。CGMP是由内皮细胞可溶性鸟苷酸环化酶(SGC)在一氧化氮(NO)刺激下产生的。CGMP通过激活蛋白激酶G(PKG)调节肺内皮细胞通透性，但对肺内皮细胞凋亡的影响尚不清楚。这一信息很重要，因为肺NO通过周期性的呼吸机拉伸而增加，ALI和吸入NO的炎症被用来改善氧合。这项拨款申请的总体目标是确定cGMP对肺内皮细胞凋亡的影响。我们的初步数据表明，人和小鼠肺内皮细胞cGMP浓度的增加显著减弱了ROS引发的细胞凋亡。在目的1中，将在人和小鼠肺微血管内皮细胞中检测调节内皮cGMP浓度对ROS诱导的细胞死亡的影响。通过给予膜途径类似物、用BAY 41-2772直接刺激sGC和5%周期牵张的生理性sGC刺激，内皮细胞单层cGMP将增加；cGMP的耗竭将通过sGC抑制完成。目的2探讨cGMP抗细胞凋亡的分子机制。PKG的作用将通过人的肺动脉内皮细胞和来自野生型和PKG基因敲除小鼠的肺微血管细胞来确定。将探索下游机制，包括可能上调促生存基因硫氧还蛋白(TRX)和血红素加氧酶1(HO-1)。急性肺损伤是危重病人发生肺功能衰竭的常见原因。在这一过程中，组织损伤的一部分包括可能导致肺功能障碍的程序性细胞死亡过程。了解这种现象在急性肺损伤中是如何调节的，可能会为这种严重疾病带来新的治疗方法。

DESCRIPTION (provided by applicant): Acute lung injury (ALI) is characterized by increased pulmonary endothelial permeability and pulmonary edema triggered by reactive oxygen species (ROS) and injurious cytokines. Endothelial dysfunction includes increased paracellular permeability to water and protein as well as an increase in endothelial cell death by both necrosis and apoptosis. Apoptosis is a form of programmed cell death with distinct biochemical and morphological features. Increasing evidence has implicated endothelial apoptosis as a major component of acute lung injury but the regulation of pulmonary endothelial apoptosis remains poorly understood. Studies in some systemic endothelial cells have identified the signaling molecule cyclic guanosine monophosphate (cGMP) as a pro-survival, anti-apoptotic factor whereas in other cells cGMP triggers apoptosis. cGMP is generated by endothelial soluble guanylyl cyclase (sGC) after stimulation with nitric oxide (NO). cGMP modulates pulmonary endothelial permeability through activation of protein kinase G (PKG) but any effects on pulmonary endothelial apoptosis are unknown. This information is important because lung NO is increased by cyclic ventilatory stretch and the inflammation in ALI and inhaled NO is administered to improve oxygenation. The overall goal of this grant application is to determine the effect of cGMP on pulmonary endothelial apoptosis. Our preliminary data suggest that increases in human and mouse pulmonary endothelial cGMP concentration significantly attenuate ROS-triggered apoptosis. In Aim 1, the effect of modulating endothelial cGMP concentration on ROS-induced cell death will be examined in human and mouse lung microvascular endothelial cells. cGMP will be increased in endothelial monolayers by administration of membrane-permeant analogues, direct stimulation of sGC with Bay 41-2772, and physiologic sGC stimulation from 5% cyclic stretch; depletion of cGMP will be accomplished with sGC inhibition. In Aim 2, the molecular mechanisms of the anti-apoptotic effects of cGMP will be explored. The role of PKG will be determined using human pulmonary artery endothelial cells and pulmonary microvascular cells from wild-type and PKG knock-out mice. Downstream mechanisms will be explored, including possible upregulation of the pro-survival genes thioredoxin (Trx) and heme oxygenase 1 (HO-1). Acute lung injury is a common cause of lung failure in critically ill patients. Part of the tissue injury in this process includes a process of programmed cell death that may contribute to lung dysfunction. An understanding of how this phenomenon is regulated in acute lung injury may lead to new therapies for this severe illness.