The Effect of cGMP on Pulmonary Endothelial Apoptosis

描述（由申请人提供）：急性肺损伤（ALI）的特征是由活性氧（ROS）和有害细胞因子引发的肺内皮通透性增加和肺水肿。内皮功能障碍包括细胞旁对水和蛋白质的渗透性增加以及坏死和凋亡引起的内皮细胞死亡增加。细胞凋亡是细胞程序性死亡的一种形式，具有独特的生物化学和形态学特征。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机制仍知之甚少。在一些系统性内皮细胞中的研究已经鉴定出信号分子环磷酸鸟苷（cGMP）作为促存活、抗凋亡因子，而在其他细胞中cGMP触发凋亡。cGMP由内皮可溶性鸟苷酸环化酶（sGC）在用一氧化氮（NO）刺激后产生。cGMP通过激活蛋白激酶G（PKG）调节肺内皮通透性，但对肺内皮细胞凋亡的任何影响尚不清楚。这一信息是重要的，因为肺NO通过循环性牵张增加，并且ALI中的炎症和吸入NO被施用以改善氧合。本基金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，人类和小鼠肺内皮cGMP浓度的增加显着减弱ROS触发的细胞凋亡。在目的1中，将在人和小鼠肺微血管内皮细胞中检查调节内皮cGMP浓度对ROS诱导的细胞死亡的影响。通过给予膜渗透类似物、Bay 41-2772直接刺激sGC和5%循环牵张的生理sGC刺激，内皮单层中的cGMP将增加; cGMP的消耗将通过sGC抑制完成。目的2探讨cGMP抗凋亡作用的分子机制。将使用来自野生型和PKG敲除小鼠的人肺动脉内皮细胞和肺微血管细胞来确定PKG的作用。下游机制将被探索，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）。急性肺损伤是危重患者肺衰竭的常见原因。该过程中的部分组织损伤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，可能会导致这种严重疾病的新疗法。

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.