The Effect of cGMP on Pulmonary Endothelial Apoptosis

cGMP对肺内皮细胞凋亡的影响

• 批准号: 7332394
• 负责人: Robert Scott Stephens
• 金额: $ 5.29万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7332394
• 关键词: Acute Lung Injury; Agonist; Antioxidants; Apoptosis; Apoptotic; Applications Grants; Attenuated; Biochemical; Caspase; Cell Death; Cells; Critical Illness; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Data; Endothelial Cells; Endothelium; Environmental air flow; Event; Failure; Family member; Functional disorder; Gene Expression Regulation; Gene Transfer; Genes; Goals; Human; Hydrogen Peroxide; In Vitro; Inflammation; Inhibition of Apoptosis; Injury; Ischemia; Knockout Mice; Lead; Lung; Mediating; Membrane; Membrane Potentials; Mitochondria; Molecular; Motion; Mus; Necrosis; Nitric Oxide; Nuclear; Oxidation-Reduction; Pathway interactions; Patients; Permeability; Phosphorylation; Physical condensation; Physiological; Process; Production; Propidium Diiodide; Protein-Serine-Threonine Kinases; Proteins; Pulmonary Edema; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Role; Series; Serine Protease; Signal Transduction; Signaling Molecule; Simulate; Soluble Guanylate Cyclase; Staining method; Stains; Stretching; Testing; Thinking; Thioredoxin; Time; Tissues; Transcriptional Activation; Transcriptional Regulation; Up-Regulation; Water; analog; apoptotic protease-activating factor 1; base; caspase-3; caspase-9; cytochrome c; cytokine; deprivation; genetic regulatory protein; heme oxygenase-1; improved; in vivo Model; inhaled nitric oxide; inhibitor/antagonist; lung ischemia; mitochondrial membrane; monolayer; novel; prevent; pro-apoptotic protein; pro-caspase-9; protective effect; pulmonary artery endothelial cell; receptor; transcription factor

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.

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内皮细胞坏死和凋亡导致的死亡增加。细胞凋亡是细胞程序性死亡的一种形式，具有独特的生化和形态学特征。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仍知之甚少。在一些系统性内皮细胞的研究中，已经发现信号分子环鸟苷单磷酸（cGMP）是一种促生存、抗凋亡因子，而在其他细胞中，cGMP会引发细胞凋亡。cGMP是由内皮可溶性鸟酰环化酶（sGC）在一氧化氮（NO）刺激后产生的。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影响尚不清楚。这一信息很重要，因为肺NO通过循环通气伸展而增加，ALI中的炎症和吸入NO可以改善氧合。本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cGMP浓度的增加可显著减弱ros引发的细胞凋亡。在Aim 1中，我们将在人和小鼠肺微血管内皮细胞中检测调节内皮细胞cGMP浓度对ros诱导的细胞死亡的影响。通过膜渗透类似物、Bay 41-2772直接刺激sGC和5%循环拉伸生理性刺激sGC，内皮单层细胞中的cGMP增加；cGMP的消耗将通过抑制sGC来完成。在Aim 2中，我们将探讨cGMP抗凋亡作用的分子机制。将利用野生型和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，包括促生存基因硫氧还蛋白（Trx）和血红素加氧酶1 （HO-1）的可能上调。急性肺损伤是危重患者肺功能衰竭的常见原因。这一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。