The role of lysophosphatidic acid signaling in lung ischemia/reperfusion injury

溶血磷脂酸信号在肺缺血/再灌注损伤中的作用

基本信息

批准号：
9070355
负责人：
Jose P Vazquez Medina
金额：
$ 5.8万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-15 至 2017-05-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9070355
关键词：
4 hydroxynonenal Address Agonist Alveolar Macrophages Apoptosis Biochemical Pathway Biological Assay Blood flow Cell membrane Cells Dyes Edema Endothelial Cells Endothelium F2-Isoprostanes Fluorescence Fluorescent Dyes Functional disorder Generations Goals Hilar In Situ In Situ Nick-End Labeling In Vitro Inflammation Injury Interruption Intervention Ischemia Knock-in Knock-in Mouse Knock-out Knockout Mice Knowledge Ligation Light Lipid Peroxidation Lung Lung Transplantation Lysophosphatidic Acid Receptors Lysophosphatidylcholines Lysophospholipids Measures Morbidity - disease rate Mus NADPH Oxidase Neutrophil Infiltration Nonesterified Fatty Acids Oral Oxidants Oxidases Patients Peroxidases Phospholipase A2 Phospholipase D Prevention Production Proteins Pulmonary vessels Reactive Oxygen Species Receptor Signaling Regulation Reperfusion Injury Reperfusion Therapy Role Signal Transduction Staining method Stains Technology Therapeutic Tissues Transcription Factor AP-1 Wild Type Mouse Work acetovanillone cytokine feeding flotillin in vitro Model in vivo inflammatory marker inhibitor/antagonist insight lung injury lung ischemia lysophosphatidic acid macrophage mortality mouse model neutrophil oxidative damage peroxiredoxin protein biomarkers public health relevance research study small hairpin RNA vzg-1 Receptor

项目摘要

DESCRIPTION (provided by applicant): Lung ischemia/reperfusion injury (LIRI) is the primary cause of mortality and morbidity in patients undergoing lung transplantation. Increased reactive oxygen species (ROS) generation is a central component of LIRI. NADPH oxidase 2 (Nox2) activation leads to increased ROS generation during lung ischemia/reperfusion. New findings show that peroxiredoxin 6 (Prdx6) is required for Nox2 activation. Prdx6 is a bifunctional protein that expresses peroxidase and phospholipase A2 (PLA2) activities. While the peroxidase activity scavenges ROS, the PLA2 activity activates Nox2 during agonist stimulation and ischemia/reperfusion in lung endothelium, alveolar macrophages and polymorphonuclear neutrophils (PMN). Although the requirement of Prdx6PLA2 for Nox2 activation is established, the mechanism of such activation remains elusive. The main products of Prdx6PLA2 are a lysophospholipid and a free fatty acid. Our preliminary studies show that lysophosphatidic acid (LPA) receptor blockade decreases Nox2-dependent ROS generation during agonist stimulation in pulmonary microvascular endothelial cells (PMVEC) and isolated perfused lungs, and that LPA treatment "recovers" ROS generation in PMVEC from Prdx6 null mice. Therefore, we will study the role of Prdx6PLA2- derived LPA signaling in Nox2 activation during lung ischemia/reperfusion and the contributions of LPA signaling to LIRI. Our central hypothesis is that blocking LPA signaling ameliorates LIRI. This hypothesis will be addressed through two specific aims. Aim 1 will evaluate the requirement of Prdx6-derived LPA signaling for Nox2 activation during ischemia/reperfusion using an in vitro model of flow-adapted PMVEC. Activation will be determined by the translocation of cytosolic components of Nox2 to the plasma membrane and ROS generation. Aim 2 will evaluate the contributions of LPA signaling to LIRI using in situ and in vivo murine models. ROS generation, lipid peroxidation, inflammation, apoptosis, neutrophil infiltration and edema will be measured as indicators of LIRI. The use of an in vitro model, knockin and knockout cells, pharmacological inhibitors and shRNA technology will allow us to elucidate the mechanism of LPA-driven ROS generation during ischemia/reperfusion. The use of isolated perfused lungs will help understanding the dynamics of LPA- driven intravascular ROS generation and oxidative damage, and the contributions of LPA signaling to lung ischemia/reperfusion. The use of an in vivo hilar clamp murine model will give a global perspective that could shade light on the contributions of other cells (PMN, macrophages) to LIRI, and the potential therapeutic value of LPA receptor blockers in the prevention of LIRI. Thus, the proposed work will provide valuable new information related to Nox2 activation during lung ischemia/reperfusion and therefore can offer an opportunity to influence the activation of the oxidase. Understanding the regulation of Nox2 activation will provide important insights for our understanding of the mechanisms for LIRI and could open the door to pharmacologic intervention in order to limit Nox2-driven ROS generation during LIRI.

描述（由应用提供）：肺部缺血/再灌注损伤（LIRI）是接受肺移植的患者死亡率和发病率的主要原因。活性氧（ROS）产生增加是LIRI的核心组成部分。 NADPH氧化物2（NOX2）激活导致肺部缺血/再灌注期间ROS产生增加。新发现表明，NOX2激活需要过氧蛋白6（PRDX6）。 PRDX6是一种双功能蛋白，表达过氧化物酶和磷脂酶A2（PLA2）活性。在过氧化物酶活性清除ROS的同时，PLA2活性在激动剂刺激和肺部内皮，肺泡巨噬细胞和多形性嗜中性粒细胞（PMN）中激活NOX2。尽管建立了PRDX6PLA2对NOX2激活的要求，但这种激活的机制仍然难以捉摸。 PRDX6PLA2的主要产物是溶物磷脂和游离脂肪酸。我们的初步研究表明，在肺微血管内皮细胞（PMVEC）和孤立的灌注肺部激动剂刺激过程中，溶物磷酸二酸（LPA）受体阻滞可降低NOX2依赖性ROS的产生，而LPA治疗”和LPA治疗“从PMVEC中恢复” PMVEC从PRDX6 NULL MICE中恢复了ROS。因此，我们将研究肺部缺血/再灌注期间NOX2激活中PRDX6PLA2衍生的LPA信号的作用，以及LPA信号对LIRI的贡献。我们的中心假设是阻断LPA信号会改善LIRI。该假设将通过两个具体目标解决。 AIM 1将使用流动适应PMVEC的体外模型评估PRDX6衍生的LPA信号传导对缺血/再灌注期间NOX2激活的需求。激活将由NOX2的胞质成分转移到质膜和ROS产生。 AIM 2将使用原位和体内鼠模型评估LPA信号对LIRI的贡献。 ROS的产生，脂质过氧化，炎症，凋亡，中性粒细胞浸润和水肿将作为LIRI的指标进行测量。使用体外模型，敲除和基因敲除细胞，药物抑制剂和SHRNA技术将使我们能够阐明缺血/再灌注期间LPA驱动的ROS产生的机理。使用孤立的灌注肺部将有助于了解LPA驱动的血管内ROS产生和氧化损伤的动力学，以及LPA信号对肺部缺血/再灌注的贡献。使用体内肺钳鼠模型将提供一个全局视角，可以使其他细胞（PMN，巨噬细胞）对LIRI的贡献以及LPA接收器阻滞剂在预防LIRI中的潜在治疗价值。这是拟议的工作将提供与肺部缺血/再灌注期间NOX2激活有关的有价值的新信息，因此可以提供机会影响氧化酶的激活。了解NOX2激活的调节将为我们理解LIRI机制提供重要的见解，并可以为药物干预打开大门，以限制LIRI期间NOX2驱动的ROS的产生。