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

 描述(由申请人提供)：肺缺血/再灌注损伤(LIRI)是肺移植患者死亡和发病的主要原因。增加的ROS生成是LIRI的一个重要组成部分。NADPH氧化酶2(NOX2)的激活导致肺缺血/再灌流时ROS生成增加。新的发现表明，NOX2的激活需要过氧化还蛋白6(Prdx6)。Prdx6是一种表达过氧化物酶和磷脂酶A2活性的双功能蛋白。在过氧化物酶活性清除ROS的同时，PLA2活性在激动剂刺激和缺血/再灌注时激活肺内皮细胞、肺泡巨噬细胞和中性粒细胞(PMN)中的NOX2。虽然Prdx6PLA2对NOX2激活的要求已经确定，但这种激活的机制仍然不清楚。Prdx6PLA2的主要产物是一种溶血磷脂和一种游离脂肪酸。我们的初步研究表明，溶血磷脂酸(LPA)受体阻断减少了在激动剂刺激下肺微血管内皮细胞(PMVEC)和离体灌流肺中依赖NOX2的ROS的产生，LPA治疗可恢复Prdx6基因缺失小鼠PMVEC中ROS的产生。因此，我们将研究Prdx6PLA2衍生的LPA信号在肺缺血/再灌注时NOX2活化中的作用以及LPA信号在LIRI中的作用。我们的中心假设是，阻断LPA信号可以改善LIRI。这一假设将通过两个具体目标加以阐述。目的1利用体外血流适应性PMVEC模型评价Prdx6来源的LPA信号在缺血/再灌注过程中对NOX2激活的需求。激活将由NOX2的胞液成分移位到质膜和ROS的产生来决定。目的2利用在体鼠模型和活体动物模型评价LPA信号对LIRI的作用。ROS生成、脂质过氧化、炎症、细胞凋亡、中性粒细胞浸润和水肿将作为LIRI的指标。使用体外模型、敲除和敲除细胞、药物抑制剂和shRNA技术将使我们能够阐明缺血/再灌注过程中LPA驱动的ROS产生的机制。使用隔离的灌流肺将有助于理解LPA驱动的血管内ROS产生和氧化损伤的动力学，以及LPA信号在肺缺血/再灌注中的作用。使用活体小鼠肝门钳夹模型将提供一个全球视角，可以揭示其他细胞(PMN、巨噬细胞)对LIRI的贡献，以及LPA受体阻滞剂在预防LIRI中的潜在治疗价值。因此，这项拟议的工作将提供与肺缺血/再灌流期间NOX2激活相关的有价值的新信息，从而可能为影响氧化酶的激活提供机会。了解NOX2激活的调控将为我们理解LIRI的机制提供重要的见解，并可能打开药物干预的大门，以限制LIRI过程中NOX2驱动的ROS的产生。

 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.