Phospholipase A2-mediated surfactant hydrolysis and dysfunction in ALI and ARDS

描述(申请人提供)：表面活性物质缺乏和损伤是急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)发病机制的重要组成部分。ALI/ARDS患者的支气管肺泡灌洗(BAL)样本分析显示多种异常，包括总表面活性物质磷脂下降，磷脂酰甘油(PG)下降不成比例。PG是通过与表面活性蛋白B(SP-B)相互作用来维持最佳表面活性的关键。分泌型磷脂酶A2(SPLA2)是肺表面活性物质损伤的重要介质，在ALI/ARDS时BAL液中sPLA2表达增加。PG耗竭是sPLA2介导的表面活性物质功能障碍的关键机制。在ARDS中，特异性sPLA2的鉴定和分泌调节机制尚不清楚。本研究将验证以下假设：ALI和ARDS炎症期释放的特异性分泌型磷脂酶A2能水解肺表面活性物质磷脂，降低PG，在限制内源性表面活性物质功能和急性呼吸衰竭的临床康复中起主要作用。这一假设将按如下方式进行检验： 具体目的1.检测肺泡磷脂酶A2和肺表面活性物质的异常。 ALI/ARDS的病程。BAL液体将从ALI/ARDS患者(n=80)病程早期获得。 将在呼吸衰竭的整个过程中获取呼吸衰竭和系列样本。将分析BAL的成分(细胞、表面活性物质颗粒和上清液)，并建立与ARDS病因、呼吸衰竭和ARDS预后的重要临床变量的关联。具体目的2.确定sPLA2表达和分泌的调控机制。将鉴定分泌到BAL中的sPLA2蛋白，并在体外测量细胞模型对sPLA2合成和释放的调节，以验证存在细胞因子驱动的刺激肺特定细胞分泌sPLA2的假设。目标1中BALS的细胞因子分析将被用于进一步指导这些研究。具体目的3.确定调节sPLA2介导的表面活性物质水解和功能障碍的机制。表面活性蛋白(SP)和肺泡磷脂形式(亚相中的大量聚集体和气液界面处的单分子膜)对sPLA2的水解、PG耗尽和表面活性物质功能障碍的影响将被检测。通过从AIM 1测量BAL中的SP水平，这些结果的体内相关性将得到加强。这项多中心、多学科的分析直接涉及NHLBI ALI研讨会的优先事项，以研究ALI的生化预测因素，并对开发涉及抑制sPLA2活性和表面活性替代的有效临床干预研究至关重要。

DESCRIPTION (provided by applicant): Surfactant deficiency and injury is an important component in the pathogenesis of acute lung injury (ALI)and acute respiratory distress syndrome (ARDS). Analysis of bronchoalveolar lavage (BAL) samples from ALI/ARDS patients reveal multiple abnormalities including decreased total surfactant phospholipid with a disproportionately greater decrease in phosphatidylglycerol (PG). PG is critical to maintain optimal surface activity through its interactions with surfactant protein B (SP-B). Secretory phospholipases A2 (sPLA2) are potent mediators of surfactant injury, and are increased in the BAL fluid in ALI/ARDS. Depletion of PG is a key mechanism in sPLA2-mediated surfactant dysfunction. Identification of specific sPLA2 and mechanisms regulating secretion are unknown in ARDS. This proposal will examine the hypothesis that specific secretory phospholipases A2 released during the inflammatory phase of ALI and ARDS hydrolyze pulmonary surfactant phospholipids, decrease PG and play a major role in limiting endogenous surfactant function and clinical recovery from acute respiratory failure. This hypothesis will be tested as follows: Specific Aim 1. Measure bronchoalveolar phospholipase A2 and surfactant abnormalities during the course of ALI/ARDS. BAL fluid will be obtained from ALI/ARDS patients (n = 80) early in the course of respiratory failure and serial samples will be obtained over the course of respiratory failure. Components of the BAL (cells, surfactant pellet and supernatant) will be analyzed and the associations with important clinical variables of ARDS etiology, respiratory failure and ARDS outcome will be established. Specific Aim 2. Determine the mechanisms regulating sPLA2 expression and secretion. sPLA2 proteins secreted into the BAL will be identified and the regulation of sPLA2 synthesis and release from cell models measured in vitro to test the hypothesis that there is a cytokine driven stimulation of sPLA2 secretion from specific cells in the lung. Cytokine analysis of the BALs in Aim 1 will be used to further direct these studies. Specific Aim 3. Determine the mechanisms that regulate sPLA2-mediated surfactant hydrolysis and dysfunction. The impact of the surfactant proteins (SP) and the alveolar phospholipid forms (bulk aggregates in the subphase and monomolecular films at the air-liquid interface) on sPLA2 hydrolysis, PG depletion and surfactant dysfunction will be examined. Interpretation of the in vivo relevance of these results will be enhanced by measuring SP levels in the BALs from Aim 1. This multicenter, multidisciplinary analysis directly addresses the NHLBI ALI Workshop priority to investigate biochemical predictors of ALI, and is essential to develop effective clinical intervention studies involving suppression of sPLA2 activity and surfactant replacement.