PULMONARY ENDOTHELIAL CELL TRANSCYTOSIS AND ACUTE LUNG INJURY AFTER HEMORRHAGIC

肺内皮是功能和结构变化的早期位点，其有助于急性肺损伤（AL 1）的发生和/或维持。我们将确定运输和关键炎症调节大分子[如S-亚硝基白蛋白（SNO-AIb）和髓过氧化物酶（MPO）]的血管作用的机制，这些大分子在失血性休克（HS）复苏后在循环中升高。因此，具体目标是： 目标1。明确SNO-AIb在培养的肺内皮细胞中转胞吞的生理机制。我们将使用真实的时间多模式成像（共聚焦和多光子扫描激光显微镜和全内反射荧光显微镜，FRET）来定义与血管细胞结合，胞吞转运和SNO-AIb介导的信号转导通过S-亚硝化和/或鸟苷酸环化酶的激活相关的分子事件。目标2。揭示活的完整肺内皮中MPO转胞吞的生理机制目标#3。探讨MPO影响肺血管细胞和完整肺中SNO-白蛋白信号的生化机制。揭示髓过氧化物酶依赖的细胞内和细胞外调节的中枢通路 调节血管NO信号，我们将研究MPO对SNO-白蛋白的作用 在日益整合的生物系统中的转移和分解动力学，包括：a）体外无细胞模型; B）大鼠肺微血管内皮细胞（RLMVEC）;和c）大鼠肺微血管平滑肌细胞和RLMVEC共培养系统。具体目标#4将SNO-白蛋白转运、氧化还原状态和MPO的病理生理作用置于从HS复苏后的AL 1的背景中。我们将对比ALl（渗透性）的程度， 指数、中性粒细胞积聚、形态学变化、肺血管调节）。

Pulmonary endothelium is an early locus of functional and structural changes that contribute to the genesis and/or maintenance of acute lung injury(ALl). We will define the mechanisms of transport and the vascular actions of key inflammatory-modulatory macromolecules [e.g. S-nitroso-albumin (SNO-AIb) and myeloperoxidase (MPO)] that are elevated in the circulation after resuscitation from hemorrhagic shock (HS). Accordingly specific aims are to: Aim #1. Define the physiological mechanism of transcytosis of SNO-AIb in cultured pulmonary endothelium. We will use real time multimode imaging (confocal and multiphoton scanning laser microscopy and total internal reflection fluorescence microscopy, FRET) to define molecular events associated with vascular cell binding, transcytosis and SNO-AIb mediatied signaling via S-nitrosation and/or activation of guanylyl cyclase. Aim #2. Reveal the physiological mechanisms of MPO transcytosis in live intact pulmonary endothelium Aim #3. Explore the biochemical mechanisms by which MPO affects SNO-albumin signaling in pulmonary vascular cells and intact lung. To reveal the central pathways for intra- and extracellular regulatory MPO-dependent modulation of vascular NO signaling, we will examine the actions of MPO on SNO-albumin transfer and decomposition kinetics in increasingly integrated biological systems including: a) cell free models in vitro; b) rat lung microvacular endothelial cells (RLMVEC); and c) rat lung microvasccular smooth muscle cell and RLMVEC co-culture system. Specific Aim #4. Place the pathophysiological contributions of SNO-albumin transport, redox status and MPO into the context of ALl after resuscitation from HS. We will contrast the extent of ALl (permeability index, neutrophil accumulation, morphological changes,pulmonary vasoregulation) in isolated perfused lungs and intact mice of wildtype and caveolin-1 and MPO homozygous null mutants.