PMN-Endothelial Cross-Talk via TRPM2 and Lung Vascular Injury

项目1的中心目标是确定新的氧化剂敏感的瞬时受体潜在的Melastafin(TRPM)2的性质，它是肺内皮细胞(ECs)中的一种钙渗透通道，它如何调节Ca(2+)信号，以及它在中性粒细胞依赖的肺血管通透性增加和炎症损伤机制中的作用。我们的方法将是确定中性粒细胞-内皮细胞相互作用在激活TRPM2通道中的关键作用，然后确定其激活机制，最后确定TRPM2激活如何导致肺内皮细胞通透性增加和中性粒细胞在黏附连接水平上的迁移。目的1验证一种假说，即PMN通过β2整合素/ICAM-1结合与肺内皮细胞相互作用，通过激活内皮细胞内TRPM2通道来增加肺血管通透性。目的#2将定义TRPM2的短剪接变体TRPM2-S的作用。在调节TRPM2介导的肺内皮细胞钙内流以及内皮高通透性和中性粒细胞迁移机制中发挥重要作用。目的#3研究核因子-kappaB依赖的ICAM-1表达在内皮细胞TRPM2活性增强中的作用，从而在介导PMN依赖的肺炎症损伤中的作用。拟议的研究将使用分子、基因组学。与中性粒细胞共培养的EC单层的生理机制 和小鼠肺模型(包括最近培育的TRPM2(-/-)小鼠)。这些数据将为急性肺损伤的机制，特别是TRPM2激活的介导肺损伤的途径提供新的见解。此外，我们相信，随着对这种跨细胞串扰的新理解，将有可能通过干扰TRPM2激活的信号通路来阻断不适当的中性粒细胞-EC相互作用和PMN介导的肺损伤。

The central objective of Project 1 is to define the properties of the novel, oxidant-sensitive transient receptor potenfial melastafin (TRPM)2, a Ca(2+)-permeable channel in lung endothelial cells (ECs), how it regulates Ca(2+) signaling, and its role in the mechanism of neutrophil-dependent increases in lung vascular permeability and infiammatory injury. Our approach will be to identify the essenfial role of neutrophil-EC interactions in activafing the TRPM2 channel, then its mechanism of activation, and finally define how TRPM2 activation leads to increased lung endothelial permeability and transmigration of PMNs at the level of adherens juncfions. Aim #1 will test the hypothesis that PMN interaction with the lung endothelium via beta2-integrin/ICAM-1 binding increases lung vascular permeability through the activation of TRPM2 channels in ECs. Aim #2 will define the role of the short splice variant of TRPM2, TRPM2-S. in regulafing TRPM2-mediated Ca2+ entry in lung ECs and in the mechanism of endothelial hyper-permeability and PMN transmigration. Aim #3 will determine the role of NF-kappaB-dependent ICAM-1 expression in amplifying TRPM2 acfivity in ECs and thereby in mediating PMN-dependent lung infiammatory injury. The proposed studies will use molecular, genefic. and physiological approaches in EC monolayers co-cultured with PMNs and mouse lung models (including the recentiy developed TRPM2(-/-) mice). These data will provide new insights into the mechanisms of acute lung injury and specifically theTRPM2-activated pathways that mediate lung injury. Furthermore, we believe that it will be possible, with a new understanding of this transcellular cross-talk, to block inappropriate neutrophil-EC interacfions and PMN-mediated lung injury by interfering with TRPM2-activated signaling pathways.