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

项目1的中心目标是确定新型氧化剂敏感性瞬时受体电位melastafin（TRPM）2（肺内皮细胞（EC）中的Ca（2+）渗透通道）的特性，它如何调节Ca（2+）信号传导，以及它在肺血管通透性和炎症损伤中的嗜中性粒细胞依赖性增加机制中的作用。我们的方法将是确定在激活TRPM 2通道中的嗜中性粒细胞-EC相互作用的本质作用，然后其激活机制，并最终确定TRPM 2激活如何导致肺内皮通透性增加和粘附连接水平的PMN的迁移。目的#1将检验以下假设：PMN通过β 2-整联蛋白/ICAM-1结合与肺内皮的相互作用通过激活EC中的TRPM 2通道增加肺血管通透性。目标#2将定义TRPM 2的短剪接变体TRPM 2-S的作用。在调节TRPM 2介导的肺ECs内Ca ~（2+）内流以及内皮细胞通透性增高和PMN迁移机制中的作用。目的#3将确定NF-κ B依赖性ICAM-1表达在增强EC中TRPM 2活性中的作用，从而介导PMN依赖性肺炎症损伤。拟议的研究将使用分子，遗传。与中性粒细胞共培养的EC单层的生理学方法 和小鼠肺模型（包括最近开发的TRPM 2（-/-）小鼠）。这些数据将为急性肺损伤的机制提供新的见解，特别是TRPM 2激活的介导肺损伤的途径。此外，我们相信，这将是可能的，与这种跨细胞的串扰的新的理解，阻止不适当的嗜中性白细胞-EC interacfions和PMN介导的肺损伤，通过干扰TRPM 2激活的信号通路。

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.