The overall objective of the proposed studies in this renewal application (yr. 13-17) is to address the critical signaling pathways by which pro- inflammatory cytokines such as TNFalpha mediate the expression of adhesion molecule, ICAM-1, in endothelial cells and thereby induce firm neutrophil (PMN) adhesion. We have shown an important function of oxidant signaling in endothelial cells in activating transcription of ICAM-1 and also a critical role of the NADPH oxidase complex in signaling ICAM-1 expression. In addition, we have identified signaling pathways involving PKC zeta and PI3 kinase/Akt that may activate oxidant signaling via NADPH oxidase. As stable and firm ICAM-1 dependent adhesion of PMN to endothelial cells will require rapid-onset protein synthesis independent of ICAM-1 expression as well as delayed protein synthesis-dependent ICAM-1 expression, we will explore both the early course of its expression involving cell surface alterations in the constitutive ICAM-1 in endothelial cells as well as delayed expression requiring de novo protein synthesis. In the proposed studies, we will determine (1) oxidant signaling of the gp91/phox and p41/phox NADPH oxidase subunits in mediating ICAM-1 expression and PMN adhesion to endothelial cells, (2) role of PKCzeta in activating oxidant signaling, and thereby in NF-kappaB activation and ICAM-1 expression, and finally (4) role of GTPases in oxidant signaling and mediating the early-onset protein synthesis-independent component of ICAM-1 expression and PMN adhesion. Studies will utilize molecular approaches to dissect the signaling pathways as well as physiological assessments of PMN sequestration and migration in lungs as well as pulmonary microvascular permeability and edema formation. With the completion of these studies, we will advance the understanding of the mechanisms by which TNFalpha induces endothelial cell ICAM-1 expression, and thereby mediates inappropriate PMN adhesion and migration across the pulmonary microvessel barrier. We hope to define the role of oxidant signaling and the signaling pathways in mediating ICAM-1 expression, and in thereby promoting endothelial adhesivity to PMN and their migration across the vessel wall. These studies will be important in providing a better understanding of the basis of inflammatory disease states such as the adult respiratory distress syndrome (ARDS) associated with increased PMN sequestration and migration so that agents can be developed to block specific signaling pathways.
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