The goal of this proposal is to explore the contribution of the enzyme xanthine oxidoreductase (XOR), via its ability to generate reactive oxygen species (ROS), to the profound vascular leakage that characterizes ventilator-associated lung injury (VALI). Our laboratory has demonstrated transcriptional up-regulation of XOR by stimuli prevalent in VALI (e.g., hypoxia, endotoxin and cytokines) and has implicated this enzyme in the development of pulmonary capillary permeability in acute lung injury. Recently, we also demonstrated post-translational modification of XOR related to phosphorylation of the protein by the p38 Mitogen Activated Protein kinase (MAP kinase) pathway. We have further demonstrated that activation of the Rho Kinase/Myosin Light Chain Kinase (MLCK) pathway and downstream effectors of the p38 MAP kinase (MAPK-activated protein kinase or MK2 and HSP27) are linked to profound re-arrangement of the actin cytoskeleton. This proposal will utilize a blend of cell biologic, molecular, biochemical, and proteomic approaches to explore the role of mechanical stress in the transcriptional regulation and post-translational modification of XOR in oxidant-mediated VALI, the role of novel XOR signaling in activation of the Rho kinase/MLCK and p38 MAP kinase pathways, and their contribution to cytoskeletal rearrangement, endothelial barrier dysfunction, and vascular leakage.
Specific Aim 1 will determine the contribution of XOR to increased permeability and oxidant injury in VALI. Using cultured PMEC we will define the mechanisms of XOR activation in response to cyclic stretch, and the contribution of XOR to endothelial barrier dysfunction. Using a mouse model of VALI, we will determine the mechanisms of upregulation of XOR in vivo and define the contribution of XOR to pulmonary capillary permeability by altering XOR expression.
Specific Aim 2 will focus on the mechanisms and sites of XOR posttranslational modification (i.e., phosphorylation) and their contribution to cyclic stretch-induced barrier dysfunction.
In Specific Aim 3, we will define novel XOR-mediated signaling in response to cyclic stretch, focusing specifically on two key regulatory pathways, the MLCK/Rho kinase and p38 MAP kinase pathways, which produce profound cytoskeletal rearrangement and barrier dysfunction and are potential targets of oxidative stress by XOR. We speculate that this project will demonstrate the specific role of XOR and XOR-derived ROS in the pathogenesis of VALI, and identify novel signaling pathways linked to barrier dysfunction in this syndrome, which may lead to targeted therapeutic approaches. ? ?
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