Tumor Necrosis Factor-Alpha (TNF) is an inflammatory mediator implicated in the pathogenesis of sepsis syndrome and Adult Respiratory Distress Syndrome (ARDS). We have demonstrated that TNF induces edema mediated by neutrophils (PMN) in lungs isolated from guinea pigs pretreated with TNF. Our preliminary data suggest that the edema is mediated by Platelet Activating Factor (PAF), Thromboxane A2 (TxA2), Reactive Oxygen Species (ROS) and the activation of Protein Kinase C (PKC). The central hypothesis of the proposal is that TNF induces a sequence of mediators which result in pulmonary edema. TNF induces endothelial PAF, which may cause (i) endothelial PKC activation, (ii) lung TxA2 generation, (iii) PMN production of reactive oxygen species (ROS), and (iv) PMN production of additional PAF. Endothelial PKC activation may be induced directly by TNF through activation of phospholipase C (PLC), and may also be mediated indirectly by PAF. Endothelial PKC activation may lead to PAF release and cytoskeletal abnormalities with associated increases in vascular permeability. The increased permeability, in conjunction with the TxA2-mediated increase in capillary pressure, results in pulmonary edema. The isolated perfused guinea pig and rat lung will be used to study the changes in pulmonary hemodynamics (capillary filtration coefficient, vascular compliance, arterial and capillary pressures), lung permeability to albumin, lung weight gain, PMN uptake, in situ actin morphology and releases of mediators (PAF, TxA2, ROS). The pulmonary arterial endothelial monolayer (PAEM) will be used to characterize the cellular mechanisms of TNF-induced pulmonary edema (PKC activation, PAF and TxA2 generation). The isolated lung and the PAEM are well-characterized; however, studies using PAEM will be limited to specific questions focused on endothelial function based on preliminary data and correlated with data obtained using the isolated lung. The studies proposed in this research plan will define the physiologic and biochemical mechanisms of TNF-induced pulmonary edema and will fill further the understanding of the mechanisms of inflammatory pulmonary edema and the vascular effects of TNF.
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