The overall goal of these studies is to improve our understanding of the biochemical basis of the lung's response to acute injury. Toward this end, this proposal seeks to elucidate the mechanisms of regulation and function of genes that have been found to exhibit notable increases in their mRNA levels in the lung after exposure to hyperoxia and administration of oleic acid, as models of acute injury. The unifying concept underlying these studies is that there may be similarities in the mechanisms that activate these genes. The rationale is that once these mechanisms are understood they may then be controlled, allowing mitigation of the lung injury. The first Specific Aim is to compare the cell-specific accumulation of lung injury-related (LIR) mRNAs during the acute phase of hyperoxic lung injury in adult and newborn rabbits, and in rabbits treated intravenously with oleic acid. In situ hybridization and immunocytochemistry will be used to test the hypothesis that LIR mRNAs are expressed in a temporally-and spatially-specific manner in cells of the injured lung, depending on the extent of localized injury. The second Specific Aim is to isolate and characterize genomic clones for rabbit genes encoding the tissue inhibitor of metalloproteinases (TIMP) and metallothionein (MT), two LIR genes. The ends of their mRNAs will also be mapped. Comparisons of the putative regulatory portions of these genes will begin to test the hypothesis that there are shared and distinct mechanisms of their induction during lung injury. These data are also essential prerequisites to later studies of the mechanisms of their regulation. The third Specific Aim is to understand the function of metallothioneins in acute lung injury. Using stable and transient gene expression systems which are currently available, two hypothetical functions of MT will be tested. The fourth Specific Aim is to study the effects of oxygen, and polypeptide growth factors and cytokines as molecular mediators of TIMP and MT induction. The isolated, perfused, and ventilated lung model will be used as a bridge, to close the gap between previous in vivo studies and additional experiments of this aim, in cultured pulmonary endothelial cells and fibroblasts, to identify the molecular mediators of LIR gene induction. The fifth Specific Aim is to elucidate the molecular mechanisms underlying the increase in TIMP and MT mRNAs in pulmonary endothelial cells and fibroblasts, respectively, during acute lung injury. In vitro DNA-protein binding and reciprocal competition experiments will more directly test the hypothesis that there are unique and overlapping mechanisms of differential and coordinate LIR gene induction.
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