Current evidence suggests that acute lung injury is secondary to an imbalance between reactive oxygen and nitrogen species and antioxidant defense mechanisms. Traditionally, studies aimed at augmenting the latter have focused on primary antioxidants. More recently, such therapeutic strategies have expanded to include manipulation of stress genes. Constitutive expression of these molecules is low, but is readily induced in response to a variety of stimuli, resulting in resistance to a broad spectrum of etiologies of cellular injury and organ dysfunction. In the previous funding period, we showed that one such gene, metallothionein (MT), protects cultured mammalian cells against the cytotoxic and DNA-damaging effects of reactive nitrogen and oxygen species. In the current proposal, we will investigate the functional role of MT as a component of pulmonary endothelial cell defense, in vivo, and determine the mechanism by which MT performs these functions in cultured murine lung endothelial cells (MLEC).
Specific Aims are to determine: I. the functional role of MT in lungs of mice exposed to 100% oxygen. We will contrast the sensitivity to hyperoxic lung injury of wildtype mice to: a) MT homozygous null mutants; b) mice after liposome-mediated somatic gene transfer of plasmid containing human MT-IIA cDNA (driven by murine CMV) to the pulmonary microcirculation; and c) transgenic mice that express MT in their vascular endothelium under the control of human von Willebrand factor promoter. II. the role of NO in hyperoxic lung injury by contrasting the pulmonary response to hyperoxia in inducible nitric oxide synthase (NOS-II) knockout mice or in mice that overexpress NOS-II in their pulmonary microcirculation after liposome-mediated gene transfer. III. the biochemical mechanisms by which MT reduces the toxicity of reactive oxygen and nitrogen species in cultured mouse lung endothelial cells.
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