We wish to identify molecular mechanisms leading to the development of acute lung injury (ALI). This devastating clinical syndrome is characterized by formation of pulmonary edema--alveolar flooding with a protein-rich exudate. Recently, our laboratory has identified the cytokine transforming growth factor-Beta (TGF-Beta) as a possible critical mediator in ALI; inhibition of TGF-Beta prevents the development of pulmonary edema in murine models. Other investigators have shown that TGF-Beta directly increases endothelial permeability in vitro. We have found that TGF-Beta directly increases epithelial permeability by a mechanism that is associated with depletion of intracellular glutathione (GSH). We hypothesize that locally activated TGF-Beta mediates the development of pulmonary edema associated with ALI by exerting specific effects on resident epithelial and endothelial cells. Furthermore, we hypothesize that TGF-Beta alters the phosphorylation state of endothelial and epithelial cell junction proteins by mechanisms involving changes in intracellular thiol redox status. Junction protein phosphorylation would affect junction integrity, and therefore, the degree of paracellular permeability. We will use TGF-Beta green fluorescent protein reporter mice to identify individual resident epithelial and endothelial cells that are stimulated by TGF-Beta in vivo. Epithelial and endothelial cell monolayers will be used to assess TGF-Beta effect on paracellular permeability. Reduced and oxidized GSH will be measured to determine thiol redox status. Epithelial and endothelial junction protein phosphorylation state will be determined and specific proteins identified. In contrast to most other potential treatments for ALT, which are effective only if administered before the onset of injury, TGF-Beta inhibition effectively attenuates ALT even after the onset of injury (previously shown in our laboratory). Therefore, identification of specific molecular mechanisms by which TGF-Beta affects epithelial and/or endothelial cells could lead to novel and improved strategies to treat ALI, a common and often lethal clinical syndrome. The proposed studies will also help me develop and acquire the necessary skills to achieve my ultimate goal of becoming a successful and independent scientist in pulmonary disease.
