The purpose of this proposal is to prepare the candidate for a career as an independent cell and molecular biology researcher in pulmonary diseases, with an emphasis on the mechanisms of lung fibrosis. The candidate is an Assistant Professor in the Section of Pulmonary Diseases and Critical Care Medicine at Tulane University Medical Center, with 80% of his time protected for research. Over the past two years he has been successful in developing expertise in cell biology methods. With the support of this K08 award, he will be able to add a working understanding of molecular biology methods necessary for exploring the pathobiology of lung fibrosis. He will gain needed experience in the disciplines of molecular biology under the guidance of established molecular biologists and pulmonary research scientists in the Tulane Lung Biology Research Program and the Tulane Molecular Biology Program. The long-term goal of this proposal is to evaluate the role that tumor necrosis factor alpha and the induction of apoptosis play in the pathogenesis of pulmonary fibrosis. He proposes to study a well-characterized murine model of Bleomycin-induced lung fibrosis consisting of a BLM-sensitive and a BLM-resistant strain in which exposure to Bleomycin (BLM) results in increased expression of lung cytokines and induction of apoptosis in alveolar macrophages. His overall hypothesis is that the upregulated expression of TNF and the Bleomycin induction of apoptosis in the lung contributes to the development of lung injury and fibrosis. To quantify TNF and TNF MRNA expression in the lungs of BLM-sensitive and BLM-resistant mice, he will study, by immunoblotting, immunohistochemistry and Northern analysis, the magnitude of the changes in the TNF and TNF MRNA observed in the lungs of mice during BLM-induced pulmonary fibrosis. Furthermore, he will determine whether or not TNF contributes to the fibrogenic effects of BLM in the lungs by exposing to BLM transgenic animals with altered or reduced TNF receptor expression. He has obtained 3 lines of transgenic mice: ablated TNF receptor expression; only TNFRI(55kDa); only TNFR2 (75kDa). These animals provide the means to determine the biological consequences of TNF expression in BLM-exposed animals. To determine the role of apoptosis in the pathogenesis of pulmonary fibrosis, he will study the apoptotic process by DNA, TUNEL and morphologic analysis in the lungs and alveolar macrophages of normal (BLM-sensitive and -resistant) and transgenic (TNF receptor and p53 knockout) mice. These studies should provide insight into the role that these mediators have in the pathogenesis of pulmonary fibrosis.
