The purpose of this proposal is to prepare myself for a career as an independent cell and molecular biology researcher in pulmonary diseases with a focus on the mechanism of lung fibrosis. The hypothesis to be tested directly in this proposal is that human lung fibroblast proliferation is mediated in vitro by platelet-derived growth factor (PDGF) A-chain through an autocrine proliferative mechanism. Furthermore, I postulate that this PDGF-A chain autocrine proliferative mechanism is an integral component of developing fibrogenic pulmonary disease. I have previously identified asbestos-induced PDGF-dependent autocrine fibroblast proliferation in rat lung fibroblasts in vitro. Investigation into PDGF- dependent autocrine mitogenesis appears to have importance because many growth factors induce fibroblast proliferation through this PDGF-dependent mechanism in vitro. In this proposal I will first validate my preliminary data that human lung fibroblast also proliferate via a PDGF-dependent autocrine mechanism, using cell proliferation assays, Western and Northern analysis, radioreceptor assays. Antisense oligonucleotides will also be used to block the PDGF A-chain ligand and thus, confirm the presence of human autocrine PDGF-A dependent proliferation in vitro. Next, I will establish whether the receptor and ligand components of the PDGF-dependent autocrine loop are present in lung mesenchymal cells during the process of scar formation in vivo using our morphologically characterized murine model of asbestos-induced lung fibrosis, histochemical immunostaining and in situ hybridization. Then, I will manipulate the autocrine PDGF-dependent fibroblast proliferation at the level of the PDGF alpha receptor. Initially, I will study the effects of a 50% reduction in the PDGF alpha receptor by comparing fibroblast proliferation in response to asbestos and growth factors in vitro, and lung scar development following aerosolized asbestos exposure in vivo , between normal C-57 and C-57 """"""""patch"""""""" mice (""""""""patch"""""""" mice express 50% fewer PDGF alpha receptors in smooth muscle cells than normal C-57 mice). Finally, I will confirm the significance of the PDGF-dependent autocrine fibroblast proliferation in vitro using a dominant negative PDGF alpha receptor mutant. The proposed experiments will enhance our understanding of the role of PDGF in fibroproliferative lung disease.
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