The long term objectives of this project are to determine the mechanisms by which myofibroblasts arise de novo in lung injury and fibrosis, and to characterize the phenotype and roles of these cells in pulmonary fibrosis. There is ample evidence that the myofibroblast is a key source of the extracellular matrix whose deposition in fibrotic lesions is a hallmark of pulmonary fibrosis. The presence of fibrotic foci, which are comprised of myofibroblasts and other fibroblast phenotypes, is now thought to be diagnostic of idiopathic pulmonary fibrosis and found to be most associated with increased mortality. Hence understanding how these cells arise and the mechanisms regulating their survival may provide novel insight into the pathogenesis of pulmonary fibrosis. A number of mediators are known to induce myofibroblast differentiation, including TGFbeta, IL-4, IL-13 and endothelin. Since TGFbeta is well-characterized as a key cytokine in driving fibrosis, and in view of its potent effects in inducing myofibroblast differentiation, the central hypothesis of the proposal is that TGFbeta induced/dependent transcription factors, and their cognate elements in the alpha-smooth muscle actin promoter are important in myofibroblast differentiation. Additionally since expression of alpha-smooth muscle actin correlates with heightened TGFbeta and procollagen I gene expression, but with decreased cell proliferation, a secondary hypothesis proposes that the myofibroblast is a terminally differentiated cell, whose survival may be a key factor in progressive fibrosis instead of resolution of lung injury and normal healing. To test these hypotheses, four specific aims are proposed. First, the ability of various lung cells to induce myofibroblast differentiation will be studied and the responsible mediators identified. Second, the transcriptional regulation of the (-smooth muscle actin promoter by TGFbeta1 will be examined, and relevant trans acting factors and cis elements will be identified. Third, key fibrotic phenotypic characteristics of the lung myofibroblast will be identified and its proliferative capacity specifically analyzed with respect to terminal differentiation and other fibrotic phenotypic characteristics. The role of alpha-smooth muscle actin in inducing this fibrotic phenotype will be identified by examining the effects of specific inhibitors of its expression and transfection with a gene construct into cells not usually known to express this actin isoform. Finally, the roles of TGFbeta and NO in selective (vis-a-vis fibroblasts) myofibroblast apoptosis will be studied and the effects of NOS inhibition will be examined in vivo in a model of bleomycin-induced lung injury and fibrosis.
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