The importance of myofibroblasts (as defined by alpha [a] smooth muscle actin [ASMA] expression), in pulmonary fibrosis is suggested by their presence in sites of active fibrosis, and their disappearance of fibrosis subsides. Progress during the current granting period further demonstrates that these cells are the primary source of interstitial collagen during the active fibrosis phase of bleomycin-induced lung injury and fibrosis, and that they are also important source of inflammatory and fibrogenic cytokine (e.g. transforming growth factor B1 [TGFB1] and monocyte chemoattractant protein-1 [MCP-1] expression, especially at more chronic phases when the inflammatory cells become less prominent as a source of these same cytokines. These studies and preliminary data also uncovered novel findings which suggest that the myofibroblast can undergo apoptosis with a suggestion that is more susceptible to induction of apoptosis than the fibroblast from which it is derived. These studies further show increases in apoptotic cells at a time point when active fibrosis subsides, accompanied by reduction in TGFB1 and ASMA expression, as well as myofibroblast numbers. These observations, plus the ability of TGFB1 to up-regulate expression of ASMA, provide the basis for the central hypothesis of this renewal project. Myofibroblasts emerge from fibroblasts under the influence of enhanced lung TGFB1 expression is reduced when active fibrosis subsides, resulting in their subsequent disappearance is reduced when active fibrosis subsides, resulting in their subsequent disappearance. To test this hypothesis, 5 specific aims are proposed: a) to ascertain the relationship between ASMA expression and cytokine and collagen expression, b) to use antisense approaches to suppress ASMA expression and examine the impact on cytokine/collagen expression and apoptosis, c) to determine if the myofibroblast is terminally differentiated, d) to examine the mechanism of interleukin-1B (IL-1B) induces apoptosis in myofibroblasts by identification of the apoptotic pathway proteins involved and to see if this is unique to IL-1B or can be extended to other inhibitors of ASMA expression as well, e) to confirm the in vivo significance of the in vitro findings in d), using the bleomycin model of lung injury and fibrosis. This combined in vitro and in vivo approach using immunological, biochemical, cellular and molecular biological techniques, are expected to provide novel insights into the genesis and disappearance of myofibroblasts, and their significance to pulmonary fibrosis.
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