The ultimate objective of this research proposal is to understand how endogenously produced (autocrine) versus exogenously generated (paracrine) growth factors regulate pulmonary smooth muscle in health and disease. Smooth muscle hypertrophy and/or hyperplasia often occurs during the repair of lung injury in response to pulmonary diseases such as bronchopulmonary dysplasia (BPD), persistent pulmonary hypertension in newborns (PPH) and idiopathic pulmonary hypertension. However, the precise mechanisms regulating pulmonary smooth muscle cell (SMC) growth and differentiation in these disease states remains unclear.
The first aim of this project is to determine the role of autocrine/paracrine interactions in mediating pulmonary smooth muscle growth and differentiation. The role of TGFbeta1 or bFGF in lung smooth muscle cell differentiation and gene expression will be assessed in both developing and oxygen injured lungs. We will make use of trangenic models kindly provided by Dr. Thomas Doetschman to determine whether the lack of or the over expression of TGF-beta1 or bFGF alter pulmonary smooth muscle development/function or are involved in the pathogenesis and severity of lung injury following acute or chronic hyperoxia. We also will direct the over expression of TGFbeta1 and bFGF to smooth muscle in transgenic mice using sequences within the smooth muscle gamma actin (SMGA) and smooth muscle alpha actin (SMAA) gene to assess the role of endogenous TGFbeta1 and bFGF. Since SMGA is a smooth muscle specific protein, emphasis will be placed on elucidating the regulation of the SMGA gene during lung morphogenesis and repair after oxygen injury. Selected portions of the SMGA gene will be linked to chloramphenicol acetyl transferase (CAT) and the activity of these chimeric genes will be tested in transgenic mice and cultured pulmonary smooth muscle cells to localize those sequences within the SMGA gene that influence its expression in different smooth muscles, especially those associated with the airway and pulmonary vasculature. The function of putative regulatory sequences will be assessed further using site directed mutagenesis and testing in vitro and in vivo. Fine mapping of the cis- acting regulatory elements will be done by gel shift assays and nuclease protection. Our third goal is to determine the role of bFGF and TGFbeta1 and oxygen in SMGA gene expression in vitro. We hypothesize that TGFbeta1 is a positive regulator of SMGA gene transcription and while bFGF and hyperoxia are negative regulators of SMGA gene transcription and that these effects are mediated through specific cis-acting elements. Overall, this line of study should lead to a better understanding of the pathogenesis of disorders of pulmonary smooth muscle associated with pulmonary diseases which involve the proliferation and dedifferentiation of smooth muscle cells (SMCs).
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