Pulmonary hypertension and pseumonectomy are examples of pulmonary conditions that implicate mechanical stress as a stimulus for lung cell proliferation and extracellular matrix production. In the present study, calcyclin gene expression is proposed to be an essential event in the mechanotransduction pathway signaling new cell division. More specifically, calcyclin is thought to function as a regulatory protein controlling changes in cell shape and cytoskeletal organization required for the cell cycle to progress. To test this hypothesis an isolated-perfused lung model of increased mechanical strain will be used to correlate the level and location of calcyclin and related lung S100 gene expression with pulmonary cell proliferation. Calcyclin and S100 gene expression will also be measured in fibroblasts stimulated to proliferate in response to mechanical skeletal organization and cell shape. Cytoskeletal involvement will be confirmed by measuring calcyclin levels in the cytoskeletal subcellular fraction and association with tropomyosin. Furthermore, transcriptional activation via cis-acting promoter elements and PDGF autocrine activity will be tested as possible regulatory control steps in this pathway. Pulmonary fibroblasts are also proposed to play an important role in strain-induced ECM remodeling. Therefore, 1) Total pro-collagen and tropoelastin synthesis will be measured in isolated perfused rat lungs ventilated at high or low states of lung inflation 2) The gene expression of a1 (I) pro-collagen and tropoelastin will be measured in mechanically strained fibroblasts cultured on ECM proteins (fibronectin elastin, laminin( 3) The role of cell shape and cytoskeletal organization will be analyzed by disruption of microfilaments, microtubules or intermediate filaments; estimating the number of ECM- cell contact sites; and identifying the abundance and specificity of ECM receptors (integrins) present on the cell surface. The functional role of specific ECM-fibroblast interactions will be determined by inhibition with fibronectin, laminin, or elastin peptides. 4) Finally, the ability of ECM peptide ligands to down regulate strain induced pro-collagen and tropoelastin expression will be tested in our mechanical strain lung model. This research will give further insight into possible strain induced proliferation and ECM remodeling observed in several physiological models currently under investigation in our integrative respiratory physiology division.
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