Patients with persistent pulmonary hypertension of newborn exhibit pulmonary vascular smooth muscle (PVSM) cell hyperplasia, enhanced vascular tone, and increased accumulation of extracellular matrix (ECM) components in their lung vessels. This proposal is based on the hypothesis that in ECM may contribute to the etiology of this disease by regulating PVSM cell contractility and growth in response to soluble vasoregulators. This concept is based on the recent finding that ECM molecules control the ~set point~ of intracellular chemical signaling pathways (e.g., Na +/H + exchange, phosphoinositide turnover, cGMP levels) which are used by soluble vasoconstrictors (e.g., endothelin-1, PDGF) and vasorelaxants (e.g., nitric oxide) to alter PVSM cell contractility and growth. PVSM cells will be cultured in chemically- defined medium on dishes that are coated with different densities of purified ECM molecules (e.g., fibronectin, laminin, different collagen types, synthetic RGD-containing peptides) or ECM-coated microbeads to vary cell-ECM contact formation and integrin binding in a controlled manner. ECM-dependent control of vasomotor tone (cytoskeletal stiffness) in the presence or absence of different vasoconstrictors and vasodilators will be measured directly in cultured PVSM cells using magnetic twisting cytometry. Results of these studies will be compared and contrasted with effects on DNA synthesis and on intracellular signaling pathways (e.g., Na +/H + exchange, inositol lipid synthesis and breakdown, Ca2+ release, cGMP levels, myosin light chain kinase activation, myosin phosphorylation, protein tyrosine kinase activation). Anti-integrin antibodies and synthetic peptides will be used to map out specific paths of transmembrane signaling and to determine the importance of specific integrin receptors during control of vasoconstriction versus growth. Immunofluorescence microscopy will be used to identify changes in the distribution of signaling molecules and their recruitment to the site of integrin binding within the focal adhesion complex in response to varying cell-ECM contact formation and addition of vasoactive agents. Finally, a newly developed method for isolating biochemically active focal adhesion complexes will be used to study how signals transmitted by integrins and receptors for vasoconstrictors integrate inside the cell. Studies also will be initiated to explore the utility of integrin antagonists as inhibitors of pulmonary vascular remodeling in vivo. In summary, we hope to better understand how pulmonary hypertension may develop in response to neonatal injury and to develop new approaches for treatment of this disease by characterizing the mechanism by which ECM regulates signal transduction and hence, controls PVSM cell growth and contractile responses.
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