Excessive smooth muscle cell (PASMC) proliferation causes abnormal pulmonary artery remodeling and hypertension in newborns with pulmonary hypertension and in infants and children with many forms of congenital heart disease. NO donors decrease mitogen-stimulated SMC proliferation in vitro. Recently inhaled nitric oxide (NO) treatment has been observed to decrease pulmonary artery cell proliferation in animals with vascular injury. Although studies suggest that NO-signaling decreases SMC proliferation via activation of cGMP-dependent protein kinase (PKG), the mechanism is incompletely understood. The BROAD, LONG-TERM OBJECTIVE of this proposal is to elucidate molecular mechanisms by which PKG activation inhibits PASMC proliferation.
Specific aim 1 examines how PKG activation modulates the cell cycle progression of proliferating PASMC and identifies specific PKG-sensitive cell cycle regulators. Using flow cytometry, studies of [3H]thymidine-incorporation into DNA, and assays of the activity and / or expression of cell cycle regulatory proteins, the antiproliferative mechanisms of PKG activation will be investigated in serum-stimulated PASMC.
Specific aim 2 tests whether PKG activation modulates cascades in the mitogen-activated protein kinase (MAPK) signaling pathway. Using PASMC expressing PKG, proliferation assays, and specific inhibitors of MAPK signaling cascades, the effect of PKG signaling on the ERK, SAPK/JNK and p38-signaling will be determined.
Specific aim 3 identifies specific PKG- phosphorylation targets through which PKG decreases PASMC proliferation. For example, should PKG activation modulate the MAPK signaling cascade, the abundance and enzymatic activity of constituents of this pathway will be evaluated using PKG-expressing proliferating PASMC. These results will provide important insights into basic mechanisms of abnormal cell proliferation which is pathognomatic for pulmonary vascular disease. This award will permit the applicant to take advantage of an ideal research training environment for the acquisition of new knowledge and skills in cell and molecular biology. In addition, a carefully constructed training program has been developed that will permit successful development of an independent research career examining the basic mechanisms of pulmonary vascular diseases.
