The pathophysiology underlying neonatal pulmonary hypertension has received relatively little study compared to adult models, even though it may involve different mechanisms. Thus, the long-term goal is to improve the understanding of the pathogenesis of neonatal pulmonary hypertension so that more effective therapies can be developed. Almost all of the research regarding neonatal pulmonary hypertension has been directed at devising therapies to acutely decrease pulmonary vascular resistance. Therapies for preventing the onset or progression of neonatal pulmonary hypertension have received little attention and have largely involved manipulating the nitric oxide (NO) pathway. Yet, not all infants respond favorably to inhaled NO. Our preliminary data demonstrate that rather than a singular effect attributable to NO, derangements in the prostanoid system, which include prostacyclin (PGI2) and thromboxane (TXA2), are key to the pathogenesis of hypoxia-induced neonatal pulmonary hypertension. Our overall hypothesis is that distinct disruptions of the prostanoid signaling pathway occur with sustained hypoxia in endothelial cells and smooth muscle cells of small pulmonary arteries (SPA) and lead to inappropriate constriction of pulmonary resistance vessels and the development of pulmonary hypertension. At the core of our methodology is the use of SPA as they are the vascular site most relevant to the development of pulmonary hypertension. This proposal will investigate the following specific hypotheses: (a) the cyclooxygenase (COX) 1-PGI2 axis is disrupted in endothelial cells of SPA with sustained hypoxemia leading to decreased dilator prostanoid production; (b) the COX 2-TXA2 axis is enhanced in smooth muscle cells of SPA with sustained hypoxia so that the ratio of constrictor to dilator prostanoid production is increased; (c) the change in ratio of constrictor to dilator prostanoid production alters smooth muscle cell K+ conductance and membrane potential leading to inappropriate constriction and pulmonary hypertension.
Specific Aim 1 will (a) determine the amounts, cellular sources and relative contributions of COX 1 and COX 2 biosynthetic pathways to PGI2 and TXA2 production using enzyme immunoassay (EIA) and cannulated artery techniques; (b) evaluate the effect of PGI2 and TXA2 on SPA tone (cannulated artery technique); (c) establish the predominant cellular localization of the major prostanoid enzymes (immunostaining technique) and (d) determine the role of various K+ channels in mediating vascular responses to PGI2 (microelectrode technique).
Specific Aim 2 will (a) determine whether the amounts and/or predominant cellular sources of production of PGI2 and TXA2 are altered in SPA from piglets exposed to chronic hypoxia (EIA and cannulated artery techniques); (b) evaluate whether the sensitivity to PGI2 and TXA2 has been altered by chronic hypoxia (cannulated artery technique); (c) determine whether mRNA levels for the major enzymes (RNase protection assay), protein abundance of the major enzymes (immunoblot technique) or the cellular localization of the major enzymes (immunostaining) underlying PGI2 and TXA2 production are affected by chronic hypoxia; (d) evaluate whether the effect from K+ channels in mediating dilation and constriction from PGI2 and TXA2 is involved with the development of pulmonary hypertension (microelectrode technique). These studies should provide important information for improving treatment of infants with pulmonary hypertension.
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