The pulmonary vascular resistance in the fetus undergoes a rapid decrease at birth to facilitate gas exchange during postnatal life. Alteration in this adaptation leads to Persistent Pulmonary Hypertension of Newborn (PPHN), a condition associated with increased morbidity and mortality. The mechanism of this altered adaptation in PPHN remains unknown. Previous studies have shown that pulmonary vasodilation at birth is facilitated by release of ATP from fetal RBC and stimulation of nitric oxide (NO) release from endothelial cells by ATP. Association of Hsp90, a stress protein, with endothelial NOS (eNOS) facilitates release of NO in response to physiological stimuli. Inhibition of Hsp90-NOS interaction appears to uncouple NOS activity from release of NO to superoxide (O2""""""""), a vasoconstrictor. The proposed studies will investigate the hypotheses that (i) association of Hsp90 with eNOS facilitates NO release and vasodilation when normal fetal pulmonary vessels are exposed to ATP, (ii) dissociation of Hsp90 from eNOS during increased pressure load in PPHN shifts the balance of NOS activity from NO to O2- and (iii) O2 impairs No independent vasodilation by inhibition of K channels on vascular smooth muscle. The hypotheses will be investigated in fetal lambs with pulmonary hypertension induced by constriction of ductus arteriosus, an established model of PPHN, and in control lambs with sham ligation of ductus arteriosus. Studies will be done in pulmonary arteries isolated from control and PPHN lambs to determine the role of Hsp90-NOS interaction and Kv channels in normal relaxation response to ATP and the role of uncoupled NOS activity in the impaired response in PPHN Studies in endothelial cells from control and PPHN lambs will determine the effect of ATP on (a) balance of NO and O2- release from eNOS, (b) Hsp90 - eNOS association and (c) serine-1177-phosphorylation of eNOS, a marker of its activation. These studies will (1) delineate the role of Hsp90-eNOS association in stimulation of NO release by ATP and (2) determine if decreased Hsp90-eNOS association results in generation of O2 when NOS is activated by ATP. The proposed studies will also address two potential mechanisms for decreased Hsp90-eNOS association in PPHN: (a) decreased tyrosine phosphorylation of Hsp90 due to nitrotyrosine formation and (b) recruitment of Hsp90 to cytoskeleton proteins, actin and a-tubulin to preserve their integrity during increased pressure load. These studies will provide new information on mechanisms of impaired adaptation in PPHN.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Human Embryology and Development Subcommittee 1 (HED)
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Berberich, Mary Anne
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Medical College of Wisconsin
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