Regulation of pulmonary circulation in fetus and newborn

描述（由申请方提供）：胎儿的肺血管阻力在出生时迅速降低，以促进出生后的气体交换。这种适应的改变导致新生儿持续性肺动脉高压（PPHN），这是一种与发病率和死亡率增加相关的疾病。PPHN这种改变适应的机制尚不清楚。以往的研究表明，出生时肺血管扩张是由胎儿红细胞释放ATP和ATP刺激内皮细胞释放一氧化氮（NO）促进的。应激蛋白Hsp 90与内皮型一氧化氮合酶（eNOS）的结合促进了生理刺激后NO的释放。Hsp 90-NOS相互作用的抑制似乎将NOS活性从NO释放到超氧化物（O2-）（一种血管收缩剂）中解偶联。本研究将探讨以下假设：（i）当正常胎儿肺血管暴露于ATP时，Hsp 90与eNOS的结合促进NO释放和血管舒张;（ii）PPHN压力负荷增加时，Hsp 90与eNOS的解离将NOS活性的平衡从NO转移到O2-;（iii）O2通过抑制血管平滑肌上的K通道而损害NO独立的血管舒张。将在动脉导管缩窄诱导肺动脉高压的胎羊（一种已建立的PPHN模型）和假结扎动脉导管的对照羔羊中研究该假设。将在从对照和PPHN羔羊分离的肺动脉中进行研究，以确定Hsp 90-NOS相互作用和Kv通道在对ATP的正常舒张反应中的作用，以及未偶联的NOS活性在PPHN中受损反应中的作用。在来自对照和PPHN羔羊的内皮细胞中的研究将确定ATP对（a）eNOS的NO和O2-释放的平衡，（B）Hsp 90- eNOS结合和（c）eNOS的丝氨酸-1177-磷酸化，其活化的标志物。这些研究将（1）阐明Hsp 90-eNOS结合在ATP刺激NO释放中的作用，（2）确定当NOS被ATP激活时，Hsp 90-eNOS结合的减少是否导致O2的产生。所提出的研究还将解决PPHN中Hsp 90-eNOS缔合减少的两个潜在机制：（a）由于硝基酪氨酸形成而减少的Hsp 90的酪氨酸磷酸化和（B）Hsp 90向细胞骨架蛋白、肌动蛋白和α-微管蛋白的募集以在增加的压力负荷期间保持它们的完整性。这些研究将为PPHN适应障碍的机制提供新的信息。

DESCRIPTION (provided by applicant): 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.