Regulation of pulmonary circulation in fetus and newborn

描述(由申请人提供)：新生儿持续性肺动脉高压(PPHN)是一种由于出生时肺血管扩张失败而导致的疾病。受影响的婴儿患有低氧血症，死亡和长期残疾的风险增加。对胎羊PPHN模型的研究表明，由产前导管狭窄引起的PPHN在肺动脉中释放的NO减少，氧化应激增加。NADPH氧化酶的激活和内皮型一氧化氮合酶(ENOS)的解偶联活性是PPHN肺动脉超氧化物歧化(O2-)的来源。线粒体耗氧量是血管细胞产生O2-的重要来源。出生时氧气利用率和氧化磷酸化的增加可能会导致线粒体中活性氧物种(ROS)的增加。然而，线粒体ROS在PPHN氧化应激中的作用尚不清楚。暴露于一氧化氮合酶激动剂三磷酸腺苷和出生后氧分压可刺激正常胎羊肺动脉内皮细胞(PAEC)内eNOS与线粒体外膜蛋白孔蛋白的结合。此部位的靶向NO释放调节氧化磷酸化的速率，以减少正常胎儿PAEC中O2-的产生。在PPHN中，锰超氧化物歧化酶(MnSOD)的表达也降低。我们提出了一个新的假设，即eNOS减少对线粒体外膜的靶向，MnSOD的表达减少导致线粒体O2-的过量产生和减少猝灭。线粒体O2-反过来又会损害出生时的肺血管扩张。本研究的主要目的是(1)研究PPHN中eNOS-线粒体相互作用和MnSOD表达的变化，及其对PPHN出生后过渡过程中O2消耗、NO和O2-水平的影响；(2)探讨PPHN中eNOS靶向线粒体的机制；(3)探讨线粒体氧化应激在PPHN出生相关过渡过程中肺血管扩张和氧合功能受损中的作用。研究将在产前结扎动脉导管(PPHN)的羔羊和假结扎对照组的PAEC和肺动脉中进行。研究人员还将在有或没有足月分娩PPHN的完整胎羊中进行研究，以探讨线粒体O2-在出生时肺循环和氧合转变中的作用。这些研究将确定PPHN中一个重要的新的氧化应激源。这些观察可能导致新的靶向治疗来改善PPHN的血管扩张和氧合。 与公共卫生相关：在出生时，流向肺的血流量增加，以帮助在出生后的生活中通过肺进行气体交换。这种适应的失败会导致新生儿持续的肺动脉高压(PPHN)，并伴随着严重的低氧血症，增加死亡和残疾的风险。拟议的研究将探索这种疾病的机制和潜在的新疗法。

DESCRIPTION (provided by applicant): Persistent pulmonary hypertension of the newborn (PPHN) is a condition that results from failure of pulmonary vasodilation to occur at birth. The affected infants are hypoxemic and have increased risks of mortality and long-term disabilities. Studies in a fetal lamb model of PPHN, induced by prenatal ductal constriction demonstrated a decrease in NO release and increase in oxidative stress in pulmonary arteries. Activation of NADPH oxidase and uncoupled activity of endothelial nitric oxide synthase (eNOS) are sources of superoxide (O2-) in the pulmonary arteries in PPHN. Mitochondrial oxygen consumption is an important source of O2- generation in vascular cells. Increase in O2 availability and oxidative phosphorylation at birth may lead to increased reactive oxygen species (ROS) in mitochondria. However, the contribution of mitochondrial ROS to oxidative stress in PPHN is unknown. Exposure to ATP, a NOS agonist, and postnatal oxygen tension stimulate the association of eNOS with the mitochondrial outer membrane protein, porin in normal fetal lamb pulmonary artery endothelial cells (PAEC). Targeted NO release in this location regulates the rate of oxidative phosphorylation to decrease O2- production in normal fetal PAEC. The expression of manganese superoxide dismutase (MnSOD) is also decreased in PPHN. We propose to investigate the novel hypothesis that decreased targeting of eNOS to mitochondrial outer membrane and decreased expression of MnSOD lead to excess generation and decreased quenching of mitochondrial O2-. The mitochondrial O2- in turn impairs pulmonary vasodilation at birth. The broad specific aims of the proposed studies are to (1) Investigate the alterations in eNOS-mitochondrial interactions and MnSOD expression in PPHN and its effect on O2 consumption, NO and O2- levels during postnatal transition of PAEC, (2) Investigate the mechanism of altered eNOS targeting to mitochondria in PPHN and (3) investigate the role of mitochondrial oxidative stress in the impaired pulmonary vasodilation and oxygenation during birth-related transition in PPHN. Studies will be done in PAEC and pulmonary arteries harvested from lambs with prenatal ligation of ductus arteriosus (PPHN) and in sham ligation controls. Studies will be also done in intact fetal lambs with or without PPHN delivered at term to investigate the role of mitochondrial O2- in the transition of pulmonary circulation and oxygenation at birth. These studies will identify an important new source of oxidative stress in PPHN. These observation may lead to new targeted therapies to improve vasodilation and oxygenation in PPHN. PUBLIC HEALTH RELEVANCE: An increase in blood flow to the lung occurs at birth to help establish gas exchange by the lung during postnatal life. Failure of this adaptation results in persistent pulmonary hypertension in the newborn infant (PPHN), associated with severe hypoxemia and increased risk of death and disability. The proposed studies will investigate the mechanisms and potential new therapies for this disease.