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）是一种由出生时肺血管舒张功能失败引起的疾病。受影响的婴儿是低氧血症，死亡率和长期残疾的风险增加。在产前导管收缩诱导的PPHN胎羊模型中，研究表明肺动脉NO释放减少，氧化应激增加。NADPH氧化酶的激活和内皮一氧化氮合酶（eNOS）的解偶联活性是PPHN中肺动脉超氧化物（O2-）的来源。线粒体耗氧是血管细胞生成氧气的重要来源。出生时O2可用性和氧化磷酸化的增加可能导致线粒体中活性氧（ROS）的增加。然而，线粒体ROS对PPHN氧化应激的作用尚不清楚。在正常胎羊肺动脉内皮细胞（PAEC）中，暴露于ATP（一种NOS激动剂）和产后氧张力刺激eNOS与线粒体外膜蛋白、孔蛋白的关联。在这个位置的靶向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。锰超氧化物歧化酶（MnSOD）在PPHN中的表达也降低。我们提出了一种新的假设，即eNOS对线粒体外膜的靶向减少和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-反过来在出生时损害肺血管舒张。拟议研究的广泛具体目的是：(1)研究PPHN中eNOS-线粒体相互作用和MnSOD表达的改变及其对PAEC出生后过渡期间O2消耗、NO和O2-水平的影响；(2)研究PPHN中eNOS靶向线粒体改变的机制；(3)研究线粒体氧化应激在PPHN出生相关过渡期间肺血管舒张和氧合受损中的作用。研究将在产前结扎动脉导管（PPHN）羔羊的PAEC和肺动脉中进行，并在假结扎对照组中进行。研究还将在足月分娩的有或没有PPHN的完整胎儿羔羊中进行，以研究线粒体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中氧化应激的重要新来源。这些观察结果可能导致新的靶向治疗来改善PPHN的血管舒张和氧合。

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.