Oxidative stress in pulmonary circulation during birth related transition

描述(申请人提供)：胎儿的肺血管阻力(PVR)在出生时会急剧下降，以促进出生后的气体交换。出生时暴露在较高的氧分压下在这种适应中起着重要作用。内皮型一氧化氮合酶(ENOS)调节PVR的转化，直接受氧刺激，间接受氧化磷酸化和ATP释放增加的刺激。氧化磷酸化还会产生超氧化物(O27-)作为副产物。然而，在这一过渡过程中，调节胎肺动脉线粒体氧化应激的机制尚不清楚。出生时PVR未能降低会导致新生儿持续性肺动脉高压(PPHN)，这是一种与肺动脉氧化应激增加相关的情况。确定氧化应激源对于发展纠正PPHN血管功能障碍的特定治疗方法至关重要。对胎羊肺动脉内皮细胞(PAEC)的研究表明，暴露于一氧化氮合酶激动剂三磷酸腺苷或过渡到出生后的氧分压可刺激eNOS与线粒体外膜蛋白孔蛋白的联系。一氧化氮合酶激动剂可增加胎肺血管内皮细胞线粒体一氧化氮的释放。我们提出了一个新的假设，即在出生相关的过渡过程中，eNOS靶向线粒体外膜通过靶向释放NO来调节PAEC的氧化磷酸化和O27产生的速度。这些研究的主要目的是：(1)研究eNOS-线粒体相互作用是否调节出生相关的PAEC线粒体O27-1，并确定这种靶向的机制；(2)确定eNOS-线粒体相互作用是否调节对出生后适应至关重要的内皮功能：NO、PGI2和ET-1的合成以及出生后肺血管扩张。研究将在PAEC和完整的胎羊身上进行。将开发细胞生物学工具来(A)将eNOS靶向于胎儿PAEC中特定的亚细胞室，以及(B)抑制eNOS与参与线粒体靶向的客户蛋白-HSP90和Porin的相互作用。拟议的研究将探索一种新的线粒体氧化应激调节机制，在出生时暴露于氧气中。在PAEC中识别这种新的氧化应激源可能会导致更特异的治疗，例如线粒体靶向抗氧化剂来恢复PPHN的肺血管扩张和氧合。 公共卫生相关性：出生时将胎儿肺暴露在氧气中可以调节肺血流量的增加，而肺血流量是在出生后生活中建立气体交换所必需的。氧气促进线粒体的新陈代谢，而线粒体又能增加自由基的产生。拟议的研究将探索一种新的机制，在这种适应过程中调节自由基的形成。

DESCRIPTION (provided by applicant): Pulmonary vascular resistance (PVR) in the fetus undergoes a dramatic decrease at birth to facilitate gas exchange during postnatal life. Exposure to higher oxygen tension at birth plays a major role in this adaptation. Endothelial nitric oxide synthase (eNOS) regulates the transition in PVR and is stimulated directly by oxygen and indirectly by an increase in oxidative phosphorylation and ATP release. Oxidative phosphorylation also generates superoxide (O27-) as a byproduct. However, the mechanisms that regulate mitochondrial oxidative stress in the fetal pulmonary arteries during this transition remain unclear. Failure of PVR to decrease at birth leads to persistent pulmonary hypertension of the newborn (PPHN), a condition associated with increased oxidative stress in pulmonary arteries. Identification of the sources of oxidative stress is critical to the development of specific therapies to correct the vascular dysfunction in PPHN. Studies in fetal lamb pulmonary artery endothelial cells (PAEC) demonstrated that exposure to ATP, a NOS agonist, or transition to postnatal oxygen tension stimulate the association of eNOS with the mitochondrial outer membrane protein, porin. NOS agonists increase mitochondrial NO release in fetal PAEC. We propose to investigate the novel hypothesis that targeting of eNOS to the mitochondrial outer membrane regulates the rate of oxidative phosphorylation and O27- generation in PAEC by targeted NO release during birth- related transition. The broad specific aims of the proposed studies are to (1) Investigate whether eNOS - mitochondrial interactions regulate the mitochondrial O27- during birth related transition in PAEC and to define the mechanism of this targeting and (2) determine whether eNOS-mitochondrial interactions regulate the endothelial functions critical to postnatal adaptation: synthesis of NO, PGI2 and endothelin-1 and postnatal pulmonary vasodilation. Studies will be done in PAEC and intact fetal lambs. Cell biology tools will be developed to (A) target eNOS to specific sub-cellular compartments in fetal PAEC and (B) to inhibit the interactions of eNOS with the client proteins involved in mitochondrial targeting - hsp90 and porin. The proposed studies will investigate a new regulatory mechanism for the mitochondrial oxidative stress during oxygen exposure at birth. Identification of this new source of oxidative stress in PAEC may lead to more specific therapies, such as mitochondrial targeted anti- oxidants to restore pulmonary vasodilation and oxygenation in PPHN. PUBLIC HEALTH RELEVANCE: Exposure of the fetal lung to oxygen at birth mediates the increase in pulmonary blood flow which is necessary to establish gas exchange during postnatal life. Oxygen increases the metabolism in mitochondria which in turn can increase free radical production. The proposed studies will investigate a new mechanism that regulates the formation of free radicals during this adaptation.