Prostanoids and hypoxic neonatal pulmonary hypertension

与成人模型相比，新生儿肺动脉高压的病理生理学研究相对较少，尽管它可能涉及不同的机制。因此，长期的目标是提高对新生儿肺动脉高压发病机制的认识，以便开发更有效的治疗方法。几乎所有关于新生儿肺动脉高压的研究都是针对设计显著降低肺血管阻力的治疗方法。预防新生儿肺动脉高压发生或发展的治疗方法很少受到关注，主要涉及操纵一氧化氮(NO)途径。然而，并不是所有的婴儿对吸入NO都有良好的反应。我们的初步数据表明，前列腺素系统的紊乱，包括前列环素(PGI2)和血栓素A2(TXA2)，而不是可归因于NO的单一效应，是缺氧诱导的新生儿肺动脉高压的关键。我们的总体假设是，在持续低氧的情况下，小肺动脉内皮细胞和平滑肌细胞(SPA)发生明显的前列腺素信号通路的破坏，导致肺阻力血管的不适当收缩和肺动脉高压的发生。我们方法学的核心是使用SPA，因为它们是与肺动脉高压发展最相关的血管部位。本提案将探讨以下具体假设：(A)持续低氧血症的SPA内皮细胞环氧合酶(COX)1-PGI2轴被破坏，导致扩张剂前列腺素合成减少；(B)持续低氧SPA的平滑肌细胞COX-2-TXA2轴增强，从而使收缩与扩张前列腺素合成的比例增加；(C)收缩与扩张前列腺素生成的比率改变，改变了平滑肌细胞的K电导和膜电位，导致不适当的收缩和肺动脉高压。具体目标1将(A)利用酶免疫分析(EIA)和管状动脉技术确定COX-1和COX-2生物合成途径对PGI2和TXA2产生的数量、细胞来源和相对贡献；(B)评估PGI2和TXA2对SPA张力的影响(管状动脉技术)；(C)确定主要前列腺素酶的主要细胞定位(免疫染色技术)；以及(D)确定各种K通道在介导血管对PGI2的反应中的作用(微电极技术)。具体目标2将(A)确定慢性缺氧(EIA和动脉插管技术)下仔猪SPA中PGI2和TXA2产生的数量和/或主要细胞来源是否发生改变；(B)评估慢性缺氧是否改变了对PGI2和TXA2的敏感性(插管动脉技术)；(C)确定主要酶的mRNA水平(核糖核酸酶保护分析)、主要酶的蛋白丰度(免疫印迹技术)或导致PGI2和TXA2产生的主要酶(免疫染色)的细胞定位是否受到慢性缺氧的影响；(D)评价K通道在介导PGI2和TXA2舒缩中的作用是否参与了肺动脉高压的发生(微电极技术)。这些研究将为改善婴幼儿肺动脉高压的治疗提供重要信息。

The pathophysiology underlying neonatal pulmonary hypertension has received relatively little study compared to adult models, even though it may involve different mechanisms. Thus, the long-term goal is to improve the understanding of the pathogenesis of neonatal pulmonary hypertension so that more effective therapies can be developed. Almost all of the research regarding neonatal pulmonary hypertension has been directed at devising therapies to acutely decrease pulmonary vascular resistance. Therapies for preventing the onset or progression of neonatal pulmonary hypertension have received little attention and have largely involved manipulating the nitric oxide (NO) pathway. Yet, not all infants respond favorably to inhaled NO. Our preliminary data demonstrate that rather than a singular effect attributable to NO, derangements in the prostanoid system, which include prostacyclin (PGI2) and thromboxane (TXA2), are key to the pathogenesis of hypoxia-induced neonatal pulmonary hypertension. Our overall hypothesis is that distinct disruptions of the prostanoid signaling pathway occur with sustained hypoxia in endothelial cells and smooth muscle cells of small pulmonary arteries (SPA) and lead to inappropriate constriction of pulmonary resistance vessels and the development of pulmonary hypertension. At the core of our methodology is the use of SPA as they are the vascular site most relevant to the development of pulmonary hypertension. This proposal will investigate the following specific hypotheses: (a) the cyclooxygenase (COX) 1-PGI2 axis is disrupted in endothelial cells of SPA with sustained hypoxemia leading to decreased dilator prostanoid production; (b) the COX 2-TXA2 axis is enhanced in smooth muscle cells of SPA with sustained hypoxia so that the ratio of constrictor to dilator prostanoid production is increased; (c) the change in ratio of constrictor to dilator prostanoid production alters smooth muscle cell K+ conductance and membrane potential leading to inappropriate constriction and pulmonary hypertension. Specific Aim 1 will (a) determine the amounts, cellular sources and relative contributions of COX 1 and COX 2 biosynthetic pathways to PGI2 and TXA2 production using enzyme immunoassay (EIA) and cannulated artery techniques; (b) evaluate the effect of PGI2 and TXA2 on SPA tone (cannulated artery technique); (c) establish the predominant cellular localization of the major prostanoid enzymes (immunostaining technique) and (d) determine the role of various K+ channels in mediating vascular responses to PGI2 (microelectrode technique). Specific Aim 2 will (a) determine whether the amounts and/or predominant cellular sources of production of PGI2 and TXA2 are altered in SPA from piglets exposed to chronic hypoxia (EIA and cannulated artery techniques); (b) evaluate whether the sensitivity to PGI2 and TXA2 has been altered by chronic hypoxia (cannulated artery technique); (c) determine whether mRNA levels for the major enzymes (RNase protection assay), protein abundance of the major enzymes (immunoblot technique) or the cellular localization of the major enzymes (immunostaining) underlying PGI2 and TXA2 production are affected by chronic hypoxia; (d) evaluate whether the effect from K+ channels in mediating dilation and constriction from PGI2 and TXA2 is involved with the development of pulmonary hypertension (microelectrode technique). These studies should provide important information for improving treatment of infants with pulmonary hypertension.