Prostanoids and hypoxic neonatal pulmonary hypertension

前列腺素与缺氧新生儿肺动脉高压

• 批准号: 6686400
• 负责人: CANDICE D FIKE
• 金额: $ 25.2万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6686400
• 关键词: RNase protection assay; biological signal transduction; blood flow measurement; disease /disorder etiology; disease /disorder model; fatty acid metabolism; gene expression; hemodynamics; hypoxia; microelectrodes; muscle cells; newborn animals; potassium channel; prostacyclins; prostaglandin endoperoxide synthase; protein localization; protein structure function; pulmonary artery; pulmonary hypertension; respiratory oxygenation; smooth muscle; swine; thromboxanes; vascular endothelium; video microscopy; western blottings

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.

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，长期目标是提高对新生儿肺动脉高压发病机制的了解，以便开发更有效的治疗方法。 几乎所有有关新生儿肺动脉高压的研究都旨在设计急剧降低肺血管阻力的疗法。 预防新生儿肺动脉高压发生或进展的疗法很少受到关注，并且主要涉及控制一氧化氮（NO）途径。 然而，并非所有婴儿都对吸入一氧化氮有良好反应。我们的初步数据表明，前列腺素系统（包括前列环素 (PGI2) 和血栓素 (TXA2)）的紊乱不是 NO 的单一作用，而是缺氧诱导的新生儿肺动脉高压发病机制的关键。 我们的总体假设是，随着小肺动脉（SPA）的内皮细胞和平滑肌细胞持续缺氧，前列腺素信号通路会发生明显的破坏，并导致肺阻力血管的不适当收缩和肺动脉高压的发生。 我们方法的核心是 SPA 的使用，因为它们是与肺动脉高压的发展最相关的血管部位。 该提案将研究以下具体假设：（a）SPA内皮细胞中环氧合酶（COX）1-PGI2轴被破坏，持续低氧血症导致扩张剂前列腺素生成减少； (b)持续缺氧的 SPA 平滑肌细胞中 COX 2-TXA2 轴增强，从而增加收缩剂与扩张剂前列腺素生成的比率； (c) 收缩剂与扩张剂前列腺素生成比例的变化改变了平滑肌细胞 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) 使用酶免疫测定 (EIA) 和插管动脉技术确定 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（插管动脉技术）； (c) 确定主要前列腺素类酶的主要细胞定位（免疫染色技术），以及 (d) 确定各种 K+ 通道在介导血管对 PGI2 反应中的作用（微电极技术）。具体目标 2 将 (a) 确定暴露于慢性缺氧的仔猪 SPA 中 PGI2 和 TXA2 的数量和/或主要细胞来源是否发生改变（EIA 和插管动脉技术）； (b) 评估慢性缺氧（插管动脉技术）是否改变了对 PGI2 和 TXA2 的敏感性； (c) 确定主要酶的 mRNA 水平（RNase 保护测定）、主要酶的蛋白质丰度（免疫印迹技术）或 PGI2 和 TXA2 产生的主要酶的细胞定位（免疫染色）是否受到慢性缺氧的影响； (d) 评估 K+ 通道介导 PGI2 和 TXA2 扩张和收缩的作用是否与肺动脉高压的发生有关（微电极技术）。这些研究应该为改善婴儿肺动脉高压的治疗提供重要信息。