NITRIC OXIDE SYNTHASES IN LUNG DEVELOPMENT AND BRONCHOPULMONARY DYSPLASIA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。信号分子一氧化氮（NO）在发育中的肺气道和血管功能中起关键作用。一氧化氮由一氧化氮合成酶（NOS）、神经元一氧化氮合成酶（nNOS）、内皮一氧化氮合成酶（eNOS）和诱导型一氧化氮合成酶（iNOS）三种亚型产生。我们在正常狒狒胎儿中的初步研究表明，这三种亚型均在气道上皮中表达，并且nNOS和eNOS在妊娠后期被上调，以优化围产期No的产生。支气管肺发育不良（BPD）是一种破坏早产儿肺发育的炎症性疾病，其特征是气道和肺血管功能障碍。我们在狒狒BPD模型中的初步研究表明，在BPD发生过程中，肺NO生成和肺nNOS和eNOS表达明显减弱，吸入NO替代（iNO）导致氧合指数持续改善。本提案的总体目标是研究NOS表达的改变在狒狒BPD模型的病理生理中的作用。主要假设是肺nNOS和eNOS表达在BPD的发展过程中下调，导致NO产生减少，气道和血管结构和功能异常。第二种假设是iNO逆转了这些异常。目的1是确定早期BPD中NOS蛋白和mRNA表达的正常发生和变化，使用激光捕获显微解剖等方法评估从冷冻切片中收获的特定细胞类型中的NOS mRNA水平。目的2是在完整动物的NOS拮抗研究中确定每种NOS亚型在气道和血管功能中的作用。目的3：揭示胎儿发育和BPD过程中呼出NO水平的变化，以及各NOS亚型对呼出NO的贡献。目的4是确定一氧化氮对气道和血管功能、早期BPD肺病程和肺组织学的影响。目的5：研究nNOS和eNOS基因在狒狒气道上皮中表达的机制，以及细胞因子在其下调中的作用。所获得的结果将增加我们对NO在正常成功的出生后肺适应和BPD病理生理学中的作用的认识，从而可能导致这种毁灭性疾病的新疗法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The signaling molecule nitric oxide (NO) is critically involved in airway and vascular function in the developing lung. NO is produced by three isoforms of NO synthase (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our preliminary work in normal baboon fetuses indicates that all three isoforms are expressed in airway epithelium, and that nNOS and eNOS are upregulated during late gestation to optimize No production in the perinatal period. Bronchopulmonary dysplasia (BPD) is an inflammatory condition which disrupts the development of the preterm human lung, and it is characterized by airway and pulmonary vascular dysfunction. Our initial studies in the baboon BPD model indicate that lung NO production and lung nNOS and eNOS expression are markedly attenuated during the genesis of BPD, and that NO replacement by inhalation (iNO) results in a sustained improvement in oxygenation index. The overall objective of this proposal is to investigate the role of alterations in NOS expression in the pathopysiology of BPD in the baboon model. The primary hypothesis is that pulmonary nNOS and eNOS expression are downregulated during the development of BPD, leading to diminished NO production and abnormal airway and vascular structure and function. The secondary hypothesis is that iNO reverses these abnormalities. Aim 1 is to define the normal ontogeny and changes in NOS protein and mRNA expression in early BPD, using approaches including laser capture microdissection to evaluate NOS mRNA levels in specific cell types harvested from frozen sections Aim 2 is to determine the role of each NOS isoform in airway and vascular function in studies of NOS antagonism in intact animals. Aim 3 is to reveal the changes in exhaled NO levels with fetal development and BPD, as well as the contribution of each NOS isoform to exhaled NO. Aim 4 is to determine the effects of iNO on airway and vascular function, on the pulmonary course of early BPD, and on lung histology. Aim 5 is to determine the mechanisms underlying constitutive nNOS and eNOS gene expression in cultured in baboon airway epithelium, and the role of cytokines in their downregulation. The results obtained will increase our knowledge of the role of NO in normally successful postnatal pulmonary adaptation and in the pathophysiology of BPD, thereby possibly leading to novel therapies for this devastating disorder.