Extracellular superoxide induces Egr-1 in the hypoxic pulmonary artery

描述(申请人提供)：在患有缺氧性肺部疾病的婴儿和儿童中，合并肺动脉高压的肺动脉血管重塑是一种危及生命的并发症。对这一过程的进一步了解对于开发旨在降低这些人的肺动脉高压严重程度的新策略至关重要。越来越多的证据表明，活性氧(ROS)，包括NADPH氧化酶产生的超氧化物(O2-)，参与了血管重构。细胞外氧化剂/抗氧化剂的动态平衡是由细胞外超氧化物歧化酶(EC-SOD)维持的，它在管壁中高度表达。该方案验证了一种假设，即低氧破坏了由NADPH氧化酶产生的细胞外O2-与PA中EC-SOD清除细胞外O2-之间的平衡。我们进一步假设，在低氧肺中产生的过量细胞外O2-上调了低氧诱导和氧化还原敏感的转录因子早期生长反应-1(Egr-1)，而早期生长反应-1又刺激了Egr-1反应基因，这些基因在导致新生儿慢性低氧诱导的肺血管重构和肺动脉高压中起重要作用。目的1使用从慢性缺氧的小牛和小鼠分离的PA片段和从新生小牛体外分离的肺动脉血管细胞来评价活性氧的产生和EC-SOD的表达和活性。目的2将利用慢性低氧高表达和缺乏EC-SOD以及NADPH氧化酶gp91Phox亚基缺失的小鼠在体内提供分子和药理学证据，证明细胞外O2-调节关键的低氧反应基因，并在慢性低氧诱导的肺血管重构和发育中的肺高压中起作用。为了补充这一模型，在目标3中，我们将使用从新生小牛分离的PA外膜成纤维细胞作为体外实验的高度相关的模型系统，以测试低氧诱导的细胞外O2-对氧化还原敏感转录因子Egr-1表达的影响。慢性低氧刺激肺血管重塑和肺高压的研究是引人注目的，因为低氧是各种肺部疾病的共同特征。因此，通过提高我们的知识基础并在动物模型中测试新的治疗方法，我们将为未来与缺氧性肺部疾病相关的一系列场景中的人类临床试验提供坚实的基础，以改善患有这些困难和严重问题的患者的健康结局。项目简介：缺氧会使婴儿和儿童的严重肺部疾病复杂化，这些患者的肺血管重塑和肺动脉高压的发展会导致右心衰竭，大大增加发病率和死亡率。这一建议测试了细胞外超氧化物的产生和清除的失衡是否有助于慢性缺氧性肺动脉高压的结构重建。这项提议将为未来在与缺氧性肺部疾病相关的一系列情景中进行人类临床试验提供基础，以改善患有这些困难和严重问题的儿科患者的健康结局。

DESCRIPTION (provided by applicant): PA vascular remodeling with pulmonary hypertension is a life-threatening complication in infants and children with hypoxic lung diseases. A further understanding of this process is essential to develop new strategies aimed at reducing the severity of pulmonary hypertension in these individuals. Accumulating evidence indicates that reactive oxygen species (ROS), including superoxide (O2-) generated via NADPH oxidase, contribute to vascular remodeling. Extracellular oxidant/antioxidant homeostasis is maintained by the extracellular isoform of superoxide dismutase (EC-SOD), which is highly expressed in the vessel wall. The proposal tests the hypothesis that hypoxia disrupts the balance between the production of extracellular O2- by NADPH oxidase and its clearance by EC-SOD in the PA. We further hypothesize that excess extracellular O2- generated in the hypoxic lung upregulate a hypoxia-inducible and redox-sensitive transcription factor, early growth response-1 (Egr-1), which, in turn, stimulates Egr-1-responsive genes important in causing neonatal chronic hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension. Aim 1 will use PA segments isolated from chronically hypoxic calves and mice and in vitro pulmonary artery vascular cells isolated from the neonatal calf to evaluate production of reactive oxygen species and expression and activity of EC-SOD. Aim 2 will use chronically hypoxic mice overexpressing and lacking EC-SOD as well as mice lacking gp91phox subunit of NADPH oxidase to provide in vivo molecular and pharmacologic evidence that extracellular O2- regulates critical hypoxia-responsive genes and contributes to chronic hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in the developing lung. To complement this model, in Aim 3, we will use the PA adventitial fibroblast isolated from the neonatal calf as a highly relevant model system for in vitro experiments to test the effects of hypoxia-induced extracellular O2- on the expression of the redox-sensitive transcription factor Egr-1. The study of chronic hypoxia as a stimulus for pulmonary vascular remodeling and pulmonary hypertension is compelling, as hypoxia is a common feature of diverse lung diseases. Thus, by advancing our knowledge base and testing new therapeutic approaches in animal models, we will provide a solid foundation for future human clinical trials in a range of scenarios associated with hypoxic lung diseases to improve health outcome for patients with these difficult and serious problems. PROJECT NARRATIVE: Hypoxia complicates severe lung diseases in infants and children, and the development of pulmonary vascular remodeling and pulmonary hypertension in these patients leads to right heart failure, greatly increasing morbidity and mortality. This proposal tests whether an imbalance in production and clearance of extracellular superoxide contributes to the structural remodeling in chronic hypoxic pulmonary hypertension. This proposal will provide the basis for future human clinical trials in a range of scenarios associated with hypoxic lung diseases to improve health outcome for pediatric patients with these difficult and serious problems.