Extracellular superoxide induces Egr-1 in the hypoxic pulmonary artery

描述（由申请方提供）：肺动脉高压导致的肺动脉血管重塑是缺氧性肺病婴儿和儿童的一种危及生命的并发症。进一步了解这一过程对于制定旨在降低这些个体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），包括通过NADPH氧化酶产生的超氧化物（O2-），有助于血管重塑。细胞外氧化剂/抗氧化剂稳态由超氧化物歧化酶（EC-SOD）的细胞外同种型维持，EC-SOD在血管壁中高度表达。该提案测试的假设，缺氧破坏细胞外O2-的生产之间的平衡，由NADPH氧化酶和其清除EC-超氧化物歧化酶在PA。我们进一步假设，过量的细胞外O2-产生的缺氧肺上调缺氧诱导和氧化还原敏感的转录因子，早期生长反应-1（Egr-1），这反过来又刺激Egr-1反应基因的重要造成新生儿慢性缺氧诱导的肺血管重塑和肺动脉高压。目的1利用慢性缺氧的小牛和小鼠肺动脉段及新生小牛肺动脉血管细胞，研究肺动脉段活性氧的产生及内皮细胞超氧化物歧化酶（EC-SOD）的表达和活性。目的2利用EC-SOD过表达和缺失小鼠以及NADPH氧化酶gp 91 phox亚基缺失小鼠，为细胞外O2-调控缺氧反应关键基因，促进慢性缺氧诱导的肺血管重构和肺动脉高压提供体内分子和药理学证据。为了补充这个模型，在目标3中，我们将使用PA外膜成纤维细胞分离的新生小牛作为一个高度相关的模型系统，在体外实验中，以测试缺氧诱导的细胞外O2-对氧化还原敏感的转录因子Egr-1的表达的影响。慢性缺氧作为肺血管重构和肺动脉高压的刺激的研究是引人注目的，因为缺氧是各种肺部疾病的共同特征。因此，通过推进我们的知识基础和在动物模型中测试新的治疗方法，我们将为未来在与缺氧性肺病相关的一系列场景中进行的人体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果。项目叙述：缺氧使婴儿和儿童的严重肺部疾病复杂化，并且这些患者中肺血管重构和肺动脉高压的发展导致右心衰竭，大大增加发病率和死亡率。本研究旨在探讨慢性低氧性肺动脉高压时细胞外超氧化物生成和清除的失衡是否与肺动脉结构重塑有关。该提案将为未来在与缺氧性肺病相关的一系列场景中进行的人体临床试验提供基础，以改善患有这些困难和严重问题的儿科患者的健康结果。

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.