Extracellular superoxide induces Egr-1 in the hypoxic pulmonary artery

肺动脉高压肺动脉血管重构是婴儿和儿童缺氧肺部疾病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/抗氧化稳态由超氧化物歧化酶（EC-SOD）的细胞外异构体维持，超氧化物歧化酶在血管壁高度表达。该提议验证了缺氧破坏了NADPH氧化酶产生细胞外O2-和PA中EC-SOD清除O2-之间的平衡的假设。我们进一步假设，缺氧肺中产生的过量细胞外O2-上调了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)，这反过来刺激Egr-1应答基因，这在引起新生儿慢性缺氧诱导的肺血管重构和肺动脉高压中起重要作用。目的1将使用从慢性缺氧犊牛和小鼠中分离的PA片段以及从新生犊牛中分离的体外肺动脉血管细胞来评估活性氧的产生以及EC-SOD的表达和活性。Aim 2将使用过表达和缺乏EC-SOD的慢性缺氧小鼠以及缺乏NADPH氧化酶gp91phox亚基的小鼠，提供体内分子和药理学证据，证明细胞外O2-调节关键缺氧反应基因，并参与慢性缺氧诱导的肺血管重构和肺高压。为了补充该模型，在Aim 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高度相关的模型系统进行体外实验，以测试缺氧诱导的细胞外O2-对氧化还原敏感转录因子Egr-1表达的影响。由于缺氧是多种肺部疾病的共同特征，慢性缺氧作为肺血管重构和肺动脉高压刺激的研究是引人注目的。因此，通过提高我们的知识基础和在动物模型中测试新的治疗方法，我们将为未来在一系列与缺氧肺部疾病相关的情况下进行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。

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.