S-nitrosoglutathione reductase and airways hyperreactivity in murine bronchopulmonary dysplasia

项目摘要/摘要： 这项建议描述了一个五年的有指导的研究和培训计划，将促进发展 托马斯·拉菲博士的医学博士授予新生儿肺部疾病的独立研究员。建立在Dr。 Raffay作为临床新生儿科医生和呼吸道反应性和新生儿方面的基础科学家的背景 支气管肺发育不良(BPD)，他将获得S的专业知识-亚硝基生物学，流式细胞术，条件性 通过结构化指导，转基因动物模型、药理学和翻译科学，严谨 凯斯韦斯特大学的实验室实践经验、教学以及正式的课堂作业和技能培训 后备大学/彩虹婴儿和儿童医院、罗切斯特梅奥诊所和华盛顿大学 温莎。本杰明·加斯顿博士，S-亚硝硫醇信号转导在儿科肺部疾病中的先驱，理查德·J博士 马丁是新生儿呼吸道疾病和BPD领域的国际领先者，将提供他们的专业知识和 建立了对该项目的指导和Raffay博士向研究独立性过渡的跟踪记录。在……里面 在美国，每年有14,000例儿童肺部疾病的新诊断病例存活 早产儿，费用超过25亿美元。重症患者的治疗选择有限 支气管痉挛和终生呼吸道阻塞是BPD的特征。用一只新生的高氧小鼠 作为BPD和呼吸道高反应性的模型，Raffay博士的新数据确定了一种可行的治疗方法。用一种治疗方法 S-亚硝基谷胱甘肽单一雾化吸入逆转幼年BPD小鼠的气道高反应性 房间的空气恢复了成年人的健康。GSNO是一种有效的内源性支气管扩张剂，对呼吸道疾病至关重要 哮喘和囊性纤维症。在这个模型中，新生儿高氧增加了GSNO的分解代谢。 引起的酶的表达和活性增加，S亚硝基谷胱甘肽还原酶(GSNOR)至少 部分是通过高氧下调microRNA(miR-342-3p)。这项研究将测试整体 GSNOR活性丧失或抑制将保护青少年BPD呼吸道高反应性的假说 和成年小鼠。这项建议将评估新生儿高氧血症对BPD呼吸道高反应性的影响。 和利用全局GSNOR基因敲除小鼠的实质肺重建(目标1A)；识别肺组织 利用荧光激活的细胞分选参与GSNOR和miR-342-3p的表达 测试条件性GSNOR基因敲除小鼠(AIM 1B)；在野外测试一种药剂级GSNOR抑制剂药物 型小鼠缓解急性支气管收缩(AIM 2a)和慢性减弱BPD表型(AIM 2B)。人类翻译目标(目标3)：GSNOR水平，实时荧光GSNO分解代谢，以及 将测量暴露在高氧环境下的人胎儿呼吸道平滑肌中microRNA的表达；以及 GSNO和GSNOR抑制剂将作为减弱氧诱导的高血糖的治疗方法进行试验 伸缩性。这些研究具有重要的临床意义，因为GSNOR抑制剂目前正在进行临床试验 囊性纤维化和哮喘，最终可能成为治疗支气管肺发育不良的新方法。

Project Abstract/Summary: This proposal describes a five-year mentored research and training plan that will facilitate the development of Dr. Thomas Raffay, MD to an independent investigator in neonatal pulmonary disease. Building upon Dr. Raffay's background as a clinical neonatologist and a basic scientist in airways reactivity and neonatal bronchopulmonary dysplasia (BPD), he will attain expertise in S-nitrosothiol biology, flow-cytometry, conditional transgenic animal models, pharmacology, and translational science through structured mentorship, rigorous hands-on laboratory experiences, didactics teaching, and formal classwork and skills training at Case Western Reserve University/Rainbow Babies and Children's Hospital, the Mayo Clinic Rochester, and the University of Windsor. Dr. Benjamin Gaston, a pioneer in S-nitrosothiol signaling in pediatric lung disease, and Dr. Richard J Martin, an international leader in neonatal airways disease and BPD, will provide their expertise and an established track record of mentorship to this project and Dr. Raffay's transition to research independence. In the United States, 14,000 new diagnoses of the pediatric lung disease, BPD, are made annually in surviving premature infants, with costs exceeding $2.5 billion. Treatment options are limited for the severe bronchospasms and life-long airway obstruction that characterize BPD. Using a neonatal hyperoxia mouse model of BPD and airways hyperreactivity, Dr. Raffay's new data identify a viable therapy. Treatment with a single aerosol of S-nitrosoglutathione (GSNO) reverses airways hyperresponsiveness in juvenile BPD mice and room air recovered adults. GSNO is a potent endogenous bronchodilator, critical for the airways diseases of asthma and cystic fibrosis. In this model, neonatal hyperoxia increases the catabolic breakdown of GSNO caused by increased expression and activity of the enzyme, S-nitrosoglutathione reductase (GSNOR), at least in part through hyperoxic downregulation of a microRNA (miR-342-3p). This study will test the overall hypothesis that loss or inhibition of GSNOR activity will protect against BPD airways hyperreactivity in juvenile and adult mice. This proposal will evaluate the effects of neonatal hyperoxia on BPD airways hyperreactivity and parenchymal lung remodeling utilizing global GSNOR knockout mice (Aim 1A); identify the lung tissues involved in GSNOR and miR-342-3p expression utilizing fluorescence-activated cell sorting and develop and test conditional GSNOR knockout mice (Aim 1B); test a pharmaceutical-grade GSNOR inhibitor drug in wild- type mice to alleviate acute bronchoconstriction (Aim 2a) and chronically to attenuate the BPD phenotype (Aim 2b). In a translational human aim (Aim 3): GSNOR levels, real-time fluorescent GSNO catabolism, and microRNA expression will be measured in human fetal airway smooth muscle exposed to hyperoxia; and GSNO and GSNOR inhibitors will be tested as a treatment for attenuation of oxygen induced hyper- contractility. These studies are clinically important because GSNOR inhibitors are currently in clinical trials for cystic fibrosis and asthma which could ultimately serve as a new treatment for bronchopulmonary dysplasia.