MOLECULAR TARGETS OF NITRIC OXIDE IN CARDIAC TRANSPLANTS

心脏移植中一氧化氮的分子靶标

• 批准号: 6755178
• 负责人: GALEN M PIEPER
• 金额: $ 37.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6755178
• 关键词: apoptosis; electron spin resonance spectroscopy; enzyme induction /repression; gel electrophoresis; gene targeting; genetic transcription; genetically modified animals; heart contraction; heart transplantation; immunoprecipitation; laboratory mouse; laboratory rat; nitric oxide; nitric oxide synthase; nitroso compounds; northern blottings; nuclear factor kappa beta; oxidative stress; superoxide dismutase; transplant rejection; western blottings

DESCRIPTION (Verbatim from the application): While nitric oxide (NO) is believed to contribute to cardiac allograft rejection, me precise mechanism is not understood. We will examine the intracellular molecular mechanisms for the actions of excess NO on cardiac allograft contractile dyskinesis relating to a paradigm which includes both oxidative and nitrosative stress. We will examine the regulation of inducible NO synthase (iNOS) gene expression by the oxidant-sensitive NF-kB transcription factor and examine key candidate cytosolic and mitochondrial proteins as molecular targets of NO. These proteins include myoglobin and aconitase. In vitro nitrosylation of these proteins are known to inhibit enzyme activity and 02 binding, thus, interfering with efficient 02 utilization by this O2-demanding organ. Hypothesis: [NO derived from iNOS targets certain cellular proteins for nitrosylation within cardiac grafts that contribute to myocardial contractile dysfunction. Furthermore, reactive oxygen plays a role in regulating iNOS gene expression at the transcriptional level via activation of the oxidant-sensitive transcription factor (NF-KB).] The applicant will use advanced, state-of-the-art molecular and biophysical techniques including: transcription factor regulation of iNOS gene (gel shift assays; Northern and Western analysis); in situ sonomicrometry; measures of nitrosyl protein and function (EPR, electron paramagnetic resonance spectroscopy and immunoprecipitation); assay for apoptosis; quantitation of reactive oxygen (EPR spin trapping; salicylate trapping); evaluation with iNOS knockout and SOD1 or SOD2 transgenic mice. The applicant will show that following allogeneic cardiac transplantation: Aim #1: [Induction of iNOS leads to iron-nitrosyl complex formation within myocardium and contractile dysfunction during allogeneic cardiac transplantation.]; Aim #2: [Certain candidate proteins are molecular targets of nitrosylation by excess NO in allogeneic transplantation.]; Aim #3: [iNOS gene deletion prevents nitrosyiprotein formation and prolongs graft survival. ]; Aim #4: [iNOS gene expression during allogeneic transplantation is regulated by activation of NF-KB.]; Aim #5: [Antioxidants inhibit activation of NF-KB, induction of iNOS, formation of nitrosyl complexes and enhance contractile function.]; Aim #6: [Antioxidant transgene overexpression inhibits activation of NF-kB, induction of iNOS, formation of nitrosyl complexes and enhances contractile function during allogeneic cardiac transplantion.] These studies will provide a novel mechanism to explain the pathogenesis of dyskinesis and graft failure during cardiac transplant rejection.

描述（申请中的逐字记录）：虽然一氧化氮 (NO) 是 据信会导致心脏同种异体移植排斥，我的精确机制是 不明白。我们将研究细胞内的分子机制 过量NO对心脏同种异体移植收缩性运动障碍的作用 包括氧化应激和亚硝化应激的范例。我们将检查 诱导型一氧化氮合酶 (iNOS) 基因表达的调节 氧化剂敏感的 NF-kB 转录因子并检查关键候选因子 细胞质和线粒体蛋白作为 NO 的分子靶标。这些蛋白质 包括肌红蛋白和乌头酸酶。这些蛋白质的体外亚硝基化是 已知抑制酶活性和 02 结合，从而干扰 该需氧器官有效利用 O2。 假设：[源自 iNOS 的 NO 靶向某些细胞蛋白 心脏移植物内的亚硝基化有助于心肌收缩 功能障碍。此外，活性氧对 iNOS 基因也有调节作用 通过激活氧化剂敏感蛋白在转录水平表达 转录因子（NF-KB）。]申请人将使用先进的、 最先进的分子和生物物理技术，包括：转录 iNOS 基因的因子调节（凝胶迁移分析；Northern 和 Western 分析）;原位声测法；亚硝酰蛋白和功能的测量 （EPR、电子顺磁共振波谱和免疫沉淀）； 细胞凋亡测定；活性氧定量（EPR 自旋捕获； 水杨酸捕获）； iNOS 敲除和 SOD1 或 SOD2 转基因评估 老鼠。申请人将证明以下同种异体心脏 移植：目标#1：[诱导一氧化氮合酶（iNOS）的诱导产生亚硝酰铁复合物 同种异体期间心肌内的形成和收缩功能障碍 心脏移植。];目标#2：[某些候选蛋白质是分子蛋白质 同种异体移植中过量NO亚硝基化的靶标。]；目标#3： [iNOS 基因缺失可防止亚硝基蛋白形成并延长移植时间 生存。 ];目标 #4：[同种异体移植过程中 iNOS 基因的表达是 受 NF-KB 激活调节。]；目标#5：[抗氧化剂抑制 NF-KB、iNOS 诱导、亚硝酰复合物形成并增强 收缩功能。]；目标#6：[抗氧化剂转基因过度表达抑制 NF-kB 的激活、iNOS 的诱导、亚硝酰复合物的形成以及 增强同种异体心脏移植期间的收缩功能。] 这些 研究将提供一种新的机制来解释疾病的发病机制 心脏移植排斥期间的运动障碍和移植失败。

项目成果

Hierarchical change in antioxidant enzyme gene expression and activity in acute cardiac rejection: role of inducible nitric oxide synthase.

急性心脏排斥反应中抗氧化酶基因表达和活性的层次变化：诱导型一氧化氮合酶的作用。

• DOI: 10.1007/s11010-005-3639-2
• 发表时间: 2005
• 期刊: Molecular and cellular biochemistry
• 影响因子: 4.3
• 作者: Nilakantan,Vani;Zhou,Xianghua;Hilton,Gail;Roza,AllanM;Adams,MarkB;Johnson,ChristopherP;Pieper,GalenM
• 通讯作者: Pieper,GalenM