eNOS: Metabolism & Vascular Biology in Health & Disease

eNOS：新陈代谢

• 批准号: 6916181
• 负责人: M HAROLD LAUGHLIN
• 金额: $ 77.64万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916181
• 关键词: adipose tissue; atherosclerosis; biological models; cardiovascular function; diabetes mellitus; dietary lipid; fibroblasts; gene targeting; genetically modified animals; glucose transport; hyperlipidemia; metabolism; model design /development; nitric oxide synthase; nuclear transfer; oxygen transport; peripheral blood vessel disorder; phenotype; respiratory system; smooth muscle; striated muscles; swine; transfection /expression vector; vascular endothelium

DESCRIPTION (provided by applicant): The proposed research has three major goals. First (AIM 1); develop genetically modified pigs (GMPs) that overexpress or under-express endothelial nitric oxide synthase (eNOS). We will develop pigs that over-express eNO by adding a construct (composed of an endothelial-specific Tie 2-promoter, full length eNOS cDNA and a selectable marker) to Yucatan fetal fibroblast cells and create the pigs using nuclear transfer technology. Such a transgenic animal will over express eNOS specifically in endothelial cells. We will develop pigs that lack eNOS (eNOS-/-) by replacin exon 12 with a neomycin cassette by homologous recombination in Yucatan fetal fibroblast cells. We will produce heterozygous eNOS (+/-) pigs using targeted fibroblast cells for nuclear transfer, eNOS-/- pigs, that lack functional eNOS protein in all cell types, will be generated from the breeding of eNOS pigs. Second (AIM 2); establish the metabolic and cardiorespiratory phenotype of these GMPs. We will characterize whole body metabolism ana cardiorespiratory functional capacities of GMPs and the regulation of coronary blood flow. From the same animals we will also characterize the effects of altered eNOS expression on metabolism of skeletal muscle and fat tissues, ana thephenotype of vascular endothelium and smooth muscle. Finally (AIM 3); determine the effects of altered eNOS expression on endothelial dysfunction and vascular disease produced by high fat diet-induced hyperlipidemia. Pigs share with humans important characteristics of muscle and intermediary metabolism and of cardiorespiratory function, making them another important link in our growing understanding of these processes. These animal models will be powerful tools for investigators at both public and private sector institutions and will form an important tool for our ongoing research concerning the role of eNOS in atherosclerotic vascular disease (supported by NHLBI). In addition, these GMPs will provide important animal models to determine the role of eNOS in: 1) the control of metabolic processes in muscle and fat tissue (NIAMS), 2) diabetes and diabetic vascular disease (NIDDK), and 3) reproductive biology (NICHD). Further development of porcine models for biomedical research will reduce the growing pressure Ion the use of nonhuman primate models for research involving genetic modifications of metabolic and cardiorespiratory systems. The strong group of investigators submitting this application is uniquely qualified and prepared to accomplish these goals.

描述（由申请人提供）： 拟议的研究有三个主要目标。第一（目标 1）；开发过度表达或低表达内皮一氧化氮合酶（eNOS）的转基因猪（GMP）。 我们将通过向尤卡坦胎儿成纤维细胞添加构建体（由内皮特异性 Tie 2 启动子、全长 eNOS cDNA 和选择标记组成）来培育过度表达 eNO 的猪，并使用核移植技术培育猪。这种转基因动物将在内皮细胞中特异地过度表达eNOS。我们将通过在尤卡坦胎儿成纤维细胞中同源重组，用新霉素盒替换外显子 12，培育出缺乏 eNOS (eNOS-/-) 的猪。我们将使用靶向成纤维细胞进行核移植来生产杂合性 eNOS (+/-) 猪，所有细胞类型都缺乏功能性 eNOS 蛋白的 eNOS-/- 猪将通过 eNOS 猪的育种产生。第二（AIM 2）；建立这些 GMP 的代谢和心肺表型。 我们将描述 GMP 的全身代谢和心肺功能能力以及冠状动脉血流的调节。我们还将从相同的动物中表征 eNOS 表达改变对骨骼肌和脂肪组织代谢以及血管内皮和平滑肌表型的影响。最后（目标 3）；确定 eNOS 表达改变对高脂饮食引起的高脂血症引起的内皮功能障碍和血管疾病的影响。猪与人类具有相同的肌肉和中间代谢以及心肺功能的重要特征，这使它们成为我们不断加深对这些过程的理解的另一个重要环节。这些动物模型将成为公共和私营机构研究人员的强大工具，并将成为我们正在进行的有关 eNOS 在动脉粥样硬化性血管疾病中的作用的研究的重要工具（由 NHLBI 支持）。此外，这些 GMP 将提供重要的动物模型来确定 eNOS 在以下方面的作用：1) 肌肉和脂肪组织代谢过程的控制 (NIAMS)，2) 糖尿病和糖尿病血管疾病 (NIDDK)，以及 3) 生殖生物学 (NICHD)。用于生物医学研究的猪模型的进一步开发将减少使用非人类灵长类动物模型进行涉及代谢和心肺系统遗传修饰的研究所面临的日益增长的压力。提交本申请的强大研究人员团队具有独特的资格并准备好实现这些目标。