Development of a Porcine Model of Atherosclerosis

描述（由申请人提供）：动脉粥样硬化是心血管疾病的主要原因，心血管疾病是美国最常见的死亡原因。动脉粥样硬化的特征是脂质、胆固醇、钙沉积和细胞碎片在血管壁上积聚，并导致斑块形成、动脉阻塞和器官血流量减少。这些斑块经常破裂，引起心肌梗死、中风或死亡。主要的危险因素包括血脂水平升高、高血压和糖尿病。目前的治疗策略是通过药理学方法改变患者的生活方式/饮食和降低胆固醇。手术干预与医疗设备，如支架用于晚期病例。虽然这些治疗方法使许多患有这种疾病的患者受益，但它们远非理想。其中一个原因是，目前还没有一种药物或设备被开发出来，并在一个模型系统中进行测试，以准确地再现所治疗的疾病。因此，早期临床前研究和人体药物试验之间存在显著差距。缺乏能够准确复制人类动脉粥样硬化所有表现的动物模型，一直是开发这种致命疾病的有效疗法和干预措施的主要障碍。已经建立了几种具有脂蛋白代谢重要基因突变的小鼠模型，尽管这些模型提供了信息，但它们不能发展为典型的人类疾病的复杂动脉粥样硬化病变。与小鼠相比，猪心血管系统的生理和解剖结构与人类非常相似。事实上，猪长期以来一直被用作心血管疾病的模型，并且猪的低密度脂蛋白受体（LDLR）基因自然发生突变，因此具有升高的低密度脂蛋白，已经有报道。虽然高胆固醇血症猪是一个有吸引力的模型，但突变的温和性质、疾病的高变异性、其他研究人员的有限获取以及费用都阻碍了它在研究界的广泛应用。因此，该项目的最终目标是开发和商业化一种基因靶向的猪动脉粥样硬化模型。我们在I期开发的LDLR胎儿成纤维细胞将用作体细胞核移植的核供体。核移植胚胎将被移植到雌性受体体内进行妊娠。由此产生的仔猪将有一个靶向LDLR基因。我们将在分子和生化水平上对LDLR靶向猪进行表征。我们将测定ldlr靶猪的脂质和脂蛋白谱，并进行形态计量学分析，以确定动脉粥样硬化的存在和程度。最后，我们将建立种猪群，以产生LDLR-/-猪，并扩大和繁殖群体。这个项目将产生一个猪动脉粥样硬化模型，这将为学术和行业研究人员提供一个机会，以更好地了解这种疾病，并开发和测试新的治疗方法和预防策略。因此，这项工作将加速这种昂贵而致命的疾病的新疗法的发现。

DESCRIPTION (provided by applicant): Atherosclerosis is the primary cause of cardiovascular disease, which is the most common cause of death in the United States. Atherosclerosis is characterized by the accumulation of lipids, cholesterol, calcium deposits, and cellular debris in vessel walls, and results in plaque formation, arterial obstruction, and diminished blood flow to organs. These plaques often rupture, causing myocardial infarction, stroke, or death. The main risk factors include elevated lipid levels, hypertension, and diabetes. Current treatment strategies are directed at changing patient lifestyle/diet and decreasing cholesterol via pharmacological methods. Surgical interventions with medical devices such as stents are used for advanced cases. While these therapeutic approaches have benefited many patients with this disease, they are far from ideal. One reason is that no drug or device is actually developed and tested in a model system that accurately recreates the disease being treated. Thus, there is a significant gap between early phase preclinical studies and human drug trials. The lack of an animal model that accurately replicates all of the manifestations of human atherosclerosis has been a major barrier to the development of effective therapies and interventions for this deadly disease. Several mouse models have been generated with mutations in genes important for lipoprotein metabolism, and while these models have been informative, they fail to develop the complex atherosclerotic lesions that are typical of the human disease. In contrast to mice, the physiology and anatomy of the porcine cardiovascular system closely resembles that of humans. In fact, pigs have long been used as models of cardiovascular disease, and pigs with naturally occurring mutations in their LDL receptor (LDLR) gene, and therefore possessing elevated LDL, have been reported. Although the hypercholesterolemic pig is an attractive model, the mild nature of the mutation, the high variability of the disease, the limited access by other researchers, and the expense prevent its wide use in the research community. Therefore, the ultimate goal of this project is to develop and commercialize a gene- targeted porcine model of atherosclerosis. LDLR fetal fibroblasts that we developed in Phase I will be used as nuclear donors for somatic cell nuclear transfer. Nuclear transfer embryos will be transferred to recipient females for gestation. Resulting piglets will have one targeted LDLR gene. We will characterize the LDLR- targeted pigs at the molecular and biochemical level. We will determine the lipid and lipoprotein profile in LDLR-targeted pigs and perform morphometric analysis to determine the presence and extent of atherosclerosis. Finally, we will establish breeding herds to generate LDLR-/- pigs and to expand and propagate the colony. This project will produce a porcine model of atherosclerosis that will provide academic and industry researchers with an opportunity to better understand the disease and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this costly and deadly disease. PUBLIC HEALTH RELEVANCE: This proposal specifically outlines the development, characterization and propagation of a genetically engineered porcine model of atherosclerosis. This project is relevant to the NIH's mission because it will provide a resource to stimulate discovery, therapeutic application, and the development of new diagnostic tools.