Zebrafish models of vascular inflammation and atherosclerosis

本提案的目的是建立斑马鱼（Danio rerio）模型，适用于动脉粥样硬化形成的某些过程的体内研究。人们普遍认为，高胆固醇血症导致动脉中的脂质积聚，这反过来又诱导血管炎症并引发动脉粥样硬化的发展，动脉粥样硬化是人类心脏病发作和中风的主要原因。因此，目前动脉粥样硬化的实验动物研究使用小鼠和兔子，其中遗传修饰和高脂肪饮食的组合诱导极端高胆固醇血症和动脉粥样硬化的快速发展。在这些研究中使用小鼠和家兔的一个重要限制是，动脉粥样硬化病变的显微镜检查只能在死后进行。使用斑马鱼的一个重要优势是其幼虫的光学透明度。我们建议使用斑马鱼研究动脉粥样硬化的某些方面，将能够监测动脉中的病理过程在活体动物。 具体而言，在目标1中，我们将建立血管炎症和动脉粥样硬化的模型，其中给斑马鱼喂食高胆固醇饮食导致血管壁中的高胆固醇血症、脂蛋白氧化和脂质积累。我们将利用成年斑马鱼建立一种新的动物模型来研究体内脂蛋白氧化。斑马鱼幼体的光学透明度将能够监测脂质积累和巨噬细胞募集到血管壁中的动力学。在这些研究中将使用在内皮细胞和巨噬细胞中表达荧光蛋白的转基因斑马鱼。开发高胆固醇血症的幼虫模型将使我们能够建立体内功能测定，以测量活斑马鱼幼虫中基质金属蛋白酶，组织蛋白酶和磷脂酶的活性（目的2）。为了证明这种独特的模型的实用性，我们将解决一个特定的机制，在动脉粥样硬化的发展至关重要。在目标3中，我们将测试脂蛋白氧化在血管脂质积聚和炎症中的重要性。具体而言，将抗氧化剂添加到鱼饲料中或溶解在鱼缸水中，并研究其对转基因斑马鱼血管系统中脂质和巨噬细胞沉积的影响，以及特定的酶活性。综上所述，我们认为，建立血管炎症和动脉粥样硬化的斑马鱼模型将有助于显着提高动脉粥样硬化的机制研究，以及新的治疗方法的设计和筛选。

The aim of this proposal is to establish zebrafish (Danio rerio) models suitable for in vivo studies of certain processes of atherogenesis. It is widely accepted that hypercholesterolemia leads to lipid accumulation in arteries, which in turn induces vascular inflammation and initiates the development of atherosclerosis, the major cause of heart attacks and stroke in humans. Hence, current experimental animal studies of atherosclerosis use mice and rabbits in which a combination of genetic modification and high-fat diet induce extreme hypercholesterolemia and rapid development of atherosclerosis. An important limitation of using mice and rabbits in these studies is that microscopic examination of atherosclerotic lesions is possible only postmortem. An important advantage of using zebrafish is the optical transparency of its larvae. We propose that using zebrafish for studying certain aspects of atherogenesis will enable monitoring the pathologic processes in arteries in a live animal. Specifically, in Aim 1 we will establish a model of vascular inflammation and atherosclerosis in which feeding zebrafish a high-cholesterol diet causes hypercholesterolemia, lipoprotein oxidation and lipid accumulation in the vascular wall. Using adult zebrafish, we will develop a novel animal model to study in vivo lipoprotein oxidation. The optical transparency of zebrafish larvae will enable monitoring the kinetics of lipid accumulation and macrophage recruitment into the vascular wall. Transgenic zebrafish with fluorescent proteins expressed in endothelial cells and in macrophages will be used in these studies. Developing the hypercholesterolemic larvae model will allow us to set up in vivo functional assays to measure the activity of matrix metalloproteinases, cathepsins and phospholipases in live zebrafish larvae (Aim 2). To demonstrate the utility of this unique model we will address a specific mechanism, critical in the development of atherosclerosis. In Aim 3, we will test the importance of lipoprotein oxidation in vascular lipid accumulation and inflammation. Specifically, antioxidants will be added to the fish diet or dissolved in the fish tank water and their effects on lipid and macrophage deposition in the vasculature of transgenic zebrafish, and specific enzyme activities will be studied. In summary, we propose that establishing zebrafish models of vascular inflammation and atherosclerosis will help significantly enhance mechanistic studies of atherosclerosis as well as the design and screening of new therapies.