Zebrafish models of vascular inflammation and atherosclerosis

这项建议的目的是建立适合于体内研究某些动脉粥样硬化形成过程的斑马鱼(Danio Rerio)模型。人们普遍认为，高胆固醇血症会导致动脉中的脂质堆积，进而引发血管炎症，并引发动脉粥样硬化的发展，而动脉粥样硬化是人类心脏病发作和中风的主要原因。因此，目前对动脉粥样硬化的实验动物研究使用了小鼠和兔子，在这些动物中，基因改造和高脂饮食的组合导致了极端的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兔子的一个重要限制是，只有在死后才能对动脉粥样硬化病变进行显微镜检查。使用斑马鱼的一个重要优势是其幼体的光学透明性。我们认为，利用斑马鱼研究动脉粥样硬化形成的某些方面，将能够监测活体动物动脉的病理过程。 具体地说，在目标1中，我们将建立一个血管炎症和动脉粥样硬化的模型，在该模型中，以高胆固醇饲料喂养斑马鱼会导致高胆固醇血症、脂蛋白氧化和血管壁脂沉积。利用成年斑马鱼，我们将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使监测脂质积累和巨噬细胞重新聚集到血管壁的动力学成为可能。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。发展高胆固醇血症幼虫模型将使我们能够建立体内功能分析来测量活斑马鱼幼虫中基质金属蛋白酶、组织蛋白和磷脂酶的活性(目标2)。为了证明这一独特模型的实用性，我们将讨论在动脉粥样硬化发展中至关重要的特定机制。在目标3中，我们将测试脂蛋白氧化在血管脂质堆积和炎症中的重要性。具体地说，抗氧化剂将被添加到鱼的饲料中或溶解在鱼缸水中，以及它们对转基因斑马鱼血管中脂肪和巨噬细胞沉积的影响，并将研究特定的酶活性。总之，我们认为，建立斑马鱼的血管炎症和动脉粥样硬化模型将有助于显著加强动脉粥样硬化的机制研究以及新疗法的设计和筛选。

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.