Zebrafish models of vascular inflammation and atherosclerosis

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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.