Abstract 19716: Zebrafish Larvae as a Model System For High-throughput Screens of Early-stage Atherosclerosis

Abstract

Introduction: A high-cholesterol diet has been reported to induce vascular deposition of lipids and oxidized LDL cholesterol in macrophages and other cell types in 15 day-old zebrafish larvae. However, results were based on small samples, and little is known about the heterogeneity of effect within and between conditions, the image quantification procedure used, and the influence of dietary energy. We intend to perform large-scale genetic and compound screens for coronary heart disease, and aimed to confirm or refute if zebrafish larvae are a suitable model system. Methods: In total, 940 transgenic zebrafish larvae were fed on one of six diets between 5 and 10 days post-fertilization (dpf), to examine main effects of, and interactions between dietary energy and cholesterol. At 10 dpf, we assessed body length and acquired optical sections of the dorsal aorta and caudal vein using an automated positioning system, fluorescence microscope and CCD camera. Atherogenic traits were quantified using a custom-written image analysis pipeline in Ilastik, CellProfiler and ImageJ, and larvae were used to assess whole body levels of total cholesterol, triglycerides and glucose using enzymatic assays. Associations were adjusted for body length, ether - used to prepare the high-cholesterol diet - and batch. Results: A high-energy diet resulted in elevated triglyceride levels (p=7.8E-3) - especially in the presence of high dietary cholesterol (p-interaction=0.01) - and in more vascular infiltration by lipids (p=1.0E-12), and co-localising lipids and macrophages (p=0.04), independently of dietary cholesterol. A high-cholesterol diet resulted in higher glucose levels (p=2.1E-3), and in more vascular infiltration by lipids (p=0.01), and co-localising lipids and neutrophils (p=0.04), and lipids, neutrophils and macrophages (p=0.01), independently of dietary energy. Qualitative analyses showed that vascular lipid deposits typically contain oxidised LDL cholesterol, and are located at bifurcation sites of the endothelium. Conclusions: Zebrafish larvae represent a promising model system for high-throughput, image-based screens of early-stage atherosclerosis, and dietary energy and cholesterol induce distinct atherogenic traits as early as 10 dpf.