Objectives:
Published results show that overfeeding zebrafish larvae on a high-cholesterol diet (HCD) can result in hypercholesterolemia and sub-endothelial lipid deposition in macrophages and other cell types. However, results are so far based on small samples, and the atherogenic response has been heterogeneous. We aim to use zebrafish larvae for large-scale, CRISPR-Cas9-based genetic screens, using results from genome wide association studies for coronary heart disease as a starting point. Firstly however, we need to ensure the model system is appropriate and robust. Therefore, we examined the effect of a high-energy diet (HED) and HCD on vascular lipid deposition in a larger number of larvae (n=241).
Methods:
Starting at 5 days post fertilization (dpf), ~30 larvae/tank were fed 2x/day on: 1) 5 mg control diet (CD; n=33); 2) 15 mg control diet (HED; n=90); or 3) 15 mg control diet enriched with 4% cholesterol (HCD; n=94). At 14-17 dpf, larvae were soaked in monodansylpentane cadaverase - a lipid staining dye - for 45 min, before imaging the dorsal aorta and caudal vein with a Leica SP5 confocal microscope. We used a custom written script in Cell Profiler to quantify the surface area of lipid deposits in the vasculature.
Results:
Manual annotation of vascular lipid deposition in 30 images (10 randomly selected images per dietary condition) allowed us to calculate the sensitivity (36%) and specificity (71%) of the Cell Profiler script. Subsequent analyses showed that HED (p=0.004) and HCD (p=0.001) fed larvae have significantly more vascular lipid deposition than CD fed larvae after adjusting for age, batch and vessel length. There was no difference in vascular lipid deposition between HED and HCD fed larvae (p=0.11).
Discussion and conclusion:
Our results confirm that zebrafish larvae represent a promising model system for early-stage atherosclerosis. In addition, they show that enriching the diet with cholesterol is not required to prompt atherogenesis.
Future directions:
In the next few months, we will examine if overfeeding also triggers vascular infiltration by macrophages, neutrophils and oxidized LDL cholesterol, and if atherogenesis can be prevented or reduced by treating larvae with statins and/or ezetimibe, using our new, automated imaging setup.
Characterization, Variables, and Antioxidant Activity of the Maillard Reaction in a Fructose–Histidine Model System