In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish
Longhou Fang, Simone R. Green, Ji Sun Baek, Sang-Hak Lee, Felix Ellett, Elena Deer, Graham J. Lieschke, Joseph L. Witztum, Sotirios Tsimikas, Yury I. Miller
Longhou Fang, Simone R. Green, Ji Sun Baek, Sang-Hak Lee, Felix Ellett, Elena Deer, Graham J. Lieschke, Joseph L. Witztum, Sotirios Tsimikas, Yury I. Miller
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Technical Advance Cardiology

In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish

Abstract

Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock–induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP–expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans.

Authors

Longhou Fang, Simone R. Green, Ji Sun Baek, Sang-Hak Lee, Felix Ellett, Elena Deer, Graham J. Lieschke, Joseph L. Witztum, Sotirios Tsimikas, Yury I. Miller

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Figure 6

Expression of IK17-EGFP attenuates vascular lipid accumulation.

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Expression of IK17-EGFP attenuates vascular lipid accumulation. (A) hsp7...
(A) hsp70:IK17-EGFP and hsp70:TT-EGFP zebrafish larvae were fed a HCD or control diet for 10 days. One group of HCD-fed zebrafish was subjected to heat shock 2 days before the start of feeding and then every 4–5 days to sustain IK17-EGFP or TT-EGFP expression levels. The other group was not subjected to heat shock at any time and, thus, did not express the transgene. Two days before imaging, the diet was switched to a diet supplemented with 10 μg/g cholesteryl BODIPY 576/589 C11, and then fluorescent lipid deposits were quantified. The results are expressed as the percentage of the area of lipid deposits normalized to the area of the caudal vein segment. Mean ± SEM (21–34 animals in each hsp70:IK17-EGFP zebrafish group and 10–19 animals in each hsp70:TT-EGFP group). *P < 0.001 for IK17/HCD versus either IK17/control or IK17/HCD/heat shock. (B) Zebrafish larvae were fed a HCD for 5 days, and lipid deposits were imaged and quantified. The animals were subjected to heat shock after the imaging session on the fifth day and then again on the eighth day. The animals were imaged again on the tenth day, and lipid deposits were quantified. The results are expressed as the percentage area of lipid deposits per caudal vein segment. Mean ± SEM (12–21 animals in each group at each time point). *P < 0.05.