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 1

Binding of recombinant IK17 to vascular lipid deposits in HCD-fed larvae.

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Binding of recombinant IK17 to vascular lipid deposits in HCD-fed larvae...
(A) Binding of fluorescently labeled IK17-Alexa488 and TT-Alexa488 to MDA-LDL and OxLDL was tested in a microplate immunoassay with anti-HA antibody detection, as described in Methods. Two independent experiments were performed in quadruplicates. nLDL, native LDL. (B) Zebrafish larvae were fed a HCD or control diet supplemented with 10 μg/g cholesteryl BODIPY 576/589 C11 for 14 days. At the end of the feeding period, larvae were injected into the caudal vein with 2 nl 10 ng/μl IK17-Alexa488 or TT-Alexa488. Twenty-four hours after injection, larvae were anesthetized and imaged live under a confocal microscope. Lipid deposits (bright red) and sites of IK17-Alexa488 binding (green) partially colocalize (yellow) in the wall of the caudal vein. Diffuse red fluorescence in the lumen is from circulating lipid marker associated with plasma lipoproteins; its intensity varies depending on the cholesterol content in diet. DA, dorsal aorta; CV, caudal vein. Scale bar: 25 μm. Representative images from 4 animals in each group.