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 2

Transgenic hsp70:IK17-EGFP and hsp70:TT-EGFP zebrafish.

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Transgenic hsp70:IK17-EGFP and hsp70:TT-EGFP zebrafish. (A) HEK293 c...
(A) HEK293 cells were transiently transfected with EGFP or IK17-EGFP, and 48 hours after transfection, the supernatants were analyzed for binding with MDA-LDL and OxLDL in a microplate assay with an anti-GFP detection antibody. Two independent experiments were performed in quadruplicates. (B) Diagram showing the hsp70:IK17-EGFP and hsp70:TT-EGFP constructs used to generate transgenic lines. Tol2, a transposable DNA element; ss, secretion signal; pA, polyA sequence. (C) Founder hsp70:IK17-EGFP and hsp70:TT-EGFP zebrafish were genotyped with IK17 and TT primers. The hairline splices denote lanes that were run on the same gel but were noncontiguous. NC, negative control; PC, positive control. (D) Two dpf F1 larvae of hsp70:IK17-EGFP and hsp70:TT-EGFP zebrafish were subjected to heat shock (HS). EGFP fluorescence was accessed 24 hours after heat shock. NHS, the larvae that were not subjected to heat shock. Dotted lines trace contours of the larvae that were not subjected to heat shock. Scale bar: 100 μm. (E) One group of 3 dpf hsp70:IK17-EGFP and hsp70:TT-EGFP larvae was subjected to heat shock, while the other group was not subjected to heat shock. Two days later, 30 fish from each group were homogenized; the homogenates were cleared by centrifugation and filtration, diluted 1:50, and tested for binding to MDA-LDL and OxLDL in a microplate assay with an anti-GFP detection antibody. Two independent experiments were performed in quadruplicates.