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Oxidized Cholesteryl Esters and Phospholipids in Zebrafish Larvae Fed a High Cholesterol Diet MACROPHAGE BINDING AND ACTIVATION* * This work was supported, in whole or in part, by National Institutes of Health Grants HL093767 (to Y. I. M. and L. F.), HL081862 (to Y. I. M.), GM069338 (to E. A. D., J. L. W., R. H., and Y. I. M.), and HL088093 (to J. L. W. and Y. I. M.). This work was also supported by University of California Tobacco-related Disease Program Fellowship 18FT-0137 (to L. F.) as well as a grant from the Leducq Foundation (to J. L. W. and Y. I. M.). The University of California, San Diego Cancer Center Shared Imaging Resource is funded by National Institutes of Health Specialized Support Grant P30 CA23100.

UC San Diego Previously Published Works (2010)

A novel hypercholesterolemic zebrafish model has been developed to study early events of atherogenesis. This model utilizes optically transparent zebrafish larvae, fed a high cholesterol diet (HCD), to monitor processes of vascular inflammation in live animals. Because lipoprotein oxidation is an important factor in the development of atherosclerosis, in this study, we characterized the oxidized lipid milieu in HCD-fed zebrafish larvae. Using liquid chromatography-mass spectrometry, we show that feeding an HCD for only 2 weeks resulted in up to 70-fold increases in specific oxidized cholesteryl esters, identical to those present in human minimally oxidized LDL and in murine atherosclerotic lesions. The levels of oxidized phospholipids, such as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine, and of various lysophosphatidylcholines were also significantly elevated. Moreover, lipoproteins isolated from homogenates of HCD-fed larvae induced cell spreading as well as ERK1/2, Akt, and JNK phosphorylation in murine macrophages. Removal of apoB-containing lipoproteins from the zebrafish homogenates with an anti-human LDL antibody, as well as reducing lipid hydroperoxides with ebselen, resulted in inhibition of macrophage activation. The TLR4 deficiency in murine macrophages prevented their activation with zebrafish lipoproteins. Using biotinylated homogenates of HCD-fed larvae, we demonstrated that their components bound to murine macrophages, and this binding was effectively competed by minimally oxidized LDL but not by native LDL. These data provide evidence that molecular lipid determinants of proatherogenic macrophage phenotypes are present in large quantities in hypercholesterolemic zebrafish larvae and support the use of the HCD-fed zebrafish as a valuable model to study early events of atherogenesis.

Cover page: Oxidized Cholesteryl Esters and Phospholipids in Zebrafish Larvae Fed a High Cholesterol Diet MACROPHAGE BINDING AND ACTIVATION* * This work was supported, in whole or in part, by National Institutes of Health Grants HL093767 (to Y. I. M. and L. F.), HL081862 (to Y. I. M.), GM069338 (to E. A. D., J. L. W., R. H., and Y. I. M.), and HL088093 (to J. L. W. and Y. I. M.). This work was also supported by University of California Tobacco-related Disease Program Fellowship 18FT-0137 (to L. F.) as well as a grant from the Leducq Foundation (to J. L. W. and Y. I. M.). The University of California, San Diego Cancer Center Shared Imaging Resource is funded by National Institutes of Health Specialized Support Grant P30 CA23100.