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J. Biol. Chem., Vol. 283, Issue 16, 10241-10251, April 18, 2008

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Cholesteryl Ester Hydroperoxides Are Biologically Active Components of Minimally Oxidized Low Density Lipoprotein^*

Richard Harkewicz, Karsten Hartvigsen, Felicidad Almazan, Edward A. Dennis, Joseph L. Witztum, and Yury I. Miller¹

From the Departments of Pharmacology, Chemistry and Biochemistry and Medicine, University of California, San Diego, La Jolla, California 92093

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE^–/– mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.

Received for publication, November 1, 2007 , and in revised form, February 7, 2008.

^* This work was supported by LIPID MAPS Large Scale Collaborative Grant GM069338 (to R. H., E. A. D., J. L. W., and Y. I. M.), Grant HL081862 (to Y. I. M.) from the NHLBI, National Institutes of Health, Grants 0530159N (to Y. I. M.) and 0630228N (to K. H.) from the American Heart Association, and a grant from the Leducq Fondation (to J. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3.

¹ To whom correspondence should be addressed: University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Fax: 858-534-2005; E-mail: yumiller{at}ucsd.edu.

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