Animal experimental and human atherosclerosis studies have convincingly demonstrated that low- density lipoprotein (LDL) undergoes oxidation, which greatly enhances its atherogenicity. One of the major pathways of LDL oxidation in vivo is the oxidation catalyzed by 12/15-lipoxygenase (12/15LO). Because oxidized LDL (OxLDL) induces many inflammatory responses in the vascular wall, high levels of so-called oxidation-specific epitopes in atherosclerotic lesions often indicate that these lesions are prone to rupture, inducing myocardial infarction or stroke. Identification of vulnerable atherosclerotic plaques prone to rupture is a major challenge for cardiovascular imaging, as current imaging techniques provide little information on plaque composition. Our group is currently developing new imaging approaches using antibodies that specifically bind oxidation-specific epitopes. In addition to imaging applications, oxidation-specific antibodies are emerging as a therapeutic treatment of atherosclerosis because they prevent OxLDL's inflammatory effects. In this application, we propose to develop new zebrafish (Danio rerio) models to study mechanisms and pathologic effects of lipoprotein oxidation, new imaging techniques targeting oxidation-specific epitopes in the vasculature of live animals, as well as novel therapeutic strategies to diminish lipoprotein oxidation and its pathologic effects. Specifically, we propose: (1) To develop imaging and analytic techniques for in vivo and ex vivo study of lipoprotein oxidation and detection of oxidation-specific epitopes in vascular lesions. We will target three common classes of oxidation-specific epitopes: malondialdehide, oxidized phospholipids and oxidized cholesteryl esters. In one approach, we will inject i.v. zebrafish larvae with fluorescently labeled antibodies and use a confocal microscope to detect antibodies binding in vascular lesions. The second approach will be to generate transgenic zebrafish with conditional expression of GFP-labeled oxidation-specific antibodies. We will also use mass spectrometry techniques to identify oxidized lipids in apoB and apoA1 lipoproteins as well as those bound to oxidation-specific antibodies. (2) To create a model of 12/15LO-induced lipoprotein oxidation and to study in vivo its role in vascular lipid accumulation and inflammation. Vascular lesions in transgenic zebrafish with endothelial or myeloid cell specific expression of human 12/15LO will be analyzed for accumulation of oxidized lipids, macrophage recruitment and foam cell formation. (3) To test the therapeutic potential of oxidation-specific antibodies, 12/15LO inhibitors and antioxidants. In summary, in order to contribute to "enhancing zebrafish research with research tools and techniques", we propose to use the zebrafish model, including new transgenic lines as well as state-of-art imaging and mass spectrometry techniques, to study an important atherogenic process - lipoprotein oxidation.
In this application we propose to create a zebrafish model of vascular inflammation and atherosclerosis, the disease that causes heart attack and stroke. The optical transparency of zebrafish larvae will enable dynamic monitoring of the processes of lipoprotein oxidation, vascular lipid accumulation and inflammation in a live animal. Successful development of a zebrafish model of atherosclerosis will help significantly enhance mechanistic studies of atherosclerosis as well as radically advance screening for new therapies.
|Willeit, Peter; Kiechl, Stefan; Kronenberg, Florian et al. (2014) Discrimination and net reclassification of cardiovascular risk with lipoprotein(a): prospective 15-year outcomes in the Bruneck Study. J Am Coll Cardiol 64:851-60|
|Fang, Longhou; Liu, Chao; Miller, Yury I (2014) Zebrafish models of dyslipidemia: relevance to atherosclerosis and angiogenesis. Transl Res 163:99-108|
|Miller, Yury I; Tsimikas, Sotirios (2013) Oxidation-specific epitopes as targets for biotheranostic applications in humans: biomarkers, molecular imaging and therapeutics. Curr Opin Lipidol 24:426-37|
|Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun et al. (2013) Control of angiogenesis by AIBP-mediated cholesterol efflux. Nature 498:118-22|
|Miller, Yury I; Choi, Soo-Ho; Wiesner, Philipp et al. (2011) Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity. Circ Res 108:235-48|
|Fang, Longhou; Green, Simone R; Baek, Ji Sun et al. (2011) In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish. J Clin Invest 121:4861-9|
|Fang, Longhou; Harkewicz, Richard; Hartvigsen, Karsten et al. (2010) Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high cholesterol diet: macrophage binding and activation. J Biol Chem 285:32343-51|
|Stoletov, Konstantin; Fang, Longhou; Choi, Soo-Ho et al. (2009) Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish. Circ Res 104:952-60|
|Miller, Yury I; Choi, Soo-Ho; Fang, Longhou et al. (2009) Toll-like receptor-4 and lipoprotein accumulation in macrophages. Trends Cardiovasc Med 19:227-32|