Oxidized LDL (OxLDL) is atherogenic because unregulated uptake in macrophages leads to foam cell formation. However, oxidized lipids of minimally modified (oxidized) LDL (mmLDL), representative of the early stages of LDL oxidation generated by exposure to cells, cause profound proinflammatory and proatherogenic changes. We generated a model mmLDL by exposure of LDL to cells overexpressing 15-lipoxygenase and demonstrated this has profound biological effects on macrophages. Yet little is known of the specific oxidized lipids responsible for these properties. In parallel studies, we will identify oxidized lipids that mediate binding of OxLDL to macrophage scavenger receptors, such as CD36. The goals of Bridge D are to identify specific oxidized lipids and synthesize stable, biologically active analogs that have proinflammatory and proatherogenic properties on macrophages, which would then be recommended for generalized study by the LIPID MAPS project. Our specific goals are: To identify and characterize biologically active oxidized lipid moieties in mmLDL and to determine their effects on relevant aspects of macrophage biology: HPLC fractions of lipids of mmLDL will be studied for biological effects on macrophages and studied by mass spectroscopy to identify candidate oxidized lipid moieties. Based on work to date, our initial focus will be on specific oxidized cholesteryl esters. We will study oxidized methyl fatty acid esters as models, as well as isolated cholesteryl esters. We will document their relevance by demonstrating their presence in atherosclerotic lesions. As time permits, we will also study other oxidized lipids in mmLDL. To identify and synthesize stable analogs of oxidized lipids that mediate binding of OxLDL to macrophage scavenger receptors and to determine their biological effects. We will identify oxidized lipids responsible for the binding of OxLDL to CD36 and SR-A. Together with Core F(B), we will design, synthesize and validate stable, small molecule analogs that mimic the activity of the natural ligand. We will prepare analogs that can (or cannot) be internalized, and thus allow assessment of the role of the receptor in cell signaling, e.g. either directly or via internalization of its ligand or (both). Our initial focus will be on synthetic analogs of the oxidized phospholipid POVPC, which binds to CD36. As time permits, we will study analogs of malondialdehyde, which binds to SR-A. Unique products with relevant biological activity will be recommended for generalized study by the LIPID MAPS project.
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