Cardiovascular disease and stroke are the two leading causes of death in the United States. Atherosclerosis, being the central cause of cardiovascular disease and stroke, is the process by which macrophages accumulate oxidized lipids, cholesterol, and other fatty substances under the endothelium of blood vessels. While the clinical consequences of atherosclerosis are clear, the complex biochemical pathways that cause it are not well understood. The oxidation of low-density lipoproteins (LDL) is a key step in the build up of atherosclerotic plaques. Oxidized lipid signaling initiates inflammatory activation of vascular endothelial cells, which promotes the recruitment of monocytes. As these mononcytes mature into macrophages, they accumulate large amounts of oxidized lipid. Recent research has shown that peroxisome proliferator-activated receptors (PPARs) are key activators of genes involved in lipid uptake and cholesterol efflux from macrophages. Thiazolidinediones, a class of insulin-sensitizing drugs, are high-affinity PPAR activators and thought to reduce atherosclerosis in mouse models. While oxidized fatty acids are low affinity activators of PPAR, no high-affinity natural oxidized lipid ligand has been identified. The purpose of this research is to search for natural ligand activators of PPARs. First, arachidonic acid was auto-oxidized for 24-48 hours, which was monitored by periodic analysis using flow injection negative ion electrospray ionization-mass spectrometry (ESI-MS). Once the arachidonic acid (Ox-AA) reached an appropriate level of oxidation, it was resuspended in methanol/water (1:1, v/v) containing 1 mM ammonium acetate and separated by liquid chromatography. Once the individual Ox-AA products were collected, they were tested for their ability to activate PPAR. The PPAR activation used modified HeLa cells that contained PPAR receptors linked to a luciferase reporter system. The degree of activation was assessed by the luminescence produced by luciferin. The exact chemical structures of the Ox-AA products that are high-affinity ligands of PPAR were analyzed by mass spectrometry. Elucidating the complex biochemical pathways of atherosclerosis is important to the treatment of cardiovascular disease and stroke. Identifying the natural ligands of PPARs is a crucial step in understanding atherosclerosis, which will give us insight into the physiological role of PPAR and may potentially provide new targets for therapies.
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