The goal of these studies is to determine the importance of specific phospholipid oxidation products, 1- palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) as regulators of endothelial cell inflammatory responses. These phospholipids have been shown to increase monocyte-endothelial and inhibit neutrophil-endothelial interactions by non-classical signal transduction pathways. The in vivo importance of these phospholipids is suggested by their accumulation in atherosclerotic lesions and at other sites of chronic inflammation. We have identified candidate signal transduction pathways by which these bioactive lipids alter endothelial cell function and have obtained evidence for the involvement of more than one receptor in their effects on transcription of IL-8 and MCP-1 and monocyte binding. A major goal of this proposal is to complete identification of these receptors and signal transduction pathways.
In Aim 1 we will test the hypothesis that PEIPC binds to a receptor complex, composed of a GPI anchored protein and a modified form of Toll 4, which alters caveolar function. These caveolar changes are hypothesized to result in activation of Src and/or SREBP. As a result LEF and/or SREBP are translocated into the nucleus resulting in activation of IL-8 transcription. We have identified cAMP and its downstream effector R-Ras as important signals activating endothelial cells to bind monocytes.
In Aim 2 we will express both known and orphan lipid-binding G-protein coupled receptors to identify the PEIPC receptor mediating monocyte binding. The identified receptors will be overexpressed in mouse aorta, using adenovirai transfection, and their effect on fatty streak formation determined. We will also determine, using quantitative morphology and immunohistochemistry, if the proposed signal transduction pathways are altered in atherosclerosis.
In Aim 3 we will test the hypothesis that the accumulation of phosholipid oxidiation products at sites of inflammation blocks neutrophil entry. For these studies we will use cell culture and also a mouse model involving bacterial endopthalmitis. To test the role of oxidized phospholipid accumulation, we will over and underexpress PON-2, an enzyme that hydrolyzes these oxidized phospholipids. The proposed studies will determine the importance of POVPC and PEIPC as mediators of atherosclerosis and other chronic inflammatory processes.
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