The overall goal of this proposal is to synthesize and use for biologic studies a novel bioactive epoxy isoprostane phospholipid that may play an important role the development of atherosclerotic lesions. 1-Palmitoyl-2- (5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC), mass 828.5, is present in minimally oxidized LDL (MM-LDL) and increased in atherosclerotic lesions. PEIPC is one of five 828.5 isomers in MM-LDL and in preliminary studies causes a strong activation (compared to the other relatively inactive isomers) of endothelial cell to bind monocytes in vitro. The proposed studies will test the hypothesis that PEIPC activates endothelial cells by interacting with a PAF-like receptor and will characterize binding to this receptor. Most of our preliminary studies of molecules of this mass have been done with mixed isomers. Isomers of 828.5 were shown to highly accumulate in HDL (compared to other bioactive phospholipids) probably because HDL enzymes less easily degrade isomers of 828.5 than other bioactive phospholipids. In preliminary studies we have demonstrated that HDL, shown by others to be less protective against endothelial activation, contained increased levels of 828.5. HDL has been shown to bind at least one of the scavenger receptors. Studies by the Witztum group have shown that l-Palmitoyl-2- (oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), another bioactive phospholipid with antigenic similarity to PEIPC, also binds to macrophage scavenger receptors. We will test the hypothesis that, in endothelial cells, PEIPC binds to the major scavenger receptor LOX-l blocking its normal interaction with HDL. Finally there is evidence that some oxidized phospholipids may bind to proteins and interact with cells in this adducted form. The structure of PEIPC suggests that it may bind to proteins under physiological or pathophysiological conditions. Such adducted phospholipids may be more stable than free bioactive phospholipids. We will test the hypothesis that PEIPC forms adduct with proteins and peptides; and we will perform limited studies using these adducts. Key to carrying out these studies is the availability of a reliable supply of PEIPC and the ability to obtain radiolabeled molecules. This proposal is a combined submission from a chemist with extensive experience in the synthesis of epoxide containing molecules, a cell biologist experienced with testing lipid endothelial interactions and a coinvestigator experienced in both areas. In summary, this interdisciplinary proposal will provide important information about the receptor interactions of the novel epoxy isoprostane phospholipid PEIPC.
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