The goal of these studies is to complete synthesis of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-snglycero-3-phosphoryl choline (PEIPC), identify the mechanism by which PEIPC interacts with proteins and to test the hypothesis that covalent binding of PEIPC to its receptor is responsible for its potent inflammatory activity. We have shown that isomers of PEIPC potently activate endothelial cells to synthesize chemokines IL-8 and MCP-1 and express the monocyte binding molecule CS-1 containing fibronectin. Supporting an inflammatory role in vivo, PEIPC has been demonstrated to accumulate in lipoproteins from animals fed a high fat diet, in atheroscierotic lesions, in dying cells and in cells treated with cytokines. PEIPC is a difficult molecule to isolate from lipid mixtures in sufficient quantity for experimentation. We have therefore developed methods for the synthesis of the 5,6 epoxy isomer of PEIPC. We have completed the most complex aspect of the synthesis of the epoxyisoprostane group of this molecule. The proposed studies will complete total synthesis of 5,6 epoxy isomer by linking the isoprostane to commercially available lysophosphatidylcholine. During the current grant period we have made the important observation that PEIPC at neutral pH can covalently link to a number of cell membrane proteins, one of which is likely its receptor. We will identify the reactive functionality of PEIPC by a) determining the structure of the products of its reaction with candidate amino acids (e.g. thiol, amine and guanidine) b) identifying the peptide sequence of selected proteins to which PEIPC binds. We will prepare several close structural analogues of PEIPC in which the reactive functional groups are replaced by structurally similar but less reactive groups in order to maintain reasonable binding to the receptor without reacting with it. These compounds will be tested for their ability to stimulate endothelial inflammatory activities and inhibit PEIPC activation of endothelial cells to synthesize chemokines (MCP1 and IL-8) and to induce deposition of the monocyte binding molecule fibronectin on the endothelial cell surface. The successful completion of these studies will provide insight into the mechanisms of action of PEIPC a potent inflammatory mediator and will provide a potential therapeutic approach to limit the inflammatory effects of this lipid in atherooscierosis and other inflammatory diseases.
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