: The goal of this proposal is to obtain proof for and to further characterize a novel epitope on oxidized LDL (a cyclized derivative of peroxidized poly- unsaturated fatty acid) that binds to amine groups, that is antigenic and biologically active. Evidence for the presence of the adduct was published in a 1992 paper and has been extended in the extensive pilot studies in the present submission. This evidence includes increased fluorescence as well as incorporation of isotopically labeled fatty acid into protein fractions.The applicant proposes to study mechanisms by which apolipoprotein B (apoB) and amino phospholipids (phosphatidylethanolamine) of LDL play a role in the propagation of lipid peroxidation and in the generation of oxidized LDL. He hypothesizes that modification of protein by lipid peroxides generates novel antigenic epitopes. There are 4 Specific Aims.
Specific Aim 1 is to demonstrate that apoB plays an active role in the oxidation process by initiating as well as propagating lipid peroxidation. Peroxidase mediated oxidation of LDL may involve the generation of apoB-derived radicals which may initiate peroxidation of LDL lipids. Experiments are proposed to determine optimum conditions for the formation of fluorescent epitopes between peroxidized lipids (LOOH) and """"""""amino compounds"""""""". He proposes to establish the components of the modification process: the peroxide moiety and the amino acceptor. A variety of potentially relevant peroxides will be tested, of which 7 compounds are listed in Box 1 of the proposal. These will be incubated with various amine-containing compounds including soluble proteins (e.g., BSA), polyamino acids, amino acids, apolipoproteins and alkylamines. Some incubations will be conducted in the presence of antioxidants. A number of peptides from apo B and albumin, especially from lysine-rich regions of these proteins, will be tested for their ability to form fluorescent products. Controls will include 9 compounds: non-lysine amino acids non-amino containing lipids, among others. Reactions will be conducted in the presence or absence of metal ion chelators. As a functional correlate, he will assess the ability of these peptides to induce IL- 1 synthesis in monocytes and to elicit chemotaxic responses from monocyte/macrophages. Simple, water soluble amino compounds will be tested for their ability to foster LDL oxidation. These experiments will address the question of whether amine groups in LDL can participate in LDL oxidation, and whether buried amino groups in close proximity to fatty acyl chains get modified in preference to surface exposed amino groups. The role of nucleophilicity in this reaction will be tested using tertiary amines (e.g., trimethylamine). Other experiments will characterize the contribution of LDL-derived radicals in the peroxidase-mediated oxidation of LDL. Metal ion catalyzed oxidation, the latter being probably more physiologically relevant.
Specific Aim 2 is to test the proposal that phosphatidylethanolamine is oxidatively modified during the oxidation of LDL. Using phospholipid analogs and specific phospholipases, he proposes to establish the nature of the modified phosphatidylethanolamine. He hypothesizes that close proximity of the amino group and the acyl chain renders this molecule especially prone to oxidative modification. Box 4 of the application lists 14 phospholipids, some containing and some lacking amino groups. The ability of oxidized phosphatidlycholine and peroxidized fatty to form fluorescent compounds with the amino compounds will be tested. Enzymatically modified lipids (e.g., by immobilized soybean lipoxygenase modified lineoyl phosphatidylethanolamine. The scheme will be tested by a comparison of the products with authentic synthesized lipid standards, and by analysis of reaction of the products with phospholipase A2 and C.
Specific Aim 3 is to define two major unexplored phospholipid modifications: 1) the modification of the amino function of phosphatidylethanolamine by lipid peroxides, analogous to protein modification that generates a fluorescent chromophore; and 2) a phosphatidylethanolamine or -choline molecule with a shortened dicarboxylic acid function at the sn-2 position. He proposes, further, that such anionic phospholipid compounds may have antigenic and biological properties. Box 5 of the proposal lists 9 such compounds for study. Modified lipids will be reconstituted into vesicles with dipalmitoyl lecithin (no double bonds). And the ability of these vesicles to interact with the scavenger receptor on macrophages will be assessed. In other experiments, he will monitor the accumulation of fluorescent lipids in macrophages. In collaboration with Dr. Russell Medford, he will study the induction of VCAM- 1 caused by oxidized LDL and the specific oxidized phospholipids.
Specific Aim 4 is to generate and characterize antibodies that are directed towards the fluorescent LOOH-modified amino groups in proteins and in phosphatidylethanolaniine. He also proposes to generate antibodies against specific dicarboxylic acid containing phospholipids. He will then determine whether such antibodies react against epitopes in oxidized LDL, plasma and atherosclerotic plaques.
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