MOLECULAR BASIS OF OXIDATIVE MODIFICATION OF LDL

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Substantial evidence suggests that the accumulation and subsequent poor processing of oxidatively modified low density lipoprotein (oxLDL) by macrophages in the arterial wall contributes to the initial stages of atherogenesis. Oxidative modification of LDL involves the derivatization of its constituent apolipoprotein B and aminophospholipids by aldehydic breakdown products of lipid peroxidation, especially 4-hydroxy-2- nonenal (HNE). Many properties of oxLDL, such as development of lipofuscin-like fluorescence and the tendency toward aggregation, can be reproduced by direct treatment of LDL with HNE. The chemistry of HNE-protein adduction is highly complex, being heterogeneous, in part subject to reversible equilibria, and associated with time- and oxidation-dependent adduct "aging" and crosslinking reactions, a subset of which was elucidated during the past grant period. Our overall working hypothesis is that changes in the nature of lipoxidation-dependent adducts over time and following uptake of oxLDL into macrophages, are important determinants of foam cell formation. Moreover, since deficient intracellular processing may be tied more to one oxLDL scavenger receptor (e.g., CD36) than another, it is critically important to ascertain which oxLDL adducts most elicit recognition by this receptor. We have developed immunochemical tools to monitor the progression of adduct chemistry during in vitro oxidation and following intracellular accumulation in macrophage cells, relative to the end-stage adducts detected in atherosclerotic lesions obtained at surgery. The antibodies are also useful for monitoring the progression of lipoxidation-dependent changes on blood proteins in patients with cardiovascular and renal disease. The new work proposed, which continues to take advantage of the pooled multi-disciplinary expertise of three senior investigators at neighboring research institutions, furthers the development of novel structurally-specific analytical tools (immunochemical, mass spectroscopic, and isotopic) and their application to cell biological studies. The key feature of unambiguous structural determination is viewed as a crucial advantage for clarifying which modifications occurring in oxLDL are most associated with atherogenic properties and what environmental factors foster production of those toxic modifications. These studies will provide a basis for designing therapeutic countermeasures.