MOLECULAR BASIS OF OXIDATIVE MODIFICATION OF LDL

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Substantial evidence suggests that the over-accumulation of oxidatively- modified low density lipoprotein (ox-LDL) by macrophages in the arterial wall contributes to the pathogenesis of atherosclerotic.plaques. Oxidative modification of LDL involves the derivatization of its constituent apolipoprotein B by breakdown products of lipid peroxidation, to a large degree by trans-4-hydroxy-2-nonenal (HNE). Many properties of ox-LDL, 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-dependent and autoxidation-dependent adduct "aging" and intermolecular crosslinking potential. Our overall working hypothesis is that such evolution in HNE adduct structure can be identified immunochemically by changes in epitope expression and, in part, through biochemical studies using group-specific reagents and tritiated HNE probes. Ox-LDL which has been internalized by macrophages appears to undergo a similar shift in HNE adduct structure, as does ox-LDL present in the extracellular space of human atherosclerotic lesions. The work proposed, which reflects the pooled multi-disciplinary expertise of three senior investigators at neighboring research institutions, involves a detailed characterization of HNE adduction chemistry in model physiomimetic studies, followed by the development of antibodies which recognize individual types of HNE-LDL modifications. These antibodies will then be used for immunochemical detection of specific HNE adducts on HNE-LDL. arid ox-LDL in vitro, on ox-LDL which has been internalized in cultured macrophages, and in atherosclerotic lesions obtained at surgery. The spatiotemporal pattern of HNE-LDL adduct appearance will be correlated with functional consequences of LDL modification in terms of accumulation in macrophages via specific receptor uptake and susceptibility to lysosomal degradation therein. The key features of (i) unambiguous determination of adduct structure and (ii) antibody specificity, are viewed as crucial requirements for clarifying which modifications occurring in ox-LDL are most associated with atherogenic properties, and which are specific to HNE.