Immunohistochemical localization of different epitopes of advanced glycation end products in human atherosclerotic lesions

To better understand the role of advanced glycation end products (AGEs) in atherogenesis, we developed specific antibodies against different immunolog ical epitopes of AGE structures, including N-epsilon-(carboxymethyl)lysine- protein adduct (CML) and a structure(s) other than CML (nonCML), and demons trated the immunohistochemical localization of CML- and nonCML-epitopes in atherosclerotic lesions of human aorta, which were obtained at autopsy from 20 nondiabetic patients (12 males and eight females; mean age, 60.8 +/- 16 .7 years). Monoclonal anti-CML antibody (6D12) recognized not only AGE-modi fied proteins, but also CML-modified proteins. On the other hand, polyclona l anti-nonCML antibody reacted to AGE-modified proteins, but not to CML-mod ified proteins. Both antibodies were unreactive to the early-stage products of glycation, including fructose-modified butyloxycarbonyl-lysine and fruc tose-epsilon-aminocaproic acid. Atherosclerotic lesions included diffuse in timal thickening (DIT), fatty streaks (FS), atherosclerotic plaques (AP) an d complicated lesions. An immunohistochemical analysis showed both CML- and nonCML-epitopes to be found along the collagen fibers in DIT in subjects m ore than 40 years old, but not in subjects less than 40 years old. CML-epit opes accumulated mainly in the cytoplasm of macrophage/foam cells, while no nCML-epitopes accumulated exclusively in the extracellular spaces in FS. AP s showed the CML-epitope stored macrophage/foam cells, and the accumulation of both CML- and nonCML-epitopes in the lipid-rich fibrous area. An immuno histochemical analysis with a monoclonal antibody against oxidized low dens ity lipoprotein (FOH1a/DLH3) showed the presence of this antigen within the cytoplasm of the macrophage/foam cells in atherosclerotic lesions, which w ere also positive for the CML-epitopes. These findings thus suggest that th e heterogeneous localization of AGEs in atherosclerotic lesions depends on their different epitopes, and that a close link, therefore, exists between the peroxidation of LDL and the formation of AGEs in atherosclerotic lesion s. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.