Urs P. Steinbrecher

Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts. (ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation--for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration--LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).

Modification of low density lipoproteins (LDL) by oxidation has been shown to permit recognition by the acetyl-LDL receptor of macrophages. The extensive oxidation of LDL that is required before interaction occurs with this receptor produces major alterations in both the lipid and protein components of LDL. Several chemical modifications of LDL also lead to recognition by this receptor; all of these involve derivatization of lysine residues of apolipoprotein B by adducts that neutralize the positively charged epsilon-amino group. The present studies show that oxidation also results in derivatization of LDL lysine residues. Analysis of amino acid composition indicated that 32% of lysine residues were modified after oxidation of LDL by exposure to 5 microM CuSO4 for 20 h. About one-half of the derivatized lysines were labile under the conditions of acid hydrolysis. Fluorescence of LDL protein was greatly increased by oxidation, with excitation maximum at 350 nm and emission maximum at 433 nm. When LDL containing phosphatidylcholine with isotopically labeled arachidonic acid in the sn-2 position was oxidized, there was a 5-fold increase in radioactivity bound to protein compared to nonoxidized LDL or oxidized LDL labeled with 2-[1-14C]palmitoyl phosphatidylcholine. Prior methylation of LDL prevented the rapid uptake and degradation by macrophages that normally accompanies oxidation. These findings suggest that oxidation of LDL is accompanied by derivatization of lysine epsilon-amino groups by lipid products and that these adducts may be important in the interaction of oxidized LDL with the acetyl-LDL receptor.

Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.