Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, proerties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was de--graded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component ofthe fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conijugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to >4096) can be demonstrated in rabbit and human sera.A growing body of evidence suggests that oxidative modification of low density lipoprotein (LDL) enhances its atherogenicity (for review see ref. 1). Monocyte-derived macrophages, the precursor of most foam cells in early atherosclerotic lesions, cannot take up native LDL rapidly enough to cause lipid loading (2). Oxidative modification converts LDL to a form recognized by the macrophage acetyl-LDL receptor (1, 2) and possibly by other receptors as well (3). This is true whether the oxidation is effected by incubation under appropriate conditions with cultured cells or by autooxidation catalyzed by Cu2+ ions in the absence of cells. Oxidative modification of LDL is accompanied by extensive degradation of its polyunsaturated fatty acids, generating a complex array of shorter chain-length fragments (4). During the oxidation, some of these fragments become covalently linked to apolipoprotein B (5), and much ofthis conjugation involves the E-amino groups of lysine residues. This chemically modified form of apolipoprotein is recognized by the acetyl-LDL receptor (6). Thus we can generate models for oxidized LDL by conjugating the apolipoprotein with single compounds generated during oxidation. Fogelman et al. (7) demonstrated that malondialdehyde (MDA)-conjugated LDL is so recognized. If oxidized LDL contains lysine residues conjugated with a variety of fatty acid fragments of different chain lengths, it should react with antibodies against a variety of such lysine derivatives. We previously showed that immunization of animals with autologous LDL modified by conjugation of lysine groups with glucose yields antisera directed specifically against glucitollysine (8) and that antibodies generated by injection of carbamoylated autologous LDL generates antisera that recognize the carbamoyllysine-not only in LDL but in other conjugated proteins as well (9). In other words, the specificity of these antisera is for very narrowly defined "X"-lysine adducts.The present studies, which use immunochemical methods, offer three lines of evidence that oxidation of LDL occurs in vivo.

Oxidative modification of LDL renders it immunogenic and autoantibodies to epitopes of oxidised LDL, such as malondialdehyde (MDA)-lysine, are found in serum and recognise material in atheromatous tissue. However, there has been no prospective study to assess the importance of oxidised LDL among patients with vascular disease. We compared the titre of autoantibodies to MDA-modified LDL and native LDL in baseline serum samples of 30 eastern Finnish men with accelerated two-year progression of carotid atherosclerosis and 30 age-matched controls without progression. Neither group had specific antibody binding to native LDL. A titre was defined as a ratio of antibody binding to MDA-LDL/binding to native LDL. Cases had a significantly higher titre to MDA-LDL (2.67 vs 2.06, p = 0.003). Cases also had a greater proportion of smokers (37% vs 3%), higher LDL cholesterol (4.2 mmol/l vs 3.6 mmol/l), and higher serum copper concentration (1.14 mg/l vs 1.04 mg/l). Even after adjusting for these variables and the severity of baseline atherosclerosis, the difference in antibody titre remained significant in a multifactorial logistic model (p = 0.031). Thus, the titre of autoantibodies to MDA-LDL was an independent predictor of the progression of carotid atherosclerosis in these Finnish men. Our data provide further support for a role of oxidatively modified LDL in atherogenesis.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to Induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydlc fragments of oxidized fatty acids capable of conjugating with lyslne residues of apoprotein B. By Immunizing animals with homologous malondlaldehyde-modlfled LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu ++ -oxldlzed LDL, we developed polyvalent and monoclonal antibodies against three epitopes found In oxldatively modified LDL The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lyslne, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lyslne. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epltope generated during Cu ++ oxldatJon of LDL With these antibodies, we demonstrated that MDA-lyslne and 4-HNE-lyslne adducts develop on apo-llpoproteln B during copper-Induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemlcal demonstration of oxidized lipoprotelns In atherosclerotic lesions of progressive severity Is described in the companion article. These antibodies should prove useful In studying the role of oxldatively modified lipoprotelns as well as other oxldatively modified proteins In atherogenesls.

Atherosclerotic lesions contain relatively large quantities of IgG. We have previously shown that both human and rabbit sera contain autoantibodies against epitopes of oxidized (Ox) low-density lipoprotein (LDL) and that LDL isolated from atherosclerotic lesions contains small amounts of tightly bound IgG. However, it is not known whether IgG isolated from atherosclerotic lesions recognizes epitopes present in native LDL or Ox-LDL. IgG was isolated from Watanabe heritable hyperlipidemic (WHHL) rabbit atherosclerotic lesions by sequential salt extractions, purified by fast protein liquid chromatography on protein G, and used in a solid-phase radioimmunoassay. IgG and immune complexes were also isolated from the saline extracts of human lesions by adsorption onto latex beads coated with anti-human IgG antibodies or protein A. IgG isolated from rabbit lesions showed significant titers against malondialdehyde (MDA)-modified LDL and LDL oxidized by copper ions for 4 and 18 hours but not against native LDL. On Western blots, lesion IgG stained MDA-LDL and fragments of Ox-LDL. Western blots of immune complexes isolated from human lesions revealed the presence in the isolated complexes of both apoprotein B and apoprotein B fragments, which reacted with antibodies to MDA-lysine. Furthermore, rabbit lesion IgG immunostained epitopes of Ox-LDL present in human atherosclerotic lesions. Immunostains obtained with rabbit lesion IgG were similar to those obtained with a monoclonal antibody specific for MDAlysine. The results show that human and rabbit atherosclerotic lesions contain IgG that recognizes epitopes characteristic of Ox-LDL. These data suggest that immunologic processes may be an important component of the atherogenic process. (Arttriosder Thromb. 1994;14J2-40.)