Circulating levels of oxidized LDL are strongly associated with angiographically documented coronary artery disease, particularly in patients 60 years of age or younger. These data suggest that the atherogenicity of Lp(a) lipoprotein may be mediated in part by associated proinflammatory oxidized phospholipids.

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.( J. Clin. Invest. 1996. 98:800-814.)

Atherosclerotic lesions contain oxidized LDL (OxLDL), immunoglobulins, and immune-competent cells. Low levels of circulating autoantibodies against malondialdehyde (MDA)-modified lysine, an epitope of OxLDL, occur in several species, and immune complexes between such autoantibodies and OxLDL are present in lesions. To study the potential role of autoantibodies against OxLDL in the atherogenic process, we prospectively hyperimmunized LDL receptor-deficient rabbits with homologous MDA-LDL and determined the effects of this intervention on the development of atherosclerosis. Immunization with MDA-LDL generated high titers of antibodies with similar specificity as naturally occurring autoantibodies. Immunized animals showed a significant reduction in the extent of atherosclerotic lesions in the aortic tree after 6.5 months, compared with "saline"-immunized controls (48% vs. 68%, P < 0.005). Immunization with keyhole limpet hemocyanin produced no change in lesion formation. Although the mechanisms by which immunization led to a protective effect are unknown, these results suggest an important role for the immune system in modulating the atherogenic process and may indicate a novel approach for inhibiting the progression of atherosclerosis.Substantial evidence indicates that oxidized LDL (OxLDL) contributes to atherogenesis by a number of mechanisms (1, 2). Furthermore, even minor modifications of LDL render it highly immunogenic (3), and circulating autoantibodies recognizing several forms of OxLDL, in particular malondialdehyde (MDA)-modified lysine, are prevalent in humans and other species (4-6). These autoantibodies are capable of binding to epitopes of OxLDL in lesions (4, 5, 7), and immunoglobulins isolated from lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits and humans recognize OxLDL and are present in lesions, in part as immune complexes with OxLDL (8).It is currently unknown if the titer of autoantibodies against epitopes of OxLDL is merely an indicator of lipoprotein modification or if such autoantibodies could modulate the atherogenic process. To address this question, we hyperimmunized WHHL rabbits with homologous MDA-LDL and determined the effect of this intervention on atherosclerosis. METHODSImmunization and Antibody Determination. LDL was isolated from healthy WHHL donor rabbits by sequential ultracentrifugation in the presence of antioxidants and antiproteolytic agents and was modified with MDA (9). MDA-LDL in which 70-85% of the lysine residues were modified was used as the immunogen and for the determination of antibody titers (9). Immunization of WHHL rabbits was begun either at an age of 6 weeks or 6 months. The primary immunization consisted of a subcutaneous injection of 160 jig of MDA-LDL (protein) per kg of body weight dissolved in phosphatebuffered saline (PBS) and suspended in an equal volume of Freund's complete adjuvant. Booster immunizations consisted of antigen in Freund's incomplete adjuvant injected intramuscularly 3 and 5 weeks after the primary immunization and ...

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Circulating OxLDL-specific markers strongly reflect the presence of ACS, implying immune awareness to newly exposed oxidation-specific epitopes and possible release of OxLDL in the circulation. The OxLDL-E06 measurements provide novel insights into plaque rupture and the potential atherogenicity of Lp(a).