Inhibition of Atherosclerosis in ApoE-Null Mice by Immunization with ApoB-100 Peptide Sequences

Abstract

Objective-LDL oxidation is believed to play an important role in the development of atherosclerosis, and oxidized LDL particles have been shown to become targets for the immune system. Immunization of animals with oxidized LDL results in reduction of atherosclerosis, suggesting an atheroprotective effect of this immune response. Methods and Results-Using a polypeptide library covering the complete sequence of apoB-100, a large number of native and malondialdehyde-modified peptide sequences in apoB-100 that are recognized by antibodies in human plasma were identified. We report here that immunization with apoB-100 peptide sequences, against which high levels of IgG and IgM antibodies are present in healthy human controls, reduce atherosclerosis in apoE-null mice by about 60%.Immunizations with these peptides were also found to increase the collagen content of subvalvular lesions. Conclusions-These studies have identified peptide sequences in apoB-100 that induce immune responses, which inhibits atherosclerosis. This suggests a way of developing an immunization therapy for coronary heart disease. Key Words: apolipoproteins Ⅲ atherosclerosis Ⅲ immunization Ⅲ mice Ⅲ peptides A ccumulation, aggregation, and modification of LDL particles in the arterial intima are believed to be among the most important initiating factors in atherosclerosis. 1,2 Oxidative modification of LDLs trapped in the vascular extracellular matrix is associated with generation of a number of highly reactive compounds, such as lysophosphatidylcholine, lipid peroxides, aldehydes, and oxysterols, that cause cell damage and local inflammation. 2,3 In general terms, the development of raised fibromuscular plaques can be said to represent a repair response to the vascular injury and oxidized lipids may be one factor causing such injury. 4 Several protective mechanisms exist to limit injury caused by oxidatively damaged LDL particles. One involves the removal of oxidized LDL by macrophage scavenger receptors. 5,6 Recent studies suggest a second protective mechanism involving specific immune responses against epitopes present in oxidized LDLs. These were initially identified in studies of hypercholesterolemic rabbits, in which immunization with oxidized LDL was found to reduce atherosclerosis by 40% to 60%. 7,8 Similar observations were subsequently also made in apoE-null and LDL receptor-null mice, 9 -11 as well as in balloon-injured hypercholesterolemic rabbits. 12 In apoE-null mice, induction of hypercholesterolemia by a high-fat diet results in a dramatic increase in autoantibodies against oxidized LDLs. Circulating autoantibodies against oxidized LDLs are also abundant in humans and have been shown to correlate with severity of disease in cardiovascular patients. [13][14][15][16] These findings suggest the possibility of developing new treatments against atherosclerosis based on selective activation of atheroprotective immune responses against oxidized LDL antigens.Oxidation of LDL is associated with formation of reactive aldehydes, such as malondi...

Recombinant Human Antibodies Against Aldehyde-Modified Apolipoprotein B-100 Peptide Sequences Inhibit Atherosclerosis

“…11 Immunization of apoE Ϫ/Ϫ mice with some of these peptide sequences resulted in inhibition of atherosclerosis to a similar extent as that observed after immunization with oxLDL and was also associated with an increase in peptide-specific IgG. 12 The present findings suggest that specific antibodies constitute an important component of atheroprotective immunity but do not exclude the involvement of cell-mediated immunity. Support for the existence of atheroprotective humoral immunity also comes from studies in apoE Ϫ/Ϫ mice demonstrating inhibi- tion of atherosclerosis by repeated injections of polyclonal IgG and by B-cell rescue of splenectomized mice.…”

“…12 A protocol similar to that for macrophage staining was used for detection of oxLDL epitopes in the plaques with IEI-E3 (100 g/mL) as the primary antibody and a biotinylated mouse anti-human IgG1 antibody (25 g/mL; ImmunKemi F&D AB) diluted in PBS as the secondary antibody.…”

“…ApoB-containing lipoproteins were precipitated with MgCl 2 and dextran sulfate as previously described. 12 Preparation of Unlabeled and 125 I-Native LDL or oxLDL LDL was isolated from blood by sequential preparative ultracentrifugation in a narrow density range (1.034 to 1.054 kg/L). Coppermediated oxidation was achieved by incubating freshly prepared LDL in PBS with a sterile solution of CuCl 2 at a final concentration of 10 mol/L.…”

“…Immunization of apoE Ϫ/Ϫ mice with the corresponding human apoB peptides was found to result in reduced plaque formation and a stable plaque phenotype, as indicated by increased collagen content. 12 This effect was associated with increased formation of IgG against the respective apoB-100 peptides. To further study the role of these IgG antibodies in the atheroprotective response and to test whether specific MDA-apoB-100 antibodies could be used for direct inhibition of atherosclerosis in apoE Ϫ/Ϫ mice, we produced recombinant human IgG1 that specifically recognizes 2 MDA-modified sequences in human apoB-100.…”

“…Active immunization with these peptides has previously been shown to reduce atherosclerosis by Ϸ50% in mice. 12 …”