Background-Repeated doses of recombinant apolipoprotein A-I Milano phospholipid complex (apoA-I m ) reduce atherosclerosis and favorably change plaque composition in rabbits and mice. In this study, we tested whether a single high dose of recombinant apoA-I m could rapidly mobilize tissue cholesterol and reduce plaque lipid and macrophage content in apoE-deficient mice. Methods and Results-High cholesterol-fed, 26-week-old apoE-deficient mice received a single intravenous injection of saline (nϭ16), 1080 mg/kg dipalmitoylphosphatidylcholine (DPPC; nϭ14), or 400 mg/kg of recombinant apoA-I m complexed with DPPC (1:2.7 weight ratio; nϭ18). Blood was sampled before and 1 and 48 hours after injection, and aortic root plaques were evaluated for lipid content and macrophage content after oil-red O and immunostaining, respectively. One hour after injection, the plasma cholesterol efflux-promoting capacity was nearly 2-fold higher in recombinant apoA-I m -treated mice compared with saline and DPPC-treated mice (PϽ0.01). Compared with baseline values, serum free cholesterol, an index of tissue cholesterol mobilization, increased 1.6-fold by 1 hour after recombinant apoA-I m injection, and it remained significantly elevated at 48 hours (PϽ0.01). Mice receiving recombinant apoA-I m had 40% to 50% lower lipid content (PϽ0.01) and 29% to 36% lower macrophage content (PϽ0.05) in their plaques compared with the saline-and DPPC-treated mice, respectively. Conclusions-A single high dose of recombinant apoA-I m rapidly mobilizes tissue cholesterol and reduces plaque lipid and macrophage content in apoE-deficient mice. These findings suggest that this strategy could rapidly change plaque composition toward a more stable phenotype. (Circulation. 2001;103:3047-3050.)

Objective-Atherosclerosis is associated with an immune response against oxidized LDL, which modulates the progression of the disease process. Methods and Results-Using a library of polypeptides covering the complete sequence of apoB-100, the only major protein of LDL, we have identified over 100 different human antibodies reacting against aldehyde-modified apoB-100 sequences. IgM antibody titer levels decreased with age and were associated with the intima-media thickness of the carotid artery in subjects younger than 60 years. There were also inverse associations between IgM levels and oxidized LDL in plasma. In prospective clinical studies, antibody levels against several aldehyde-modified apoB-100 sites were associated with cardiovascular disease in this age group. Whether this immune response is adaptive (protective) or maladaptive (causal) in atherosclerosis requires further investigation. Conclusions-We have characterized a large number of epitopes within the apoB-100 component of oxidized LDL that provoke an immune response in humans. These findings will make it possible to study the role of immune responses against specific sites in oxidized LDL and to determine whether these immune responses influence the risk for future cardiac events.

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...