Passive immunization with hypochlorite-oxLDL specific antibodies reduces plaque volume in LDL receptor-deficient mice

Abstract

Aims: New strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.

Methods and results: Monoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr(-/-) mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr(-/-) mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.

Conclusion: Our data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.

Figure 1. Schematic representation of the collar model and the experimental design.

A. Atherosclerotic lesions develop caudal to the collar, cross-sections were made of the common right carotid artery in a caudal direction from the collar and collected in a parallel series of slides, B. Time schedule of the experiment to study the effect of passive immunization using Moab A7S8 or PBS on atherosclerotic plaque development.

Figure 2. IgM Monoclonal antibody (Moab A7S8).

A. Reactivity to native, HOCL-oxLDL and MDA-oxLDL. Measured by ELISA, OD 405 at different concentrations (100, 50, 25, 10 and 1 µg/ml IgM concentration). B. Sequence IgM hybridoma. The sequence of Moab A7S8 was compared with germline VH genes by using the international ImMunoGeneTics information system/−QUEry and Standardization (IMGT/V-Quest). Moab A7S8 is composed of IgHV1–78*01, IGHJ3*01 and IGHD1-1*02 alleles, with 100% identity and no gaps. Except for the N-addition, the alleles are in frame germline sequences.

Figure 3. Atherosclerotic plaque volume at the collar and aortic root.

A. Representative lesions of PBS and Moab A7S8 treated mice of collar-induce atherosclerosis in LDLr^−/− mice. Scale bar, 100µm (haematoxylin-eosin). B. Percentage mice with atherosclerotic plaque formation after certain distances from collar. Closed bars: PBS treated mice. Grey bars: control IgM treated mice. Open bars: Moab A7S8 treated mice. C. Representative lesions in the aortic root of PBS and Moab A7S8 treated LDLr^−/− mice. Scale bar, 100 µm (toluidin blue). D. Atherosclerotic plaque volume in the aortic root. Plaque volume, expressed in mm2. E. Collar-induced atherosclerotic plaque volume, measured at every 50 µm from collar, in PBS (▪), control IgM (○) and Moab A7S8 (□) treated mice. A dot represents the mean ± SEM.

Figure 4. Collagen content in the atherosclerotic plaque.

Percentage positive collagen of the total plaque area. In PBS (▪) and Moab A7S8 (□) treated mice.

Figure 5. Cholesterol levels.

Serum cholesterol levels are expressed as mM (± SEM). Closed bars: PBS treated mice. Grey bars: control IgM treated mice. Open bars: Moab A7S8 treated mice.

Figure 6. Total IgM and HOCl and MDA anti-oxLDL IgM titers.

A. Total IgM titers, B. anti-HOCl oxLDL IgM titers and C. anti-MDA oxLDL IgM in PBS treated mice (▪), control IgM treated mice (o) and Moab A7S8 treated mice (□).

Figure 7. Cu-anti-oxLDL IgM titers, T15/EO6 IgM antibodies and IgM-apoB immune complexes.

A. Anti-Cu-oxLDL IgM titers, B. T15/EO6 IgM antibodies and C. IgM-apoB immune complexes expressed as IgM/apoB. Mice treated with PBS (▪), control IgM treated mice (o) and in mice treated with Moab A7S8 (□).

Figure 8. Anti-HOCl, MDA and Cu-oxLDL IgG titers.

A. Anti-HOCl-oxLDL IgG titers, B. anti-MDA-oxLDL IgG titers and C. anti-Cu-oxLDL IgG titers in mice treated with PBS (▪), control IgM treated mice (o) and in mice treated with Moab A7S8 (□).