Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect

Abstract

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.

Fig 1. Identification of LDL-IgG-IC within the density range of LDL.

Lipoproteins were separated by a self-generated iodixanol gradient single-step ultracentrifugation. Density of the obtained 25 subfractions and LDL region (A). Cholesterol levels of collected fractions from 3 healthy subjects (B). Distributions of cholesterol (solid lines), MDA (dotted lines) and LDL-IgG-IC detected by a specific anti-human-IgG antibody (insert at the top right) in patients with PAOD. MDA levels represent protein bound MDA (C).

Fig 2. Identification of in vitro produced small LDL-IgG-IC by density shift.

Small LDL-IgG-IC were produced by in vitro assembly of LDL-subfraction #8 and an anti-human apoB antibody (IgG). After self-generated iodixanol gradient single-step ultracentrifugation (fractionation step size: 3.0 mm) the cholesterol content was measured in the obtained subfractions and the presence of small LDL-IgG-IC was assessed by DELFIA (A). LDL-IgG-F(ab')₂-IC consisting of an HRP-antibody fragment (targeting the F(ab)₂ fragment of human IgG) and small LDL-IgG-IC were produced by incubation of an LDL-IgG-IC rich subfraction (#11) with the HRP-antibody fragment (antibody/LDL particle ratio of 1:100). After self-generated iodixanol gradient single-step ultracentrifugation fractionation was carried out with a step size of 1.0 mm. The density shift of LDL-IgG-IC (peak to peak difference) due to formation LDL-IgG-F(ab')₂-IC is detected by measurement of HRP activity and immunodetection (dot-blot) of IgG in the control experiment (incubation without HRP-antibody fragment) (B).

Fig 3. Estimation of LDL-IgG-IC particle size.

Protein staining (left panel; Ponceau staining) and IgG immunodetection (right panel) of LDL-subfractions after electrophoresis in 3% polyacrylamide slab gel (migration is possible for particles < 35 nm). The small LDL-IgG-IC particles of subfractions #11, #12 and #13 (IgG) show a similar mobility like the major LDL-subfraction #7 (protein stain) (A). High performance gel permeation chromatography (TSK 5000 PW column; 600 mm x 4 mm) of a small LDL-IgG-IC enriched LDL-subfraction. Fractions of 1 min in the elution region of LDL were collected (solid line: preparative isolation; sample = 80 μL of LDL-IgG-IC enriched subfraction). The dotted line represents an analytical run sample = 20 μL of LDL-IgG-IC enriched subfraction). The distribution of LDL-IgG-IC (preparative run; fractions of min 25–31) in the eluted fractions was visualized after dot-blot analysis (immunodetection of IgG) (insert) (B).

Fig 4. Small LDL-IgG-IC are not precipitable with PEG.

Lipid electrophoresis of LDL-subfractions prepared (iodixanol gradient ultracentrifugation) from original serum and PEG supernatants after PEG 8000 precipitation (fractionation step size: 3.0 mm). Bands represent small LDL-IgG-IC present in the indicated LDL-subfractions visualized by immunodetection of IgG. Day 0 (fresh serum): LDL-subfractions #8–11 (from PEG supernatant) and LDL-subfractions #9–12 (original serum diluted with borate buffered saline to the same final volume). Band C: undiluted control (LDL-IgG-IC rich) subfraction isolated earlier from the same donor. Day 1: Repetition of the experiment with serum stored for 24 h at 4°C. The numbering of bands (PEG supernatant and original serum) is slightly different due to the density contribution of PEG. (A). Cholesterol amounts of combined LDL-subfractions (iodixanol ultracentrifugation) after PEG precipitation. Bars represent cholesterol concentrations (transformed to serum concentrations) of original serum (cholesterol of combined LDL-subfractions without PEG precipitation), PEG supernatant (cholesterol of combined LDL-subfractions after PEG precipitation) and PEG pellet (cholesterol content of precipitate) on day 0 (fresh serum) and day 1 (stored serum). *p < 0.001; increase of pellet cholesterol vs. day 0 (B).

Fig 5. Simvastatin lowers small LDL-IgG-IC levels more effectively than cholesterol and apoB in patients with CAD.

The reduction of total small LDL-IgG-IC levels is presented as percentage change from baseline for the 6 individual LDL-subfractions. Total amounts of small LDL-IgG-IC per fraction were calculated by conversion of the LDL-IgG-IC DELFIA counts. For each subject and each LDL fraction the baseline value and the value after statin therapy were used to calculate the difference as a percentage (post-statin minus pre-statin). Each bar represents the mean difference of total small LDL-IgG-IC per fraction on a percentage basis (*p < 0.05; **p < 0.01). (A). Comparison of the reduction of LDL-cholesterol, LDL-apoB levels and total small LDL-IgG-IC (average of reduction of LDL-subfractions) expressed as percentage change from baseline. Bars for LDL-cholesterol and LDL-apoB represent the percentage differences (post-statin minus pre-statin) determined in the entire LDL fractions. The reduction of the total amount of small LDL-IgG-IC of LDL is presented as average of the percentage differences calculated for LDL-subfractions 1–6 (shown in Fig 5A). Data represent means ± SD (B).