Incubation of MDCO-216 (ApoA-IMilano/POPC) with Human Serum Potentiates ABCA1-Mediated Cholesterol Efflux Capacity, Generates New Prebeta-1 HDL, and Causes an Increase in HDL Size

Abstract

MDCO-216 is a complex of dimeric ApoA-IMilano and palmitoyl oleoyl phosphatidylcholine (POPC), previously shown to reduce atherosclerotic plaque burden. Here we studied the effect of incubation of human plasma or serum with MDCO-216 on cholesterol efflux capacity from J774 cells, on prebeta-1 high density lipoprotein (prebeta-1 HDL) and on HDL size assessed by proton nuclear magnetic resonance (1H-NMR). MDCO-216 incubated in buffer containing 4% human serum albumin stimulated both ABCA1-mediated efflux and ABCA1-independent cholesterol efflux from J774 macrophages. When incubated with human serum a dose- and time-dependent synergistic increase of the ABCA1-mediated efflux capacity were observed. Using a commercially available ELISA for prebeta-1 HDL, MDCO-216 as such was poorly detected (12–15% of nominal amount of protein). Prebeta-1 HDL was rapidly lost when human plasma alone is incubated at 37°C. In contrast, incubation of human plasma with MDCO-216 at 37°C produced a large amount of new prebeta-1 HDL. Native 2D electrophoresis followed by immunoblotting with an apoA-I antibody, which also detects ApoA-I Milano, confirmed the increase in prebeta-1 HDL upon incubation at 37°C. With the increase of prebeta-1 HDL, the concomitant disappearance of the small alpha-3 and alpha-4 HDL and MDCO-216 and an increase in the large alpha-1 and alpha-2 HDL were observed. Immunoblotting with Mab 17F3 specific for ApoA-I Milano showed the appearance of ApoA-I Milano in alpha-1 and alpha-2, but not in prebeta-1 HDL. 1H-NMR analysis of plasma incubated with MDCO-216 confirmed rapid disappearance of small-sized HDL particles and increase of medium- and large-sized HDL particles accompanied with a decrease in total HDL particle number. In conclusion, incubation of human plasma or serum with MDCO-216 strongly enhanced ABCA1-mediated cholesterol efflux, caused a strong increase of prebeta-1 HDL, and drastically changed the distribution of HDL subpopulations. Overall, the results are in line with the hypothesis that MDCO-216 fuses with small alpha-migrating HDL particles forming larger particles containing both apoA-I WT and ApoA-I Milano, meanwhile liberating the endogenous wild-type apoA-I which enriches prebeta-1 HDL subpopulation.