ApoA-I dissociated from human HDL retains its acceptor properties in ABCA1-mediated cholesterol efflux from RAW 264.7 macrophages in coronary artery disease

Abstract

BackgroundThe significance of cholesterol efflux as a predictor of coronary artery disease (CAD) remains controversial. The primary route for intracellular cholesterol export via the ABCA1 transporter involves the acceptance of cholesterol by lipid-free apolipoprotein A-I and high-density lipoproteins (HDL). A separate estimate of the efficiencies of two reactions is thus required. MethodsHDL from plasma of 63 control and 76 CAD male patients was obtained by PEG precipitation of apoB-containing lipoproteins and denatured by 4.25 M urea at the transition midpoint. We measured apoA-I dissociation concomitant with HDL denaturation, the expression of 65 preselected genes by real-time PCR, and the efficiency of ABCA1-mediated cholesterol efflux from RAW 264.7 macrophages to dissociated lipid-free apoA-I. The dissociation parameter D was calculated from the accumulation of apoA-I with pre{beta}-mobility at HDL separation by agarose gel electrophoresis followed by immunodetection. ResultsThe phospholipid:apoA-I and cholesterol: apoA-I ratios for CAD patients were consistently higher than those for control patients in a whole range of plasma HDL-cholesterol levels. ApoA-I partitioned 1.5-fold higher into the water phase for HDL from CAD patients relative to controls. For CAD patients, the dissociation parameter D was inversely correlated with phospholipid and cholesterol levels and the cholesterol:apoA-I ratio in HDL. For control patients, the D parameter was positively correlated with CETP and ABCA1 gene expression levels. For CAD patients, the D parameter was negatively correlated with CUBN and ALB gene expression levels that may be associated with the increased catabolism of lipid-free apoA-I. The Vm and Km values showed that apoA-I functionality in ABCA1-mediated cholesterol efflux from RAW 264.7 macrophages to lipid-free apoA-I generated at urea-induced HDL denaturation was similar for HDL from both groups. ConclusionsThe enrichment of HDL with cholesterol, with the concomitant increase of competition between apoA-I and cholesterol for the binding to phospholipid molecules adjacent to apoA-I, could be involved in the increased apolipoprotein dissociation from HDL in CAD. The obtained estimates for the ABCA1-mediated cholesterol efflux to lipid-free apoA-I may be the prerequisite for the detailed study of cholesterol efflux kinetics with two apoA-I forms that could allow to be described them as CAD predictors.