Differential Expression of Subsets of Genes Related to HDL Metabolism and Atherogenesis in the Peripheral Blood in Coronary Artery Disease.

Differential expression of genes (DEGs) in coronary artery disease (CAD) and the association between transcript level and high-density lipoprotein cholesterol (HDL-C) were studied with 76 male patients with CAD and 63 control patients. The transcript level of genes related to HDL metabolism (24 genes) and atherosclerosis-prone (41 genes) in RNA isolated from peripheral blood mononuclear cells was measured by real-time RT-PCR. Twenty-eight DEGs were identified. The expression of cholesterol transporters, ALB, APOA1, and LCAT was down-regulated, while the expression of AMN, APOE, LDLR, LPL, PLTP, PRKACA, and CETP was up-regulated. The systemic inflammation in CAD is evidenced by the up-regulation of IL1B, TLR8, CXCL5, and TNFRSF1A. For the controls, TLR8 and SOAT1 were negative predictors of the HDL-C level. For CAD patients, PRKACG, PRKCQ, and SREBF1 were positive predictors, while PRKACB, LCAT, and S100A8 were negative predictors. For CAD patients, the efficiency of reverse cholesterol transport is 73-79%, and intracellular free cholesterol seems to accumulate at hyperalphalipoproteinemia. Both atheroprotective (via S100A8) and proatherogenic (via SREBF1, LCAT, PRKACG, PRKACB, and PRKCQ) associations of gene expression with HDL-C determine HDL functionality in CAD patients. The selected key genes and involved pathways may represent HDL-specific targets for the diagnosis and treatment of CAD and atherosclerosis.

Different Pathways of Cellular Cholesterol Efflux.

Cholesterol efflux is the first and rate-limiting step of reverse cholesterol transport (RCT) from peripheric cells to the liver. The involvement of high-density lipoprotein (HDL) in RCT determines the atheroprotective properties of HDL. Cholesterol efflux from different membrane pools includes both passive and energy-dependent processes. The first type of route consists of cholesterol desorption from the cell membrane into the unstirred layer adjacent to the cell surface and diffusion in the water phase. Moreover, the selective uptake and facilitated diffusion of cholesterol and cholesteryl ester molecules through the hydrophobic tunnel in the scavenger receptor BI molecule does not require energy consumption. The second type of route includes active cholesterol export by the ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Several cholesterol acceptors specifically bind cholesterol and phospholipid molecules, and cholesterol binding to the albumin molecule, which acts as a shuttle, significantly increases cholesterol movement between acceptors and red blood cells, thus functioning as a sink for cholesterol. Cholesterol and phospholipid molecules effluxed from macrophages by ABCA1 are accepted exclusively by the lipid-free apolipoprotein apoA-I, which is the major protein moiety of HDL, whereas those effluxed by ABCG1 are accepted by HDL. ABCA1- and ABCG1-mediated cholesterol transport, together with cholesterol diffusion, largely determine cholesterol turnover at the physiological level of intracellular cholesterol. However, at cholesterol overload, ABCA1-mediated efflux prevails over other routes. The exchange of apoA-I between lipid-free and lipid-associated states and the synergism of nascent and mature HDL contribute to cholesterol efflux efficiency. Moreover, extracellular cholesterol deposits and microvesicles may be involved in RCT.

Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling.

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1-43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185-243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (-10 to -28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (-52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.

Denaturation of human plasma high-density lipoproteins by urea studied by apolipoprotein A-I dissociation.

We studied the mechanism of HDL denaturation with concomitant apoA-I dissociation with HDL preparations from 48 patients with a wide range of plasma HDL-C and evaluated the contribution of lipid-free apoA-I into cholesterol efflux from macrophage, in particular, mediated by cholesterol transporter ABCA1. We prepared HDL by precipitation of apoB-containing lipoproteins by polyethylene glycol and used the chaotropic agent urea to denature HDL preparations. Apo-I dissociation from urea-treated HDL was assessed by the increase of preß-band fraction with agarose gel electrophoresis followed by electro transfer and immunodetection and by the increase of ABCA1-mediated efflux of fluorescent analogue BODIPY-Cholesterol from RAW 264.7 macrophages. The HDL denaturation is governed by a single transition to fully dissociated apoA-I and the transition cooperativity decreases with increasing HDL-C. The apoA-I release depends on phospholipid concentration of HDL preparation and HDL compositional and structural heterogeneity and is well described by apolipoprotein partition between aqueous and lipid phases. Dissociated apoA-I determines the increase of ABCA1-mediated efflux of BODIPY-Cholesterol from RAW 264.7 macrophages to patient HDL. The increase in apoA-I dissociation is associated with the increase of ABCA1 gene transcript in peripheral blood mononuclear cells from patients. The low level of plasma HDL particles may be compensated by their increased potency for apoA-I release, thus suggesting apoA-I dissociation as a new HDL functional property.