Plasma turnover of HDL apoC-I,apoC-III,and apoE in humans: in vivo evidence for a link between HDL apoC-III and apoA-I metabolism

Numerous factors are known to affect the plasma metabolism of HDL, including lipoprotein receptors, lipid transfer protein, lipolytic enzymes and HDL apolipoproteins. In order to better define the role of HDL apolipoproteins in determining plasma HDL concentrations, the aims of the present study were: a) to compare the in vivo rate of plasma turnover of HDL apolipoproteins [i.e., apolipoprotein A-I (apoA-I), apoC-I, apoC-III, and apoE], and b) to investigate to what extent these metabolic parameters are related to plasma HDL levels. We thus studied 16 individuals with HDL cholesterol levels ranging from 0.56-1.66 mmol/l and HDL apoA-I levels ranging from 89-149 mg/dl. Plasma kinetics of HDL apolipoproteins were investigated using a primed constant (12 h) infusion of deuterated leucine. Plasma HDL apolipoprotein levels were 41.8 +/- 1.5, 9.7 +/- 0.5, 4.9 +/- 0.5, and 0.7 +/- 0.1 micromol/l for apoA-I, apoC-I, apoC-III and apoE. Plasma transport rates (TRs) were 388.6 +/- 24.7, 131.5 +/- 12.5, 66.5 +/- 9.1, and 31.4 +/- 3.3 nmol.kg-1.day-1; and residence times (RTs) were 5.1 +/- 0.4, 3.7 +/- 0.3, 3.6 +/- 0.3, and 1.1 +/- 0.1 days, respectively. HDL cholesterol and apoA-I levels were significantly correlated with HDL apoA-I RT (r = 0.69 and r = 0.56), and were not significantly correlated with HDL apoA-I TR. In contrast, HDL apoC-I, apoC-III, and apoB levels were all positively related to their TRs and not their RTs. HDL apoC-III TR was positively correlated with levels of HDL apoC-III (r = 0.73, P < 0.01), and with those of HDL cholesterol and apoA-I (r = 0.54 and r = 0.53, P < 0.05, respectively). HDL apoC-III TR was in turn related to HDL apoA-I RT (r = 0.51, P < 0.05). Together, these results provide in vivo evidence for a link between the metabolism of HDL apoC-III and apoA-I, and suggest a role for apoC-III in the regulation of plasma HDL levels.     

Behaviour of phospholipase-modified HDL towards cultured hepatocytes. I. Enhanced transfers of HDL sterols and apoproteins

Human HDL subfractions (HDL2, HDL3, or HDL separated by heparin affinity chromatography) were labelled either on their apolipoprotein moiety with 125I or on their sterols: unesterified [14C]cholesterol and [3H]cholesteryl linoleyl ether, a non-hydrolysable analog of esterified cholesterol. HDL subfractions were then treated with or without phospholipase A2 from Crotalus adamanteus in presence of albumin leading to a 72-82% phosphatidylcholine degradation. Control and treated HDL were reisolated and then addressed to cultured rat hepatocytes. (A) During incubations, unesterified [14C]cholesterol from HDL3 readily appeared in hepatocytes. The specific uptake of HDL esterified cholesterol calculated from [3H]cholesteryl ether was 2-4-times less important. Uptake of HDL cholesterol tended to saturate at 150-200 micrograms/ml HDL protein. A prior phospholipase treatment of HDL3 stimulated by 2-5-fold the uptake of [3H]cholesteryl ether, whereas the transfer of free [14C]cholesterol was minimally increased. The uptake of 3H/14C-labelled sterols from HDL2 was 2-3-times higher than from HDL3. (B) Parallel experiments were conducted with 125I-labelled HDL subfractions. At 37 degrees C, the specific uptake and degradation of HDL3 125I-apolipoprotein were about 2-fold enhanced following treatment of HDL3 with phospholipase A2. Uptakes of apolipoprotein and of esterified cholesterol were compared, indicating a preferential delivery of the sterol over apoprotein (X5). The dissociation was still more pronounced with phospholipase-treated HDL3. Competition experiments showed that 12-times more unlabelled HDL3 were required to half reduce the uptake of HDL3 [3H]cholesteryl ether than to impede similarly the HDL 125I-apolipoprotein recovered in cells. Uptake of 125I-labelled apolipoprotein from HDL2 was quantitatively comparable to that from HDL3. (C) Binding of 125I-HDL subfractions was followed at 4 degrees C. A specific binding was observed for HDL2 and HDL3, although kinetic parameters were quite different (KD of 9 and 25 micrograms/ml, respectively). Following phospholipolysis, both the specific and non-specific contributions to total binding were increased. Hence, hepatocytes take up more 125I-labelled apolipoprotein and 3H/14C-labelled sterols from lipolysed HDL than from unmodified particles. This is associated to changes in the binding characteristics.     

Role of apolipoprotein A-II in the structure and remodeling of human high-density lipoprotein (HDL): protein conformational ensemble on HDL

High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and genetic determinants of HDL metabolism and hepatic lipase activity in normolipidemic females.

The metabolic and genetic determinants of HDL cholesterol (HDL-C) levels and HDL turnover were studied in 36 normolipidemic female subjects on a whole-food low-fat metabolic diet. Lipid, lipoprotein, and apolipoprotein levels, lipoprotein size, and apolipoprotein turnover parameters were determined, as were genetic variation at one site in the hepatic lipase promoter and six sites in the apolipoprotein AI/CIII/AIV gene cluster. Menopause had no significant effect on HDL-C or turnover. Stepwise multiple regression analysis revealed that HDL-C was most strongly correlated with HDL size, apolipoprotein A-II (apoA-II), and apolipoprotein A-I (apoA-I) levels, which together could account for 90% of the variation in HDL-C. HDL size was inversely correlated with triglycerides, body mass index, and hepatic lipase activity, which together accounted for 82% of the variation in HDL size. The hepatic lipase promoter genotype had a strong effect on hepatic lipase activity and could account for 38% of the variation in hepatic lipase activity. The apoA-I transport rate (AI-TR) was the major determinant of apoA-I levels, but AI-TR was not associated with six common genetic polymorphism in the apoAI/CIII/AIV gene cluster.A simplified model of HDL metabolism is proposed, in which A-I and apoA-II levels combined with triglycerides, and hepatic lipase activity could account for 80% of the variation in HDL-C.     

Human high density lipoprotein (HDL3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL3 to purified rat liver plasma membranes was studied. 125I-labeled HDL3 bound to the membranes with a dissociation constant of 10.5 micrograms protein/ml and a maximum binding of 3.45 micrograms protein/mg membrane protein. The 125I-labeled HDL3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-labeled HDL3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL3. The 125I-labeled HDL3-binding activity of the membranes was not sensitive to pronase or phospholipase A2; however, prior treatment of the membranes with phospholipase A2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100 degrees C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL3-binding activity.     

Interaction of human plasma high-density lipoprotein HDL2b with discoidal complexes of dimyristoylphosphatidylcholine and apolipoprotein A-I

The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.     

HDL modification by secretory phospholipase A(2) promotes scavenger receptor class B type I interaction and accelerates HDL catabolism

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney.We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.     

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL₃ or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Oxidized HDL are much less cytotoxic to lymphoblastoid cells than oxidized LDL.

The possible effect of oxidized HDL was investigated on lymphoblastoid cells, in comparison to the cytotoxic effect of oxidized LDL. Oxidation of HDL was promoted by UV-C irradiation, or by copper ion (5 microM) or the combination of the two treatments. HDL extensively treated by UV-C for 20 h did not exhibit any cytotoxic effect on cultured lymphoblastoid cells even at a concentration of 500 micrograms apolipoprotein A-I/ml. In contrast to UV-treated (2 h) LDL, which were highly cytotoxic (already at a concentration of 100 micrograms apolipoprotein B/ml), HDL treated by copper or copper + UV were oxidized, as shown by TBARS formation and PUFA content decrease, but were slightly cytotoxic.     

Purified HDL-apolipoproteins, A-I and C-III, substitute for HDL in promoting the growth of SV40-transformed REF52 cells in serum-free medium

The lipid-free apolipoproteins of human high density lipoprotein (HDL) have been assayed for their ability to substitute for native HDL in promoting the growth of a SV40-transformed REF52 cell line in serum-free medium. Total HDL-apolipoproteins (apoHDL) were found to mimic almost exactly the growth promoting effects of whole HDL. The apoHDL-associated growth promoting activity eluted from a Sephacryl S-200 column in two separate fractions coinciding with the protein peaks of apolipoprotein A-I and the C group of apolipoproteins. These two fractions, designated S-II and S-IV, respectively, acted additively in promoting WT1A cell growth when tested at saturating concentrations. The active component in the S-II fraction maximally stimulated WT1A cell growth at 40-60 micrograms/ml and was identified as apolipoprotein A-1 by NaDodSO4 polyacrylamide gel electrophoresis and affinity chromatography on anti-(apoA-I). The active component in the S-IV fraction was maximally active at 1-2 micrograms/ml and was identified as apolipoprotein C-III by DEAE ion exchange high pressure liquid chromatography and polyacrylamide gel electrophoresis (at pH 8.3) in 6 M urea. These results indicate that the growth promoting effect of HDL on WT1A cells is mediated via the HDL-apolipoproteins, A-I and C-III, and that the mechanism responsible does not necessarily involve their participation in the uptake (or utilization) of HDL-associated lipids.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

Testosterone replacement in hypogonadal men alters the HDL proteome but not HDL cholesterol efflux capacity

The effects of androgens on cardiovascular disease (CVD) risk in men remain unclear. To better characterize the relationship between androgens and HDL, we investigated the effects of testosterone replacement on HDL protein composition and serum HDL-mediated cholesterol efflux in hypogonadal men. Twenty-three older hypogonadal men (ages 51-83, baseline testosterone < 280 ng/dl) were administered replacement testosterone therapy (1% transdermal gel) with or without the 5α-reductase inhibitor dutasteride. At baseline and after three months of treatment, we determined fasting lipid concentrations, HDL protein composition, and the cholesterol efflux capacity of serum HDL. Testosterone replacement did not affect HDL cholesterol (HDL-C) concentrations but conferred significant increases in HDL-associated paraoxonase 1 (PON1) and fibrinogen α chain (FGA) (P = 0.022 and P = 0.023, respectively) and a decrease in apolipoprotein A-IV (apoA-IV) (P = 0.016). Exogenous testosterone did not affect the cholesterol efflux capacity of serum HDL. No differences were observed between men who received testosterone alone and those who also received dutasteride. Testosterone replacement in older hypogonadal men alters the protein composition of HDL but does not significantly change serum HDL-mediated cholesterol efflux. These effects appear independent of testosterone conversion to dihydrotestosterone. Further research is needed to determine how changes in HDL protein content affect CVD risk in men.     

Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI

Apolipoprotein A-I (apoA-I) is an important ligand for the high density lipoprotein (HDL) scavenger receptor class B type I (SR-BI). SR-BI binds both free and lipoprotein-associated apoA-I, but the effects of particle size, composition, and apolipoprotein conformation on HDL binding to SR-BI are not understood. We have studied the effect of apoA-I conformation on particle binding using native HDL and reconstituted HDL particles of defined composition and size. SR-BI expressed in transfected Chinese hamster ovary cells was shown to bind human HDL(2) with greater affinity than HDL(3), suggesting that HDL size, composition, and possibly apolipoprotein conformation influence HDL binding to SR-BI. To discriminate between these factors, SR-BI binding was studied further using reconstituted l-alpha-palmitoyloleoyl-phosphatidylcholine-containing HDL particles having identical components and equal amounts of apoA-I, but differing in size (7.8 vs. 9.6 nm in diameter) and apoA-I conformation. The affinity of binding to SR-BI was significantly greater (50-fold) for the larger (9.6-nm) particle than for the 7.8-nm particle. We conclude that differences in apoA-I conformation in different-sized particles markedly influence apoA-I recognition by SR-BI. Preferential binding of larger HDL particles to SR-BI would promote productive selective cholesteryl ester uptake from larger cholesteryl ester-rich HDL over lipid-poor HDL.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

HDL proteome in hemodialysis patients: a quantitative nanoflow liquid chromatography-tandem mass spectrometry approach

Aside from a decrease in the high-density lipoprotein (HDL) cholesterol levels, qualitative abnormalities of HDL can contribute to an increase in cardiovascular (CV) risk in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Dysfunctional HDL leads to an alteration of reverse cholesterol transport and the antioxidant and anti-inflammatory properties of HDL. In this study, a quantitative proteomics approach, based on iTRAQ labeling and nanoflow liquid chromatography mass spectrometry analysis, was used to generate detailed data on HDL-associated proteins. The HDL composition was compared between seven chronic HD patients and a pool of seven healthy controls. To confirm the proteomics results, specific biochemical assays were then performed in triplicate in the 14 samples as well as 46 sex-matched independent chronic HD patients and healthy volunteers. Of the 122 proteins identified in the HDL fraction, 40 were differentially expressed between the healthy volunteers and the HD patients. These proteins are involved in many HDL functions, including lipid metabolism, the acute inflammatory response, complement activation, the regulation of lipoprotein oxidation, and metal cation homeostasis. Among the identified proteins, apolipoprotein C-II and apolipoprotein C-III were significantly increased in the HDL fraction of HD patients whereas serotransferrin was decreased. In this study, we identified new markers of potential relevance to the pathways linked to HDL dysfunction in HD. Proteomic analysis of the HDL fraction provides an efficient method to identify new and uncharacterized candidate biomarkers of CV risk in HD patients.     

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.