Free Cholesterol Efflux Research Articles

Tangier Disease

Tangier disease is one of the most severe forms of familial high-density lipoprotein (HDL) deficiency. Since its discovery it has been diagnosed in about 100 patients and is characterized by severe plasma deficiency or absence of HDL, apolipoprotein A-I (apoA-I, the major HDL apolipoprotein) and by accumulation of cholesteryl esters in many tissues throughout the body. The biochemical signs of this condition are plasma HDL concentrations less than 5 mg/dL, low total plasma cholesterol (below 150 mg/dL), and normal or high plasma triglycerides. Tangier disease is caused by mutations in the 'ATP-Binding Cassette transporter A1' (ABCA1) gene, which encodes the membrane transporter ABCA1. This transporter plays a key role in the first step of reverse cholesterol transport, through which the efflux of free cholesterol from peripheral cells is transferred to lipid-poor apoA-I. The Tangier disease clinical phenotype is inherited as an autosomal recessive trait, the biochemical phenotype is inherited as an autosomal co-dominant trait. Nearly all the children affected by Tangier disease were identified on the basis of large, yellow-orange tonsils, while half of the adult patients affected by Tangier disease came to medical attention because of symptoms of neuropathy. Diagnosis in the remaining subjects was related to the clinical features of hepatomegaly, splenomegaly, premature myocardial infarction (about 30% of Tangier disease cases) or stroke, thrombocytopenia, anemia, gastrointestinal disorders, corneal opacities, hypocholesterolemia, low HDL cholesterol, or following a familial screening of Tangier patients. To date there is no specific treatment for Tangier disease. Old and recently designed drugs, known to increase HDL levels, have been shown to be ineffective in Tangier patients. The possible and more realistic therapeutic strategy should be designed to obtain a selective increase of mature HDL concentration to restore cholesterol efflux. Recently designed drugs like the cholesteryl ester transfer protein (CETP) inhibitors dalcetrapib and anacetrapib and reconstituted forms of HDL could be considered until the development of gene therapy.

SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to high-density lipoprotein 3

BackgroundThe single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated.ResultsWhen apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI.ConclusionsWe found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.

Elevated CETP Activity Improves Plasma Cholesterol Efflux Capacity From Human Macrophages in Women

We aim to identify the impact of endogenous cholesteryl ester transfer protein (CETP) activity on plasma capacity to mediate free cholesterol efflux from human macrophages. Endogenous plasma CETP activity was measured in a population of 348 women. We defined a low CETP group corresponding to subjects displaying an endogenous plasma CETP activity within the first tertile and a high CETP group corresponding to subjects with an endogenous plasma CETP activity within the third tertile. Subjects from the high CETP activity group displayed a significant increase in the capacity of their plasma (+8.2%; P=0.001) to mediate cholesterol efflux from human acute monocytic leukemia cell line human macrophages and from ATP-binding cassette transporter A1-dependent pathway (+23.4%; P=0.0001) as compared with those from the low CETP activity group. Multivariate analyses revealed that the impact of CETP activity was independent of plasma lipids levels. Pre-β1-high-density lipoprotein concentrations were significantly elevated (+29.6%; P=0.01) in the high CETP activity group as compared with the low CETP activity group. A positive correlation between pre-β1-high-density lipoprotein levels and plasma efflux efficiency from human acute monocytic leukemia cell line human macrophages was observed (r=0.29, P=0.02). CETP leading to the improvement of plasma efflux capacity, as a result of efficient pre-β-high-density lipoprotein formation and ATP-binding cassette transporter A1 efflux, should be preserved to prevent lipid accumulation in human macrophages.

Measurement of Reverse Cholesterol Transport Pathways in Humans: In Vivo Rates of Free Cholesterol Efflux, Esterification, and Excretion

BackgroundReverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC.Methods and ResultsA constant infusion of [2,3-13C2]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester 13C–enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,3-13C2]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or ≍8 g/d. Red blood cell–derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was ≍28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,3-13C2]-cholesterol in fecal bile acids and neutral sterols, respectively.ConclusionsThree components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. (J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826.)

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.

Involvement of cholesterol efflux pathway in the control of cardiomyocytes cholesterol homeostasis

Although cholesterol-rich microdomains are highly involved in the functions of cardiomyocytes, the cholesterol homeostasis is largely unknown in these cells. We developed experimental procedures to assess cholesterol synthesis, cholesterol masses and cholesterol efflux from primary cultures of cardiac myocytes obtained from 2 to 4days old Wistar rats. We first observed that cardiomyocytes poorly internalized exogenously supplied native or modified LDL and that free cholesterol (FC) efflux to free apolipoprotein AI (apo AI) and to HDL was mediated by ATP binding cassette transporter A1 (ABCA1) and likely by ATP binding cassette transporter G1 (ABCG1), respectively, which are both upregulated by liver X receptor/retinoid X receptor (LXR/RXR) activation. We then investigated the consequences of cholesterol synthesis inhibition on cholesterol homeostasis using an HMGCoA reductase inhibitor (pravastatin, 90% effective concentration (EC90): 0.11mM, 18h). We observed no impact of cholesterol synthesis inhibition on the FC or cholesteryl ester (CE) masses. Consistently with no FC mass changes, pravastatin treatment had no notable impact on LDL receptors mRNA expression or on the capacity of cardiomyocytes to uptake radiolabeled LDL. Conversely, pravastatin treatment induced a significant decrease of cholesterol efflux to both apo AI and HDL whereas the passive aqueous diffusion remained unchanged. The cholesterol efflux pathway reductions induced by cholesterol synthesis inhibition were not caused by a reduction of ABC transporter expression (mRNA or protein). These results show that cardiac myocytes down-regulate active cholesterol efflux processes when endogenous cholesterol synthesis is inhibited, allowing them to preserve cholesterol homeostasis.

Abstract 23: Identification of a Novel Intracellular Cholesteryl Ester Hydrolase (CES3) in Human Macrophages: Compensatory Increase Following CES1 Silencing

Removal of free cholesterol (FC) from lesion-associated macrophage foam cells represents the major mechanism for reducing the lipid burden of atherosclerotic plaques and intracellular cholesteryl ester (CE) hydrolysis is recognized as the rate-limiting step in this process. We have earlier reported cloning of human macrophage CE hydrolase (gene symbol CES1) that associated with cytoplasmic lipid droplets and enhanced cellular CE mobilization. Macrophage-specific transgenic expression of CES1 attenuated diet-induced atherosclerosis and lesion necrosis in ldlr -/- mice. Several enzymes have since been identified as intracellular CE hydrolases in human macrophages and the objective of the present study was to determine the relative contribution of CES1 to the total CE hydrolytic activity in human macrophages. Immune-precipitation by CES1-specific antibody resulted in 70-80% decrease in enzyme activity indicating that CES1 is responsible for &gt;70% of the total CE hydrolytic activity (Panel A). To examine the effects of CES1 depletion/knockdown on FC efflux, human monocyte/macrophages (THP1) were stably transfected with 4 different CES1-specific shRNA vectors (p81-p84) to generate THP1-shRNA cells; cells transfected with pRSL vector were used as controls. Despite significant reduction in CES1 expression both at mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel carboxylesterase, CES3 (Panel B). Human macrophage CES3 was identical to the reported CES3 except it lacked exon 8 and, in wild type THP1 cells it was only expressed at 29.2±6.1 percent of CES1. Transient over-expression of this novel variant of CES3 led to an increase in CE hydrolytic activity (&gt;4 fold), mobilization of intracellular lipid droplets and reduction in cellular CE content (108.1±7.65 vs 160.9±7.00, P=0.0005), establishing CES3 as a bona fide CE hydrolase. Reciprocal backup circuits, where deficiency of a gene leads to an increase in another gene with similar function, have been described and this study provides first evidence of functional compensation in intracellular CE hydrolysis whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. To gain insight into the mechanisms underlying the observed increase in CES3 following CES1 deficiency in silico analyses of CES3 promoter was performed and several NF-kB binding sites were identified. Based on our earlier demonstration of a decrease in NF-kB activation by CES1 over-expression, it is speculated that conversely, deficiency of CES1 increases NF-kB activation leading to compensatory increase in CES3 expression.

New Roles of HDL in Inflammation and Hematopoiesis

High-density lipoprotein (HDL) levels are inversely associated with coronary heart disease due to HDL's ability to transport excess cholesterol in arterial macrophages to the liver for excretion [i.e., reverse cholesterol transport (RCT)]. However, recent advances highlight additional atheroprotective roles for HDL beyond bulk cholesterol removal from cells through RCT. By promoting cellular free cholesterol (FC) efflux, HDL and its apolipoproteins (apoA-I and apoE) decrease plasma membrane FC and lipid raft content in immune and hematopoietic stem cells, decreasing inflammatory and cell proliferation signaling pathways. HDL and apoA-I also dampen inflammatory signaling pathways independent of cellular FC efflux. In addition, HDL lipid and protein cargo provide protection against parasitic and bacterial infection, endothelial damage, and oxidant toxicity. Here, current knowledge is reviewed regarding the role of HDL and its apolipoproteins in regulating cellular cholesterol homeostasis, highlighting recent advances on novel functions and mechanisms by which HDLs regulate inflammation and hematopoiesis.

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR(-/-) background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [(14)C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [(14)C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6C(lo)) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.

Functionality of postprandial larger HDL2 particles is enhanced following CETP inhibition therapy

ObjectiveTo evaluate the impact of CETP inhibition on the capacity of individual postprandial HDL subspecies to promote key steps of the reverse cholesterol transport pathway. MethodsThe capacity of HDL particles to mediate cellular free cholesterol efflux and selective hepatic uptake of cholesteryl esters was evaluated throughout postprandial phase (0–8h) following consumption of a standardised mixed meal before and after treatment for 6 weeks with atorvastatin alone (10mg/d) and subsequently with combination torcetrapib/atorvastatin (60/10mg/d) in 16 patients displaying low HDL-C levels (<40mg/dl). ResultsThe larger HDL2b and HDL2a subfraction displayed a superior capacity to mediate cellular free cholesterol efflux via both SR-BI and ABCG1-dependent pathways than smaller HDL3 subspecies. CETP inhibition specifically enhanced the capacity of HDL2b subfraction for both SR-BI and ABCG1 dependent efflux. However, only the SR-BI-dependent efflux to HDL2b subspecies can be further enhanced during postprandial lipemia following CETP inhibition. Concomitantly, postprandial lipemia was associated with a reduced capacity of total HDL particles to deliver cholesteryl esters to hepatic cells in a drug independent manner. ConclusionCETP inhibition specifically improves postprandial SR-BI and ABCG1-dependent efflux to larger HDL2b subspecies. In addition, CETP inhibition improves HDL-CE delivery to hepatic cells and maintains an efficient direct return of cholesteryl esters to the liver during postprandial lipemia.

Apolipoprotein A-I Inhibits CD40 Proinflammatory Signaling via ATP-Binding Cassette Transporter A1-Mediated Modulation of Lipid Raft in Macrophages

Apolipoprotein A-I (apoA-I), the major component of high-density lipoprotein (HDL), has been recently found to suppress inflammation. This study was to investigate the effects and potential mechanisms of apoA-I on the CD40/CD40 ligand (CD40L) proinflammatory signaling pathway. Human THP-1 macrophage-derived foam cells were treated with sCD40L alone or in the presence of apoA-I. Secretion of proinflammatory cytokines was performed by enzyme-linked immunosorbent assay(ELISA). The proteins and mRNA expression were examined by western-blot and real-time PCR analysis, respectly. Cholesterol efflux was assessed by liquid scintillation counting. Cholesterol depletion of macrophages was performed with methylated β-cyclodextrin. ApoA-I inhibits the inflammatory response stimulated by soluble CD40L (sCD40L) in macrophages. In addition, apoA-I inhibited the sCD40L-stimulated activation of nuclear factor-kB (NF-kB). The apoA-I-induced NF-kB deactivation was related to the decreased recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF-6), a crucial adapter protein for CD40 in macrophages, to lipid rafts after being treated by sCD40L. When interfering the expression of ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transporter for apoA-I in macrophages, it could significantly diminish the effect of apoA-I on the sCD40L-stimulated inflammatory response. ApoA-I suppresses CD40 proinflammatory signaling in macrophages by preventing TRAF-6 translocation to lipid rafts through ABCA1-dependent regulation of free cholesterol (FC) efflux, which may present a novel mechanism of apoA-I-mediated inflammation inhibition in macrophages.

ATP-binding cassette transporter G1 protects against endothelial dysfunction induced by high glucose

IntroductionIt is known that hyperglycaemia can contribute to defects in endothelial function, which is the early characteristic of atherosclerosis. ATP-binding cassette transporter G1 (ABCG1), a regulator of reversing cholesterol efflux,...

Interleukin-6 Protects Human Macrophages from Cellular Cholesterol Accumulation and Attenuates the Proinflammatory Response

Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions.

Identification of an amino acid residue in ATP-binding cassette transport G1 critical for mediating cholesterol efflux

The ATP-binding cassette transporter G1 (ABCG1) mediates free cholesterol efflux onto lipidated apolipoprotein A-I (apoA-I) and plays an important role in macrophage reverse cholesterol transport thereby reducing atherosclerosis. However, how ABCG1 mediates the efflux of cholesterol onto lipidated apoA-I is unclear. Since the crystal structure of ABCG family is not available, other approaches such as site-directed mutagenesis have been widely used to identify amino acid residues important for protein functions. We noticed that ABCG1 contains a single cysteine residue in its putative transmembrane domains. This cysteine residue locates at position 514 (Cys514) within the third putative transmembrane domain and is highly conserved. Replacement of Cys514 with Ala (C514A) essentially abolished ABCG1-mediated cholesterol efflux onto lipidated apoA-I. Substitution of Cys514 with more conserved amino acid residues, Ser or Thr, also significantly decreased cholesterol efflux. However, mutation C514A had no detectable effect on protein stability and trafficking. Mutation C514A also did not affect the dimerization of ABCG1. Our findings demonstrated that the sulfhydryl group of Cys residue located at position 514 plays a critical role in ABCG1-mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).

The origin and metabolism of a nascent pre-β high density lipoprotein involved in cellular cholesterol efflux.

The pre-β HDL fraction constitutes a heterogeneous population of discoid nascent HDL particles. They transport from 1 to 25 % of total human plasma apo A-I. Pre-β HDL particles are generated de novo by interaction between ABCA1 transporters and monomolecular lipid-free apo A-I. Most probably, the binding of apo A-I to ABCA1 initiates the generation of the phospholipid-apo A-I complex which induces free cholesterol efflux. The lipid-poor nascent pre-β HDL particle associates with more lipids through exposure to the ABCG1 transporter and apo M. The maturation of pre-β HDL into the spherical α-HDL containing apo A-I is mediated by LCAT, which esterifies free cholesterol and thereby forms a hydrophobic core of the lipoprotein particle. LCAT is also a key factor in promoting the formation of the HDL particle containing apo A-I and apo A-II by fusion of the spherical α-HDL containing apo A-I and the nascent discoid HDL containing apo A-II. The plasma remodelling of mature HDL particles by lipid transfer proteins and hepatic lipase causes the dissociation of lipid-free/lipid-poor apo A-I, which can either interact with ABCA1 transporters and be incorporated back into pre-existing HDL particles, or eventually be catabolized in the kidney. The formation of pre-β HDL and the cycling of apo A-I between the pre-β and α-HDL particles are thought to be crucial mechanisms of reverse cholesterol transport and the expression of ABCA1 in macrophages may play a main role in the protection against atherosclerosis.

Extracellular Disulfide Bonds Support Scavenger Receptor Class B Type I-Mediated Cholesterol Transport

Scavenger receptor class B type I (SR-BI) binds high-density lipoprotein (HDL) and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesized that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite for SR-BI-mediated cholesterol transport. Thus, single-Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S-, and C384S-SR-BI), as well as Cys-less SR-BI, a mutant SR-BI receptor void of all Cys residues, were created, and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, and C334S-SR-BI and Cys-less SR-BI mutant receptors displayed weakened HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI's ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.

Atheroprotective Reverse Cholesterol Transport Pathway Is Defective in Familial Hypercholesterolemia

Low high-density lipoprotein (HDL) cholesterol levels are frequently observed in familial hypercholesterolemia (FH) and might be associated with functional alterations of HDL particles that may influence their efficaciousness in the reverse cholesterol transport pathway. We evaluated key steps of the reverse cholesterol transport, ie, cellular free cholesterol efflux, cholesteryl ester transfer protein-mediated cholesteryl ester (CE) transfer from HDL to apolipoprotein B-containing lipoproteins, and hepatic HDL-CE uptake, in patients displaying FH (n = 12) and in healthy normolipidemic control subjects (n = 12). Large HDL2 particles isolated from FH patients displayed a reduced capacity to mediate free cholesterol efflux via both scavenger receptor-BI- and ABCG1-dependent pathways. A significant inverse relationship between scavenger receptor-BI-dependent HDL2 efflux capacity and carotid intima-media thickness (r = -0.473; P = 0.0186), as well as between ABCG1-dependent HDL2 efflux capacity and carotid intima-media thickness (r = -0.485; P = 0.0212), was detected. We also observed an elevated cholesteryl ester transfer protein-mediated CE transfer from HDL2 and HDL3 particles to low-density lipoprotein and a reduced capacity of HDL particles to deliver CEs to the liver. We demonstrated that the centripetal movement of cholesterol from peripheral tissues, including the vessel wall, to feces is defective in FH, thereby emphasizing its atherogenicity.

PPARγ activation redirects macrophage cholesterol from fecal excretion to adipose tissue uptake in mice via SR-BI

PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/ 3H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change 3H-tracer plasma appearance, but surprisingly decreased fecal 3H-free sterol excretion by 43% ( P < 0.01) over 48 h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with 3H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived 3H-CE uptake by adipose tissue ( P < 0.005) with concomitant 22% decrease in HDL derived 3H-CE uptake by the liver ( P < 0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived 3H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.