ABCA1 Pathway Research Articles

Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPARγ–LXRα–ABCA1 pathway

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50=0.09μM). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ; EC50=5.2–9.3μM). Liver X receptor α (LXRα) is a target gene of PPARγ and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXRα protein expression in HepG2 cells. Addition of PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPARγ. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPARγ–LXRα–ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

Possibility of increasing cholesterol efflux by adiponectin and its receptors through the ATP binding cassette transporter A1 in HEK293T cells

A decrease in adiponectin secretion leads to the early stage of atherosclerosis. Discoidal high-density lipoproteins (HDL) accept the cholesterol that effluxes from cells expressing the ATP binding cassette transporter A1 (ABCA1) in the first step of reverse cholesterol transport (RCT). Recently, a new therapeutic strategy involving reconstituted (r)HDL has been shown to enhance RCT. Therefore, we hypothesized that adiponectin may increase the efflux associated with ABCA1 and also enhance rHDL-induced efflux in human embryonic kidney 293 (HEK293T) cells. We transfected adiponectin receptor 1 and 2 (AdipoR1 and AdipoR2) cDNA into cells. The transfected cells were labeled with [3H]cholesterol following cholesterol loading with or without adiponectin for 24h. The levels of cholesterol efflux were analyzed using a liquid scintillation counter. Treatment with adiponectin was associated with significantly higher levels of efflux in AdipoR1- and AdipoR2-transfected cells. Interestingly, rHDL-induced cholesterol efflux was enhanced in the presence of adiponectin. The down-regulation of adiponectin receptors using short-hairpin RNA decreased rHDL-induced cholesterol efflux with the down-regulation of ABCA1. In summary, adiponectin and its receptors increased cholesterol efflux and also enhanced rHDL-induced efflux at least partially through an ABCA1 pathway. These results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic effect.

Impact of HDL oxidation by the myeloperoxidase system on sterol efflux by the ABCA1 pathway

Protein oxidation by phagocytic white blood cells is implicated in tissue injury during inflammation. One important target might be high-density lipoprotein (HDL), which protects against atherosclerosis by removing excess cholesterol from artery wall macrophages. In the human artery wall, cholesterol-laden macrophages are a rich source of myeloperoxidase (MPO), which uses hydrogen peroxide for oxidative reactions in the extracellular milieu. Levels of two characteristic products of MPO-chlorotyrosine and nitrotyrosine-are markedly elevated in HDL from human atherosclerotic lesions. Here, we describe how MPO-dependent chlorination impairs the ability of apolipoprotein A-I (apoA-I), HDL's major protein, to transport cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. Faulty interactions between apoA-I and ABCA1 are involved. Tandem mass spectrometry and investigations of mutated forms of apoA-I demonstrate that tyrosine residues in apoA-I are chlorinated in a site-specific manner by chloramine intermediates on suitably juxtaposed lysine residues. Plasma HDL isolated from subjects with coronary artery disease (CAD) also contains higher levels of chlorinated and nitrated tyrosine residues than HDL from healthy subjects. Thus, the presence of chlorinated HDL might serve as a marker of CAD risk. Because HDL damaged by MPO in vitro becomes dysfunctional, inhibiting MPO in vivo might be cardioprotective.

Familial high-density lipoprotein deficiency states and premature coronary heart disease

High-density lipoprotein cholesterol (HDL-C) values <40 mg/dL have been shown to be an independent risk factor for coronary heart disease (CHD). Rare genetic disorders associated with marked human HDL deficiency include apolipoprotein A-I (apoA-I) deficiency with undetectable plasma apoA-I, which can be due to defects within the APOA1 gene resulting in lack of apoA-I secretion. Such patients have marked HDL deficiency, normal levels of triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C), and they can have xanthomas and premature CHD. ApoA-I variants with amino acid substitutions, especially in the region of amino acid residues 50–93 and 170–178, have been associated with amyloidosis. Patients with homozygous Tangier disease have defective cellular cholesterol efflux due to mutations in the adenosine-5′-triphosphate (ATP)-binding cassette transporter A1, detectable plasma apoA-I levels, and only pre-β1 HDL in their plasma. They have decreased LDL-C levels and can develop neuropathy and premature CHD. Patients with lecithin:cholesterol acyltransferase deficiency have both pre-β1 and α4 HDL present in their plasma and develop corneal opacities, anemia, proteinuria, and kidney failure. HDL deficiency has been associated with hypertriglyceridemia, obesity, insulin resistance, and diabetes. Common familial disorders associated with premature CHD and low HDL are: (1) dyslipidemia, seen in 15% of families, (2) combined hyperlipidemia, seen in 14% of families, and (3) hypoalphalipoproteinemia, seen in 4% of families. A common feature of all these disorders associated with premature CHD is a marked deficiency of very large α1 HDL. Niacin is currently the most optimal treatment strategy to raise HDL-C and normalize the HDL particle profile in these patients. Hot Topics in Cardiometabolic Disorders Volume 2 • Issue 3 • Year 2011 © FBCommunication Modena (Italy) Cite this article as: Schaefer EJ, Santos RD, Tani M, Schaefer PM, Asztalos BF. Familial high-density lipoprotein deficiency states and premature coronary heart disease. Hot Topics Cardiomet Disord 2011;2(3):7-16. E-mail: ernst.schaefer@tufts.edu Hot Topics in Cardiometabolic Disorders 2011;3:7-16 Copyright © 2011 FBCommunication s.r.l. a socio unico Downloaded from www.hottopicsin.com As shown in Figure 2-upper panel pre-β1 HDL is a very small disk with two molecules of apoA-I forming a belt (in yellow) around approximately 16 molecules of phospholipid (in blue), with a diameter of about 5.6 nm and a molecular weight of about 70 kDa. This particle is converted to small α4 HDL, which is a small disk with 2 molecules of apoA-I, but has about 26 molecules of phospholipid, and about 12 molecules of free cholesterol (in green), with a diameter of about 7.4 nm, and a molecular weight of about 80 kDa. Figure 2-lower panel depicts a model of medium spherical α3 HDL with 2 molecules of apoA-I, 1 molecule of apoA-II, with phospholipid and free cholesterol on its surface, and cholesteryl ester (in green) and TG (in purple) in the HDL core with a diameter of about 8.0 nm. The large spherical α2 HDL contains 4 molecules of apoA-I; 2 molecules of apoA-II, phospholipid, and free cholesterol on its surface; and cholesteryl ester and TG in its core, with a diameter of about 9.2 nm (see Figure 2–lower panel). The α1 HDL is very large and spherical with 8 molecules of apoA-I, phospholipid, and free cholesterol on its surface, and cholesteryl ester and TG in the core of HDL with a diameter of about 11.0 nm (see Figure 2–lower panel) [19]. In collaboration with George Rothblat’s laboratory, the authors studied the roles of HDL particles in ABCA1-, ABCG1-, and scavenger receptor class B type 1 (SRB1) mediated lipid flux [20-23]. It has been documented that only the small precursor HDL (pre-β1) was able to remove lipids (phospholipid and free cholesterol) from cells via the ABCA1 pathway, and that the bidirectional cell-lipid flux via the SRB1 mechanism was mediated by the larger HDL

Modifying Apolipoprotein A-I by Malondialdehyde, but Not by an Array of Other Reactive Carbonyls, Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.

Polyphenols and Cholesterol Efflux

Despite strong evidence for an inverse association between high-density lipoprotein cholesterol (HDL-C) levels and cardiovascular risk,1 successful therapeutic strategies to target HDL have remained elusive. A recent Phase III clinical trial failed to show clinical benefit with the cholesteryl ester transfer protein inhibitor torcetrapib despite markedly increased HDL-C levels.2,3 This outcome reinforced a growing consensus that measurement of HDL-C alone may be an incomplete surrogate for the in vivo functionality of HDL and the clinical efficacy of targeting HDL. The careful mechanistic assessment of HDL function has thus emerged as a potential way forward.4 Macrophage reverse cholesterol transport (RCT), the process by which cholesterol is transported from macrophage “foam” cells to the liver for ultimate fecal excretion, has been postulated to play a major role in HDL-mediated atheroprotection.5 Indeed, quantitative measures of macrophage RCT are more strongly associated with atherosclerosis than plasma HDL-C concentrations in mice.6 The first critical step of macrophage RCT involves efflux of cellular cholesterol to circulating HDL particles. Research in recent years has documented a specific role for the macrophage transporters ATP-binding cassette subfamilies A1 and G1 (ABCA1 and ABCG1) in cholesterol efflux (see the Figure). These findings have stimulated efforts to target the macrophage at the cellular level as a means of enhancing overall RCT. For example, pharmacological agonism of the liver X receptor (LXR) upregulates both ABCA1 and ABCG1 expression,7,8 promotes macrophage cholesterol efflux ex vivo and RCT in vivo, …

LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.

HIV hijacks cholesterol transporter ABCA1 to get access to cellular cholesterol

Cholesterol plays an important role in the HIV life cycle, and cholesterol depletion impairs both production and infectivity of HIV virions. Such dependence on cholesterol suggests that HIV may have evolved mechanisms to ensure access to cellular cholesterol during viral assembly. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits activity of the cellular cholesterol transporter ABCA1 and impairs reverse cholesterol transport (RCT) from infected cells. In this study, we examined interaction between Nef and ABCA1 and investigated the role of ABCA1 in cholesterol delivery to nascent HIV virions. ABCA1 is a 12-transmembrane domain protein, and co-immunoprecipitation analysis using Nef and various fragments of ABCA1 identified multiple sites of interaction. Importantly, Nef was found to interact with the 46-aminoacid carboxyl-terminal domain of ABCA1, which was previously implicated in RCT. Mutation of leucines to alanines in positions 2230, 2233 and 2235 impaired interaction of this C-terminal domain of ABCA1 with Nef. When these mutations were introduced in the full-length ABCA1, such mutant protein was able to support RCT but lost sensitivity to Nef. To further characterize the role of ABCA1 inhibition in HIV biology, we analyzed cholesterol content of lipid rafts and HIV virions produced in cells expressing or not ABCA1. This analysis demonstrated that ABCA1 expression significantly reduced lipid raft cholesterol content, resulting in a corresponding reduction of virus-associated cholesterol and viral infectivity. This result is consistent with ABCA1-mediated redirection of cholesterol from lipid rafts (sites of HIV assembly) to non-raft regions of the plasma membrane (physiological localization of ABCA1). Intriguingly, Nef expression induced re-localization of ABCA1 to lipid rafts, suggesting that HIV hijacks the ABCA1 pathway to deliver cholesterol to the sites of HIV assembly.

HDL, lipid peroxidation, and atherosclerosis

Genetic, clinical, and pharmacological studies implicate elevated levels of LDL in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies (1). Paradoxically, native LDL fails to exert potentially atherogenic effects in vitro, suggesting that it must be modified to promote vascular disease. Indeed, many lines of evidence support the LDL oxidation hypothesis, which suggests that oxidative damage to LDL is one important mechanism for rendering lipoproteins atherogenic (as reviewed in Refs. 2, 3).

The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis

Reverse cholesterol transport (RCT) is a term used to describe the efflux of excess cellular cholesterol from peripheral tissues and its return to the liver for excretion in the bile and ultimately the feces. It is believed to be a critical mechanism by which HDL exert a protective effect on the development of atherosclerosis. In this paradigm, cholesterol is effluxed from arterial macrophages to extracellular HDL-based acceptors through the action of transporters such as ABCA1 and ABCG1. After efflux to HDL, cholesterol may be esterified in the plasma by the enzyme lecithin:cholesterol acyltransferase and is ultimately transported from HDL to the liver, either directly via the scavenger receptor BI or after transfer to apolipoprotein B-containing lipoproteins by the cholesteryl ester transfer protein. Methods for assessing the integrated rate of macrophage RCT in animals have provided insights into the molecular regulation of the process and suggest that the dynamic rate of macrophage RCT is more strongly associated with atherosclerosis than the steady-state plasma concentration of HDL cholesterol. Promotion of macrophage RCT is a potential therapeutic approach to preventing or regressing atherosclerotic vascular disease, but robust measures of RCT in humans will be needed in order to confidently advance RCT-promoting therapies in clinical development.

A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL

Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

An Amphipathic Helical Region of the N-terminal Barrel of Phospholipid Transfer Protein Is Critical for ABCA1-dependent Cholesterol Efflux

Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.

The roles of different pathways in the release of cholesterol from macrophages

Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins. We determined the effect of enriching cells with free cholesterol (FC) on the release of FC to human serum. Loading Fu5AH cells with FC had no effect on fractional efflux, whereas enriching mouse peritoneal macrophages (MPMs) resulted in a doubling of fractional efflux. Efflux from cholesterol-normal MPM and Fu5AH cells to 15 human sera correlated well with HDL parameters. However, these relationships were reduced or lost with cholesterol-loaded MPMs. Using macrophages from scavenger receptor class B type I (SR-BI)-, ABCA1-, and ABCG1-knockout mice, together with inhibitors of SR-BI- and ABCA1-mediated efflux, we were able to quantitate efflux upon loading macrophages with excess cholesterol and to establish the contributions of the various efflux pathways in cholesterol-normal and -enriched cells. The removal of ABCA1 had essentially no effect on the total efflux when cell cholesterol levels were normal. However, in cholesterol-enriched cells, the removal of ABCA1 reduced efflux by 50%. Approximately 20% of the efflux stimulated by FC-loading MPM is attributable to ABCG1. The SR-BI contribution to efflux was small. Another pathway that is present in all cells is aqueous diffusion. Our studies demonstrate that this mechanism is one of the major contributors to efflux, particularly in cholesterol-normal cells.

Dual effects of oxidized low-density lipoprotein on LXR-ABCA1-apoA-I pathway in 3T3-L1 cells

Background The adipocyte has been proven to recognize and degrade oxidized low-density lipoprotein (oxLDL), while cholesterol efflux from adipocytes to clear excess cholesterol loaded by oxLDL is essential to maintain its normal function. Thus, it is intriguing to explore the effects of oxLDL on cholesterol efflux in adipocytes. Methods Fully differentiated 3T3-L1 cells were incubated in the medium containing various concentrations of oxLDL (0 to 50 μg/mL) for 8 or 24 h. 10μmol/L 22(R)-hydroxycholesterol was exposed to preconditioned adipocytes with 25μg/mL oxLDL for 24 h. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate adipocytes mRNA expression. Cholesterol efflux rate was determined through measuring release of radioactivity from 3H-cholesterol prelabeled cells into medium containing apolipoprotein A-I (apoA-I). Results Low concentrations of oxLDL caused a significant increase in apoA-I-mediated cholesterol efflux via enhancement of ATP binding cassette transporter A1 (ABCA1) pathway, whereas higher concentrations were incapable. In adipocytes preincubated with 25 μg/mL oxLDL for 24 h, 22(R)-hydroxycholesterol could increase ABCA1 and LXRα mRNA levels and apoA-I-mediated cholesterol efflux. Conclusion OxLDL has dual effects on ABCA1 pathway in adipocytes. It depends on the concentration and exposure time. The new action of low levels of oxLDL may provide further understanding to its atheroprotective effects.

Adiponectin accelerates reverse cholesterol transport by increasing high density lipoprotein assembly in the liver

Plasma high density lipoprotein (HDL)–cholesterol levels are negatively correlated with the incidence of coronary artery disease. HDL plays an important role in protecting against atherosclerosis by removing cholesterol from atheroma and transporting it back to the liver. The ATP-binding cassette transporters (ABCA1 and ABCG1) and scavenger receptor BI (SR-BI) are thought to be one of the rate-limiting factors to generate HDL in the liver. Adiponectin (APN) secreted from adipocytes is also one of the important molecules to inhibit the development of atherosclerosis. Recently, it has been reported that plasma HDL–cholesterol levels are positively correlated with plasma APN concentrations in humans. Therefore, we investigated the association of APN with HDL assembly in the liver. Human hepatoma cell line, HepG2 cells, were incubated for 24 h in the culture medium with the indicated concentrations of recombinant APN. APN enhanced the mRNA level of apolipoprotein A-I (apoA-I) in HepG2 cells and increased the secretion of apoA-I from the cells to the medium. Furthermore, APN increased both mRNA and protein levels of ABCA1, but not ABCG1 and SR-BI, in HepG2 cells. Taken together, the current study demonstrates that APN might protect against atherosclerosis by increasing HDL assembly through enhancing ABCA1 pathway and apoA-1 synthesis in the liver.

Unsaturated fatty acids phosphorylate and destabilize ABCA1 through a protein kinase C δ pathway

Abnormal HDL metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are increased in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. We previously reported that unsaturated fatty acids destabilize ABCA1 in murine macrophages and ABCA1-transfected baby hamster kidney cells by increasing its serine phosphorylation through a phospholipase D (PLD) pathway. Here, we examined the cellular pathway downstream of PLD that mediates the ABCA1-destabilizing effects of unsaturated fatty acids. The protein kinase C delta (PKCdelta)-specific inhibitor rottlerin and PKCdelta small interfering RNA completely abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1, implicating a role for PKCdelta in the ABCA1-destabilizing effects of fatty acids. These data indicate that unsaturated fatty acids destabilize ABCA1 by activating a PKCdelta pathway that phosphorylates ABCA1 serines.

The Modern Art of Atherosclerosis

Atherosclerosis is a disease of large arteries that accounts for more than 50% of all deaths in the developed countries1 and, according to the World Health Organization prediction, it will gain a status of the major morbidity cause worldwide by the year 2010.2 Atherosclerosis is widely viewed as an inflammatory disease with hypercholesterolemia being a dominant underlying risk factor.3,4 It is believed to be initiated by retention of LDL particles in the lesion-prone areas, which is followed by monocyte recruitment and their differentiation into cholesterol-laden macrophage foam cells.1,5 Excessive cholesterol accumulation in macrophages exaggerates innate immune response that is manifested by upregulated production and secretion of inflammatory cytokines and chemokines, thus dramatically amplifying initial signal originated from the injured artery.6 See page 519 Despite the overall consensus on the causative roles of excessive cholesterol build-up and inflammation in the development and progression of atherosclerosis, the relationship between aberrant cholesterol metabolism and exaggerated innate immune response has not been totally established. Recent reports showing reduced inflammation in association with the cholesterol-lowering statin therapy provided indirect evidence linking cholesterol metabolism with regulation of immune response.7 However, this class of cholesterol lowering drugs not only inhibits cholesterol biosynthesis but also suppresses the synthesis of isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Thus, inhibition of prenylation pathways such as those required for activation of Ras and Rho GTPase family proteins may be responsible for some of the …

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.