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Cardiovascular Research 2005 68(3):425-432; doi:10.1016/j.cardiores.2005.07.003
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Copyright © 2005, European Society of Cardiology

Oxidized LDL downregulates ATP-binding cassette transporter-1 in human vascular endothelial cells via inhibiting liver X receptor (LXR)

Yi Zhua,b,*, Hailing Liaob, Xuefen Xiea, Yuan Yuanb, Tzong-Shyuan Leeb, Nanping Wanga, Xian Wanga, John Y.-J. Shyyb and Michael B. Stemermanb

aDepartment of Physiology, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Health Science Center, Peking University, Beijing 100083, China
bDivision of Biomedical Sciences, University of California, Riverside, Riverside, CA 92521, United States

* Corresponding author. Department of Physiology and Pathophysiology, Peking University, Health Sciences Center, Beijing 100083, China. Tel.: +86 10 8280 1440; fax: +86 10 8280 1440. Email address: zhuyi{at}bjmu.edu.cn

Received 18 March 2005; revised 27 June 2005; accepted 5 July 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: ATP-binding cassette transporter-1 (ABCA1) mediates the active efflux of cholesterol and phospholipids, playing an important role in cholesterol homeostasis and atherogenesis. Oxidized low density lipoprotein (oxLDL) is an atherogenic molecule associated with the vascular endothelial dysfunction and development of atherosclerotic plaque. This report describes the effect of copper-catalyzed oxLDL on the regulation of ABCA1 in human endothelial cells (ECs).

Methods and results: oxLDL downregulated ABCA1 at both mRNA and protein levels in a dose-dependent manner. This inhibitory effect of oxLDL was observed with both minimally and extensively oxLDL. Transfection of the ABCA1 promoter luciferase revealed oxLDL to substantially decrease ABCA1 promoter activity at basal conditions and after stimulation by overexpressing the liver X receptor LXR{alpha} and retinoid X receptor RXR{alpha}. oxLDL also attenuated LXR activation by blocking LXR ligand binding and interfering with the generation of 27-hydroxycholesterol, an LXR endogenous ligand. Furthermore, oxLDL inhibited exogenous cholesterol- and oxysterol-induced endothelial ABCA1 induction.

Conclusion: oxLDL downregulated ABCA1 by inhibiting LXR activation in endothelial cells. Such an effect may contribute to endothelial dysfunction and plaque formation.

KEYWORDS Cholesterol; Endothelial function; Gene expression; Lipoprotein; Membrane transport

This article is referred to in the Editorial by Li and Mehta (pages 353–354) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Formation of an atherosclerotic plaque depends upon lipid accumulation in the artery wall. This deposition initially likely results from the transendothelial entry of low density lipoprotein (LDL), followed by LDL oxidation in the subendothelial space [1,2]. Oxidatively modified LDL (oxLDL) is implicated in the pathogenesis of atherosclerosis, because it has been observed within plaques [3] and associated with intima–media thickness in the carotid arteries of clinically healthy men [4]. Vascular endothelial cells (ECs), forming a barrier between the vessel wall and lipoproteins and lipids in circulation, play a critical role in maintaining vascular integrity. Evidence suggests that altered endothelial function predisposes the vessel wall to vasoconstriction, leukocyte adherence, platelet activation, thrombosis, vascular inflammation and atherosclerosis. Thus, endothelial dysfunction is an early atherosclerotic marker. Through a receptor-mediated mechanism, oxLDL enters ECs to cause endothelial dysfunction or injury. The pro-inflammatory and pro-atherogenic effects of oxLDL likely are mediated by numerous endogenous compounds and by the release cytokines and chemokines [5,6].

The protective effect of high-density lipoprotein (HDL) against atherosclerosis is primarily attributed to its function in reverse cholesterol transport, a process by which excess cell cholesterol is taken up by HDL particles. The HDL-processed cholesterol is delivered to the liver for metabolism and bile excretion. The first step of this process is linked to ATP binding cassette transporter 1 (ABCA1), a 254-kDa cytoplasmic membrane protein regulating lipid efflux from cells to apolipoproteins [7]. Mutations in the ABCA1 gene, discovered in patients with Tangier disease and familial HDL deficiency, cause impaired efflux of lipids, including free cholesterol (FC) and phospholipids to apoA-I, which results in a near absence of plasma HDL. Study of ABCA1 heterozygotes revealed that the impaired FC efflux is associated with reduced plasma HDL cholesterol levels and increased risk of coronary artery disease [8]. Recent studies from our laboratory showed that the expression of ABCA1 in ECs is upregulated by LDL cholesterol and oxysterols but downregulated by sterol deprivation and oscillatory flow [9,10]. As a highly atherogenic moiety, oxLDL was reported to upregulate ABCA1 through the activation of the peroxisome proliferator-activated receptor gamma (PPAR{gamma})–liver X receptor (LXR) pathway in lipid-loaded macrophages [11].

However, the regulation of ABCA1 by oxLDL in ECs remains unknown. Given the important role of oxLDL and ABCA1 in atherogenesis and cholesterol traffic in the vessel wall, the present study aims to study the role of oxLDL in the modulation of ABCA1 and the underlying mechanism in ECs. oxLDL appears to transcriptionally downregulate ABCA1 via the inhibition of LXR. Importantly, oxLDL inhibited the generation of 27-hydroxycholesterol, (27-HO), an endogenous LXR ligand in ECs. Thus, oxLDL-regulated ABCA1 may contribute to endothelial dysfunction, accumulation of lipid within the vascular wall and the subsequent development of atherosclerosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Reagents
The DNA-modifying enzymes and PCR enzymes were purchased from Promega Corp. (Madison, WI). Steroids were obtained from Steroloids (Newport, RI) or Avanti, Inc. (Alabaster, AL). 3H-cholesterol was from MP Biomedicals (Irvine, CA). Anti-ABCA1 antibody was purchased from Novus Biologicals (Littleton, CO). Recombinant human fibroblast growth factor (FGF) was a generous gift from Dr. J.A. Thompson (University of Alabama, Birmingham, AL).

2.2 Cell culture
Human umbilical vein endothelial cells (HUVECs) were isolated and maintained as described [12]. In experiments involving oxLDL treatment, M199 medium supplemented with 2% fetal bovine serum (FBS) and 5 ng/ml FGF was used. All experiments were performed with HUVECs up to passage 4. The COS-7 cell line was cultured in DMEM medium and THP-1 cell line was in RPMI-1640 medium and both supplemented with 5% FBS. The study conforms to the Declaration of Helsinki for the use of human umbilical vein endothelial cells.

2.3 LDL isolation and oxLDL preparation
The preparations of oxLDL and minimal oxidized LDL (mmLDL) from human plasma were performed as described [12,13]. Briefly, LDL was diluted to 200 mg/dL in M199 and incubated with 5 µmol/L of CuSO4, CuCl2 or copper acetate in 37 °C for 24 h as indicated. For mmLDL preparation, LDL was incubated with 2 µmol/L of CuSO4 in 37 °C for 4 h. The reactions were stopped by adding 100 µmol/L EDTA. The oxidation state of LDL was measured by use of PeroXOquantTM Quantitative Peroxide Assay kit (Pierce, Rockford, IL). Peroxidation products contained in LDL, mmLDL and oxLDL ranged from <5, 20–82 and 200–300 µmol/L of H2O2, respectively. Contamination of LDL and modified LDL preparations by endotoxin (LPS) was assessed with an LAL kit (Bio Whittaker). All the LDL preparations were with LPS <50 pg/mg protein.

2.4 Northern blotting, RT-PCR and Western blotting
Total RNA isolation and Northern blotting for ABCA1 and vWF expression were performed as described [9]. ABCA1, CYP27 and β-actin underwent reverse transcription and polymerase amplification (RT-PCR). Samples of 1.0 µg of total RNA were used for RT-PCR. The typical PCR reaction consisted of 25 cycles (94 °C, 61 °C, and 72 °C each for 60 s). The resulting products were resolved by 2% agarose gel electrophoresis and detected by ethidium bromide staining. HUVECs were solubilized and cellular membrane proteins from whole cell lysate were isolated as previously described [14]. Membrane proteins were separated by SDS-PAGE. Western blotting analysis was performed with antibodies against ABCA1.

2.5 Assessment of FC efflux
We assessed the FC efflux as described with modification [9]. Briefly, cells in 6-well plates were exposed to LDL, oxLDL or 22(R)-hydroxycholesterol (22R-HO) for 24 h and then labeled with 3H-cholesterol (0.2 µCi/mL) for 6 h. After washing, cells were incubated with or without apoA-I (10 µg/mL) for 2 h. Aliquots of medium and cell lysates were assayed for 3H-cholesterol by liquid scintillation. The results represent radioactivity of FC in the medium as a proportion of the total (medium+cell lysate).

2.6 Transient transfection
In the promoter activation study, HUVECs were co-transfected with pABCA1(–928)-luc, a reporter plasmid of the hABCA1 promoter [15], or LXREx3 TK-luc, a reporter construct containing 3 copies of LXRE [16] with CMX-hLXR{alpha} and CMX-hRXR{alpha}, the expression plasmids of human LXR{alpha} and RXR{alpha}, respectively. The in vivo LXR/RXR ligand binding systems, CMX-GAL-hLXR{alpha} or CMX-GAL-hRXR{alpha} with a GAL4 reporter, were co-transfected into HUVECs with the use of the Targefect F2 (Targeting Systems, San Diego) [16]. pRSV-β-gal was co-transfected as a transfection control. The results were normalized against β-galactosidase.

2.7 Determination of 27-Hch by gas chromatography–mass spectrometry (GC–MS)
Cellular lipids were extracted and oxysterol fractions purified and analyzed by GC–MS as described [17]. Briefly, cellular lipids were extracted with hexane/isopropyl alcohol/water (3:2:0.1, v/v/v). Internal control 22S-HO was added and the samples were dried under argon. The lipid extract was then purified on Isolute Silica columns, TMS derivatized, and then GC–MS was performed with use of a ThermoQuest GCQ instrument equipped with a DB1 capillary column. The gas chromatography program was 180 °C for 1 min, followed by 20 °C/min to 300 °C and a final elution at 300 °C for 20 min. The injector was operated in the split mode (1:10 split). The relative peak areas of 27-HO in the sample were used to calculate the amount of 27-HO by comparing with the standard 22S-HO.

2.8 Statistics
Quantitative data were expressed as mean ± S.D. Statistical significance of the data was evaluated by Student's t-test. P values less than 0.05 were considered significant. For nonquantitative data, results represent those from at least 3 independent experiments.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 oxLDL downregulates the level of ABCA1 expression in ECs
To study the regulation of ABCA1 by oxLDL in ECs, we examined the mRNA level of ABCA1 in HUVECs. Confluent cells were exposed to 20 mg/dL of oxLDL in EC medium with 2% FBS for 24 h. Under this condition, cells were with normal morphology and no cell death was detected. We have previously reported that LDL at 180 mg/dL and cholesterol at 5 µg/mL increased ABCA1 in ECs [9,10], therefore, LDL and cholesterol at these concentrations were used as positive controls. Northern blotting results showed that the ABCA1 mRNA level was increased after the exposure of LDL and cholesterol; it was greatly inhibited when ECs were exposed to oxLDL in 2% FBS medium (Fig. 1A). Further, Western blotting analysis revealed that ABCA1 was decreased after oxLDL exposure for 24 h (Fig. 1B). Consistent with a previous report [11], results in Fig. 1C showed that ABCA1 mRNA was upregulated by oxLDL in THP-1 cells. This observation suggested that the mechanism of ABCA1 regulation by oxLDL in ECs differs from macrophages. Furthermore, exposure of ECs to different concentrations of oxLDL caused a dose-dependent decrease in the level of ABCA1 mRNA (Fig. 2A). To ascertain whether different preparations of oxLDL can cause ABCA1 downregulation, LDL was oxidized with different copper solutions, including CuSO4, CuCl2 and copper acetate. Northern blotting results in Fig. 2B showed that the downregulation of ABCA1 mRNA by oxLDL, under these conditions, did not depend on treatment with different copper solutions. In addition, different degrees of oxidation of LDL by CuSO4 [13] in same concentration of LDL revealed that both mmLDL and oxLDL have similar ABCA1 inhibition only in low serum (2% FBS) condition but not in high serum (20% FBS) medium, suggesting that it did not depend on the degree of oxidation (data not shown).


Figure 1
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  		Fig. 1 Effects of oxLDL on ABCA1 expression in HUVECs and THP-1 cells. HUVECs (A and B) or THP-1 cells (C) were incubated with LDL (180 mg/dL), oxLDL (20 mg/dL) or cholesterol (CHL, 10 µg/mL) in 2% FBS-containing medium for 24 h. (A, C) Total RNA (15 µg) was resolved by agarose gel electrophoresis and then hybridized with [{alpha}-32P]-labeled hABCA1 or vWF cDNA. (B) The membrane proteins (40 µg/lane) in each group were separated by electrophoresis and the ABCA-1 protein was detected by Western blot analysis, 22R-HO (5 µM) was used as a positive control.

 

Figure 2
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  		Fig. 2 Effects of different preparations of oxLDL on ABCA1 mRNA in ECs. HUVECs were incubated with (A) different concentrations of oxLDL, and (B) different preparations of oxLDL at 20 mg/dL in 2% FBS-containing medium for 24 h. Total RNA (15 µg) was resolved by agarose gel electrophoresis and then hybridized with [{alpha}-32P]-labeled hABCA1 or vWF cDNA.

 
3.2 oxLDL inhibits apoA-I-mediated FC efflux from ECs
To study whether the inhibition of ABCA1 by oxLDL affected apoA-I-mediated FC efflux from ECs, we exposed confluent ECs to LDL, oxLDL in medium containing 2% FBS for 24 h. After 3H-cholesterol labeling, FC efflux was determined in the presence or absence of apoA-I. An oxysterol LXR agonist, 22R-HO, was used as a positive control. As shown in Fig. 3, LDL and 22R-HO promoted a 30% and 137% increase, respectively, in FC efflux in the presence of apoA-I. Given that lipoprotein loading may increase cholesterol content and FC exchange, both LDL and oxLDL exposure increased the level of basic FC efflux, which did not increase further in the presence of apoA-I in oxLDL-exposed cells. Therefore, oxLDL not only downregulated ABCA1 in mRNA and protein but also inhibited the function of ABCA1 in ECs.


Figure 3
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  		Fig. 3 Effect of oxLDL on ApoA-I-mediated cholesterol efflux in ECs. HUVECs cultured in 12-well plates were treated with LDL (180 mg/dL), oxLDL (20 mg/dL), 22R-HO (5 µM) for 24 h. Cells were labeled with 3H-cholesterol (0.2 µCi/mL) for 6 h and then incubated with or without apoA-I (10 µg/mL) for 2 h. The radioactivity in the medium and the cell lysates was assayed by liquid scintillation. The results were radioactivity in medium as a proportion of medium+cell lysate. Results are mean ± S.D. of 3 independent experiments, each in triplicate.

 
3.3 oxLDL inhibits ABCA1 promoter activities in ECs
To characterize the effect of oxLDL on ABCA1 expression, we examined the upstream regulatory regions by transfecting HUVECs with ABCA1 promoter-driven reporter constructs. As shown in Fig. 4A, cholesterol and 22R-HO increased and oxLDL decreased promoter activities of the construct pABCA1(–928)-luc.


Figure 4
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  		Fig. 4 Effect of oxLDL on the activation of hABCA1 promoter and LXRE in ECs. HUVECs were transfected with hABCA1-Luc only in (A), co-transfected with pCMX-hLXR{alpha} or pCMX-hRXR{alpha} in (B) or co-transfected with LXREx3 TK-luc and pCMX-hLXR{alpha} or pCMX-hRXR{alpha} in (C) for 18 h. Then, the transfected cells were incubated with 180 mg/dL of LDL, 20 mg/dL of oxLDL, 10 µg/mL of cholesterol, or 5 µM of 22R-HO for 24 h. Promoter activities were measured by use of luciferase and normalized with β-galactosidase. Data are mean ± S.D. of the relative luciferase activities in 4 independent experiments, each performed in triplicate.

 
A DR4 element (LXRE) is located at the noncoding strand between –70 and –55 bp within the ABCA1 promoter. This DR4 site is the sterol-responsive element and is regulated by LXR and RXR heterodimers and LDL/cholesterol [15]. To investigate further the effect of oxLDL on LXR-mediated activation of the ABCA1 promoter, we co-transfected pABCA1(–928)-luc with expression plasmids for LXR{alpha} and/or RXR{alpha} and then treated the transfected cells with oxLDL or cholesterol. As shown in Fig. 4B, overexpression of both LXR{alpha} and RXR{alpha} significantly increased ABCA1 promoter activities while cholesterol further enhanced this activation. In contrast, oxLDL inhibited the promoter activity in all conditions. Similar results were obtained with LXRE-luc transfection (Fig. 4C). Co-transfection with LXR{alpha} alone increased the basal LXRE-mediated luciferase activity by 4-fold and cholesterol by 8-fold. LXRE activity was increased up to 60-fold after co-transfection with both LXR{alpha} and RXR{alpha}, and cholesterol increased the activity further. However, oxLDL suppressed the basal and LXR/RXR-induced LXRE activities. Thus, oxLDL downregulated ABCA1 by inhibiting LXR in ECs.

3.4 oxLDL inhibits the generation of endogenous LXR ligands in ECs
To study LXR ligand binding, the plasmids of a CMX-GAL-hLXR{alpha} or a CMX-GAL-hRXR{alpha} and a GAL4 reporter were co-transfected in ECs or COS-7 cells. The effect of oxLDL was assessed by measuring GAL4 reporter luciferase. 22R-HO, an oxysterol LXR ligand, and 9-cis retinoic acid (9CRA), an RXR ligand, were used as positive controls. Fig. 5A shows that ligand-dependent LXR activation in both ECs and COS-7 cells was significantly increased by CHL and 22R-HO. oxLDL inhibited the LXR activation in ECs but not in COS-7 cells. In contrast, oxLDL did not affect the ligand-dependent RXR activation in both cell types (Fig. 5B).


Figure 5
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  		Fig. 5 Effects of oxLDL on LXR-LBD activation. HUVECs or COS-7 cells were co-transfected with GAL4 reporter with CMX-GAL-hLXR{alpha} in (A) or CMX-GAL-hRXR{alpha} in (B). pRSV-β-gal was a control. Post-transfected cells were incubated with 20 mg/dL of oxLDL, 10 µg/mL of cholesterol for 24 h. 9CRA (10 µM) in (A) and 22R-HO (5 µM) in (B) were positive controls. GAL4 reporter activity was measured by use of the reporter luciferase which was normalized to that of β-galactosidase. Data are mean ± S.D. of the relative luciferase activities in 3 independent experiments, each performed in triplicate.

 
High levels of sterol 27-hydroxylase (CYP27) can actively generate 27-HO from cholesterol in ECs [18]. 27-HO was reported to be an endogenous ligand for LXR in cholesterol-loaded macrophages [17]. To further explore the mechanism by which oxLDL inhibits LXR activation, we studied the modulation of sterol CYP27 and 27-HO in ECs. The mRNA level of CYP27 was not changed by oxLDL, as revealed by RT-PCR (Fig. 6A). However, GC–MS results demonstrated that oxLDL attenuated the production of 27-HO in ECs by 78% compared with untreated controls. Native LDL and cholesterol increased the production of 27-HO by 0.7- and 1.0-fold, respectively (Fig. 6B). To confirm that 27-HO can upregulate ABCA1, we showed that 24-h incubation with 22R-HO and 27-HO could increase ABCA1 mRNA (Fig. 6C). Interestingly, the addition of oxLDL blocked the effect of these oxysterols on ABCA1 upregulation. Thus, Figs. 5 and 6 demonstrate that oxLDL downregulates ABCA1 by inhibiting the generation of LXR ligand in ECs.


Figure 6
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  		Fig. 6 Effect of oxLDL on levels of CYP27 mRNA and 27-HO syntheses in ECs. HUVECs were incubated with LDL (180 mg/dL), oxLDL (20 mg/dL) or cholesterol (10 µg/mL) for 24 h. (A) Total RNA from each sample (1 µg) were used for RT-PCR with the primers for CYP27, ABCA1 or β-actin. (B) Cellular lipids were extracted and oxysterol fractions were purified and analyzed by GC-MS. Data are mean ± S.D. of the amount of normalized 27-HO from 4 independent experiments. (C) HUVECs were incubated with 22R-HO (5 µM) or 27-HO (7.5 µM) with or without oxLDL (20 mg/dL) for 24 h. Total RNA (15 µg) were subjected to Northern blotting with [{alpha}-32P]-labeled hABCA1 or vWF cDNA as probes. Results represent 3 independent experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we investigated the role of oxLDL on the regulation of endothelial ABCA1 and its underlying mechanism. Our findings demonstrate that oxLDL suppresses ABCA1 at the protein and mRNA levels in HUVECs in a tissue-specific manner; oxLDL can inhibit apoA-I-mediated FC efflux in ECs; oxLDL decreases ABCA1 promoter activity via a ligand-dependent inhibition of LXR; and LDL reduces the production of 27-HO, an LXR endogenous ligand. These results suggest that downregulation of ABCA1 by oxLDL may play an important role in endothelial dysfunction and cholesterol deposition in the arterial wall.

oxLDL plays a substantial role in endothelial dysfunction, including activation of NF-{kappa}B, inhibition of eNOS, and increased monocyte adhesion and apoptosis [5,19–21]. ECs express receptors for oxLDL that are not subject to negative feedback regulation by cholesterol and thereby implicated in the formation of foam cells. However, ECs do not show the lipid-loaded phenotype, as seen in macrophages and smooth muscle cells in the atherosclerotic plaque. Thus, ECs may have unique mechanisms such as their strong ability to efflux cellular cholesterol to HDL [22] to control lipid homeostasis. Oxysterol formation may be another means for removing excess cholesterol from ECs [23]. Indeed, ECs were reported to have high levels of CYP27, which could actively generate 27-HO from FC [18]. Fu and colleagues reported that 27-HO was an endogenous ligand for LXR to modulate ABCA1 in macrophages [17]. Further, the CYP27-mediated cholesterol elimination may compete for HDL-mediated efflux from the cells [23]. Thus, the impaired lipid metabolism in ECs may also contribute to atherogenesis. Fig. 6 suggests that oxLDL may interrupt the function of ECs to convert cholesterol to oxysterols (i.e., 27-HO), which, in turn, inhibits LXR activation and ABCA1 expression. This oxLDL downregulated ABCA1 is evidence of an alteration of endothelial function predisposing to lipid accumulation within the vessel wall.

ABCA1 plays an important role in reverse cholesterol transfer and, thus, offers atherogenic protection [24]. This protein is expressed in many organs and cultured cells [25–27], as well as within atherosclerotic lesions [28]. However, there have been conflicting reports on the mechanisms of ABCA1 and cell types contributing to HDL generation and athero-protection. Most in vitro studies involving ABCA1 function and regulation were performed with the use of cultured macrophages, fibroblasts and hepatocytes. Although the dysfunction of lipid metabolism in ECs and the ensuing oxidation and deposition in the subendothelial space impose important pathophysiological consequences, only a few documented reports exist on ABCA1 regulation in ECs. We and others reported that ABCA1 was expressed in human aortic ECs, HUVECs, porcine brain capillary ECs, and rat liver ECs [9,29,30]. Recently, Langmann and colleagues showed that the low level of ABCA1 expressed in ECs was not required for apoA-I-mediated cholesterol efflux [25]. However, we observed that oscillatory flow activated SREBPs and inhibited the expression of ABCA1 in ECs. This observation suggests that lipid/cholesterol homeostasis was impaired in the curves or branches of the arterial tree, and can promote atherogenesis [10]. These disparities in the above findings are likely due to cell source and culture conditions, which may affect the abundance of ABCA1 and hence the extracellular cholesterol loading. We isolated HUVECs by ourselves, used early passages and did not rely on hydrocortisone and growth factors other than FGF during cell culture. Using this approach, ABCA1 was detected in the culture medium. Under these conditions, serum deprivation lowered cholesterol content and ABCA1 mRNA levels [10]. Results from the current study reveal that oxLDL downregulated ABCA1 via the inhibition of EC LXR. In vitro, ECs cultured on plastic plates, responding to cellular FC levels, ABCA1 was expressed and the presence of apoA-I could only efflux cholesterol into the culture medium. Using trans-well cultures, apoA-I-mediated FC efflux increased in the upper but not lower chambers (Zhu et al. unpublished data, 2005), which suggests that endothelial ABCA1 may be involved in the physiological redistribution of lipids by delivering its lipid cargo into the vascular lumin. Whether such a scenario occurs in vivo requires further investigation.

Various studies have shown that the ABCA1 gene is tightly regulated by the cholesterol contents in the cell. This effect of cholesterol was suggested to be mediated by target genes of LXR activated by oxysterol ligands [31]. ABCA1 is transcriptionally regulated by LXR/RXR heterodimers via a DR4 site in its promoter region [15]. ABCA1 activity can also be greatly influenced by post-translational processes [32,33]. Here, we show oxLDL suppresses ABCA1 transcription through the inhibition of LXR in a ligand-dependent mechanism. Previous work has implicated that oxLDL induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by PPAR{gamma}–LXR{alpha} [11]. Indeed, Fig. 1C confirms the upregulation of ABCA1 by oxLDL in THP-1 cells. Since ECs are rarely observed to accumulate lipid or undergo foam cell formation as macrophages do, unique mechanisms in ECs are likely to control lipid homeostasis. One such example is the differential regulation of ABCA1 by oxLDL in ECs in comparison to macrophages.

Cholesterol efflux is activated when cells are loaded with cholesterol: three mechanisms are proposed [34,35]. The first two are simple diffusion and protein-dependent but ATP-independent facilitated diffusion. The third mechanism is ABCA1-mediated active transport to assemble HDL. Caveolin-1 has been proposed to be involved in FC transport to membrane caveolae to facilitate FC efflux to HDL [35,36]. We found that elevated LDL increased intracellular cholesterol content and increased caveolin-1 and cholesterol translocation to caveolae [37]. oxLDL further affects the subcellular localization of caveolin-1 by modifying caveolae cholesterol content [30]. Fig. 3 showed that both LDL and oxLDL increased basic FC efflux, suggesting that lipoprotein loading also increased cholesterol exchange. However, oxLDL did not increase FC efflux in the presence of apoA-I. Therefore, oxLDL can functionally inhibit ABCA1 in ECs.

In summary, this study shows not only the downregulation of ABCA1 by oxLDL in an LXR ligand-dependent fashion but also the inhibition of 27-HO, an endogenous LXR ligand, by oxLDL in ECs. Thus, as our hypothetical model shown in Fig. 7, the mechanism by which oxLDL regulates ABCA1 would constitute an important part of endothelial dysfunction and subsequent atherogenesis.


Figure 7
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  		Fig. 7 A hypothetical model for cholesterol homeostasis in ECs under physiological and pathophysiological conditions. (A) Endothelium under normal conditions can efflux LDL-derived or synthesized cholesterol to maintain cholesterol homeostasis in ECs. The cholesterol efflux is through ABCA1 on the luminal surface into circulation and caveolae on the basal lateral membrane to its acceptors, e.g., HDL or albumin. (B) When ECs are exposed to hyperlipidemia, high levels of LDL enter the vessel wall and the infiltrated LDL is oxidized. Disturbed flow and oxLDL inhibit ABCA1-mediated cholesterol efflux. As a result, cholesterol efflux through other pathways (e.g., caveolae) is increased, which may lead to lipid deposition in the subendothelial space.

 


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported in part by the National Natural Science Foundation of China 30440003, 30470631 (Y.Z.) and 30330250 (X.W.) and National Institutes of Health grant HL33742 (M.B.S.).


   Notes
 
Time for primary review 30 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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