Skip Navigation

Cardiovascular Research 2006 72(3):473-482; doi:10.1016/j.cardiores.2006.08.024
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Marcil, V.
Right arrow Articles by Levy, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marcil, V.
Right arrow Articles by Levy, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

Oxidative stress influences cholesterol efflux in THP-1 macrophages: Role of ATP-binding cassette A1 and nuclear factors

Valérie Marcila,b, Edgard Delvina,c, Alain Théophile Sanéa,b, André Tremblaya,d and Emile Levya,b,*

aCentre de Recherche, CHU-Sainte-Justine, Université de Montréal, 3175, Côte Ste-Catherine, Montréal (Québec), Canada H3T 1C5
bDepartment of Nutrition, Université de Montréal, 3175, Côte Ste-Catherine, Montréal (Québec), Canada H3T 1C5
cDepartment of Biochemistry, Université de Montréal, 3175, Côte Ste-Catherine, Montréal (Québec), Canada H3T 1C5
dDepartment of Obstetrics and Gynecology, Université de Montréal, 3175, Côte Ste-Catherine, Montréal (Québec), Canada H3T 1C5

* Corresponding author. Tel.: +1 514 345 4626; fax: +1 514 345 4999. Email address: emile.levy{at}recherche-ste-justine.qc

Received 17 April 2006; revised 17 August 2006; accepted 30 August 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Understanding the mechanisms involved in oxidative stress-induced foam cell formation is of fundamental importance for atherosclerosis. Our aim was to characterize the effects of oxidative stress on key receptors of macrophage cholesterol homeostasis, on the nuclear transcription factors PPAR and LXR regulating their expression, and on macrophage cholesterol handling.

Methods and results: The incubation of macrophages derived from the human monocyte cell line THP-1 with iron (100 µm)/ascorbate (1000 µm) for a period of 4 h induced a strong peroxidation, as demonstrated by the elevation of malondialdehyde (220%, P<0.001). The production of lipid peroxidation affected cholesterol efflux, which was probably due to decreased ABCAI gene and protein expression. On the other hand, cholesterol influx remained unchanged as did the mRNA and protein levels of SR-BI and CD36, important protein receptors that participate in cholesterol import. Experiments using RT-PCR showed that the ABCAI modulation was orchestrated by the nuclear receptors LXR{alpha}, LXRβ, PPAR{alpha}, and PPAR{gamma}. Treatment with powerful antioxidants (Trolox and BHT) prevented the adverse effects of iron-ascorbate on cholesterol movement, conceivably supporting the role of oxidative stress.

Conclusion: Our results show that oxidative stress can directly be induced in macrophages and concomitantly impairs the expression of receptors involved in cholesterol flux, which could influence foam cell formation and atherosclerosis development.

KEYWORDS ABCA1; SR-BI; CD36; PPAR; LXR; Macrophage; Oxidative stress


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiovascular complications are characterized by endothelial dysfunction and accelerated atherosclerosis, and remain among the leading causes of morbidity and mortality. Reactive oxygen species generated in and around the vascular endothelium appear as important causative factors in endothelial dysfunction underlying the development of atherosclerosis [1]. In fact, oxidized lipids and lipoproteins in the arterial wall increase adhesion molecules, recruitment of mononuclear cells to the endothelium, and inflammation response and foam cell formation, potentially leading to acute plaque rupture, thrombosis and ischemic heart disease [2].

A key determinant of the atherosclerotic lesion occurrence consists in foam cell formation, which is associated with enhanced macrophage cholesterol [3] and probably reflects the imbalance between lipid influx and efflux. The acquisition and evacuation of cholesterol by macrophages is mediated by a number of cell surface receptors, including scavenger receptor class B type I (SR-BI), CD36 and ATP-binding cassette A1 (ABCA1) [4–6]. The first step in the reverse cholesterol transport is linked to ABCA1, a transmembrane protein mediating lipid efflux from cells to apolipoproteins (apo). Mutations in the ABCA1 gene, discovered in patients with Tangier disease and familial high-density lipoprotein (HDL) deficiency, caused impaired cholesterol efflux to apo A-1, which results in a near absence of plasma HDL [7]. It has been concluded that ABCA1 plays an important role in cholesterol homeostasis and atherogenesis.

SR-BI, a member of the CD36 superfamily, is predominantly expressed in the liver and steroidogenic tissues, where it mediates selective uptake of cholesteryl ester from HDL [8,9]. SR-BI is also expressed in macrophages, including tissue macrophages, monocyte-derived macrophages, and macrophages in atherosclerotic lesions [10,11]. SR-BI plays a dual role in macrophages, as it has been described that it can bind and internalize modified lipoproteins [6] and also mediate the efflux of free cholesterol to HDLs [12]. The regulation of SR-BI in response to sterol loading is still a matter of debate [13,14].

CD36 consists in a scavenger receptor which is expressed, among other cell types, in monocytes and macrophages. It has been identified to bind and internalize oxidized low-density lipoproteins (oxLDL), but also a broad variety of ligands including anionic phospholipids, apoptotic cells, long-chain fatty acids and other modified LDL [14–16]. CD36 recognizes lipid moieties of oxLDL and seems to be the dominant scavenger receptor in the uptake of oxLDL by macrophages during foam cell [14,15,17–21]. Additionally, growing evidence suggests that peroxisome proliferator-activated receptors (PPARs) exert antiatherogenic effects by enhancing cholesterol efflux via activation of the liver X receptor (LXR)–ABCA1 pathway [22,23]. Although numerous reports have emphasized the remarkable capacity of oxLDL to trigger proatherogenic events, little is known about the direct effect of oxidative stress per se on atherogenesis without the involvement of LDL, which may not be obligatory in the induction of vascular lesions [24]. The main objective of the present investigation was, therefore, to determine the role of iron-ascorbate-mediated lipid peroxidation in cholesterol influx and efflux in macrophages. Furthermore, the gene and protein expression of SR-BI, ABCA1 and CD36 were examined. Finally, the gene expression of PPAR{alpha}, PPARβ and PPAR{gamma}, as well as LXR{alpha} and LXRβ was assessed.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. THP-1 cell culture
THP-1 human monocytes (American Type Culture Collection (ATCC) TIB 202) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.05 mM 2-mercaptoethanol. The cell line was cultivated at 37 °C, 95% humidity and 5% CO2 and used between passages 4 and 12. Cells (1x106 cells/ml) were differentiated into macrophages in 100 mm-dishes by the addition of 100 ng/ml phorbol 12-mysistate 13-acetate (Sigma) for a 72-hour period.

2.2. Isolation of monocytes
Monocytes were isolated from buffy coats as described previously [25]. Purified monocytes were differentiated into macrophages by culturing them with 100 ng/ml phorbol 12-myristate 13-acetate and 10% human serum (v/v) for 9–10 do those prevailing in THP-1. This part of the study was approved by the Ethics Review Board of CHU Ste-Justine in accordance to the Declaration of Helsinki.

2.3. Estimation of lipid peroxidation
THP-1 cells were incubated for 4 h in the presence or absence of Fe2+ (100 µM)-ascorbate (1000 µM) and the antioxidants Trolox (6-Hydroxy-2,5,7,9-tetramethyl-chroman-2-carboxylic acid, 0.5 mM) and butylated hydroxytoluene (2,6-di-t-butyl-p-cresol, BHT, 0.5 mM). The reaction was terminated by the addition of 0.2% BHT to measure malondialdehyde (MDA), as an index of lipid peroxidation. The amount of free MDA formed during the reaction was determined by HPLC as described previously [26]. Briefly, proteins were first precipitated with a 10% sodium tungstate (Na2WO4) (Aldrich Chemical) solution, and protein-free supernatant was then reacted with an isovolume of 0.5% thiobarbituric acid (Sigma) solution at 90 °C for 60 min. After cooling to room temperature, chromogene was extracted with 1-butanol and dried over a stream of nitrogen at 37 °C. The dry extract was then resuspended in KH2PO4/methanol (70:30, pH 7.0) mobile phase before MDA detection by HPLC.

2.4. Isolation and modification of lipoproteins
Human LDL (1.019<d<1.063 g/ml), HDL3 fraction (1.125<d<1.210 g/ml) and lipoprotein-deficient serum (LPDS, d>1.125 g/ml) were prepared from plasma of healthy human subjects and isolated by differential ultracentrifugation as described previously [27–29]. The lipoprotein fractions were dialyzed intensively against phosphate buffered saline (PBS, pH 7.4) containing 150 mM NaCl and 0.3 mM ethylene diaminetetraacetic acid (EDTA). In order to generate oxLDL, plasma LDL (3 mg apo B/ml) was extensively dialyzed against PBS (pH 7.4) containing 150 mM NaCl and 5 µM EDTA and then incubated with 10 µM CuSO4 for 18 h at 37 °C. Its modification was verified by its mobility on agarose gel electrophoresis (Paragon, Beckman Instruments). All lipoprotein fractions were filtered through a 0.2 µM Millipore membrane and stored at 4 °C.

2.5. Cholesterol-HDL3 uptake
THP-1 cells were cultured in 12-well plates at 1.8x106 cells per well. After the 72-hour differentiation period, cells were incubated for 4 h at 37 °C with or without Fe2+-ascorbate and antioxidants in 0.5 ml RPMI. Then, cells were supplemented with 5% v/v LPDS containing [3H]-cholesteryl hexadecyl ether-HDL3 (25 µg/ml) for 4 h. To determine the non-specific binding, cells and labeled-HDL3 were incubated in the presence of a 50-fold excess of unlabeled HDL3. The assays were essentially carried out as previously described [30].

2.6. Cholesterol efflux
The differentiated cells were loaded with radiolabeled cholesterol by incubation for 24 h in 0.5 ml of supplemented RPMI with 5% v/v LPDS and 2.64x106 dpm/ml [14C]-cholesteryl oleate oxLDL (50 µg apo B/ml). After a 16-hour equilibration period of time without radioactivity, cells were washed with PBS and treated for 4 h with or without Fe2+-ascorbate and antioxidants. Cells were washed again and incubated with HDL3 (25 µg/ml) for 24 h. The media were centrifuged at 4000 g for 10 min to remove any suspended or dead cells. The assays were performed as previously described [30]. Briefly, after incubation, cells were washed twice with cold PBS, detached from the plate by gentle pipetting with PBS solution containing anti-proteases (phenylmethylsulfonyl fluoride, pepstatin, EDTA, aminocaproic acid, chlorampehnicol, leupeptin, glutathione, benzamidine, dithiothreitol, sodium azide, and trasylol, all at a final concentration of 1 mM) and homogenized by sonication on ice (3x10 s, lowest power setting). For each well, radioactivity was counted and protein was determined by Bio-Rad protein assay kit with BSA as a standard. The specific uptake of [3H]-cholesteryl hexadecyl ether-HDL3 by cells was calculated by subtracting the non-specific value.

2.7. Western blot analysis
To determine the protein expression of SR-BI, ABCA1 and CD36, cells were homogenized and proteins (30 µg) were denatured at 95 °C for 5 min in SDS, dithiothreitol and β-mercaptoethanol-containing sample buffer, separated on a 4–7.5% gradient SDS-PAGE, and electroblotted onto Hybond-C Extra nitrocellulose membranes (Amersham) [30]. The blots were then incubated overnight at 4 °C in blocking solution with the antibodies for SR-BI (Novus Biologicals) (1:2000), ABCA1 (Novus Biologicals) (1:1000), CD36 (Santa Cruz Biotechnology) (1:200), and β-actin (Sigma-Aldrich) (1:5000). The relative amount of primary antibody was detected with species-specific horseradish peroxidase-conjugated secondary antibody. Blots were developed and the mass of the aforementioned proteins was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software.

2.8. RT-PCR expression analysis
Levels of specific mRNAs were semi-quantitatively assessed by the reverse transcription-polymerase chain reaction (RT-PCR). Briefly, complementary DNA was synthesized in a total volume of 20 µl, from RNA samples by mixing 2 µg of total RNA, 2 µl of reverse transcriptase buffer (10x) supplemented with dNTPs (0.5 mM each), oligo(dT) primers (2.5 µM), RNase inhibitor (10 U) and Omniscript Reverse Transcriptase (Qiagen). The first strand DNA synthesis was carried out at 37 °C for 60 min. PCR amplification was performed in 50 µl volume using 5 µl PCR Buffer Hot Star (10x), 10 µl Q Solution (5x), dNTPs (200 µM), 0.4 µM of each corresponding primer and 2.5 U of HotStarTM DNA Polymerase (Qiagen). The PCR amplifications were performed using a GeneAmp PCR System 9700 (Applied Biosystems) under the following profile: 33–36 cycles of amplification were used at 95 °C for 30 s, 58 °C for 30 s and 72 °C for 40 s. Amplicons were visualized on standard ethidium bromide stained 1.5% agarose gel and analysed using Scion Image software.

2.9. Apolipoproteins
The levels of apolipoproteins A-I and B were estimated by nephelometry as described previously [31].

2.10. Statistical analysis
Data from the experiments were analysed by using a student's t-test. Reported values are expressed as means±SEM. Statistical significance was accepted at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. MDA generation after iron-ascorbate exposure
The effectiveness of iron-ascorbate in initiating lipid peroxidation was tested after incubation with THP-1 cells. At the end of a 4-hour culture period, the degree of lipid peroxidation was determined by measuring MDA in cells. As illustrated in Fig. 1A, iron-ascorbate promotes the production of peroxidation above control values, and the formation of MDA increased with rising iron-ascorbate concentrations in a dose-dependent manner. Neither iron nor ascorbate alone could induce marked lipid peroxidation (data not shown). The concentration–response relationship for THP-1 cell peroxidation for 100/1000 µM (iron-ascorbate complex) was 9.2-fold higher (P<0.0001) in the presence than in the absence of iron-ascorbate. We, therefore, selected this concentration of iron-ascorbate for the following studies since it does not represent a pharmacological dose. The efficiency of powerful antioxidants in preventing or reducing lipid peroxidation induced by iron-ascorbate was then evaluated (Fig. 1B). Trolox and BHT significantly (P<0.0001) suppressed cellular peroxidation (7.9-fold and 15.3-fold, respectively) induced by iron-ascorbate at the concentrations tested. Thus, they were chosen for the subsequent experiments. It is important to note that other antioxidants such as vitamin E (0.5 mM) and N-acetylcysteine (5 mM) were far less effective than BHT and Trolox in counteracting iron-ascorbate-mediated lipid peroxidation (data not shown).


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Lipid peroxidation in THP-1 cells challenged with iron-ascorbate. THP-1 cells were incubated with increasing concentrations of iron-ascorbate for 4 h at 37 °C. Iron concentrations shown on the x-axis represent actual iron concentration along with a corresponding 10-fold higher ascorbate concentration at each iron amount. Lipid peroxidation was monitored by measuring malondialdehyde (MDA) formation in cells. A concentration-dependent increase in equivalent MDA formed was observed between 50 and 200 µM of Fe2+ in cells (A). The effects of antioxidants (Trolox and BHT) on iron-ascorbate-mediated lipid peroxidation were also tested (B). To this end, cells were incubated for 4 h with Trolox or BHT in addition to iron-ascorbate. Cells were then treated to determine MDA by HPLC. Values are means±SEM for 4–6 different experiments. *P<0.0001 vs. controls.

 
3.2. Cholesterol influx and efflux
We next investigated if cholesterol flux is influenced by iron-ascorbate-mediated lipid peroxidation. THP-1 macrophages were exposed to iron-ascorbate for 4 h and were incubated for 4 h with 25 µg of protein/ml HDL3-[3H]-cholesteryl ether and cholesterol influx was then measured. As shown in Fig. 2A, the incorporation of [3H]-cholesteryl ether remained unchanged either with iron-ascorbate or antioxidants. On the other hand, cholesterol efflux to HDL3 after loading cells with oxLDL-[14C]-cholesteryl oleate and washing was decreased by iron-ascorbate (Fig. 2B) and restored with Trolox or BHT. We concluded that lipid peroxidation affected the efflux process only.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of iron-ascorbate on macrophage cholesterol influx (A) and cholesterol efflux (B). Macrophages were treated with LPDS and 25 µg/ml HDL3 [3H]-cholesteryl hexadecyl ether at 37 °C for 4 h. The cholesterol influx content was calculated by assessing radioactivity in cell lysate. The cholesterol efflux content was assessed after loading cells with 50 µg apo B/ml oxLDL [14C]-cholesteryl oleate. Values are means±SEM for 5 separate experiments. *P<0.05 vs. controls.

 
3.3. Assessment of gene and protein expression of ABCA1, SR-BI and CD36
We attempted to define the mechanisms for the alterations in receptor-mediated cell efflux from THP-1 macrophages following exposure to iron-ascorbate. To this end, ABCA1 was characterized as the rate limiting unidirectional cellular cholesterol exporter. Similarly, we evaluated mRNA and protein mass of SR-BI that may promote the bidirectional flux of free cholesterol between macrophages and lipoproteins and is particularly of importance for macrophage efflux, together with CD36 implicated in oxidized lipid uptake. Iron-ascorbate significantly reduced the protein (Fig. 3A). However, Trolox and BHT were able to prevent iron-ascorbate-induced ABCA1 protein fall. Similar trends were noted with respect to ABCA1 mRNA, but BHT was less efficient than Trolox in opposing lipid peroxidation (Fig. 3B). Interestingly, BHT alone was able to significantly increase ABCA1 mRNA and its combination with iron-ascorbate enhanced ABCA1 protein levels. No significant alterations were recorded in mRNA and protein mass of SR-BI in the presence of iron-ascorbate (Fig. 4), but CD36 protein mass was reduced by peroxidation without changing the gene transcription (Fig. 5).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Influence of gene and protein expression of ABCA1. THP-1 macrophages were exposed to iron-ascorbate and antioxidants for 4 h at 37 °C. Thereafter, protein and mRNA levels of the receptors were determined by Western blotting (A) and RT-PCR (B). Values are means±SEM for 3 separate experiments. *P<0.05, {dagger} P<0.01 and {ddagger}P<0.001 vs. controls. A representative blot is shown.

 

Figure 4
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Influence of gene and protein expression of SR-BI. THP-1 macrophages were exposed to iron-ascorbate and antioxidants for 4 h at 37 °C. Thereafter, protein and mRNA levels of the receptors were determined by Western blotting (A) and RT-PCR (B). Values are means±SEM for 3 separate experiments. *P<0.05, {dagger} P<0.01 and {ddagger}P<0.001 vs. controls. A representative blot is shown.

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Influence of gene and protein expression of CD36. THP-1 macrophages were exposed to iron-ascorbate and antioxidants for 4 h at 37 °C. Thereafter, protein and mRNA levels of the receptors were determined by Western blotting (A) and RT-PCR (B). Values are means±SEM for 3 separate experiments. *P<0.05, {dagger} P<0.01 and {ddagger}P<0.001 vs. controls. A representative blot is shown.

 
3.4. Gene expression of nuclear factors
We further tested PPAR and LXR, which represent nuclear receptors extensively involved in the control of lipid metabolism. The mRNA levels of PPAR{alpha}, PPARβ/{delta}, PPAR{gamma}, LXR{alpha}, and LXRβ were quantified by RT-PCR. Iron-ascorbate-mediated lipid peroxidation down-regulated the expression of PPAR{alpha} (Fig. 6A), PPAR{gamma} (Fig. 6C), LXR{alpha} (Fig. 6D), and LXRβ (Fig. 6E) in comparison with controls, whereas the incubation with BHT prevented its decline or even enhanced it. PPARβ/{delta} mRNA was not changed following the treatment of THP-1 with iron-ascorbate (Fig. 6B).


Figure 6
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Regulation of nuclear factors by iron-ascorbate. Iron-ascorbate was administered to THP-1 in the presence or absence of Trolox or BHT. Following the period culture (4 h) the mRNA levels of PPAR{alpha} (A), PPARβ/{delta} (B), PPAR{gamma} (C), LXR{alpha} (D), and LXRβ (E) were examined by RT-PCR as described in the Methods section. Values are means±SEM for 4 separate experiments. *P<0.05, {dagger} P<0.01 vs. controls. A representative blot is shown.

 
3.5. Protein expression in human monocyte-derived macrophages
Studies were also carried out to determine whether iron-ascorbate was able to induce lipid peroxidation and caused changes in ABCA1, SR-B1 and CD36 in monocytes isolated from human blood and differentiated in culture. Using the same experimental conditions as employed with THP-1, we could observe that human macrophages exhibited high MDA levels in the presence of iron-ascorbate (Fig. 7). Besides, the protein expression of ABCA1 and CD36 was lowered whereas the protein mass of SR-BI remained unchanged (Fig. 8). Therefore, data on human monocyte-derived macrophage confirmed the findings in THP-1.


Figure 7
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Lipid peroxidation in monocytes-derived macrophages following incubation with iron-ascorbate. Lipid peroxidation was assessed by the measurement of malondialdehyde (MDA). Values are means±SEM for 4 separate experiments. {dagger} P<0.01 vs. controls.

 

Figure 8
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Effects of iron-ascorbate-mediated lipid peroxidation on the protein expression of ABCA1, SR-BI and CD36. Protein mass of ABCA1 (A), SR-BI (B) and CD36 (C) was estimated by Western blot as described in the Methods section. Values are means±SEM for 4 separate experiments. *P<0.05 vs. controls.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Experimental data support a role for oxLDL in the pathogenesis of atherosclerosis. This is based on several lines of evidence, including the demonstration that (1) oxidatively modified LDL exists in atherosclerotic lesions [32–35]; (2) inhibition of oxidation by several antioxidants can slow the progression of the disease [36–38]; (3) the susceptibility of LDL to oxidation correlates with the severity of coronary atherosclerosis [39]; and (4) the ability of oxLDL to transform macrophages into foam cells [40]. However, the direct impact of oxidative stress, without the implication of oxLDL, as a causal factor for the alterations of cholesterol metabolism in macrophages, has poorly been examined. Noticing this prompted us to explore whether iron-catalyzed free radical-mediated lipid peroxidation provoked abnormalities in cholesterol trafficking in THP-1 with an attention on the mechanisms associated. In this study we showed that exposure of THP-1 macrophages to iron-ascorbate: (i) induced lipid peroxidation as assessed by the rise of MDA; (ii) decreased cholesterol efflux without effect on cholesterol influx; (iii) diminished the gene and protein expression of ABCA1 among other unaffected receptors that regulate cholesterol homeostasis investigated; and (iv) down-regulated the expression of PPAR{alpha}, PPAR{gamma}, LXR{alpha}, and LXRβ. Treatment with powerful antioxidants (Trolox and BHT) prevented the adverse effects of iron-ascorbate, conceivably supporting the role of oxidative stress.

We employed iron-ascorbate, a well-established model for the induction of lipid peroxidation [26]. It initiates peroxidation, as demonstrated by the increased values of MDA, probably by producing highly reactive hydroxyl radicals from hydrogen peroxide via Fenton-type reactions. Ascorbic acid can amplify the oxidative potential of iron by promoting metal ion-induced lipid peroxidation [41]. In the experiments described herein, the iron-ascorbate complex was very effective in inducing lipid peroxidation, as demonstrated by high MDA levels, a well-established measure of lipid peroxidation. Supporting evidence was provided by the scavenger activity of Trolox and BHT antioxidants. Furthermore, we employed THP-1 cells that represent a well-known model of human macrophages [42], widely used in studies of foam cell formation [43–47], but the cholesterol regulation by oxidative stress in this model has not yet been established.

The integrity of cholesterol movement in THP-1 seems compromised under the iron-ascorbate prooxidant effect, which suggests that oxidation of LDL is an important, but not obligatory, event in foam cell formation. Interestingly, only the efflux path was disturbed. The important clinical corollary is that inhibition of oxidation can inhibit atherosclerosis independent of lowering plasma cholesterol levels. Inconclusive results have been obtained with clinical trials, but remarkable efforts are still necessary to design the appropriate combination of antioxidant molecules.

As mentioned before, foam cell formation from macrophages is a critical event in the initiation of atherosclerosis. The modulation by oxidative stress of the expression of genes involved in the import and export of cholesterol may have a great impact on foam cell formation and lesion development. We, therefore, decided to simultaneously investigate the regulation of genes and receptor proteins that participate in cholesterol influx and efflux. CD36 is a major class of scavenger receptors that internalize modified LDL [47,48]. Our results show that only CD36 protein expression in THP-1 macrophages was significantly modified by exposure to iron-ascorbate. The mechanism of induction of CD36 by oxLDL was shown to be due to the ability of oxLDL to activate the transcription factor PPAR{gamma} [49, 50], potentially leading to foam cell formation, cell death, release of lipids and matrix-metalloproteinases, all features of unstable plaques. Interestingly, in our study, PPAR{gamma} gene expression was down-regulated by iron-ascorbate while no change in CD36 gene expression could be noticed.

Similarly, the iron-ascorbate stimulus could not promote changes in the gene and protein expression of SR-BI that mediates the bidirectional flux of cholesterol across the plasma membrane [12]. The expression of SR-BI in macrophages appears to be regulated by cholesterol loading [10], as well as by the PPAR{alpha}, PPAR{gamma} and LXR{alpha} pathways [11,51]. However, its regulation is incompletely understood as it is also inversely correlated with ABCA1 expression [52,53]. Under our experimental conditions, no significant regulation of SR-BI by oxidative stress has been noticed even if the PPAR and LXR pathway has been modified.

In contrast, oxidative stress reduced the gene and protein expression of ABCA1, a crucial unidirectional cholesterol exporter in macrophages, which could account for the decrease in cholesterol efflux. Therefore, cholesterol outflow could not keep pace with uptake in order to avoid intracellular cholesterol accumulation given the down-regulation of ABCA1 expression elicited by oxidative stress. Expression of the ABCA1 gene is transcriptionally regulated. PPAR was shown recently to induce the expression of ABCA1 in macrophages through a transcriptional cascade mediated by the nuclear receptor LXR that heterodimerizes with retinoic-X-receptor (RXR) [22,23,54–56]. Therefore, we determined whether the oxidative stress-mediated decreases in ABCA1 mRNA expression and cholesterol efflux were dependent on the inactivation of PPAR and/or LXR. Our data clearly demonstrated that iron-ascorbate down-regulated the LXR- and PPAR-dependent transcription, since decreased mRNA levels of PPAR{alpha}, PPAR{gamma}, LXR{alpha}, and LXRβ were recorded.

Studies have provided evidence that the nuclear receptors LXR{alpha} and LXRβ, which bind with the RXR as obligate heterodimers, mediate the lipid induction of ABCA1 [54,55]. PPAR{alpha} and PPAR{gamma} have also been demonstrated to stimulate cholesterol efflux in cultured macrophages by inducing the expression of LXR{alpha} [22,23]. However, other studies demonstrated that PPAR{gamma} enhances HDL-dependant cholesterol efflux from macrophages in a LXR-independent mechanism [57].

Since caution should be taken in directly extrapolating the results obtained in THP-1 to human macrophages, we have also carried out experiments in human monocytes-derived macrophages. This approach allowed us to confirm that iron-ascorbate-mediated lipid peroxidation provoked a decline of ABCA1 and CD36 proteins.

As a result of oxidative stress, cholesterol accumulation in THP-1 should be viewed as reflecting the imbalance between influx and efflux, which originates from decreased delivery. The diagram in Fig. 9 depicts the possible mechanisms for such an occurrence: oxidative stress limits cholesterol outflow through a molecular cascade involving inhibition of LXR gene expression, leading to a decline in PPAR mRNA, which in turn decreased ABCA1 expression and lower cholesterol efflux. In conclusion, iron-ascorbate catalyzed lipid peroxidation appears to play an important role in regulating cholesterol efflux from THP-1 macrophages.


Figure 9
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 Diagram of the main players influencing cholesterol accumulation in THP-1 macrophages. Oxidative stress could influence ABCA1 gene transcription by repressing PPAR{alpha} and {gamma}, which would influence LXR{alpha} and β, or by diminishing directly the LXR receptors. This reduction in gene transcription could in term limit the protein translation and expression and then cholesterol efflux, leading to its cellular accumulation.

 


   Acknowledgement
 
The authors thank Mrs Schohraya Spahis for her technical assistance.


   Notes
 
* This paper was supported by research grants from the Canadian Institutes of Health Research (CIHR).

Time for primary review 23 days


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

  1. Stocker R., Keaney J.F. Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev (2004) 84:1381–1478.[Abstract/Free Full Text]
  2. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med (1999) 340:115–126.[Free Full Text]
  3. Tabas I. Apoptosis and plaque destabilization in atherosclerosis: the role of macrophage apoptosis induced by cholesterol. Cell Death Differ (2004) 11(Suppl_1):S12–S16.[CrossRef][ISI][Medline]
  4. Kunjathoor V.V., Febbraio M., Podrez E.A., Moore K.J., Andersson L., Koehn S., et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem (2002) 277:49982–49988.[Abstract/Free Full Text]
  5. Linsel-Nitschke P., Tall A.R. HDL as a target in the treatment of atherosclerotic cardiovascular disease. Nat Rev Drug Discov (2005) 4:193–205.[CrossRef][ISI][Medline]
  6. Acton S.L., Scherer P.E., Lodish H.F., Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem (1994) 269:21003–21009.[Abstract/Free Full Text]
  7. Hayden M.R., Clee S.M., Brooks-Wilson A., Genest J. Jr., Attie A., Kastelein J.J. Cholesterol efflux regulatory protein, Tangier disease and familial high-density lipoprotein deficiency. Curr Opin Lipidol (2000) 11:117–122.[CrossRef][ISI][Medline]
  8. Krieger M. Charting the fate of the "good cholesterol": identification and characterization of the high-density lipoprotein receptor SR-BI. Annu Rev Biochem (1999) 68:523–558.[CrossRef][ISI][Medline]
  9. Krieger M. Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. J Clin Invest (2001) 108:793–797.[CrossRef][ISI][Medline]
  10. Fluiter K., van der Westhuijzen D.R., van Berkel T.J. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells. J Biol Chem (1998) 273:8434–8438.[Abstract/Free Full Text]
  11. Chinetti G., Gbaguidi F.G., Griglio S., Mallat Z., Antonucci M., Poulain P., et al. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. Circulation (2000) 101:2411–2417.[Abstract/Free Full Text]
  12. Ji Y., Jian B., Wang N., Sun Y., Moya M.L., Phillips M.C., et al. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J Biol Chem (1997) 272:20982–20985.[Abstract/Free Full Text]
  13. Hirano K., Yamashita S., Nakagawa Y., Ohya T., Matsuura F., Tsukamoto K., et al. Expression of human scavenger receptor class B type I in cultured human monocyte-derived macrophages and atherosclerotic lesions. Circ Res (1999) 85:108–116.[Abstract/Free Full Text]
  14. Yu L., Cao G., Repa J., Stangl H. Sterol regulation of scavenger receptor class B type I in macrophages. J Lipid Res (2004) 45:889–899.[Abstract/Free Full Text]
  15. Febbraio M., Hajjar D.P., Silverstein R.L. CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest (2001) 108:785–791.[CrossRef][ISI][Medline]
  16. Podrez E.A., Febbraio M., Sheibani N., Schmitt D., Silverstein R.L., Hajjar D.P., et al. Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species. J Clin Invest (2000) 105:1095–1108.[ISI][Medline]
  17. Batt K.V., Patel L., Botham K.M., Suckling K.E. Chylomicron remnants and oxidised low density lipoprotein have differential effects on the expression of mRNA for genes involved in human macrophage foam cell formation. J Mol Med (2004) 82:449–458.[ISI][Medline]
  18. Osterud B., Bjorklid E. Role of monocytes in atherogenesis. Physiol Rev (2003) 83:1069–1112.[Abstract/Free Full Text]
  19. Parthasarathy S., Fong L.G., Otero D., Steinberg D. Recognition of solubilized apoproteins from delipidated, oxidized low density lipoprotein (LDL) by the acetyl-LDL receptor. Proc Natl Acad Sci U S A (1987) 84:537–540.[Abstract/Free Full Text]
  20. Tsukamoto K., Kinoshita M., Kojima K., Mikuni Y., Kudo M., Mori M., et al. Synergically increased expression of CD36, CLA-1 and CD68, but not of SR-A and LOX-1, with the progression to foam cells from macrophages. J Atheroscler Thromb (2002) 9:57–64.[Medline]
  21. Han J., Hajjar D.P., Febbraio M., Nicholson A.C. Native and modified low density lipoproteins increase the functional expression of the macrophage class B scavenger receptor, CD36. J Biol Chem (1997) 272:21654–21659.[Abstract/Free Full Text]
  22. Chawla A., Boisvert W.A., Lee C.H., Laffitte B.A., Barak Y., Joseph S.B., et al. A PPAR gamma-LXR–ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell (2001) 7:161–171.[CrossRef][ISI][Medline]
  23. Chinetti G., Lestavel S., Bocher V., Remaley A.T., Neve B., Torra I.P., et al. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med (2001) 7:53–58.[CrossRef][ISI][Medline]
  24. Witztum J.L., Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med (2001) 11:93–102.[CrossRef][ISI][Medline]
  25. He Y., Kwan W.C., Steinbrecher U.P. Effects of oxidized low density lipoprotein on endothelin secretion by cultured endothelial cells and macrophages. Atherosclerosis (1996) 119:107–118.[CrossRef][ISI][Medline]
  26. Courtois F., Suc I., Garofalo C., Ledoux M., Seidman E., Levy E. Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins. Am J Physiol Gastrointest Liver Physiol (2000) 279:G12–G19.[Abstract/Free Full Text]
  27. Levy E., Thibault L., Roy C.C., Letarte J., Lambert M., Seidman E.G. Mechanisms of hypercholesterolaemia in glycogen storage disease type I: defective metabolism of low density lipoprotein in cultured skin fibroblasts. Eur J Clin Invest (1990) 20:253–260.[ISI][Medline]
  28. Levy E., Thibault L., Garofalo C., Messier M., Lepage G., Ronco N., et al. Combined (n-3 and n-6) essential fatty deficiency is a potent modulator of plasma lipids, lipoprotein composition, and lipolytic enzymes. J Lipid Res (1990) 31:2009–2017.[Abstract]
  29. Levy E., Rizwan Y., Thibault L., Lepage G., Brunet S., Bouthillier L., et al. Altered lipid profile, lipoprotein composition, and oxidant and antioxidant status in pediatric Crohn disease. Am J Clin Nutr (2000) 71:807–815.[Abstract/Free Full Text]
  30. Suc I., Brunet S., Mitchell G., Rivard G.E., Levy E. Oxidative tyrosylation of high density lipoproteins impairs cholesterol efflux from mouse J774 macrophages: role of scavenger receptors, classes A and B. J Cell Sci (2003) 116:89–99.[Abstract/Free Full Text]
  31. Stan S., Lambert M., Delvin E., Paradis G., O'loughlin J., Hanley J.A., et al. Intestinal fatty acid binding protein and microsomal triglyceride transfer protein polymorphisms in French–Canadian youth. J Lipid Res (2005) 46:320–327.[Abstract/Free Full Text]
  32. Palinski W., Rosenfeld M.E., Yla-Herttuala S., Gurtner G.C., Socher S.S., Butler S.W., et al. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A (1989) 86:1372–1376.[Abstract/Free Full Text]
  33. Yla-Herttuala S., Palinski W., Rosenfeld M.E., Parthasarathy S., Carew T.E., Butler S., et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest (1989) 84:1086–1095.[ISI][Medline]
  34. Haberland M.E., Fong D., Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science (1988) 241:215–218.[Abstract/Free Full Text]
  35. Rosenfeld M.E., Palinski W., Yla-Herttuala S., Butler S., Witztum J.L. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis (1990) 10:336–349.[Abstract/Free Full Text]
  36. Heinecke J.W. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis (1998) 141:1–15.[ISI][Medline]
  37. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem (1997) 272:20963–20966.[Free Full Text]
  38. Sasahara M., Raines E.W., Chait A., Carew T.E., Steinberg D., Wahl P.W., et al. Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation? J Clin Invest (1994) 94:155–164.[ISI][Medline]
  39. Regnstrom J., Nilsson J., Tornvall P., Landou C., Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet (1992) 339:1183–1186.[CrossRef][ISI][Medline]
  40. Chisolm G.M., Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med (2000) 28:1815–1826.[CrossRef][ISI][Medline]
  41. Bachowski G.J., Thomas J.P., Girotti A.W. Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: cholesterol product analysis as a probe of reaction mechanism. Lipids (1988) 23:580–586.[ISI][Medline]
  42. Auwerx J. The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte–macrophage differentiation. Experientia (1991) 47:22–31.[CrossRef][ISI][Medline]
  43. Argmann C.A., Sawyez C.G., McNeil C.J., Hegele R.A., Huff M.W. Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins. Arterioscler Thromb Vasc Biol (2003) 23:475–482.[Abstract/Free Full Text]
  44. Fu Y., Luo N., Lopes-Virella M.F., Garvey W.T. The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages. Atherosclerosis (2002) 165:259–269.[CrossRef][ISI][Medline]
  45. Llaverias G., Jove M., Vazquez-Carrera M., Sanchez R.M., Diaz C., Hernandez G., et al. Avasimibe and atorvastatin synergistically reduce cholesteryl ester content in THP-1 macrophages. Eur J Pharmacol (2002) 451:11–17.[CrossRef][ISI][Medline]
  46. Llaverias G., Noe V., Penuelas S., Vazquez-Carrera M., Sanchez R.M., Laguna J.C., et al. Atorvastatin reduces CD68, FABP4, and HBP expression in oxLDL-treated human macrophages. Biochem Biophys Res Commun (2004) 318:265–274.[CrossRef][ISI][Medline]
  47. Llaverias G., Lacasa D., Vinals M., Vazquez-Carrera M., Sanchez R.M., Laguna J.C., et al. Reduction of intracellular cholesterol accumulation in THP-1 macrophages by a combination of rosiglitazone and atorvastatin. Biochem Pharmacol (2004) 68:155–163.[CrossRef][ISI][Medline]
  48. Nicholson A.C., Hajjar D.P. CD36, oxidized LDL and PPAR gamma: pathological interactions in macrophages and atherosclerosis. Vascul Pharmacol (2004) 41:139–146.[CrossRef][ISI][Medline]
  49. Barbier O., Torra I.P., Duguay Y., Blanquart C., Fruchart J.C., Glineur C., et al. Pleiotropic actions of peroxisome proliferator-activated receptors in lipid metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol (2002) 22:717–726.[Abstract/Free Full Text]
  50. Nagy L., Tontonoz P., Alvarez J.G., Chen H., Evans R.M. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell (1998) 93:229–240.[CrossRef][ISI][Medline]
  51. Malerod L., Juvet L.K., Hanssen-Bauer A., Eskild W., Berg T. Oxysterol-activated LXRalpha/RXR induces hSR-BI-promoter activity in hepatoma cells and preadipocytes. Biochem Biophys Res Commun (2002) 299:916–923.[CrossRef][ISI][Medline]
  52. Chen W., Silver D.L., Smith J.D., Tall A.R. Scavenger receptor-BI inhibits ATP-binding cassette transporter 1-mediated cholesterol efflux in macrophages. J Biol Chem (2000) 275:30794–30800.[Abstract/Free Full Text]
  53. Yancey P.G., Kawashiri M.A., Moore R., Glick J.M., Williams D.L., Connelly M.A., et al. In vivo modulation of HDL phospholipid has opposing effects on SR-BI- and ABCA1-mediated cholesterol efflux. J Lipid Res (2004) 45:337–346.[Abstract/Free Full Text]
  54. Costet P., Luo Y., Wang N., Tall A.R. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem (2000) 275:28240–28245.[Abstract/Free Full Text]
  55. Repa J.J., Turley S.D., Lobaccaro J.A., Medina J., Li L., Lustig K., et al. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science (2000) 289:1524–1529.[Abstract/Free Full Text]
  56. Venkateswaran A., Laffitte B.A., Joseph S.B., Mak P.A., Wilpitz D.C., Edwards P.A., et al. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A (2000) 97:12097–12102.[Abstract/Free Full Text]
  57. Li A.C., Binder C.J., Gutierrez A., Brown K.K., Plotkin C.R., Pattison J.W., et al. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha, beta/delta, and gamma. J Clin Invest (2004) 114:1564–1576.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Marcil, V.
Right arrow Articles by Levy, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Marcil, V.
Right arrow Articles by Levy, E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?