The FASEB Journal • Research Communication

Aspirin regulates expression and function of scavenger receptor-BI in macrophages: studies in primary human macrophages and in mice Ivan Tancevski,*,1 Andreas Wehinger,*,1 Wilfried Schgoer,* Philipp Eller,* Salvatore Cuzzocrea,† Bernhard Foeger,* Josef R. Patsch,* and Andreas Ritsch*,2 *Department of Internal Medicine, Innsbruck Medical University, Innsbruck, Austria; and † Dipartimento Clinico e Sperimentale di Medicina e di Farmacologia, Centro per lo Studio ed il Trattamento dei Neurolesi Lungodegenti, Facolta` di Medicina e Chirurgia, Universita` di Messina, Messina, Italy ABSTRACT

Scavenger receptor class B type I (SR-BI) has been shown to be expressed in human atherosclerotic plaque macrophages, where it is believed to reduce atherosclerosis by promoting cholesterol efflux. In this study we investigated the influence of aspirin and other NSAIDs on SR-BI expression and function in cultured human macrophages as well as in different mouse strains. Incubation of human macrophages with 0.5 mmol/l aspirin resulted in increased SR-BI protein expression and increased uptake of HDL-associated [3H]cholesteryl oleate without changes of SR-BI mRNA levels. In contrast, using 5 mmol/l of aspirin, SR-BI expression and function were significantly decreased. Sodium salicylate exerted similar effects on SR-BI expression, whereas no effects were observed using known COX1/2 inhibitors ibuprofen and naproxen, respectively. In in vivo studies low-dose aspirin treatment (6 mg/kg䡠day) induced SR-BI expression in wildtype and PPAR-␣ knockout mice, respectively, whereas the opposite effect was observed upon high-dose aspirin treatment (60 mg/kg䡠day) in these animals. We could show that COX-independent effects of aspirin were able to enhance expression of SR-BI in macrophages in a post-transcriptional, PPAR-␣ independent way, suggesting a novel pharmacologic effect of aspirin.—Tancevski, I., Wehinger, A., Schgoer, W., Eller, P., Cuzzocrea, S., Foeger, B., Patsch, J. R., Ritsch, A. Aspirin regulates expression and function of scavenger receptor-BI in macrophages: studies in primary human macrophages and in mice. FASEB J. 20, 1328 –1335 (2006)

to be expressed in macrophages within atherosclerotic plaques (6, 7). Aspirin (acetyl-salicylic acid) is an established widely used agent for therapy of inflammatory diseases, as well as for the prevention of vascular events such as myocardial infarction and stroke (8, 9). This protective effect has mostly been attributed to platelet inhibitory and anti-inflammatory effects (8, 10). It was recently shown that aspirin affects the expression of peroxisome proliferator-activated receptor-␣ (PPAR-␣), a nuclear receptor involved in regulation of lipid metabolism (11). In mice, activation of PPAR-␣ by fenofibrate was shown to result in up-regulation of SR-BI in macrophages of atherosclerotic lesions. In differentiated primary human macrophages, activation of PPAR-␣ was also shown to cause induction of SR-BI protein (6). In the present study we investigated the influence of aspirin on SR-BI mRNA and protein levels of primary human macrophages, and on uptake of HDL-associated [3H]cholesteryl oleate into these cells. To further study mechanisms possibly involved in aspirin-dependent regulation of SR-BI, we performed incubation experiments of human macrophages with different NSAIDs and analyzed whether known COX-independent effects of aspirin were involved in this process. We also investigated the involvement of PPAR-␣ in this scenario and were able to confirm our in vitro data in corresponding in vivo experiments using wild-type (WT) and PPAR-␣ knockout (PPAR-␣ KO) mice.

Key Words: SR-BI 䡠 PPAR-␣ 䡠 KO mice 䡠 NF-␬B

MATERIALS AND METHODS Reagents

The first hdl receptor to be discovered was the scavenger receptor class B type I (SR-BI) (1). The human homologue of SR-BI was initially identified as CLA-1 and mapped to chromosome 12 (2, 3). SR-BI mediates high-affinity binding of HDL facilitating bidirectional flux of cholesterol across the plasma membrane (4, 5). Besides its main sites of expression, namely steroidogenic tissues and liver, SR-BI was shown 1328

Chemicals used for experiments were aspirin (Alexis, Gru¨nberg, Germany), sodium salicylate, ibuprofen, naproxen 1

These authors equally contributed to this work. Correspondence: Department of Internal Medicine, Innsbruck Medical University, Anichstrasse 35, Innsbruck 6020, Austria. E-mail: [email protected] doi: 10.1096/fj.05-5368com 2

0892-6638/06/0020-1328 © FASEB

(Sigma, St. Louis, MO, USA), and fenofibrate (Nycomed, Austria). Isolation and culture of primary human macrophages and culture of the THP-1 cell line Peripheral blood mononuclear cells were prepared from forearm venous blood of healthy volunteers by Biocoll density gradient centrifugation as described (12). Monocytes (CD14⫹) were selected by the use of paramagnetic beads according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured in RPMI 1640 medium containing 10% of homologous or AB-serum on poly-L-lysine-coated 6-well plates at a density of 2 million cells per well and incubated at 37°C and 5% CO2. All cell culture experiments were performed after 8 days of differentiation. For use of cells in different experimental settings, supplemented RPMI 1640 was replaced by serum-free RPMI 1640. Macrophage differentiation of primary human monocytes was monitored morphologically by light microscopy and by fluorescence-activated cell sorting (FACS) analysis using a monoclonal antibody specific for CD163. THP-1 cells were obtained from the American Type Culture Collection (ATCC) (ATCC, Manassas, VA, USA) and cultivated by standard procedures. Differentiation into macrophages was achieved in supplemented RPMI 1640 cell culture medium containing 100 nmol/l of phorbol 12-myristate 13-acetate (PMA) (Promega, Madison, WI, USA) for 72 h. Calcein fluorescence staining To determine cell viability, cells were incubated for 40 min in dark surroundings with 1 ␮mol/l calcein-acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) and percentage of fluorescence emitting (viable) cells was determined by fluorescence microscopy (Olympus, Germany). RNA isolation, reverse transcription, and quantitative real-time polymerase chain reaction (PCR) Total RNA from human macrophages was prepared using High Pure RNA Isolation Kit (Roche, Mannheim, Germany) and reverse transcribed with Omniscript-RT Kit (Qiagen, Hilden, Germany). The modulation of SR-BI was investigated by quantitative Real-Time Taqman PCR using the Mx4000® Multiplex Quantitative PCR System (Stratagene, Amsterdam, The Netherlands) as described. GAPDH was used as reference gene (Applied Biosystems, Foster City, CA, USA). Average cycle (Ct) was calculated by Mx4000® software (Stratagene, Amsterdam, Netherlands). Protein extraction and Western blot analysis Preparation of whole cell extracts and Western blot analysis were performed as described (14). Immunodetection of SR-BI was carried out by the use of a monoclonal murine antibody (Ab) against CLA-1 (BD Biosciences, Franklin Lakes, NJ, USA) and a secondary horseradish-peroxidaseconjugated goat anti-mouse IgG Ab (Dako, Glostrup, Denmark). Immunodetection of PPAR-␣ was performed with a polyclonal goat anti-PPAR-␣ Ab and a secondary horseradishperoxidase-conjugated rabbit anti-goat (H⫹L) Ab (Jackson Immunoresearch Inc., Soham, UK). Antibodies against human cytoplasmatic ERK1/2 and phosphorylated ERK1/2, c-Jun NH2-terminal kinase (JNK), and p38 were purchased from Cell Signaling Technology (Beverly, MA, USA). The chemoluminescent reaction was performed using Super SigASPIRIN INFLUENCES MACROPHAGE SR-BI EXPRESSION

nal West Dura Reagent (Pierce, Rockford, IL, USA) and blots were visualized by Fluor-S-Imager using Quantity One V4.1 software (Bio-Rad, Hercules, CA, USA). After stripping with Re-Blot Plus Mild Solution (Chemicon, Temecula, CA, USA), immunodetection of ␤-actin was performed by using a monoclonal anti-␤-actin Ab (Sigma, St. Louis, MO, USA). HDL cholesterol uptake HDL was isolated by zonal ultracentrifugation (15) and labeled with [3H]-cholesteryl oleate as described (16). HDL uptake assays were carried out essentially as described previously (14). Preparation of nuclear extracts Nuclear extracts from differentiated primary human monocyte-derived macrophages were isolated after 40 h of treatment with aspirin as described (17). Briefly, cell culture plates were set on ice and cells were washed twice with ice-cold PBS. Cells were harvested, vortexed, and lysed in nuclei EZ lysis buffer (Sigma, St. Louis, MO, USA) for 5 min. Cell lysates were centrifuged at 500 g for 5 min at 4°C, and resuspended in an appropriate amount of nuclei EZ storage buffer (Sigma, St. Louis, MO, USA). Concentration of nuclei was determined spectrophotometrically according to the following equation: concentration (mg/ml) ⫽ 1.55 ⫻ A (280 nm) – 0.76 ⫻ A (260 nm), where A denotes absorbance. Nuclei were frozen at –70°C until EMSA was performed. Electrophoretic mobility shift assay (EMSA) Hybridization of equimolar amounts of single-stranded DNA oligonucleotides from the PPAR-␣ consensus sequence (5⬘-GGTAAA-GGT-CAA-AGG-TCA-ATC-GGC-3⬘) (GenXpress, Wiener Neudorf, Austria) (17) into double-stranded DNA oligonucleotides was performed in a T3 thermocycler® (Montreal Biotech, Guthrie, Canada). Double-stranded DNA probe specific for NF-␬B complexes was purchased from Promega (Promega, Madison, WI, USA). Probes were labeled by T4 polynucleotide kinase (New England Biolabs, Frankfurt, Germany) with [␥-32P]ATP (3000 Ci/mmol at 10 mCi/ml) (Amersham, Uppsala, Sweden) and purified using G-25 sephadex columns (Roche, Mannheim, Germany). Equal amounts of protein from nuclear extracts (5 ␮g) were mixed with gel shift binding buffer [10 mmol/l HEPES (pH 7.9), 50 mmol/l KCl, 0.1 mmol/l EDTA, 2.5 mmol/l DTT, 10% glycerol, and 0.05% Nonidet P-40] and incubated for 10 min at room temperature. 1 ␮l of 32P-labeled double-stranded oligonucleotides were added and the whole mixture was incubated for 30 min at 25°C. DNA-protein complexes were separated from unbound DNA probe on native polyacrylamide gels [37.5:1 acrylamide:bisacrylamide (40%, w/v)] in TBE buffer (pH 8.3) containing (in mmol/l) Tris 89.0, borate 89.0, and EDTA 2.0 (pH 8) by electrophoresis at 350 V for 2 h at room temperature. Then, gels were placed on filter paper and dried on a gel dryer for 2 h, before overnight analysis via X-ray films (Kodak, Rochester, NY, USA) at –70°C was performed. In vivo studies Certified Balb/c mice were obtained from the Central Facility of Laboratory Animals in Innsbruck. PPAR-␣ knockout (PPAR-␣ KO) mice were described previously (18). All procedures and care of animals were approved by the Austrian Animal Care and Use Committee. After 1 wk of acclimatiza1329

tion, mice received either normal drinking water or drinking water containing 60 mg/l aspirin (low-dose) and 600 mg/l (high-dose), respectively, which was replaced every other day. Considering that each animal drinks on average 2 to 3 ml of water per day, low-dose treatment would be equal to 120 to 180 ␮g aspirin per day for a mouse of 20 g of wt. On a body scale-adjusted scale, this amount would be equal to 360 to 540 mg/day if the animals weighed 60 kg (19). After 7 days of treatment, animals were sacrificed by neck dissection. Resident peritoneal macrophages were isolated by peritoneal lavage with supplemented Dulbecco’s modified Eagle medium and seeded onto 100 mm cell culture dishes. After 2 h, nonadherent cells were washed off with PBS, adherent macrophages were scraped and proteins for immunoblotting were extracted as described above.

RESULTS Influence of aspirin on SR-BI protein expression and function in human macrophages In our first experiments using human THP-1-derived macrophages, increasing doses of aspirin of up to 0.5 mmol/l led to enhancement of SR-BI expression, whereas concentrations equally or higher than 1 mmol/l showed the opposite effect (Fig. 1A). In the following experiments we therefore exclusively used the two concentrations of aspirin leading to maximum stimulatory and inhibitory effects, 0.5 mmol/l and 5 mmol/l aspirin, respectively. In cultured primary human macrophages SR-BI protein was found markedly

increased with 0.5 mmol/l of aspirin and diminished after treatment with 5 mmol/l (Fig. 1B). These effects were observed after 20 h of incubation, reaching maximum levels after 40 h (data not shown). To exclude any toxic effects of aspirin, we monitored cell morphology and viability of differentiated macrophages using light microscopy and esterase-mediated calcein fluorescence staining, respectively (20). Neither technique revealed any indication of toxicity, even at highest doses used in our experiments (Fig. 1C). To evaluate data from SR-BI expression experiments for internal consistence, we analyzed influence of aspirin treatment on uptake of HDL-[3H]-cholesteryl esters into primary human macrophages, reflecting SR-BI function in these cells. Indeed, HDL cholesteryl ester uptake into cells was found to be increased in primary human macrophages treated with 0.5 mmol/l aspirin and was reduced upon treatment with 5 mmol/l aspirin, respectively (Fig. 1D). The PPAR-␣ pathway Chinetti and co-workers recently showed that SR-BI protein expression was found increased on fenofibrateinduced activation of PPAR-␣ in human and murine macrophages (6). To evaluate the involvement of PPAR-␣ in the effects of aspirin observed by us, we attempted to answer this question by 1) coincubation experiments using the hypolipidemic drug fenofibrate, a well-defined PPAR-␣ activator, in addition to aspirin

Figure 1. A) Western blot analysis showing the influence of aspirin on SR-BI expression within THP-1-derived macrophages. A representative blot and quantification of 3 independent experiments by Quantity One V4.1 software are shown. Data are given as means ⫾ sd. Given P value corresponds to multiple comparison using 1-way ANOVA (F test). B) Western blot analysis of primary human macrophages treated for 40 h with vehicle (DMSO) or indicated amounts of aspirin. A representative blot and quantification of 3 independent experiments are shown. Data are given as means ⫾ sd (*P⬍0.05; ***P⬍0.001). C) Cell viability of primary human macrophages was determined by light microscopy (left) and calcein fluorescence (right) after 40 h of treatment with aspirin. D) Uptake of [3H]-labeled HDL cholesteryl esters by primary human macrophages. After 40 h of treatment with vehicle (DMSO) or aspirin and incubation with [3H]-labeled HDL for 5 h, cell-associated [3H]-HDL was measured by liquid scintillation counting. Data are given as means ⫾ sd (*P⬍0.05; ***P⬍0.001). 1330

Vol. 20

July 2006

The FASEB Journal

TANCEVSKI ET AL.

treatment; and by 2) studying effects of aspirin on PPAR-␣ expression and PPAR-␣ DNA binding activity in primary human macrophages. As illustrated in Fig. 2A, coincubation of 0.5 mmol/l aspirin and 100 ␮mol/l fenofibrate led to a further increase of SR-BI protein expression compared with corresponding levels of expression by treatment with aspirin or fenofibrate alone, respectively. However, down-regulation of SR-BI by 5 mmol/l of aspirin could not be reversed by coincubation with 100 ␮mol/l fenofibrate. The observed additive effects of 0.5 mmol/l aspirin and 100 ␮mol/l fenofibrate on SR-BI protein expression were confirmed in HDL cholesteryl ester uptake experiments, where increased levels of uptake due to incubation with 0.5 mmol/l of aspirin were further increased by the concomitant addition of fenofibrate (data not shown). To investigate the influence of aspirin and/or fenofibrate in more detail, we looked at PPAR-␣ protein expression and PPAR-␣ DNA binding activity in our system. Western blot experiments in human macrophages showed no change in PPAR-␣ expression after incubation with 0.5 mmol/l aspirin, whereas a drastically reduced expression of PPAR-␣ could be found on 5 mmol/l aspirin treatment (Fig. 2B, left three lanes). Addition of fenofibrate to the same experimental settings had no further effect on PPAR-␣ expression (Fig. 2B, right three lanes). However, we found increased levels of PPAR-␣ DNA binding capacity in nuclear extracts of the cells after 40 h of incubation with 0.5 mmol/l aspirin and no effect with 5 mmol/l aspirin, respectively (Fig. 2C). Influence of aspirin on SR-BI mRNA transcription To investigate the influence of enhanced PPAR-␣ DNA binding activity on transcription of SR-BI, we decided

to measure SR-BI mRNA levels in our experimental settings. As illustrated in Fig. 3, Taqman Real-Time PCR measurements showed no change of SR-BI mRNA upon incubation with 0.5 mmol/l aspirin compared with vehicle. The same results were observed on concomitant addition of fenofibrate. Enhanced PPAR-␣ DNA binding activity through 0.5 mmol/l aspirin did not affect SR-BI transcription in human primary macrophages, suggesting a post-transcriptional regulation of SR-BI. In contrast, treatment with 5 mmol/l of aspirin drastically reduced SR-BI mRNA levels to 30%. In agreement with data from our Western blot experiments shown in Fig. 2, reduction of SR-BI mRNA by 5 mmol/l of aspirin could not be reversed by concomitant incubation with fenofibrate (Fig. 3). Aspirin influences SR-BI expression in macrophages of wild-type and PPAR-␣ KO mice To confirm results observed in our cell culture experiments, we decided to perform in vivo experiments. Male Balb/c mice were randomized to receive placebo, low-dose aspirin (60 mg/l), or high-dose aspirin (600 mg/l) in their drinking water, respectively. Based on the daily water intake of 2–3 ml for each mouse, the estimated daily intake of aspirin was calculated to be 6 –9 mg/kg (low-dose) and 60 –90 mg/kg (high-dose), respectively. No apparent differences concerning mobility, behavior, or food intake could be observed during the entire study. After 1 wk of treatment, resident peritoneal macrophages were isolated by peritoneal lavage. In agreement with our cell culture studies, we found increased levels of SR-BI expression in macrophages from mice treated with low-dose aspirin compared with the placebo-treated animals; the opposite effect was observed after treatment with high-dose

Figure 2. A) Western blot analysis showing the influence of aspirin with or without fenofibrate on SR-BI expression within primary human macrophages. Cells were treated for 40 h with the indicated amounts of aspirin/fenofibrate. A representative blot and quantification of 3 independent experiments are shown. Data are given as means ⫾ sd (*P⬍0.05; ***P⬍0.001). B) Western blot analysis showing the influence of aspirin with or without fenofibrate on PPAR-␣ expression within primary human macrophages. Cells were treated for 40 h with aspirin and/or fenofibrate. A representative blot and quantification of 3 independent experiments are shown. Data are given as means ⫾ sd (**P⬍0.01). C) Autoradiograph showing activation of PPAR-␣ in nuclear extracts from primary human macrophages 40 h after incubation with indicated amounts of aspirin (lane 1: vehicle; lane 2: 0.5 mmol/l aspirin; lane 3: 5 mmol/l aspirin). PPAR-␣ binding activity was determined by EMSA. The arrow indicates the position of PPAR-␣-specific complexes, F indicates free probe. ⫹, positive control; ⫺, negative control. ASPIRIN INFLUENCES MACROPHAGE SR-BI EXPRESSION

1331

shown to be enhanced on 0.5 mmol/l aspirin and decreased upon 5 mmol/l aspirin treatment, respectively (Fig. 6B).

DISCUSSION

Figure 3. Taqman Real-Time PCR analysis of SR-BI mRNA expression in primary human macrophages. Cells were treated for 40 h with aspirin and/or fenofibrate. GAPDH mRNA was used as reference. Data are given as means ⫾ sd from 3 independent experiments (***P⬍0.001).

aspirin (Fig. 4A). Same results were observed within female Balb/c mice, indicating that the aspirin effect on SR-BI was not gender-specific (data not shown). To clarify the involvement of PPAR-␣ in this scenario, we repeated the same experiments in PPAR-␣ KO mice. As seen in our experiments performed in WT mice, SR-BI expression in macrophages of PPAR-␣ KO mice was induced by in vivo treatment with low-dose aspirin and decreased after treatment with high-dose aspirin (Fig. 4B). These results indicated that PPAR-␣ is not required for either basal expression or induction of macrophage SR-BI by aspirin.

In our experiments, aspirin at a concentration of 0.5 mmol/L indeed enhanced SR-BI protein expression in primary human macrophages, but at a concentration of 5 mmol/L induced the opposite effect. These results agree with recently published data by Vin ˜ als et al. showing increased SR-BI protein expression in differentiated THP-1 on treatment with 0.6 mmol/l aspirin (25). In our experiments, changes in expression were accompanied by corresponding changes in SR-BI function, respectively. Similar effects were observed after incubation with sodium salicylate, but not with nonsalicylic NSAIDs (Fig. 5). These results clearly point toward a COX-independent effect for aspirin in our experimental settings. The observed opposite effects of 0.5 mmol/l and 5 mmol/l aspirin in our experiments are pointing to two different scenarios of regulation. On the one hand, stimulatory effects of 0.5 mmol/l aspirin as well as of fenofibrate were not accompanied by changes in SR-BI mRNA levels, respectively. On the other hand, 5 mmol/l aspirin treatment of primary human macrophages resulted in markedly decreased levels of both SR-BI protein and mRNA. This was accompanied by a

Influence of different NSAIDs on SR-BI regulation To evaluate whether the observed effects of aspirin on SR-BI were mediated by inhibition of cyclooxygenase (COX), human macrophages were also incubated with its active metabolite, sodium salicylate. Sodium salicylate exerted similar effects on SR-BI expression in macrophages as aspirin, with 0.5 mmol/l being the strongest inducing and 5 mmol/l being the strongest down-regulating concentration, respectively (Fig. 5A). These data indicated that aspirin-dependent regulation of SR-BI occurred in a COX-independent way. To strengthen this view and to clarify whether the observed effects were inherent to aspirin and sodium salicylate, cells were incubated with two well-known COX1/2 inhibitors, ibuprofen and naproxen. Corresponding to previous reports, we used increasing concentrations of ibuprofen and naproxen up to 100 ␮mol/l and 300 ␮mol/l, respectively (21–23). Neither compound was shown to influence SR-BI expression (Fig. 5B, C). In agreement with results from our sodium salicylate experiments, these data suggested a COX-independent effect of aspirin on SR-BI expression in human macrophages. We therefore analyzed known COX-independent effects in our setting, including activation of NF-␬B and phosphorylation of MAP kinases Erk1/2, JNK and p38, respectively (24). No activity alterations within the MAP kinase cascade could be observed, as shown in Fig. 6A. Nuclear translocation of NF-␬B was 1332

Vol. 20

July 2006

Figure 4. Western blot analysis showing the influence of aspirin on SR-BI expression within resident peritoneal macrophages of mice treated for 7 days with indicated doses of aspirin. A) Results from Balb/c mice. B) Results from PPAR-␣ KO mice. Each lane represents pooled protein extracts from 3 mice treated with vehicle, 6 mg/kg and 60 mg/kg aspirin, respectively. Detection of ␤-actin within protein extracts served as reference.

The FASEB Journal

TANCEVSKI ET AL.

decrease in PPAR-␣ protein concentration, suggesting a yet unknown effect of highly concentrated aspirin on the expression of proteins involved in lipoprotein metabolism. In murine liver cells SR-BI has been shown to be regulated in a post-transcriptional fashion (26, 27). Data from our experiments suggest that a similar mode of regulation takes place in human primary macrophages when treated with 0.5 mmol/l aspirin or fenofibrate. A post-transcriptional up-regulation of SR-BI through 0.5 mmol/l aspirin might be attributed to increased expression of PDZK1, which has been proposed to affect post-transcriptional processing and/or stability of SR-BI in liver cells (26, 27). Since macrophages do not express PDZK1 (unpublished data from

Figure 5. Western blot analysis showing the influence of sodium salicylate, ibuprofen and naproxen on SR-BI expression within THP-1-derived macrophages. Cells were treated for 40 h with vehicle (DMSO) or indicated amounts of the different drugs. A) A representative blot and quantification of 3 independent experiments using sodium salicylate are shown (P⬍0.001). Representative blots of 2 independent experiments using ibuprofen (B) and naproxen (C) are shown. Detection of ␤-actin served as loading control. ASPIRIN INFLUENCES MACROPHAGE SR-BI EXPRESSION

Figure 6. A) Effects of aspirin on phosphorylation of mitogenactivated protein kinases. Nuclear extracts from THP-1-derived macrophages incubated with vehicle (DMSO) or indicated amounts of aspirin for 40 h were analyzed by Western blot analysis using antibodies specific for phosphorylated ERK1/2, JNK, and p38, respectively. Bands detected after incubation with Ab against nonphosphorylated ERK1/2 served as loading control. Representative blots of 3 independent experiments are shown. B) Autoradiograph showing different activation of NF-␬B in nuclear extracts from THP1derived human macrophages, 40 h after incubation with different amounts of aspirin (lane 1: vehicle; lane 2: 0.5 mmol/l aspirin; lane 3: 5 mmol/l aspirin). NF-␬B DNAbinding activity was determined by EMSA. The arrow indicates the position of NF-␬B-specific complexes, F indicates free [␥-32P]ATP-labeled double-stranded DNA oligo probe. Representative EMSA from 3 independent experiments is shown. ⫹, positive control (nuclear extracts from venous smooth muscle cells); –, negative control.

1333

our laboratory), our findings might be explained by a PPAR-␣-independent regulation of a yet unrecognized protein through aspirin influencing macrophage SR-BI protein stability. This unknown protein could be a downstream-target of NF-␬B, since NF-␬B activity was markedly increased upon 0.5 mmol/l aspirin treatment, whereas it was decreased on 5 mmol/l aspirin. However, we cannot rule out that regulation of NF-␬B might be a parallel event in our scenario. NF-␬B has been shown to be involved in numerous signaling cascades in atherogenesis. Besides its role as regulator of proinflammatory and anti-inflammatory genes, it has been shown to be involved in initiation of atherosclerosis and foam cell formation (28). However, the role of NF-␬B in macrophages has not been studied thoroughly. Aspirin concentrations used in our tissue culture experiments are markedly higher than those reached in plasma of patients treated with low-dose aspirin. However, these dosages are well within the range or even lower than those used in previous tissue culture experiments (24, 25, 29 –34). To confirm data from our in vitro experiments and to determine a possible physiological role of aspirin, animal studies were performed using a dosage comparable to that routinely used in the clinical setting. In our in vivo experiments, the tested concentrations are corresponding to a daily intake of 360 –540 mg aspirin for a 60 kg individual. Indeed, this regimen enhanced SR-BI expression in resident peritoneal macrophages. On the other hand, the 10-fold dose of aspirin down-regulated SR-BI expression in resident macrophages. Same results were obtained in male and female mice, demonstrating that the effects of aspirin on macrophage SR-BI expression were not due to any gender-specific properties. Moreover, same results were observed in both WT and PPAR-␣ KO mice, respectively, indicating that this transcription factor is not involved in regulation of SR-BI by aspirin. After administration, aspirin is rapidly converted to its active metabolite salicylate, emphasizing our assumption that the effects of aspirin on SR-BI experiments are based on a COX-independent action of this compound (35, 36). In the clinical setting, aspirin treatment is one of the major pharmacologic interventions against atherosclerosis with its platelet-inhibitory and anti-inflammatory effects as the rationale for its use. Our results suggest an additional mechanism underlying the atheroprotective effect of aspirin, namely that aspirin enhances SR-BI expression and function. Accordingly, Chinetti et al. proposed that increased expression of SR-BI in atherosclerotic lesion macrophages could enhance the removal of unesterified cholesterol in these foam cells resulting in the regression of the fatty streak (6), since SR-BI is able to bind HDL with high affinity and to promote cholesterol efflux in the presence of a favorable cholesterol gradient (37–39). Van Berkel and co-workers recently demonstrated the protective role of SR-BI in advanced atherosclerotic lesions in mice (40). Thus, we speculate that enhancement of cholesterol efflux from atherosclerotic lesions might represent an 1334

Vol. 20

July 2006

auxiliary effect in addition to the known protective effects of aspirin against vascular diseases. In conclusion, we demonstrated in this study that 0.5 mmol/l aspirin enhanced SR-BI expression and function in human macrophages in a COX-independent post-transcriptional way, suggesting a novel pharmacologic effect for aspirin that might contribute to the atheroprotective properties of this agent. ¨ sterThis work was supported by the Jubila¨umsfond der O reichischen Nationalbank (grant 9340 to A.R.), by the Austrian Fonds zur Fo¨rderung der wissenschaftlichen Forschung (grant P16121-B07 to B.F.), and the Diabetes- und Atherosklerosezentrum Innsbruck (DAZ).

REFERENCES 1.

2. 3.

4.

5. 6.

7.

8. 9. 10.

11.

12. 13.

14.

Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271, 518 – 520 Calvo, D., and Vega, M. A. (1993) Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J. Biol. Chem. 268, 18929 –18935 Cao, G., Garcia, C. K., Wyne, K. L., Schultz, R. A., Parker, K. L., and Hobbs, H. H. (1997) Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1. J. Biol. Chem. 272, 33068 –33076 Ji, Y., Jian, B., Wang, N., Sun, Y., Moya, M. L., Phillips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J. Biol. Chem. 272, 20982–20985 Krieger, M. (2001) Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. J. Clin. Invest. 108, 793–797 Chinetti, G., Gbaguidi, F. G., Griglio, S., Mallat, Z., Antonucci, M., Poulain, P., Chapman, J., Fruchart, J. C., Tedgui, A., Najib-Fruchart, J., and Staels, B. (2000) CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. Circulation 101, 2411–2417 Hirano, K., Yamashita, S., Nakagawa, Y., Ohya, T., Matsuura, F., Tsukamoto, K., Okamoto, Y., Matsuyama, A., Matsumoto, K., Miyagawa, J., and Matsuzawa, Y. (1999) Expression of human scavenger receptor class B type I in cultured human monocytederived macrophages and atherosclerotic lesions. Circ. Res. 85, 108 –116 Awtry, E. H., and Loscalzo, J. (2000) Aspirin. Circulation 101, 1206 –1218 Cronstein, B. N., and Weissmann, G. (1995) Targets for antiinflammatory drugs. Annu. Rev. Pharmacol. Toxicol. 35, 449 – 462 Antiplatelet Trialists’ Collaboration. (1994) Collaborative overview of randomised trials of antiplatelet therapy-I៮: prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. Br. Med. J. 308, 81–106 Planaguma, A., Titos, E., Lopez-Parra, M., Gaya, J., Pueyo, G., Arroyo, V., and Claria, J. (2002) Aspirin (ASA) regulates 5-lipoxygenase activity and peroxisome proliferator-activated receptor alpha-mediated CINC-1 release in rat liver cells: novel actions of lipoxin A4 (LXA4) and ASA-triggered 15-epi-LXA4. FASEB J. 16, 1937–1939 Recalde, H. R. (1984) A simple method of obtaining monocytes in suspension. J. Immunol. Methods 69, 71–77 Tancevski, I., Frank, S., Massoner, P., Stanzl, U., Schgoer, W., Wehinger, A., Fievet, C., Eller, P., Patsch, J. R., and Ritsch, A. (2005) Increased plasma levels of LDL cholesterol in rabbits after adenoviral overexpression of human scavenger receptor class B type I. J. Mol. Med. 83, 927–932 Ritsch, A., Tancevski, I., Schgoer, W., Pfeifhofer, C., Gander, R., Eller, P., Foeger, B., Stanzl, U., and Patsch, J. R. (2004)

The FASEB Journal

TANCEVSKI ET AL.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25.

26. 27.

Molecular characterization of rabbit scavenger receptor class B types I and II: portal to central vein gradient of expression in the liver. J. Lipid Res. 45, 214 –222 Patsch, J. R., Sailer, S., Kostner, G., Sandhofer, F., Holasek, A., and Braunsteiner, H. (1974) Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation. J. Lipid Res. 15, 356 –366 Kaser, S., Ebenbichler, C. F., Wolf, H. J., Sandhofer, A., Stanzl, U., Ritsch, A., and Patsch, J. R. (2001) Lipoprotein profile and cholesteryl ester transfer protein in neonates. Metabolism 50, 723–728 Dichtl, W., Nilsson, L., Goncalves, I., Ares, M. P., Banfi, C., Calara, F., Hamsten, A., Eriksson, P., and Nilsson, J. (1999) Very low-density lipoprotein activates nuclear factor-kappaB in endothelial cells. Circ. Res. 84, 1085–1094 Cuzzocrea, S., Di Paola, R., Mazzon, E., Genovese, T., Muia, C., Centorrino, T., and Caputi, A. P. (2004) Role of endogenous and exogenous ligands for the peroxisome proliferators activated receptors alpha (PPAR-alpha) in the development of inflammatory bowel disease in mice. Lab. Invest. 84, 1643–1654 Cyrus, T., Sung, S., Zhao, L., Funk, C. D., Tang, S., and Pratico, D. (2002) Effect of low-dose aspirin on vascular inflammation, plaque stability, and atherogenesis in low-density lipoprotein receptor-deficient mice. Circulation 106, 1282–1287 Bell, E., Cao, X., Moibi, J. A., Greene, S. R., Young, R., Trucco, M., Gao, Z., Matschinsky, F. M., Deng, S., Markman, J. F., Naji, A., and Wolf, B. A. (2003) Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets. Diabetes 52, 2731–2739 Mouithys-Mickalad, A., Deby-Dupont, G., Dogne, J. M., de Leval, X., Kohnen, S., Navet, R., Sluse, F., Hoebeke, M., Pirotte, B., and Lamy, M. (2004) Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1). Biochem. Biophys. Res. Commun. 325, 1122–1130 Jans, D. M., Martinet, W., Fillet, M., Kockx, M. M., Merville, M. P., Bult, H., Herman, A. G., and De Meyer, G. R. (2004) Effect of non-steroidal anti-inflammatory drugs on amyloid-beta formation and macrophage activation after platelet phagocytosis. J. Cardiovasc. Pharmacol. 43, 462– 470 Martel-Pelletier, J., Mineau, F., Fahmi, H., Laufer, S., Reboul, P., Boileau, C., Lavigne, M., and Pelletier, J. P. (2004) Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids. Arthritis Rheum. 50, 3925–3933 Tegeder, I., Pfeilschifter, J., and Geisslinger, G. (2001) Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J. 15, 2057–2072 Vinals, M., Bermudez, I., Llaverias, G., Alegret, M., Sanchez, R. M., Vazquez-Carrera, M., and Laguna, J. C. (2005) Aspirin increases CD36, SR-BI, and ABCA1 expression in human THP-1 macrophages. Cardiovasc. Res. 66, 141–149 Silver, D. L. (2004) SR-BI and protein-protein interactions in hepatic high density lipoprotein metabolism. Rev. Endocr. Metab. Disord. 5, 327–333 Yesilaltay, A., Kocher, O., Rigotti, A., and Krieger, M. (2005) Regulation of SR-BI-mediated high-density lipoprotein metabo-

ASPIRIN INFLUENCES MACROPHAGE SR-BI EXPRESSION

28. 29.

30. 31.

32. 33.

34. 35.

36.

37. 38.

39.

40.

lism by the tissue-specific adaptor protein PDZK1. Curr. Opin. Lipidol. 16, 147–152 de Winther, M. P., Kanters, E., Kraal, G., and Hofker, M. H. (2005) Nuclear factor kappaB signaling in atherogenesis. Arterioscler. Thromb. Vasc. Biol. 25, 904 –914 Garcia-Martinez, J. M., Fresno Vara, J. A., Lastres, P., Bernabeu, C., Betes, P. O., and Martin-Perez, J. (2003) Effect of metamizol on promyelocytic and terminally differentiated granulocytic cells. Comparative analysis with acetylsalicylic acid and diclofenac. Biochem. Pharmacol. 65, 209 –217 Ranganathan, S., Joseph, J., and Mehta, J. L. (2003) Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53. Biochem. Biophys. Res. Commun. 301, 143–146 Voisard, R., Fischer, R., Osswald, M., Voglic, S., Baur, R., Susa, M., Koenig, W., and Hombach, V. (2001) Aspirin (5 mmol/L) inhibits leukocyte attack and triggered reactive cell proliferation in a 3D human coronary in vitro model. Circulation 103, 1688 –1694 Matasic, R., Dietz, A. B., and Vuk-Pavlovic, S. (2000) Cyclooxygenase-independent inhibition of dendritic cell maturation by aspirin. Immunology 101, 53– 60 Kagawa, A., Azuma, H., Akaike, M., Kanagawa, Y., and Matsumoto, T. (1999) Aspirin reduces apolipoprotein(a) (apo(a)) production in human hepatocytes by suppression of apo(a) gene transcription. J. Biol. Chem. 274, 34111–34115 Kopp, E., and Ghosh, S. (1994) Inhibition of NF-kappa B by sodium salicylate and aspirin. Science 265, 956 –959 Cronstein, B. N., Montesinos, M. C., and Weissmann, G. (1999) Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFkappaB. Proc. Natl. Acad. Sci. U. S. A. 96, 6377– 6381 Pillinger, M. H., Capodici, C., Rosenthal, P., Kheterpal, N., Hanft, S., Philips, M. R., and Weissmann, G. (1998) Modes of action of aspirin-like drugs: salicylates inhibit erk activation and integrin-dependent neutrophil adhesion. Proc. Natl. Acad. Sci. U. S. A. 95, 14540 –14545 Connelly, M. A., and Williams, D. L. (2004) Scavenger receptor BI: a scavenger receptor with a mission to transport high density lipoprotein lipids. Curr. Opin. Lipidol. 15, 287–295 de La Llera-Moya, M., Connelly, M. A., Drazul, D., Klein, S. M., Favari, E., Yancey, P. G., Williams, D. L., and Rothblat, G. H. (2001) Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL. J. Lipid Res. 42, 1969 –1978 Jian, B., de la Llera-Moya, M., Ji, Y., Wang, N., Phillips, M. C., Swaney, J. B., Tall, A. R., and Rothblat, G. H. (1998) Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors. J. Biol. Chem. 273, 5599 –5606 Van Eck, M., Bos, I. S., Hildebrand, R. B., Van Rij, B. T., and Van Berkel, T. J. (2004) Dual role for scavenger receptor class B, type I on bone marrow-derived cells in atherosclerotic lesion development. Am. J. Pathol. 165, 785–794 Received for publication December 13, 2005. Accepted for publication February 27, 2006.

1335