PPAR activators and COX inhibitors selectively block cytokine-induced COX-2 expression and activity in human aortic smooth muscle cells

European Journal of Pharmacology 606 (2009) 121–129

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Cardiovascular Pharmacology

PPAR activators and COX inhibitors selectively block cytokine-induced COX-2 expression and activity in human aortic smooth muscle cells Yves Rival ⁎, Laurence Puech, Thierry Taillandier, Nathalie Benéteau, Anne Rouquette, Fabrice Lestienne, Elisabeth Dupont-Passelaigue, Isabelle Le Roy, Jean-François Patoiseau, Didier Junquéro Centre de Recherche Pierre Fabre-17, Avenue Jean Moulin-81106 Castres Cédex, France

a r t i c l e

i n f o

Article history: Received 27 June 2008 Received in revised form 16 December 2008 Accepted 9 January 2009 Available online 21 January 2009 Keywords: Atherosclerosis Inflammation PPAR (peroxisome proliferator-activated receptor) COX Smooth muscle cell

a b s t r a c t Atherosclerotic complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells (SMCs), and an accumulation of inflammatory cells. Regulation of this inflammatory response is an essential element in chronic inflammatory diseases such as atherosclerosis. Nuclear receptors and particularly peroxisome proliferator-activated receptors (PPARs) have emerged as therapeutic targets with a widespread impact on the treatment of metabolic disorders because they can modulate gene expression involved in lipid and glucose homeostasis and can exert anti-inflammatory properties. However, little is known about nuclear receptor effects on SMC inflammation, which produces large amounts of IL-6 and prostanoids. The aim of this study was to evaluate anti-inflammatory properties of nuclear receptor activators in a human physiological SMC model. We show that PPAR activators, as well as liver X receptor α, farnesoid X receptor and retinoid X receptor α activators, inhibit IL-1β-induced SMC 6-keto PGF1α synthesis, an index of cyclooxygenase (COX)-2 activity, with IC50 between 1 and 69 µM. In contrast, PPARγ activators, as exemplified by rosiglitazone and pioglitazone, were unable to inhibit cytokine-induced 6-keto PGF1α synthesis. We also demonstrate for the first time that the COX-2 inhibitor rofecoxib can reduce 6-keto PGF1α production by both enzymatic inhibition and transcriptional repression. These results show that some nuclear receptor activators have SMC anti-inflammatory properties due to COX-2 inhibition which could participate in their anti-atherosclerotic properties beyond lipid impacts. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Atherosclerosis is a multi-step vascular disease initiated by endothelial injury, monocyte and T lymphocyte recruitment in the subendothelial space, followed by differentiation into macrophages and cholesterol deposition. Formation of foam cells, smooth muscle cell (SMC) proliferation and migration to the intima are the next following crucial steps. The presence of inflammatory cells as well as numerous cytokines in the atherosclerotic lesion strongly suggests an important immunological component in the pathogenesis of atherosclerosis. Regulation of this inflammatory response is an essential element in the pathogenesis of chronic inflammatory diseases such as atherosclerosis. The synthesis and activity of the prostaglandins have occupied a prominent position in inflammation research since the demonstration in 1971 that aspirin, the first non-steroidal anti-inflammatory drug (NSAID) blocked the activity of cyclooxygenase (COX). COX catalyses the conversion of arachidonic acid into the prostaglandin (PG) H2, ⁎ Corresponding author. Tel.: +33 5 63 71 42 12; fax: +33 5 63 71 43 63. E-mail address: [email protected] (Y. Rival). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.01.010

leading to other PGs following the action of a variety of PG synthases. Inhibition of prostaglandins can be a double-edged sword since prostaglandins contribute to various homeostatic functions such as maintenance of normal gastric mucosa and fluid/electrolyte balance. NSAID research changed radically with the discovery that COX existed in two isoforms: the constitutively expressed COX-1, mainly responsible for homeostatic prostaglandins, and an inducible COX-2 isoform localized in inflammatory cells and tissues and up-regulated during inflammatory response (Englesbe et al., 2004; Staels et al., 1998; Yan et al., 2000). COX-2 selective inhibitors like rofecoxib or celecoxib (Gierse et al., 1996; Niederberger et al., 2004) have been developed and approved by the FDA for the treatment of arthritis and acute pain (Chan et al., 1999; Mamdani et al., 2004). Nevertheless, VIGOR (Vioxx Gastrointestinal Outcomes Research, 2000) suggested an increased risk of cardiovascular events after 18 months of treatment, which was further confirmed with the clinical study APPROVe (Adenomatous Polyp Prevention on VIOXX, 2004; Ai-Wen et al., 2004) and resulted in a worldwide withdrawal of VIOXX the same year. Celecoxib is currently marketed but the CLASS (Celecoxib Longterm Arthritis Safety Study) clinical trial has revealed a similar tendency, and has stimulated a discussion on the molecular basis of

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such adverse effects. The exact mechanism(s) that results in these side effects due to COX-2 inhibitors is not clear; nevertheless it has been suggested that the selective prostacyclin inhibition in vascular cells without affecting COX-1-mediated formation of TXA2 in platelets tips the balance toward a prothrombotic state (Mukherjee et al., 2001; Fitzgerald, 2004). PPARs have emerged as therapeutic targets with widespread impacts on the treatment of metabolic disorders because they are ligand-dependent transcription factors (Kersten et al., 2000) that (1) modulate the expression of genes involved in lipid and glucose homeostasis (Willson et al., 2000) (2) exert anti-inflammatory properties by antagonizing the AP-1, NF-kB, and STAT pathways in vascular cells (Delerive et al., 1999; Ryoo et al., 2004; Rival et al., 2002) (3) inhibit the proliferation and migration of vascular smooth cells (Marx et al., 1998; Wang et al., 2006; Zahradka et al., 2006). PPARs promote transcription by forming heterodimers with members of the retinoid X receptor α receptor family and binding to specific DNA motifs termed PPAR-response elements (PPRE). Three PPAR subtypes were identified; the α isoform is the primary subtype expressed in the liver, but also in the heart and the kidney, and acts as a major regulator of metabolism of fats, catabolism of fatty acids, and synthesis and catabolism of lipoproteins (Barbier et al., 2002). PPARα is also involved in cholesterol efflux from peripheral tissues and the high-density lipoprotein reverse cholesterol-transport pathway (Chinetti et al., 2001). The PPARγ isoform is essentially expressed in adipocytes where it stimulates lipoprotein lipase and promotes fatty acid uptake and storage in mature adipocytes. These effects, along with increased insulin sensitivity, decrease circulating free fatty acids and triglycerides (Reginato and Lazar, 1999). Like PPARα, PPARγ also influences lipid metabolism in macrophages from the arterial wall by upregulating the expression of ABCA1 transporter which results in an increased cholesterol efflux (Chawla et al., 2001). The functions of the ubiquitous PPARδ isoform were more recently elucidated by the demonstration of its role in cell differentiation in cells such as preadipocytes, keratinocytes (Wahli, 2002) and enterocytes. PPARδ activation also stimulates fatty acid oxidation and energy uncoupling in skeletal muscle (promoting slow-twitch fibers) and in brown adipose tissue leading to decreased fatty acid storage in white adipose tissue (retarding weight gain) (Oliver et al., 2001; Wang et al., 2003; Barish et al., 2006). At the level of the vascular wall, PPARδ activation regulates the availability of BCL-6, an inflammatory suppressor protein released upon ligation of PPARδ (anti-inflammatory switch), to control macrophage-elicited inflammation and atherogenesis (Gross et al., 2005). PPARδ has also anti-thrombotic effects since Ali et al. (2006) have demonstrated that PPARδ ligands synergize with nitric oxide to inhibit human platelet aggregation. The liver X receptor, farnesoid X receptor and retinoid X receptor α are also members of the nuclear hormone receptor super-family which have recently been implicated as potential novel pharmacological targets for the treatment of metabolic disorders. Liver X receptor α agonists induce cholesterol efflux, improve glucose metabolism and inhibit macrophage-derived inflammation (Bruemmer and Law, 2005). In apoE−/− mouse atherosclerosis model, the synthetic liver X receptor agonist T0901317 reduces aortic atherosclerotic lesion area by around 60% and results in a reduction in macrophage content (Dai et al., 2008). Farnesoid X receptor is now recognized as a bile acid receptor with ligands including chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA). Its activation leads to induction of an orphan nuclear receptor, small heterodimer partner (SHP), that mediates some of the inhibitory effects of farnesoid X receptor ligands on bile acid and lipid metabolism (Lu et al., 2000). A common feature of these various metabolic pathways is their control by retinoid X receptor α heterodimers. Hence, retinoid X receptor is able to heterodimerize with PPAR, liver X receptor α and retinoid X receptor to activate key gene transcrip-

tion. In apoE−/− mice, a well established animal model of atherosclerosis, the retinoid X receptor agonist LG100364 drastically reduces atherosclerosis development (Claudel et al., 2001). Liver X receptor, farnesoid X receptor and retinoid X receptor have been described in vascular SMCs and their activation can reduce inflammation and remodelling, as well as promote plaque stability (Delvecchio et al., 2007; Yoyo et al., 2007; Antonio et al., 2003). Although PPAR levels are much lower in vascular cells, each of the three PPAR isoforms has also been detected in vascular SMCs (Marx et al., 2004; Zahradka et al., 2006). However, little is known about the effects of various nuclear receptor activators on the inflammation of SMCs which produce large amounts of IL-6 and prostanoids. IL-6, a pleiotropic cytokine which has been detected in human atherosclerotic lesions, is secreted by endothelial cells, monocytes/macrophages and SMCs (Loppnow and Libby, 1990; Browatzki et al., 2000). IL-6 controls macrophage and T cell activation, SMC proliferation and migration but is also a major regulator of the hepatic acute phase response (Yudkin et al., 2000). This acute phase response is associated with elevated concentrations of C-reactive protein and elevated levels of fibrinogen and platelet number/activity resulting in an increased blood viscosity. Moreover, circulating IL-6 stimulates the hypothalamic–pituitary–adrenal axis, whose activation may induce central obesity, hypertension and insulin resistance (Yudkin et al., 2000). For these reasons the cytokine IL-6 is considered as a good marker of vascular inflammation. Primary human aortic SMCs (HAoSMCs) represent a suitable model to address vascular inflammation and related mechanisms but are difficult to obtain and have a short life-span. The aim of this investigation was to develop a SMC model to evaluate the anti-inflammatory properties of nuclear receptor activators. We therefore studied IL-6 production and cyclooxygenease-2 (COX-2) expression, especially in the presence of IL-1β, as a model of the cytokine network. We show that PPAR activators but also farnesoid X receptor, liver X receptor α and retinoid X receptor α activators inhibit IL-1β induced SMC inflammation. Moreover, our study demonstrates that PPAR activator effects are mediated at least in part by interfering negatively via COX-2 expression. 2. Materials and methods 2.1. Antibodies and reagents Primary goat polyclonal antibodies to human COX-1 (C20, sc1752) and COX-2 (C20, sc-1745) and secondary peroxidase-linked donkey anti-goat IgG antibody (sc-2033) were purchased from Santa Cruz. Protein standard bands (MagicMark™XP Western Protein Standard, LC5602) came from InVitrogen. Human recombinant IL-1β (expressed in Escherichia coli, N5 × 107 U/mg) was purchased from Roche Applied Science. Fenofibric acid, 2,2-Dichloro-12-(4-chloro-phenyl)dodecanoic acid (BM-17.0744, purityN 98%), [4-Chloro-6-(2,3-dimethylphenylamino)-pyrimidin-2-ylsulfanyl]-acetic acid (Wy-14643, purity N 98%), rosiglitazone, pioglitazone, (S)-2-Ethoxy-3-{4-[2-(4methanesulfonyloxy-phenyl)-ethoxy]-phenyl}-propionic acid (AZ-242, purityN 97%), ((4-Methoxy-phenoxycarbonyl)-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]-benzyl}-amino)-acetic acid (BMS-298585, purity N 98%), {2-Methyl-4-[4-methyl-2-(4-trifluoromethyl-phenyl)thiazol-5-ylmethylsulfanyl]-phenoxy}-acetic acid (GW-501516, purity N 98%), (3-{3-[(2-Chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid (GW-3965, purity N 98%), 3-((E)-2-{2-Chloro-4-[3-(2,6-dichloro-phenyl)-5-isopropyl-isoxazol-4-ylmethoxy]-phenyl}-vinyl)-benzoic acid (GW-4064, purity N 98%)), rofecoxib and celecoxib were synthesized or extracted by a Medicinal Chemistry Division of Pierre Fabre Research Centre. 9-cis retinoïc acid and indomethacin were provided by Sigma; SC-560 and NS-398 were provided by Calbiochem. All culture medium and additives came from Promocell or InVitrogen.

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2.2. HAoSMCs culture

2.5. IL-6 and 6-keto PGF1α quantifications in HAoSMC supernatants

HAoSMCs (Promocell) were grown in Smooth Muscle Cell Growth Medium 2 (C-22062) (Promocell) containing 5% fetal calf serum, growth factors (EGF 0.5 ng/ml, bFGF 2 ng/ml, insulin 5 µg/ml), 50 ng/ml amphotericin B and 50 µg/ml gentamicin in a water-saturated 5% CO2 atmosphere. The cells up to passage number 10 were used in the study. In the following protocols, HAoSMCs were treated with either compound or vehicle [0.1% dimethyl sulfoxide (DMSO)] in MEM [(InVitrogen 42360-024) containing 5% charcoal-dextran treated fetal bovine serum (HyClone), growth factors (EGF 0.5 ng/ml, bFGF 2 ng/ml, insulin 5 µg/ml), 50 ng/ml amphotericin B and 50 µg/ml gentamicin] and simultaneously activated with IL-1β (3 U/ml) for 24 h (cell viability, 6-keto PGF1α and IL-6 measurements, western blot analysis) or 16 h (quantitative PCR analysis).

At the end of cell treatment and activation, culture supernatants were collected and centrifuged (1500 g,10 min,15 °C) for IL-6 and 6-keto PGF1α determinations, the latter used as a marker of COX-2 activation. Both were determined using commercially available kits (6-Keto Prostaglandin F1α Enzyme-immunoassay Biotrak (EIA) System and Interleukin-6 [(h)IL-6] Human, Biotrak ELISA System, products code RPN221 and RPN 2754, Amersham Biosciences) according to the manufacturer's instructions.

2.3. HAoSMC viability Lactate dehydrogenase (LDH) release assay (Cytotox 96® Assay, G1780, Promega) was used to measure cell death (48-well plate, in duplicate). At the end of cell treatment and activation, culture supernatants were collected after centrifugation for immunoassays. Cells were washed with PBS with Ca2+ and Mg2+ and then directly lysed by −80 °C freezing-thawing cycles (100 µl of PBS with Ca2+ and Mg2+/well). After thawing, appropriate dilutions of cell lysates (50 µl) were transferred to enzymatic plates for LDH quantification. LDH measurement was performed by adding 50 µl of substrate mix/well. After a 30 min incubation, reaction was stopped by the Stop Solution and OD490 nm was measured on a microplate reader (Molecular Devices). Results were expressed as % of LDH decrease as compared to control wells (cells treated with the vehicle, 0.1% DMSO). A compound was considered as toxic from 30% of LDH decrease. 2.4. Quantitative PCR analysis For human COX-1 (hCOX-1) and COX-2 (hCOX-2) mRNA determinations, HAoSMCs were seeded at 1.8 to 3.5 × 103 cells/well (12-well plates) and were grown to sub-confluence at 37 °C in 5% CO2 in the culture medium. At the end of cell treatment and activation they were washed with Dulbecco's phosphate-buffer saline without Ca2+ and Mg2+, dried, and frozen at −80 °C. Total RNA was isolated from cells using an RNeasy Mini Kit according to manufacturer specifications (QIAGEN, Valencia, CA). Contaminating DNA in the RNA preparation was removed by DNaseI treatment on-column at room temperature for 15 min. The quality and the amount of extracted RNA were determined using the 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA). For reverse transcription, 500 ng of total RNA was used to generate cDNA in a total volume of 20 µl, using iScript (Bio-Rad, Hercules, CA) during 40 min at 42 °C. Reaction was stopped by a 5 min step at 85 °C. Real-time PCR was carried out on the iCycler iQ Real Time PCR Detection System (Bio-Rad) using gene-specific primers and iQ SYBR green Supermix (Bio-Rad). The sequences of the primers are as follows: hCOX-1 forward 5′-CAC AGT GCG CTC CAA CCT TA-3′; hCOX-1 reverse 5′-TGG AGA AAG ACT CCC AGC TGA-3′; hCOX-2 forward 5′GCT GGA ACA TGG AAT TAC CCA-3′; hCOX-2 reverse 5′-CTT TCT GTA CTG CGG GTG GAA-3′; hCyclophilin forward 5′-CCA ACA CAA ATG GTT CCC AGT TT-3′ and hCyclophilin reverse 5′-ATT CAT GCC TTC TTT CAC TTT GCC-3′. The PCRs were performed in a final volume of 25 µl, as follows: 5 min at 95 °C to activate “hot start” enzyme and 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, concluded by a melt curve from 65 °C to 95 °C (0.5 °C every 10 s), to determine specific temperature melting of each amplicon. Cyclophilin was used as internal reference normalization for relative quantifications of genes of interest. Ratios were determined by ΔΔCt method (Pfaffl, 2001) and corrected by efficacy.

2.6. Western blot analysis After treatment and activation, cells were trypsinised and pelleted by centrifugation (10 min, 300 g, 4 °C). Cell pellets were lysed during 30 min in lysing buffer (NaCl 150 mM, Triton X-100 1%, IGEPAL 1% and protease inhibitors using Complete™ EDTA-free Protease Inhibitor Cocktail Tablets, Boehringer Mannheim). Cell extracts were centrifuged (10 min, 14,000 g, 4 °C) and the supernatants defined as the Triton X-100 soluble fraction were assayed for protein content using a modified Bradford Coomassie assay (Pierce). Proteins were finally stored at −80 °C in aliquots containing between 25 and 40 µg of proteins for further western blot analysis. Protein samples were resolved on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels overnight at 60 V and trans-blotted onto nitrocellulose membrane (Biorad) in 25 mM Tris, 192 mM glycine, SDS 0.1%, 20% methanol for 1 h 30 at 75 V. Electrophoretic transfer of proteins was checked by Ponceau S staining. Membrane was blocked with 10% low fat dried milk in PBS (blocking buffer) for 2 h. The blot was incubated for 2 h with the appropriate first antibody (anti human COX-1 or anti-human COX-2) in blocking buffer with 0.1% Tween 20 and then for 1 h with horseradish peroxidase-labeled secondary antibody. Between the various incubation steps, nitrocellulose membrane was washed three times with PBS containing 0.1% Tween-20. Immunoreactive antigens were revealed by enhanced chemiluminescence (ECL) and analyzed on the CCD-camera ImageMaster VDS-CL (Amersham Pharmacia Biotech). Quantifications were performed using the software program ImageMaster TotalLab (no statistical tests were carried out because of critical steps as protein transfer and immunologic detection). 2.7. In vitro COX-1 and COX-2 activity assays COX-1 and COX-2 enzymatic activities were determined using commercially available EIA kits (Colorimetric COX inhibitor screening assay, product code 760111, Cayman Chemical) according to the manufacturer's instructions. Inhibition of COX activity, by a variety of selective and non-selective inhibitors, was tested and showed similar potencies to those observed with other in vitro methods (De Leval et al., 2001). 2.8. Statistical analysis Results were expressed as mean± S.E.M and were compared by oneway ANOVA combined with comparison to zero (Fig. 1 and 2) or Dunnett's test (Fig. 3). When equal variance test was failed (Levene test), a nonparametric Kruskall–Wallis test followed by Dunn's test were performed. P b 0.05 values were considered statistically different. ⁎ or # or ¤: P b 0.05. ⁎⁎ or ## or ¤¤: P b 0.01. ⁎⁎⁎ or ### or ¤¤¤: P b 0.01. 3. Results 3.1. IL-6 and 6-keto PGF1α secretions were induced by IL-1β in HAoSMCs When confluent HAoSMCs were exposed to 3 U/ml of IL-1β, an increase in IL-6 secretion over controls was monitored in the supernatant, reaching values of up to 75 ng/well after 72 h. IL-6

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and cells were washed and re-stimulated with IL-1β to assess biosynthesis of 6-keto PGF1α directly from the given time point after IL-1β. As a result, qualitatively, the same time course as in Fig. 2A was observed (data not shown). 3.2. Nuclear receptor activators and COX inhibitors reduced 6-keto PGF1α production in HAoSMCs Activation of HAoSMCs with IL-1β-induced 6-keto PGF1α metabolite accumulation in the supernatant. HAoSMCs were treated with PPARα activators (fenofibric acid, BM-17.0744 and Wy-14643), PPARγ activators (rosiglitazone and pioglitazone), PPARδ activator GW-501516, dual PPARα/γ agonists (AZ-242 and BMS-298585), liver X receptor α agonist GW-3965, farnesoid X receptor agonist GW-4064, natural retinoid X receptor α agonist 9-cis retinoïc acid, and COX inhibitors (non selective COX inhibitor indomethacin; COX-1 inhibitor SC-560; COX-2 inhibitors NS-398, rofecoxib and celecoxib) (Fig. 3 and Table 1). As shown in Fig. 3A, IL-1β-induced 6-keto PGF1α was reduced in a concentration-dependent manner by the PPARα activator fenofibric acid, the dual PPARα/γ activators (AZ-242 and BMS-298585) and the PPARδ activator GW-501516. In contrast, the PPARγ activator rosiglitazone was ineffective up to 30 µM, as well as the other thiazolidinedione pioglitazone (Fig. 3 and Table 1). PPARα activation-mediated 6-keto PGF1α reduction was further confirmed beyond fenofibric acid through

Fig. 1. Formation of IL-6 by HAoSMCs. A/ Time course of IL-6 secretion from unstimulated or IL-1β (3 U/ml)-stimulated HAoSMCs (end point detection). B/ Concentration-response of IL-6 secretion by IL-1β−stimulated HAoSMCs after 24 h. IL-6 formation was measured by ELISA. Data are shown as the mean±S.E.M of three independent experiments, each experimental point performed in triplicate. Asterisks represent significant comparison to zero (for further details see Materials and methods).

secretion was significantly increased after only 3 h of IL-1β activation (Fig. 1A). IL-6 was not detected in the supernatant of unstimulated HAoSMCs until 72 h of culture. A concentration-response of IL-1β mediated IL-6 secretion was performed at 24 h (Fig. 1B) to better characterize this stimulation: IL-1β at 0.3 U/ml induced a significant increase of IL-6 secretion which became sub-maximal at 3 U/ml, a concentration equivalent to 60 pg/ml which was in the range of a level having a pathophysiological impact in in vivo pharmacological models (Peterson et al., 2008). The release of prostacyclin by SMCs was shown to completely depend on COX-2 expression (Schildknecht et al., 2004), whereas that of prostaglandin E2 involved both COX-1 and COX-2 isoforms. Hence, the prostacyclin-derived stable metabolite 6-keto PGF1α instead of PGE2 was then studied after IL-1β activation to characterize specifically COX-2 activity. Fig. 2A illustrates the accumulation of 6-keto PGF1α in the supernatant, with and without IL-1β stimulation. The kinetics of 6-keto PGF1α secretion was similar to IL-6 secretion, with a significant increase after 3 h of 3 U/ml IL-1β activation over controls (no 6-keto PGF1α could be detected in unstimulated cells until 72 h of activation). In Fig. 2B, the concentration response of IL-1β on 6-keto PGF1α secretion is shown at 24 h. The first significant concentration of IL-1β on 6-keto PGF1α was 1 U/ml, and 3 U/ml of IL-1β induced a sub-maximal accumulation of 6-keto PGF1α. Because the observed 6-keto PGF1α concentrations represent accumulated metabolites over time, they could have been subjected to considerable degradation. Therefore, in another set of experiments, the medium was discarded at the indicated time points,

Fig. 2. Formation of 6-keto PGF1α by HAoSMCs. A/ Time course of 6-keto PGF1α secretion from unstimulated or IL-1β (3 U/ml)-stimulated HAoSMCs (end point detection). B/ Concentration-response of 6-keto PGF1α secretion by IL-1β-stimulated HAoSMCs after 24 h. 6-keto PGF1α formation was measured by EIA. Data are shown as the mean ± S.E.M of three independent experiments, each experimental point performed in triplicate. Asterisks represent significant comparison to zero (for further details see Materials and methods).

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activator was as potent and efficacious as the farnesoid X receptor activator GW-4064 (IC50 of 10 µM). Well-known COX inhibitors were also tested in this assay (Fig. 3C and Table 1) and completely inhibited 6-keto PGF1α synthesis at concentrations between 0.03 and 0.1 µM, as expected. In all experimental conditions, HAoSMC viability (Table 1) was over 80%, demonstrating that pharmacological activity was not related to toxic effects of agents at the concentrations tested (high for the PPARα activators fenofibric acid/Wy-14643 and the dual PPARα/γ activators AZ-242). 3.3. PPAR activators and COX inhibitors selectively reduced COX-2 mRNA levels and protein contents in HAoSMCs Using HAoSMCs, we investigated by real-time PCR assays whether the decrease in the accumulation of stable prostacyclin metabolite 6-keto PGF1α was mediated by a reduction in COX-2 mRNA levels. As a control, we also tested these different compounds on COX-1 mRNA levels but none were able to modulate COX-1 mRNA levels (Fig. 4A). As shown in Fig. 4B, the PPARα activator BM-17.0744, the dual PPARα/γ activators AZ-242 and BMS-298585 and the PPARδ activator GW-501516 reduced COX-2 mRNA levels. The strongest decrease in COX-2 mRNA levels was observed with the compound BMS-298585 at 30 µM (almost 80% of inhibition versus control cells). Fenofibric acid-treated HAoSMCs contained a similar level of COX-2 mRNA as control cells. As expected, rosiglitazone treated cells did not affect mRNA COX-2 levels. Surprisingly, COX inhibitors as indomethacin, NS-398 and rofecoxib also inhibited mRNA COX-2 levels in HAoSMCs. No change was observed in the levels of the housekeeping gene cyclophilin upon various treatments (data not shown). To determine whether the inhibitory effects of selected compounds were due to an inhibition of COX-2 expression, western blot analysis of COX-1 and COX-2 were performed in HAoSMCs. As shown in Fig. 5A, COX-1 protein was present in unstimulated HAoSMCs and its expression was not modified by IL-1β stimulation or compound treatment. In contrast, IL-1β (at 3 U/ml) induced a strong expression of COX-2 protein as compared to unstimulated HAoSMCs after a 24 h cytokine activation (Fig. 5B). Immuno-bands of COX-2 were quantified using the ImageMaster TotalLab software and the results were expressed as percentage of IL-1β activation (3 U/ml). As shown Fig. 5B, COX-2 protein expression was decreased by treatment with the PPARα activator fenofibric acid, the dual PPARα/γ activator BMS298585 and the enzymatic COX inhibitors NS-398/rofecoxib. For the other compounds investigated (BM-170744, GW-501516, AZ-242 and indomethacin), a slight decrease (in the three independent western blot analyses performed) of COX-2 protein expression was only observed. In

Fig. 3. Effects of nuclear receptor activators and COX inhibitors on HAoSMC 6-keto PGF1α secretion. HAoSMCs were treated with either compounds (panels A, B and C) or vehicle (0.1% DMSO) and simultaneously activated with IL-1β (3 U/ml) for 24 h. 6-keto PGF1α formation was measured by EIA. Data are shown as the mean ± S.E.M of three independent experiments, each experimental point performed in triplicate. Symbols represent significant comparison to the lowest concentration tested (for further details see Materials and methods).

other PPARα activators such as BM-17.0744 and Wy-14643 (Table 1). The rank order of potency of PPAR agonists on 6-keto PGF1α synthesis was the following: GW-501516 N BM-17.0744 ∼ BMS-298585 N fenofibric acid N Wy-14643 N AZ-242. Other nuclear receptor activators were also able to inhibit 6-keto PGF1α (Fig. 3B), particularly the potent and efficacious liver X receptor α activator GW-3965 (IC50 of 1 µM and maximal inhibition of around 100% at 10 µM) and the farnesoid X receptor activator GW-4064 (IC50 of 8 µM and maximal inhibition of 60% at 10 µM). The natural retinoid X receptor α activator 9-cis retinoïc

Table 1 Effects of compounds (IC50 and LDH cell viability) on IL-1β stimulated 6-keto PGF1α production Treatment

6-keto PGF1α IC50, µM

LDH cell viability (% of IL-1β 3 U/ml)

IL-1β 3 U/ml Fenofibric acid BM-17.0744 Wy-14643 Rosiglitazone Pioglitazone AZ-242 BMS-298585 GW-501516 GW-3965 GW-4064 9-cis retinoic acid Indomethacin SC-560 NS-398 Rofecoxib Celecoxib

n.a. 13 8 36 i.a i.a 69 9 2 1 8 10 0.001 0.005 0.002 0.006 0.002

100 N 90 N 95 86 100 100 N 95 N 90 N 95 N 90 83 N 90 N 95 N 95 N 90 100 87

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Fig. 4. Effects of PPAR activators and COX inhibitors on HAoSMC COX-1 (A) and COX-2 (B) mRNA levels. HAoSMCs were treated with either compounds or vehicle (0.1% DMSO) and simultaneously activated with IL-1β (3 U/ml) for 16 h. Total RNA was isolated from cells, and COX-2 and COX-1 mRNA were quantified using real-time PCR and normalized with the housekeeping cyclophilin gene. Data are shown as the mean ± S.E.M of three independent experiments, each experimental point performed in triplicate.

contrast, the specific and potent PPARγ activator rosiglitazone was unable to modify COX-2 protein expression. Thus, these results demonstrate that the selective reduction of COX-2 protein is involved in HAoSMC anti-inflammatory effects of these compounds. The enzymatic COX inhibitors were also able to inhibit COX-2 protein expression confirming mRNA results and demonstrating that not only an enzymatic mechanism but also a transcriptional pathway were involved in their anti-inflammatory impacts.

3.4. COX inhibitors but not PPAR activators inhibited COX enzymatic activity In order to evaluate a putative impact of PPAR activators on COX enzymatic activity, a COX enzymatic assay was used. As shown in Table 2, NS-398 and rofecoxib were potent and efficacious inhibitors of COX-2 (IC50 of 0.7 µM and maximal inhibition of around 90% for both compounds), as expected. As is well-known, the non selective COX

inhibitors indomethacin and SC-560 decreased the activity of both COX isoforms but with a better potency towards COX-1 enzyme (COX-1 IC50 of 0.02–0.03 µM and COX-2 IC50 of 7 and 35 µM for indomethacin and SC-560 respectively). In this in vitro assay, none of the selected PPAR activators were able to inhibit COX-1 and/or COX-2 enzymatic activity.

4. Discussion Atherosclerosis development is a complex process involving several cell types including endothelial cells, macrophages and SMCs. Injury of the vascular endothelium is a critical event in the initiation of atherosclerosis. Vascular inflammation participates in disease progression involving plaque erosion and thrombus formation leading to vessel occlusion and cardiovascular events. Since it is accepted that atherosclerosis is a chronic inflammatory disease, there may be benefits of the administration of anti-inflammatory drugs. Additional interest belongs to drugs which improve metabolic

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Fig. 5. Effects of PPAR activators and COX inhibitors on HAoSMC COX 1 (A) and COX-2 (B) protein levels. HAoSMCs were treated with either compounds or vehicle (0.1% DMSO) and simultaneously activated with IL-1β (3 U/ml) for 24 h. Western blot analyses were performed using specific anti-human COX-1 and anti-human COX-2 protein. Quantification of COX-2 protein in unstimulated and IL-1β-activated HAoSMCs was conducted. Three independent western blot analyses were performed and one representative experiment was shown (Fig. 5B, panel above). Lane 1–13: stimulated with IL-1β 3 U/ml; DMSO 0.1% (lane 1, 9, 12 and 13); BM-17.0744 30 µM (lane 2); rofecoxib 0.1 µM (lane 3); indomethacin 0.01 µM (lane 4); NS-398 0.01 µM (lane 5); AZ-242 100 µM (lane 6); BMS298585 30 µM (lane 7); GW-501516 10 µM (lane 8); rosiglitazone 10 µM (lane 10); fenofibric acid 100 µM (lane 11). Lane 14: unstimulated; DMSO 0.1%.

parameters and possess anti-inflammatory effects on the vessel wall. Several reports indicate an improvement in atherosclerotic disease after treatment of atherosclerotic mice with indomethacin, aspirin or sulindac with inhibited neointimal formation after arterial injury. Rofecoxib has already been shown to reduce atherosclerosis in LDL−/− r mice, and those which also carried the COX-2−/− phenotypes developed significantly less atherosclerosis than COX-2+/+ mice (Burleigh et al., 2002). On the other hand, there are data, although highly controversial, indicating that COX-2 selective inhibitors are associated with an increased risk of cardiovascular events probably due to an imbalance between COX-1 (thromboxane) and COX-2 products (prostaglandins and prostacyclin) (Schildknecht et al., 2004; Bishop-Bailey et al., 1998; Mukherjee et al., 2001; Fitzgerald, 2004). COX-2 selective inhibitors can also increase blood pressure by several mechanisms, and suppress the formation of lipoxins, resolvins and endothelial-derived nitric oxide adding other putative reasons to support cardiovascular risks associated to these compounds (Solomon et al., 2004; Das, 2005). In this current study, we investigate PPAR activator effects on 6-keto PGF1α synthesis a selective product of COX-2 activity in HAoSMCs. Fenofibrate, rosiglitazone and pioglitazone are currently marketed for dyslipidaemia and type 2 diabetes, and the PPARα activator BM-17.0744 and the dual PPARα/γ activators AZ-242, BMS-298585 reached clinical development. To further elucidate nuclear receptor impacts on HAoSMC inflammation, we also evaluate GW-3965, GW-4064 and 9-cis retinoic acid as liver X receptor α, farnesoid X receptor and retinoid X receptor α activators, respectively.

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Our results described for the first time that not only PPAR activators but also other nuclear receptor activators are able to modulate SMC inflammation represented by the stable COX-2 metabolite 6-keto PGF1α. Nevertheless, the mechanism of liver X receptor α-, farnesoid X receptor- or retinoid X receptor α-mediated 6-keto PGF1α inhibition needs to be further explored through several experiments. The COX-2 promoter contains NF-kB binding sites and we showed previously that the radical-scavenging antioxidant pyrrolidine dithiocarbamate (PDTC) is able to reduce NF-kB activation and promotes reduction in COX-2 expression and activity (data not shown). PPAR activators but also liver X receptor α activator GW-3965, farnesoid X receptor activator GW-4064 and retinoid X receptor α activator retinoïc acid can also probably reduce COX-2 activity through an inhibition of this NF-kB pathway. In human endothelial cells, Eligini et al. (2005) have demonstrated that reactive oxygen species and altered redox status within the cell are fundamental steps for COX-2 induction (at least two independent pathways with the overlapping upstream reactive oxygen species signal). It would be interesting to explore the SMC COX-2 regulating pathways and to determine putative nuclear activator interference (Bourcier et al., 1997). The mechanism through which PPAR activators inhibit 6-keto PGF1α is likely to be transcriptional as demonstrated by RT-PCR analysis. Nevertheless, the decrease in COX-2 mRNA could also be attributable to an inhibition of COX-2 expression and/or a decrease of the stability of COX-2 mRNA as it has been described for MMP-9 mRNA (Eberhardt et al., 2002). As shown by our results, a positive relationship exists between COX-2 mRNA levels and protein expression levels except for fenofibric acid. This specific PPARα agonist induces a strong decrease of COX-2 protein as detected by western blot analysis which is not due to a reduction of COX-2 mRNA levels. The evaluation of COX-2 protein stability after fenofibric acid treatment warrants further investigation. One of the most relevant results of our study could be that SMC COX-2 mRNA levels are strongly reduced by the selective COX-2 inhibitor rofecoxib. It would be interesting to determine if this is the case in other vascular cells such as endothelial cells. COX-2 represents a critical link among vascular homeostasis, inflammatory response angiogenesis and tumor growth. The identification of anti-inflammatory compounds to treat chronic vascular disorders is therefore of great medical interest. Thus, we have shown that nuclear receptor activators (PPARα, dual PPARα/γ, liver X receptor α, farnesoid X receptor and retinoid X receptor α) can reduce the production of the COX-2 metabolite 6-keto PGF1α in SMC at least in part via a transcriptional pathway. Nevertheless, characterization of the mechanisms of action of each compound warrants further experiments (NF-kB translocation or interaction, COX-2 ARNm or protein stability, transport within the cellular compartments) and also necessitates the evaluation of other putative signalling pathways (AP-1; CD40-CD40 ligand, see Mach et al., 1997) and other potential anti-inflammatory key players (Papadaki et al., 1998). Moreover, each novel agent has to be Table 2 Effects of compounds (IC50 and maximal inhibition) on COX-1 and COX-2 enzymatic assays COX-1 Fenofibric acid BM-17.0744 Rosiglitazone AZ-242 BMS-298585 GW-501516 Indomethacin SC-560 NS-398 Rofecoxib

COX-2

IC50(µM)

Inh.max(%)

IC50(µM)

Inh.max(%)

n.d. n.d. n.d. n.d. n.d. n.d. 0.03 0.02 n.d. n.d.

i.a. i.a. i.a. i.a. i.a. i.a. 95 ± 0.4 96 ± 0.2 i.a. i.a.

n.d. n.d. n.d. n.d. n.d. n.d. 7 35 0.7 0.7

i.a. i.a. i.a. i.a. i.a. i.a. 89 ± 3 63 ± 2 89 ± 1 90 ± 0.4

n.d.: not determined; i.a.: inactive.

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evaluated in vivo to determine carefully its pharmacological impact on the prostaglandins-thromboxane/prostacyclin inflammatory balance since our in vitro study used 6-keto PGF1α only as an index of COX-2 activation but was not interpreted as a pathophysiological biomarker. Clinical beneficial effects of agents against vascular inflammation and atherosclerosis cannot be directly drawn from this model of HAoSMC-regulated COX-2. For instance, different effects have been shown in mice atherosclerosis models, most notably for dual PPARα/γ activators: Tesaglitazar (AZ-242) reduces atherosclerosis in the female LDL−/− mice via r lipid-independent mechanisms, probably at least in part by direct actions on the vessels (Chira et al., 2007). In contrast, in apoE−/− mice, the PPARα agonist gemfibrozil and the PPARγ agonist rosiglitazone reduce atherosclerosis but the dual PPARα/γ agonist compound 3q increases total plaque area and up-regulates plaque instability markers (Calkin et al., 2007). In type 2 diabetes patients with cardiovascular disease, reduced levels of inflammatory markers (matrix metalloproteinase -9, tumor necrosis factor -α, sCD40 L and IL-6) have been shown after treatment with PPARγ activator glitazone (Marx et al., 2003; Haffner et al., 2002; Dormandy et al., 2005 PROactive study) or PPARα activator fenofibrate (Libby and Plutzky, 2007; Keech et al., 2006 FIELD study) and no clinically relevant cardiovascular safety concern was reported for the latter. The development of multimodal drugs which can reduce hyperglycemia and concomitantly prevent diabetic cardiovascular complications may offer therapeutic option, but for the moment no dual agonist PPARα/γ has reached the market. These data provide an important framework for further exploring the potential utility and safety of combinatorial approaches and to demonstrate the clinical benefits of compounds with anti-inflammatory properties in chronic vascular diseases. In the near future, existent medications and novel drugs will probably be found to specifically target inflammatory pathways in atherosclerotic disease.

Acknowledgements The authors thank Cécile Bayle Rouais for GW-4064 synthesis. The authors wish to acknowledge Sophie Bréand for her help in statistical analysis of the results and Dr. Ian Rilatt for reading this manuscript.

References Ai-Wen, W., Jin, G., Zhen-Fu, L., Jia-Fu, J., Guang-Wie, X., 2004. COX-2 expression and tumor angiogenesis in colorectal cancer. World J. Gastroenterol. 10, 2323–2326. Ali, F.Y., Davidson, S.J., Moraes, L.A., Traves, S.L., Paul-Clark, M., Bishop-Bailey, D., Warner, T.D., Mitchell, J.A., 2006. Role of nuclear receptor signaling in platelets: antithrombotic effects of PPARδ. FASEB J. 20, 326–328. Antonio, V., Janvier, B., Brouillet, A., Andreani, M., Raymondjean, M., 2003. Oxysterol and 9-cis-retinoic acid stimulate the group IIA secretory phospholipase A2 gene in rat smooth-muscle cells. Biochem. J. 376, 351–360. Barbier, O., Torra, P., Duguay, Y., Blanquart, C., Fruchart, J.C., Glineur, C., Staels, B., 2002. Pleiotropic actions of peroxisome proliferator-activated receptors in lipid metabolism and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 22, 717–726. Barish, G.D., Narkar, V.A., Evans, R.M., 2006. PPARδ: a dagger in the heart of the metabolic syndrome. J. Clin. Invest. 116, 590–597. Bishop-Bailey, D., Pepper, J.R., Larkin, S.W., Mitchell, J.A., 1998. Differential induction of cyclooxygenase-2 in human arterial and venous smooth muscle. Role of endogenous prostanoids. Arterioscler. Thromb. Vasc. Biol. 18, 1655–1661. Bourcier, T., Sukhova, G., Libby, P., 1997. The nuclear factor kappa-B signaling pathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis. J. Biol. Chem. 272, 15817–15824. Browatzki, M., Schmidt, J., Kubler, W., Kranzhofer, R., 2000. Endothelin-1 induces interleukin-6 release via activation of the transcription factor NF-kappaB in human vascular smooth muscle cells. Bas. Res. Cardiol. 95, 98–105. Bruemmer, D., Law, R.E., 2005. Liver X receptors: potential novel targets in cardiovascular diseases. Curr. Drug Targets Cardiovasc. Haematol. Disord. 5, 533–540. Burleigh, M.E., Babaev, V.R., Oates, J.A., Harris, R.C., Gautam, S., Riendeau, D., Marnett, L.J., Morrow, J.D., Fazio, S., Linton, M.F., 2002. Cyclooxygenase-2 promotes early atherosclerotic lesion formation in LDL receptor-deficient mice. Circ. Res. 105, 1816–1823. Calkin, A.C., Allen, T.J., Lassila, M., Tikellis, C., Jandeleit-Dahm, K.A., Thomas, M.C., 2007. Increased atherosclerosis following treatment with a dual PPAR agonist in the ApoE knockout mouse. Atheroscler. 195, 17–22.

Chan, C.C., Boyce, S., Brideau, C., Charleson, S., Cromlish, W., Ethier, D., Evans, J., Ford-Hutchinson, A.W., Forrest, M.J., Gauthier, J.Y., Gordon, R., Gresser, M., Guay, J., Kargman, S., Kennedy, B., Leblanc, Y., Leger, S., Mancini, J., O'Neill, G.P., Ouellet, M., Patrick, D., Percival, M.D., Perrier, H., Prasit, P., Rodger, I., Tagari, P., Therien, M., Vickers, P., Visco, D., Wang, Z., Webb, J., Wong, E., Xu, L.J., Young, R.N., Zamboni, R., Riendeau, D., 1999. Rofecoxib [Vioxx, MK-0966; 4-(4′-methylsulfonylphenyl)-3phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles. J. Pharmacol. Exp. Ther. 290, 551–560. Chawla, A., Boisvert, W.A., Lee, C.H., Laffitte, B.A., Barak, Y., Joseph, S.B., Liao, D., Nagy, L., Edwards, P.A., Curtiss, L.K., Evans, R.M., Tontonoz, P., 2001. A PPARγ-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol. Cell. 7, 161–171. Chinetti, G., Lestavel, S., Bocher, V., Remaley, A.T., Neve, B., Pineda Torra, I., Teissier, E., Minnich, A., Jaye, M., Duverger, N., Brewer, H.B., Fruchart, J.C., Clavey, V., Staels, B., 2001. PPAR-α and PPAR-γ activators induce cholesterol removal from human macrophage foam cells through stimulation of ABCA1 pathway. Nat. Med. 7, 53–58. Chira, E.C., McMillen, T.S., Wang, S., Haw 3rd, A., O'Brien, K.D., Wight, T.N., Chait, A., 2007. Tesaglitazar, a dual peroxisome proliferator-activated receptor alpha/gamma agonist, reduces atherosclerosis in female low density lipoprotein receptor deficient mice. Atheroscler. 195, 100–109. Claudel, T., Leibowitz, M.D., Fiévet, C., Tailleux, A., Wagner, B., Repa, J.J., Torpier, G., Lobaccaro, J.M., Paterniti, J.R., Mangelsdorf, D.J., Heyman, R.A., Auwerx, J., 2001. Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor. PNAS 98, 2610–2615. Dai, X.Y., Ou, X., Hao, X.R., Cao, D.L., Tang, Y.L., Hu, Y.W., Li, X.X., Tang, C.K., 2008. The effects of T0901317 on ATP-Binding Cassette Transporter A1 and Niemann–Pick type C1 in ApoE−/− mice. J. Cardiovasc. Pharmacol. 23 [Electronic publication]. Das, U.N., 2005. COX-2 inhibitors and metabolism of essential fatty acids. Med. Sci. Monit. 11, RA233–RA237. Delerive, P., De Bosscher, K., Besnard, S., Vanden Berghe, W., Peters, J.M., Gonzalez, F.J., Fruchart, J.C., Tedgui, A., Haegeman, G., Staels, B.,1999. Peroxisome proliferator-activated receptor α negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-κB and AP-1. J. Biol. Chem. 274, 32048–32054. De Leval, X., Delarge, J., Devel, P., Neven, P., Michaux, C., Masereel, B., Pirotte, B., David, J.L., Henrotin, Y., Dogne, J.M., 2001. Evaluation of classical NSAIDs and COX-2 selective inhibitors on purified ovine enzymes and human whole blood. Prostaglandins leukot. essent. 64, 211–216. Delvecchio, C.J., Bilan, P., Radford, K., Stephen, J., Trigatti, B.L., Cox, G., Parameswaran, K., Capone, J.P., 2007. Liver X receptor stimulates cholesterol efflux and inhibits expression of proinflammatory mediators in human airway smooth muscle cells. Mol. Endocrinol. 21, 1324–1334. Dormandy, J.A., Charbonnel, B., Eckland, D.J., Erdmann, E., Massi-Benedetti, M., Moules, I.K., Skene, A.M., Tan, M.H., Lefèvre, P.J., Murray, G.D., Standl, E., Wilcox, R.G., Wilhelmsen, L., Betteridge, J., Birkeland, K., Golay, A., Heine, R.J., Koranyi, L., Laakso, M., Mokan, M., Norkus, A., Pirags, V., Podar, T., Scheen, A., Schernaum, W., Schernthaner, G., Schmitz, O., Skrha, J., Smith, U., Taton, J., PROactive investigators, 2005. Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial. Lancet 366, 1279–1289. Eberhardt, W., Akool, el-S., Rebhan, J., Frank, S., Beck, K.F., Franzen, R., Hamada, F.M., Pfeilschifter, J., 2002. Inhibition of cytokine-induced matrix metalloproteinase 9 expression by peroxisome proliferator-activated receptor alpha agonists is indirect and due to a NO-mediated reduction of mRNA stability. J. Biol. Chem. 277, 33518–33528. Eligini, S., Barbieri, S.S., Cavalca, V., Camera, M., Brambilla, M., De Franceschi, M., Tremoli, E., Colli, S., 2005. Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-α and ester in human endothelial cells. Cardiovasc. Res. 65, 683–693. Englesbe, M.J., Deou, J., Bourns, B.D., Clowes, A.W., Daum, G., 2004. Interleukin-1 beta inhibits PDGF-BB-induced migration by cooperating with PDGF-BB to induce cyclooxygenase-2 expression in baboon aortic smooth muscle cells. J. Vasc. Surg. 39, 1091–1096. Fitzgerald, G.A., 2004. Coxibs and cardiovascular disease. N. Engl. J. Med. 351, 1709–1711. Gierse, J.K., McDonald, J.J., Hauser, S.D., Rangwala, S.H., Koboldt, C.M., Seibert, K., 1996. A single amino acid difference between cyclooxygenase-1 (COX-1) and -2 (COX-2) reverses the selectivity of COX-2 specific inhibitors. J. Biol. Chem. 271, 15810–15814. Gross, B.S., Fruchart, J.-C., Staels, B., 2005. Peroxisome proliferator-activated receptor β/δ: a novel target for the reduction of atherosclerosis. Drug Discov. Today Therapeut. Strateg. 2, 237–243. Haffner, S.M., Greenberg, A.S., Weston, W.M., Chen, H., Williams, K., Freed, M.I., 2002. Effect of rosiglitazone treatment on nontraditional markers of cardiovascular disease in patients with type 2 diabetes mellitus. Circulation 106, 679–684. Kersten, S., Desvergne, B., Wahli, W., 2000. Roles of PPARs in health and disease. Nat 405, 421–424. Keech, A., Simes, R.J., Barter, P., Best, J., Scott, R., Taskinen, M.R., Forder, P., Pillai, A., Davis, T., Glasziou, P., Drury, P., Kesäniemi, Y.A., Sullivan, D., Hunt, D., Colman, P., d, Emden, M., Whiting, M., Ehnholm, C., Laakso, M., FIELD study investigators, 2006. Effects of longterm fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial. Lancet 366, 1849–1861. Libby, P., Plutzky, J., 2007. Inflammation in diabetes mellitus: role of peroxisome proliferator-activated receptor-alpha and peroxisome proliferator-activated receptor-gamma agonists. Am. J. Cardiol. 99, 27B–40B. Loppnow, H., Libby, P., 1990. Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6. J. Clin. Invest. 85, 731–738. Lu, T.T., Makishima, M., Repa, J.J., Schoonjans, K., Kerr, T.A., Auwerx, J., Mangelsdorf, D.J., 2000. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Moll. Cell. 6, 507–515.

Y. Rival et al. / European Journal of Pharmacology 606 (2009) 121–129 Mach, F., Schonbeck, U., Sukhova, G.K., Bourcier, T., Bonnefoy, J.Y., Pober, J.S., Libby, P., 1997. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40–CD40 ligand signalling in atherosclerosis. PNAS 94, 1931–1936. Mamdani, M., Juurlink, D.N., Kopp, A., Naglie, G., Austin, P.C., Laupacis, A., 2004. Gastrointestinal bleeding after the introduction of COX 2 inhibitors: ecological study. BMJ 328, 1415–1416. Marx, N., Schönbeck, U., Lazar, M.A., Libby, P., Plutzky, J., 1998. Peroxisome proliferatoractivated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ. Res. 83, 1097–1103. Marx, N., Froehlich, J., Siam, L., Ittner, J., Wierse, G., Schmidt, A., Scharnagl, H., Hombach, V., Koenig, W., 2003. Antidiabetic PPAR gamma-activator rosiglitazone reduces MMP-9 serum levels in type 2 diabetics patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 23, 283–288. Marx, N., Duez, H., Fruchart, J.C., Staels, B., 2004. Peroxisome proliferator-activated receptors and atherogenesis: regulators of gene expression in vascular cells. Circ. Res. 94, 1168–1178. Mukherjee, D., Nissen, S.E., Topol, E.J., 2001. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 286, 954–959. Niederberger, E., Manderscheid, C., Grösch, S., Schmidt, H., Ehnet, C., Geisslinger, G., 2004. Effects of the selective COX-2 inhibitors celecoxib and rofecoxib on human vascular cells. Biochem. Pharmacol. 68, 341–350. Oliver, W.R., Shenk, J.L., Snaith, M.R., Russell, C.S., Plunket, K.D., Bodkin, N.L., Lewis, M.C., Winegar, D.A., Sznaidman, M.L., Lambert, M.H., Xu, H.E., Sternbach, D.D., Kliewer, S.A., Hansen, B.C., Willson, T.M., 2001. A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport. PNAS 98, 5306–5311. Papadaki, M., Tilton, R.G., Eskin, S.G., McIntire, L.V., 1998. Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow. Am. J. Physiol. 274, H616–H626. Peterson, S.J., Drummond, G., Kim, D.H., Li, M., Kruger, A.L., Ikehara, S., Abraham, N.G., 2008. L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice. J. Lipid Res. 49, 1658–1669. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Ac. Res. 29, 2002–2007. Reginato, M.J., Lazar, M.A., 1999. Mechanisms by which thiazolidinediones enhance insulin action. Trends Endocrinol. Metab. 10, 9–13. Rival, Y., Beneteau, N., Taillandier, T., Pezet, M., Dupont-Passelaigue, E., Patoiseau, J.-F., Junquero, D., Colpaert, F.C., Delhon, A., 2002. PPARα and PPARδ activators inhibit cytokine-induced nuclear translocation of NF-κB and expression of VCAM-1 in EAhy926 endothelial cells. Eur. J. Pharmacol. 435, 143–151.

129

Ryoo, S., Won, M., Kim, D.-U., Kim, L., Han, G., Park, S.-K., Mukaida, N., Maeng, P., Yoo, H.-S., Hoe, K.-L., 2004. PPARα activation abolishes LDL-stimulated IL-8 production via AP-1 deactivation in human aortic smooth muscle cells. Biochem. Bioph. Res. Co. 318, 329–334. Schildknecht, S., Bachscchmid, M., Baumann, A., Ullrich, V., 2004. COX-2 inhibitors selectively block prostacyclin synthesis in endotoxin exposed vascular smooth muscle cells. FASEB J 10, 1096. Solomon, D.H., Schneeweiss, S., Levin, R., Avorn, J., 2004. Relationship between COX-2 specific inhibitors and hypertension. Hypertension 44, 140–145. Staels, B., Koenig, W., Habib, A., Merval, R., Lebret, M., Pineda Torra, I., Delerive, P., Fadel, A., Chinetti, G., Fruchart, J.C., Najib, J., Maclouf, J., Tedgui, A., 1998. Activation of human aortic smooth-muscle cells is inhibited by PPARα but not by PPARγ activators. Nat 393, 790–793. Wahli, W., 2002. Peroxisome proliferator-activated receptors (PPARs): from metabolic control to epidermal wound healing. Swiss Med. Wkly. 132, 83–91. Wang, Y.X., Lee, C.H., Tiep, S., Yu, T.Y., Ham, J., Kang, H., Evans, R.M., 2003. Peroxisomeproliferator-activated receptor δ activates fat metabolism to prevent obesity. Cell 113, 159–170. Wang, K., Zhou, Z., Zhang, M., Fan, L., Forudi, F., Zhou, X., Qu, W., Lincoff, M., Schmidt, A.M., Topol, E.J., Penn, M.S., 2006. Peroxisome proliferator-activated receptor γ downregulates receptor for advanced glycation end products and inhibits smooth muscle cell proliferation in a diabetic and nondiabetic rat carotid artery injury model. J. Pharmacol. Exp. Ther. 317, 37–43. Willson, T.M., Brown, P.J., Sternbach, D.D., Henke, B.R., 2000. The PPARs: from orphan receptors to drug discovery. J. Med. Chem. 43, 527–550. Yan, Z., Subbaramaiah, K., Camilli, T., Zhang, F., Tanabe, T., McCaffrey, T.A., Dannenberg, A.J., Weksler, B.B., 2000. Benzo[a] pyrene induces the transcription of cyclooxygenase-2 in vascular smooth muscle cells. J. Biol. Chem. 275, 4949–4955. Yoyo, T.Y.L., Swales, K.E., Thomas, G.J., Warner, T.D., Bishop-Bailey, D., 2007. Farnesoid X receptor ligands inhibit vascular smooth muscle cell inflammation and migration. Arterioscler. Thromb. Vasc. Biol. 27, 2606–2611. Yudkin, J.S., Kumari, M., Humphries, S.E., Mohamed-Ali, V., 2000. Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link? Atheroscler. 148, 209–214. Zahradka, P., Wright, B., Fuerst, M., Yurkova, N., Molnar, K., Taylor, C.G., 2006. Peroxisome proliferator-activated receptor α and γ ligands differentially affect smooth muscle cell proliferation and migration. J. Pharmacol. Exp. Ther. 317, 651–659.