Báo cáo khoa học: Visfatin is induced by peroxisome proliferator-activated receptor gamma in human macrophages

Visfatin is induced by peroxisome proliferator-activated receptor gamma in human macrophages The´ re` se He` rve´ e Mayi1,2,3,4, Christian Duhem1,2,3,4, Corinne Copin1,2,3,4, Mohamed Amine Bouhlel1,2,3,4, Elena Rigamonti1,2,3,4, Franc¸ ois Pattou1,5,6, Bart Staels1,2,3,4 and Giulia Chinetti-Gbaguidi1,2,3,4

1 Univ Lille Nord de France, France 2 Inserm, Lille, France 3 UDSL, Lille, France 4 Institut Pasteur de Lille, France 5 Service de Chirurgie Ge´ ne´ rale et Endocrinienne, Centre Hospitalier Re´ gional et Universitaire de Lille, France 6 Inserm ERIT-M 0106, Faculte´ de Me´ decine, Lille, France

Keywords adipocytokines; inﬂammation; macrophages; nuclear receptors; visfatin

exerts anti-inﬂammatory effects

Correspondence Bart Staels, Inserm UR 1011, Institut Pasteur de Lille, 1, rue du Professeur Calmette, BP 245, Lille 59019, France Fax: +33 3 20 87 73 60 Tel: +33 3 20 87 73 88 E-mail: bart.staels@pasteur-lille.fr

(Received 15 January 2010, revised 27 April 2010, accepted 3 June 2010)

doi:10.1111/j.1742-4658.2010.07729.x

Obesity is a low-grade chronic inﬂammatory disease associated with an increased number of macrophages (adipose tissue macrophages) in adipose tissue. Within the adipose tissue, adipose tissue macrophages are the major source of visfatin ⁄ pre-B-cell colony-enhancing factor ⁄ nicotinamide phos- phoribosyl transferase. The nuclear receptor peroxisome proliferator-acti- vated receptor gamma (PPARc) in macrophages by inhibiting cytokine production and enhancing alternative differentiation. In this study, we investigated whether PPARc modulates visfatin expression in murine (bone marrow-derived macrophage) and human (primary human resting macrophage, classical macrophage, alterna- tive macrophage or adipose tissue macrophage) macrophage models and pre-adipocyte-derived adipocytes. We show that synthetic PPARc ligands increase visfatin gene expression in a PPARc-dependent manner in primary human resting macrophages and in adipose tissue macrophages, but not in adipocytes. The threefold increase of visfatin mRNA was paralleled by an increase of protein expression (30%) and secretion (30%). Electrophoretic mobility shift assay experiments and transient transfection assays indicated that PPARc induces visfatin promoter activity in human macrophages by binding to a DR1–PPARc response element. Finally, we show that PPARc ligands increase NAD+ production in primary human macrophages and that this regulation is dampened in the presence of visfatin small interfering RNA or by the visfatin-speciﬁc inhibitor FK866. Taken together, our results suggest that PPARc regulates the expression of visfatin in macro- phages, leading to increased levels of NAD+.

Abbreviations AcLDL, acetylated low-density lipoprotein; AP-1, activator protein 1; ATM, adipose tissue macrophages; EMSA, electrophoretic mobility shift assay; IL, interleukin; NAMPT, nicotinamide phosphoribosyl transferase; M1, classical pro-inﬂammatory macrophage phentotype; M2, alternative anti-inﬂammatory macrophage phenotype; NF-jB, nuclear factor-kappaB; NR, nuclear receptor; PBEF, pre-B-cell colony-enhancing factor; PPARc, peroxisome proliferator-activated receptor gamma; PPRE, peroxisome proliferator-activated receptor response elements; Q-PCR, quantitative PCR; ROS, reactive oxygen species; RM, resting macrophages; RSG, rosiglitazone; RXR, retinoic X receptor; siRNA, small interfering RNA; SIRN, sirtuin (silencing mating type information regulation 2 homolog); SMC, smooth muscle cells; TNF-a, tumor necrosis factor alpha.

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Introduction

(NAMPT)

transferase

rheumatoid arthritis, obesity,

IL-1b,

such as

tumor necrosis

induce an alternative anti-inﬂammatory macrophage phenotype (M2) [17]. In macrophages, many genes are regulated by transcription factors, such as the nuclear receptors (NRs), which translate physiological signals into gene regulation. Peroxisome proliferator-activated receptor gamma (PPARc) is a NR that regulates genes controlling lipid, glucose metabolism and inﬂammation. After activation by its ligands, PPARc forms a hetero- dimer with the retinoic X receptor (RXR) [18]. The binding of this heterodimer to speciﬁc DNA sequences, called PPAR response elements (PPRE), results in the regulation of its target genes [18]. In this way PPARc modulates crucial pathways of adipocyte differentiation and lipid metabolism, thus impacting on glucose metab- olism and insulin sensitivity. Furthermore, activated PPARc inhibits inﬂammatory response genes by nega- tively interfering with the NF-jB, signal transducers and activators of transcription (STAT) and AP-1 signal- ing pathways in a DNA-binding independent manner [19]. This trans-repression activity is probably the basis for the anti-inﬂammatory properties of PPARc.

(RSG)

rosiglitazone

and pioglitazone

converts,

enzyme

that

to have

thought

Originally discovered in liver, skeletal muscle and bone marrow, and also known as pre-B-cell colony- enhancing factor (PBEF), a cytokine acting in B-cell differentiation [1], visfatin, is nicotinamide phosphori- bosyl [2,3], a rate-limiting enzyme in the synthesis of NAD+ from nicotin- synthesized and amide. Visfatin ⁄ PBEF ⁄ NAMPT is secreted in adipose tissue by adipocytes, and mostly by macrophages, and circulates in the plasma of humans and mice [4]. Plasma concentrations of visfa- tin are positively associated with cytokines such as interleukin (IL)-6 and increase in morbidly obese subjects. Elevated circulating levels of visfatin have been observed in many inﬂammatory diseases such as insulin resistance and type 2 diabetes [5–7]. Visfatin is secreted by neutrophils in response to inﬂammatory stimuli and is regulated in monocytes by pro-inﬂammatory fac- tors factor alpha (TNF-a), IL-6 via nuclear factor-kappaB (NF-jB) and AP-1-dependent mechanisms [8–10]. Visfatin acti- vates pro-inﬂammatory signalling pathways in human endothelial and vascular smooth muscle cells (SMC) (ROS)-dependent through reactive oxygen species NF-jB activation or NAMPT activity, respectively, and therefore could provide a link between obesity and atherothrombotic diseases [11,12]. Visfatin func- tions as an extracellular and intracellular NAD bio- in mammals, synthetic nicotinamide to NMN, (a form of vitamin B3) a NAD precursor. Thus, the NAD pool is maintained, at least in part, by visfatin, which is important, for instance, in b-cell insulin secretion [2]. Although still controversial, visfatin is insulin mimetic effects and, similarly to insulin, visfatin enhances glucose uptake by myocytes and adipocytes and inhibits hepatocyte glucose release in vitro [13,14]. Altogether, the pleiotropic role of visfatin suggests that the regulation of NAD+ synthesis is critical for several aspects of cell physiology [15].

PPARc is activated by natural or synthetic ligands such as GW1929 and the antidiabetic thiazolidinedi- [20]. ones PPARc expression is very low in human monocytes, but is induced upon differentiation into macrophages and is present in foam cells of atherosclerotic lesions [21–23]. More recently, PPARc has been shown to enhance the differentiation of monocytes into alterna- tive anti-inﬂammatory M2 macrophages [24,25] and to promote the inﬁltration of M2 macrophages into adi- pose tissue [26]. Consistent with these results, selective inactivation of macrophage PPARc in BALB ⁄ c mice results in an impairment in the maturation of alterna- tively activated M2 macrophages and in the exacerba- tion of diet-induced obesity, insulin resistance, glucose intolerance and expression of inﬂammatory mediators [24,27]. All these studies provide evidence that macro- phage PPARc is a central regulator of inﬂammation and insulin resistance.

signals

interferon-gamma,

such as

Here, we identify visfatin as a novel PPARc-regu- lated gene Interestingly, in human macrophages. PPARc activation enhanced visfatin gene expression in both M1 and M2 human macrophages, but not in murine macrophages or in human adipocytes. Finally, we show that intracellular NAD+ concentrations cor- relate with visfatin protein expression upon PPARc ligand activation. Reduction of visfatin expression and activity by small interfering RNA (siRNA) or a spe- ciﬁc inhibitor abolished the PPARc-mediated increase of NAD+.

Macrophages, crucial cells in the development of inﬂammatory and metabolic disorders such as athero- sclerosis and obesity, are a heterogeneous cell popula- tion that adapts and responds to a large variety [16]. The activation of microenvironmental states and functions of macrophages are regulated by several cytokines and microbial products. T helper 1 cytokines, IL-1b or lipopolysaccharide (LPS), induce activation of a classi- cal pro-inﬂammatory macrophage phenotype (M1), whereas T helper 2 cytokines, such as IL-4 and IL-13,

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Results

PPARc agonists induce visfatin gene expression in human macrophages in a PPARc-dependent manner

gene [23], was also induced to a similar extent in a dose-dependent manner (data not shown). Interest- ingly, visfatin regulation by PPARc was also observed in macrophage foam cells, obtained by acetylated loading (Fig. 1C). low-density lipoprotein (AcLDL) Moreover, GW1929 (600 nm) also regulated visfatin expression in inﬁltrated adipose tissue macrophages (ATM) derived from visceral fat depots (Fig. 1D). To determine whether PPARc agonists up-regulate visfatin expression in a PPARc-dependent manner, the effect of GW1929 (600 nm) was analysed in the presence or in the absence of the PPARc inhibitor, T0070907 (1 lm) [28]. T0070907 abolished GW1929-induced visf- atin mRNA expression (Fig. 1E). Furthermore, infec- tion of RM with PPARc-expressing adenovirus resulted in a signiﬁcant further increase of visfatin expression in the presence of the agonist (Fig. 1F). Expression of two PPARc target genes, CD36 and FABP4 (aP2), used as positive controls, was also these data increased (Fig. 1G,H). Taken together,

To investigate whether PPARc regulates visfatin gene expression, quantitative PCR (Q-PCR) analysis was performed in primary human resting macrophages (RM) upon PPARc activation. Time course experi- ments showed that visfatin induction was already observed after 9 h of stimulation with GW1929 (600 nm) or RSG (100 nm) and became maximal at 24 h (Fig. 1A), with no signiﬁcant further increase after 48 h (data not shown). Treatment of RM with increasing concentrations of the PPARc ligands GW1929 (300, 600 and 3000 nm) or RSG (50, 100 and 1000 nm) for 24 h signiﬁcantly increased visfatin mRNA levels in a concentration-dependent manner (Fig. 1B). Expression of CD36, a known PPARc target

D

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Control GW1929 RSG

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i l i

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h p o c y c / n i t a f s V

3 h

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6 h

1 2 h

2 4 h

AcLDL

ATM

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i l i

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h p o c y c / 6 3 D C

h p o c y c / 4 P B A F

h p o c y c / n i t a f s V

A d G F P

T 00709

C o ntrol

A d P P A Rγ

Fig. 1. PPARc agonists regulate visfatin gene expression in human macrophages in a PPARc-dependent manner. Primary human macrophag- es were incubated or not (control) with (A) GW1929 (600 nM) or RSG (100 nM), for the indicated periods of time, or (B) with GW1929 (300, 600 and 3000 nM) or RSG (50, 100 and 1000 nM) for 24 h, or (C) were transformed into foam cells by AcLDL (50 lgÆmL)1) loading before treat- ment with PPARc ligands. (D) Human visceral ATM were treated with GW1929 (600 nM) for 24 h. (E) Primary human monocytes were differ- entiated in macrophages in the presence or absence of GW1929 (600 nM), T0070907 (1 lM), or both, which were added at the start of the differentiation. Primary human macrophages were infected with recombinant adenovirus AdGFP or AdPPARc and treated with RSG (100 nM) for 24 h. Visfatin (F), CD36 (G) and FABP4 (H) mRNA were analyzed by quantitative PCR and normalized to cyclophilin mRNA. The results are representative of those obtained from three independent macrophage preparations and are expressed relative to the levels in untreated cells set as 1. Each bar is the mean value ± SD of triplicate determinations. Statistically signiﬁcant differences between treatments and controls are indicated (t-test; *P < 0.05; **P < 0.01; ***P < 0.001; T00709 + G929 versus GW1929 §P < 0.05).

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demonstrate that PPARc ligands induce visfatin gene expression in human macrophages through a PPARc- dependent mechanism.

in vitro, while the tion of primary pre-adipocytes expression of CD36 was strongly induced (Fig. 2C,D). Similar results were obtained in the murine pre-adipo- cyte cell line, 3T3L1, after treatment with RSG or pioglitazone (data not shown), in line with a previous report [29].

PPARc agonists do not regulate visfatin gene expression in murine macrophages or human adipocytes

PPARc regulates visfatin gene expression at the transcriptional level

identiﬁed in the

To determine whether regulation of visfatin also occurs in mouse macrophages, experiments were performed in murine bone marrow-derived macrophages that were treated with GW1929 (1200 nm) or RSG (1000 nm) for 24 h. PPARc activation did not increase visfatin gene expression, although expression of CD36 was induced (Fig. 2A,B). Similar results were observed with the murine macrophage cell line, RAW264.7, when incu- bated with increasing concentrations of GW1929 and RSG (data not shown). Furthermore, activation of PPARc by exposure to GW1929 (600 nm) for 24 h did not lead to an increased expression of visfatin in human mature adipocytes derived from the differentia-

1.6

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0.4

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h p o c y c / 6 3 D C m

To determine whether visfatin is a direct PPARc target gene, the human visfatin promoter was examined by bio-informatic analysis. Three putative DR1-like PPRE motifs were 2150-bp sequence upstream of the ATG start site of the visfatin gene [30]. Among these sites, only the putative PPRE identi- ﬁed at position -1501 ⁄ -1513 (AGGGCA A AGATCA) was found to be functional in electrophoretic mobility shift assay (EMSA) experiments (Fig. 3A). Incubation of the labeled -1501 ⁄ -1513 visfatin–PPRE oligonucleo- tide with in vitro-translated PPARc and RXRa resulted in the formation of a retarded complex (Fig. 3A, lane 6). The binding speciﬁcity of PPARc to this DR1–visfatin–PPRE site was demonstrated by competitive inhibition with excess cold unlabeled wild- type (Fig. 3A, lanes 7-11), but not mutated (Fig. 3A, lanes 12-17), visfatin–PPRE oligonucleotide, as well as by the supershift with a speciﬁc anti-human PPARc IgG1 (Fig. 3A, lane 18). Binding of RXRa and PPARc to labelled DR1-consensus PPRE was assayed as a positive control (Fig. 3A, lane 2).

h p o c y c / n i t a f s V m

1.4

Control GW1929

***

Control GW1929

1.2

2.5

D A N R m n

C A N R m n

i l i

0.8

1.5

0.6

0.4

h p o c y c / n i t

0.5

0.2

h p o c y c / 6 3 D C

a f s V

cloned in six copies

To determine whether PPARc activates transcription from the (-1501 ⁄ -1513) PPRE site, six copies of this element were cloned in front of the heterologous her- pes simplex virus thymidine kinase promoter to obtain the (DR1–visfatin–PPRE)6x-Tk-Luc luciferase reporter vector. Co-transfection of the pSG5–PPARc expres- sion vector with the (DR1–visfatin PPRE)6 reporter vector in primary human RM led to a signiﬁcant induction of transcriptional activity compared with the pSG5 empty vector, an effect enhanced in the presence of GW1929 (600 nm) (Fig. 3B). The consensus DR1– (DR1–consensus PPRE site PPRE)6, used as a positive control, was strongly induced by PPARc (Fig. 3B). Taken together, these results indicate that visfatin is a direct PPARc target gene in human macrophages.

PPARc activation induces visfatin gene expression in M1 and M2 macrophages

Fig. 2. PPARc agonists do not regulate visfatin gene expression in (A, B) Murine bone murine macrophages or human adipocytes. marrow-derived macrophages (BMDM) were incubated or not (con- trol) in the presence of PPARc ligands GW1929 (1.2 lM) or RSG (1 lM). (C, D) Human mature adipocytes derived from the differenti- ation of pre-adipocytes in vitro were incubated or not (control) in the presence of PPARc ligands GW1929 (600 nM). CD36 (A, B) and visfatin (C, D) mRNA were analyzed using quantitative PCR and normalized to cyclophilin mRNA. The results are representative of at least three independent cell preparations and are expressed rela- tive to the levels in untreated cells set as 1. Each bar is the mean triplicate determinations. Statistically signiﬁcant value ± SD of differences between treatments and controls are indicated (t-test; *P < 0.05; ***P < 0.001).

As macrophages are heterogeneous cells [16,17], we decided to investigate whether induction of visfatin

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RXRα + PPARγ

Rγ

A

B

A

Rγ

2.5

m u t

Rγ

** ***

Control GW1929

Co ld DR1 V is fa t in -PPRE

w t Co ld DR1 V is fa t in -PPRE

R α P

A b a nti- P

L y s ate

R α + P L y s ate

A N R m n

* *

***

i l i

1.5

§§

0.5

h p o c y c / n i t a f s V

M 2

F- α

L P

IL-1β

3

1

2

4

5

6

7

8 9 10 11 12 13 14 15 16 17 18

DR1-Visfatin-PPRE wt

DR1-consensus PPRE

B

(DR1-Visfatin PPRE)6

(DR1-consensus PPRE)6

Control GW1929

0 60 50

§§§

0 0 0 1 ×

0 0 1 ×

§§

determinations.

Statistically

signiﬁcant

l a g - β / U L R

Fig. 4. PPARc agonists induce visfatin gene expression in M1 and M2 macrophages. (A) Primary human monocytes were differenti- ated to RM and treated for 24 h with GW1929 (600 nM). Where indicated, RM were activated to M1 macrophages by incubation with recombinant human TNF-a (5 ngÆmL)1) or recombinant human IL-1b (5 ngÆmL)1) for 4 h or with LPS (100 ngÆmL)1) for 1 h after GW1929 treatment. (B) Primary human monocytes were differenti- ated in RM or M2 macrophages in the presence of IL-4 (15 ngÆmL)1), and the PPARc agonist GW1929 (600 nM) was added or not during the differentiation process. Visfatin mRNA was ana- lyzed using Q-PCR and normalized to cyclophilin mRNA. The results are representative of those obtained from ﬁve independent macro- phage preparations and are expressed relative to the levels in untreated cells set as 1. Each bar is the mean value ± SD of triplicate differences between treatments and controls are indicated (control versus PPARc agonists *P < 0.05, ***P < 0.001; control versus cytokines §P < 0.05, §§P < 0.01).

pSG5

pSG5-PPAR γ

indicative of

and LPS treatment were ampliﬁed in the presence of the PPARc agonist GW1929 (Fig. 4A). Under the same experimental conditions, PPARc inhibited the induction of TNF-a or IL-1b induced by inﬂammatory its anti-inﬂammatory activity stimuli, (data not shown).

Fig. 3. PPARc binds to and activates a PPRE in the human visfatin gene promoter. (A) EMSAs were performed using the end-labeled DR1–consensus–PPRE (lanes 1 and 2) or the DR1–visfatin–PPREwt oligonucleotide in the presence of unprogrammed reticulocyte lysate or in vitro-translated human PPARc and human RXRa (lanes 3–5). Competition experiments were performed in the presence of excess cold unlabeled wild-type (wt) (lanes 6–11) or mutated (mut) DR1– visfatin–PPRE oligonucleotides (lanes 12-17). Supershift assays were performed using an anti-human PPARc Ig (lane 18). (B) Primary human macrophages were transfected with the indicated reporter constructs (DR1–visfatin–PPRE)6 or (DR1–consensus–PPRE)6, in the presence of pSG5 empty vector or pSG5–PPARc. Cells were treated or not (Control) with GW1929 (600 nM) and luciferase activity was measured. Statistically signiﬁcant differences are indicated (pSG5 versus pSG5-PPARc; §§P < 0.01, §§§P < 0.001; control versus GW1929 *P < 0.05, **P < 0.01). b-gal, beta-galactosidase; RLU, relative luciferase units.

in the presence of

In parallel experiments, human monocytes were dif- ferentiated in vitro into M2 macrophages with recom- binant IL-4 (15 ngÆmL)1) in the absence or in the presence of the PPARc agonist GW1929 added at the start of the differentiation process [25]. As shown in Fig. 4B, the expression of visfatin was signiﬁcantly decreased by IL-4 stimulation. However, as with RM, the PPARc agonist GW1929 enhanced visfatin gene expression in M2 macrophages. A similar regulation was observed in monocytes differentiated into M2 macrophages IL-13 (data not shown).

PPARc activation regulates visfatin protein expression and secretion in human macrophages

also occurs after PPARc activation in classical (M1) or alternative (M2) macrophages. Human monocytes were differentiated in vitro into RM macrophages and acti- vated into inﬂammatory M1 macrophages with recom- binant human TNF-a (5 ngÆmL)1), IL-1b (5 ngÆmL)1) or LPS (100 ngÆmL)1). As expected [8], expression of visfatin was strongly induced by pro-inﬂammatory stimuli (Fig. 4A). Interestingly, the effects of TNF-a

To determine whether visfatin gene induction by PPARc agonists leads to an increased protein level,

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W 1929

C ontrol

Visfatin

β-actin

B

A

2.5

1.6

tion was followed by an increased secretion, we exam- ined the ability of PPARc to stimulate visfatin release. As shown in Fig. 5B, GW1929 markedly increased (approximately 30%) the visfatin concentration in macrophage supernatants after 24 h of treatment.

***

1.4

1.2

1.0

1.5

0.8

PPARc activation increases the intracellular NAD+ concentration in human macrophages

n i t c a - β / n i t a f s

0.6 0.4V

0.5

0.2

) 1 – L m · g n ( n o i t e r c e s n i t a f s V

o ntr ol

W 1 9 2 9

C

G

As visfatin is known as a nicotinamide phosphoribosyl transferase [2], we investigated whether the induction of visfatin by PPARc affects the concentration of NAD+. Human RM were treated or not with GW1929 (600 nm) for 24 h and intracellular NAD+ levels were determined using an enzymatic assay. Our results showed that PPARc activation signiﬁcantly enhances the cellular NAD concentration (Fig. 6), an effect in line with the observed induction of visfatin expression (Figs 1 and 5).

Fig. 5. PPARc regulates visfatin protein expression and secretion in primary human macrophages. Primary human macrophages were treated or not (control) with GW1929 (600 nM) for 24 h. (A) Intracel- lular visfatin and b-actin protein expression was analyzed by wes- tern blotting and relative signal intensities were quantiﬁed using Quantity One Software. The results are representative of four inde- pendent macrophage preparations and are expressed relative to the levels in untreated cells set as 1. (B) Secretion of visfatin protein was quantiﬁed in the macrophage supernatant using ELISA. The results are representative of three independent macrophage prepa- rations. Each bar is the mean value ± SD of triplicate determina- tions. Statistically signiﬁcant differences between treatments and controls are indicated (t-test; *P < 0.01; ***P < 0.001).

To determine whether the NAD+ enhancement by PPARc was dependent on visfatin induction, experi- ments were performed in RM macrophages in the absence or in the presence of a speciﬁc visfatin siRNA. Q-PCR analysis showed a signiﬁcant decrease in visfa- tin gene expression after siRNA (scrambled = 1 ± 0.019 versus siRNA visfatin = 0.27 ± 0.01), whereas PPARc activation increased visfatin gene expres- sion (scrambled + GW1929 = 2.04 ± 0.4 and siRNA visfatin + GW1929 = 0.51 ± 0.022). siRNA-mediated the visfatin knockdown resulted in a reduction of basal, as well as of the GW1929-induced, NAD+ con- centration (Fig. 6A). Moreover, experiments performed

western blot analysis was performed on human RM treated with GW1929 (600 nm) or dimethylsulfoxide for 24 h. Activation of PPARc caused a signiﬁcant increase (approximately 30%) of visfatin protein expression (Fig. 5A). To examine whether this induc-

)

§ §

)

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100

r a u

l l

e c a r t n I

Scrambled siRNA visfatin

e c a r t n I

AdPPARγ

Vehicle

FK866

AdGFP

§P < 0.05,

Fig. 6. PPARc activation affects intracellular NAD concentrations in primary human macrophages. Primary human macrophages were trans- fected or not with non-silencing control or silencing siRNA against human visfatin (A), or treated or not with the visfatin inhibitor FK866 (100 nM) (B), or infected or not with PPARc-expressing (AdPPARc) or GFP (AdGFP) adenovirus (C) and subsequently treated with GW1929 (600 nM), RSG (100 nM) or dimethylsulfoxide for 24 h. Cells were lysed in NAD extraction buffer and the NAD+ concentrations were mea- sured using an enzymatic cycling reaction assay, normalized to protein levels and expressed as a percentage, the control non-stimulated cells being expressed as 100%. The results are representative of those obtained from three independent macrophage preparations. The val- ues are means ± SD of triplicates. Statistically signiﬁcant differences are indicated (t-test; control versus PPARc agonists, *P < 0.05, §§P < 0.05; AdGFP + PPARc agonists versus **P < 0.01; scrambled versus siRNA visfatin or vehicle versus FK866, AdPPARc + PPARc agonists, §P < 0.05). GFP, green ﬂuorescent protein.

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in the presence of a speciﬁc noncompetitive inhibitor of visfatin (FK866) in the presence or absence of GW1929 demonstrated that the induction of NAD+ by GW1929 was inhibited in the presence of FK866 (Fig. 6B). Finally, PPARc over-expression increased NAD+ levels, an effect enhanced by its synthetic ligands GW1929 and RSG (Fig. 6C).

Discussion

at position -1501 ⁄ -1513 within the promoter. This PPRE is distinct from the described AP-1 or NF-jB– response element (RE) like elements (located at posi- tion -1757 ⁄ -1767) within the human visfatin promoter [30]. This can explain our observation that inﬂamma- tory cytokines and PPARc agonists have an additive effect on visfatin mRNA expression, an effect appar- ently in contrast to the known anti-inﬂammatory actions of PPARc in macrophages as a result of its ability to interfere with the NF-jB and AP-1 signaling pathways [19]. This is similar to what has already been reported for other nuclear receptors, such as liver X receptor, for which short-term pretreatment with liver X receptor agonists signiﬁcantly reduced the LPS- induced inﬂammatory response, whereas 24-h pretreat- ment of macrophages with agonists resulted in an enhanced inﬂammatory response [34].

nonclassical

through

secreted

inﬂammatory pathologies,

PPARc agonists induce visfatin protein expression and secretion in human primary macrophages. Visfatin is a secreted cytokine-like protein [35], although it has been speculated that the release of visfatin may be lysis or by cell death [36,37]. caused either by cell However, it has been demonstrated in adipocytes and Chinese Hamster ovary (CHO) cells that visfatin is actively (non- Golgi ⁄ endoplasmic reticulum system) secretory path- way [2]. In our experiments we did not observe any cellular toxicity after treatment with PPARc agonists, suggesting that the secretion of visfatin in human mac- rophages may be an active process.

Visfatin has been suggested to act as an inﬂamma- tory mediator, being expressed in blood monocytes and foam cell macrophages within unstable athero- sclerotic lesions where it potentially plays a role in plaque destabilization [8,31]. Visfatin induces leuko- cyte adhesion to endothelial cells by inducing the expression of the cell-adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), thus potentially contributing to endothelial dysfunction [11]. More- over, visfatin increases matrix metalloproteinase-9 activity and the expression of TNF-a and IL-8 in THP-1 monocytes [8]. These effects of visfatin were abolished when insulin receptor signalling was blocked [8], in line with the report that visfatin could bind and activate insulin receptors [14]. However, the insulin- mimetic actions of visfatin are still debated [13]. All that visfatin might be a player these data suggest linking several including obesity-associated insulin resistance, diabetes mellitus and vascular wall dysfunctions [9,32].

concentration

In addition,

shown).

In this study we showed that PPARc activation up-regulates the expression of visfatin in human mono- cyte-derived macrophages and ATM. This induction is concentration dependent and does not occur during the short incubation time generally required for macro- phage activation, but requires an incubation period of more than 9 h. The maximum effect was obtained at 24 h with no signiﬁcant further increase at 48 h (data not treatment with AcLDL induced visfatin mRNA levels, and PPARc activation further increased visfatin expression in these AcLDL- loaded macrophages.

transfection experiments

As visfatin is the rate-limiting enzyme for the con- version of nicotinamide to NAD+ in mammals, the intracellular NAD+ increased induced by PPARc agonists is probably the conse- quence of visfatin induction. NAD+ modulates vari- ous signalling pathways. For instance, it regulates the transcription and function of NAD+-dependent SIR- Ts, and increased expression of visfatin upregulates sirtuin 1(SIRT1) activity [2]. The observed variation of intracellular NAD+ concentrations after visfatin mod- ulation (by siRNA or PPARc activation) are in the same order of magnitude as previously reported in murine NIH-3T3 ﬁbroblasts transduced with visfatin- speciﬁc small hairpin RNA (shRNA). The reduction of intracellular visfatin protein in these cells led to a reduction of NAD+ levels from 20% to 40%, whereas cells over-expressing visfatin displayed a 15–25% intracellular NAD+ levels [38]. By increase in total using the pharmacological visfatin inhibitor, FK866, a signiﬁcant decrease in the intracellular NAD+ concen- tration was observed, even in the presence of PPARc ligand, conﬁrming the role of the enzymatic activity of visfatin and the possibility that PPARc can modulate

By over-expressing PPARc with adenovirus con- structs, or by inhibiting PPARc with a speciﬁc antago- nist, we demonstrated that PPARc agonists induce visfatin gene expression in a PPARc-dependent man- ner [33]. By bio-informatics analysis, we detected the presence of three DR1-like motifs that might serve as PPREs in the 2150-bp sequence upstream of the ATG codon of the human visfatin gene [30]. Using EMSA and transient in primary human macrophages, a functional PPRE was identiﬁed

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intracellular NAD+ levels via an increase of visfatin expression. Indeed, a small increase in the concentra- tion of NAD+ in response to GW1929 in siRNA visf- atin treated-macrophages was observed, suggesting that an additional PPARc-related pathway might mod- ulate NAD+ levels.

Moreover, we have shown that PPARc agonists increase the expression of visfatin in macrophages irre- spective of their M1 or M2 polarization. Visfatin-depen- dent recycling of nicotinamide to NAD+ may represent a physiologically important homeostatic mechanism to avoid depletion of the intracellular NAD+ pool during its active use as a substrate by sirtuins, cADP-ribose synthases or PARPs [15]. It has recently been shown that pharmacological SIRT1 activators exert broad anti-inﬂammatory effects in macrophages [39]. Con- versely, SIRT1 knockdown leads to an increase in the basal expression of TNF-a, monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived chemokine (KC). The activity of SIRT1 requires an increase of visfatin expression to compensate for the consumption of NAD+. Van Gool et al. have identiﬁed SIRT6, another member of the sirtuin family, as the NAD- dependent enzyme able to increase TNF-a production in macrophages by acting post-transcriptionally [40]. Taken together, these observations suggest that NAD+ can exert pro- and ⁄ or anti-inﬂammatory properties depending on the activated sirtuins.

Adipose tissue is composed not only of adipocytes, including but also of several other types of cells, macrophages, lymphocytes and endothelial cells. It has been shown that PPARc agonists induce the expression of visfatin in the visceral fat of OLETF rats [48]. The authors analyzed whole adipose tissue, and thus it cannot be determined whether PPARc regulation of visfatin occurred in macrophages or in adipocytes. Here we show that PPARc activation leads to an increased expression of visfatin in ATM. However, this regulation does not occur in human primary mature adipocytes derived from pre-adipocyte differentiation in vitro. It has been shown recently that PPARc binding in macrophages occurs at genomic locations different from those in adipocytes, showing that PPARc-binding sites are cell type-speciﬁc [49]. These results are in agreement with a previous report showing that in humans, PPARc has distinct func- tions in different cell types because treatment with pioglitazone induces apoptotic cell death speciﬁcally in macrophages, whereas differentiated adipocytes did not show any signiﬁcant increase in apoptosis [50]. Furthermore, treatment with pioglitazone for 3 weeks did not alter visfatin gene expression in adipose cells, in either non-diabetic or diabetic individuals [43]. Altogether, these results may allow some light to be shed on the regulation of visfatin expression by PPARc in human adipose tissue, an effect limited to ATM.

In conclusion, our results identify visfatin as a novel PPARc target gene in human macrophages and dem- onstrate that PPARc activation induces visfatin gene and protein secretion in different types of human macrophages. This induction of visfatin by PPARc in macrophages contributes to enhanced concentrations of intracellular NAD+.

Materials and methods

Cell culture

It is also possible that macrophage-produced visfatin has a local paracrine effect on surrounding cells, such as SMC, within atherosclerotic plaques, because in vascular SMC, over-expression of visfatin promotes cell maturation by regulating NAD+-dependent SIRT deacetylase activity [41]. Visfatin has been reported as a longevity protein that extends the life span of human SMC, suggesting that visfatin allows vascular cells to resist stress and senescence, a hallmark of atheroscle- rotic lesions [42]. The ability of visfatin to prolong the longevity of vascular SMC might contribute to the sta- bilization efﬁciency of a developing atherosclerotic lesion by SMC. Treatment of humans with PPARc ligands does not alter adipose visfatin gene expression and circulating visfatin levels, as reported in several publications [43–45]. However, other authors reported that in lean as well as in lean-HIV-infected patients, RSG treatment increased the amounts of circulating visfatin [46,47]. It thus appears that the effect of treat- ment with PPARc ligand on circulating visfatin levels is highly dependent on the patient phenotype. How- ever, in such studies the net contribution of visfatin from adipocytes or macrophages cannot be evaluated and cell-speciﬁc PPARc regulation of visfatin may have a local effect.

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Mononuclear cells were isolated from blood (buffy coats; thrombopheresis residues) of human healthy normolipidem- ic donors by Ficoll gradient centrifugation [21]. Brieﬂy, after Ficoll gradient centrifugation, peripheral blood mono- nuclear cells were suspended in RPMI-1640 (Gibco, Invitro- gen) containing gentamycin (40 lgÆmL)1) and glutamine (0.05%) (both from Gibco, Invitrogen). Cells were cultured, depending on the experiment, at a density of 1 or 2 · 106 cells per well in six-well plastic culture dishes (Pri- maria; Becton Dickinson Labware). Selection of a pure monocyte population occurred spontaneously after 2 h of two washing cell adhesion to the culture dish. After

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Visfatin induction by PPARc in human macrophages

facturer’s instructions, until conﬂuence. After conﬂuence, pre-adipocytes were cultured in pre-adipocyte differentia- tion medium (Promocell) for 72 h. To complete the differ- entiation process into mature adipocytes, cells were fed every 2–3 days for 12 days with adipocyte nutrition med- ium (Promocell). At the end of the differentiation, mature adipocytes were treated with the PPARc ligand GW1929 (600 nm).

RNA extraction and analysis

Murine bone marrow-derived macrophages were pre- pared from C57BL ⁄ 6J mice. Bone marrow cell suspensions were isolated by ﬂushing the femurs and tibias with NaCl ⁄ Pi and cells were cultured as previously described [51]. Bone marrow-derived macrophages were treated with the PPARc ligands GW1929 (1.2 lm) and RSG (1 lm) for 24 h.

steps with NaCl ⁄ Pi, cells were cultured in RPMI-1640 con- taining gentamycin (40 lgÆmL)1), glutamine (0.05%) and 10% pooled human serum (Biowest, Nuaille´ , France). Dif- ferentiation of monocytes into macrophages is completed after 7 days, characterized by immunocytochemistry or ﬂow cytometry analysis using macrophage marker anti-CD68 antibody [21]. These primary human macrophages, also called RM, were used for experiments after 7 days of differ- entiation. RM were incubated for 3, 6, 9, 12 or 24 h in the presence of the PPARc ligands GW1929 (300, 600, 3000 nm) or RSG (50, 100, 1000 nm), or with dimethylsulf- oxide as a control. Where indicated, RM were transformed to foam cells by 48-h loading with AcLDL (50 lgÆmL)1) and treated with the PPARc ligands GW1929 (600 nm) or RSG (100 nm), or with dimethylsulfoxide as a control. Where indicated, the PPARc antagonist T0070907 (1 lm) (Tocris Bioscience, Bristol, UK) or the NAMPT inhibitor FK866 (100 nm) (Cayman Chemical, Tallinn, Estonia) were added. In other experiments, RM were treated with GW1929 (600 nm) or dimethylsulfoxide for 24 h and then activated into M1 macrophages by incubation with recom- binant human TNF-a (5 ngÆmL)1) or human IL-1b (5 ngÆmL)1) (Promokines, Heidelberg, Germany) for 4 h or with LPS (100 ngÆmL)1) (Sigma, Saint-Quintin Fallavier, France) for 1 h. M2 macrophages were obtained by differ- entiating monocytes in the presence of recombinant human IL-4 (15 ngÆmL)1) (Promokines).

Adenovirus preparation and cell infection

Total cellular RNA was extracted from human macrophages using Trizol (Invitrogen, France) for RM or the RNeasy micro kit (Qiagen, Courtaboeuf, France) for ATM. For Q- PCR, total RNA was reverse transcribed and cDNAs were quantiﬁed by the Q-PCR on an MX 4000 apparatus (Strata- gene) using speciﬁc primers for human visfatin (forward, 5¢- GCC AGC AGG GAA TTT TGT TA-3¢; and reverse, 5¢- TGA TGT GCT GCT TCC AGT TC-3¢), mouse visfatin (forward, 5¢-TCCGGCCCGAGATGAAT-3¢; and reverse, 5¢-GTGGGTATTGTTTATAGTGAGTAACCTTGT-3¢), human CD36 (forward, 5¢-TCAGCAAATGCAAAGAAG GGAGAC-3¢; and reverse, 5¢-GGTTGACCTGCAGCCGT TTTG-3¢), mouse CD36 (forward, 5¢-GGATCTGAAATC GACCTTAAAG-3¢; and reverse, 5¢-TAGCTGGCTTGAC CAATATGTT-3¢), human FABP4 (forward, 5¢-TACTGG GCCAGGAATTTGAC-3¢; and reverse, 5¢-GTGGAAGT GACGCCTTTCAT-3¢) and human ⁄ mouse cyclophilin (for- ward, 5¢-GCA TAC GGG TCC TGG CAT CTT GTC C-3¢; and reverse, 5¢-ATG GTG ATC TTC TTG CTG GTC TTG C-3¢). Visfatin mRNA levels were subsequently normalized to those of cyclophilin.

instructions,

Visceral adipose tissue biopsies were obtained from consenting obese patients undergoing bariatric surgery. This study was approved by the Ethics Committee of the University Hospital of Lille, France. After removing all ﬁbrous materials and visible blood vessels, adipose tissue was cut into small pieces and digested in Krebs buffer, pH 7.4, containing collagenase (1.5 mgÆmL)1; Roche Diagnos- tic, Meylan, France). The cell suspension was ﬁltered through a 200-lm pore-size ﬁlter and centrifuged at 300 g for 15 min to separate ﬂoating adipocytes. The stromal vascular fraction was pelleted, treated with erythrocyte lys- ing buffer (131 mm NH4Cl, 9 mm NH4CO3, 1 mm EDTA, pH 7.4) for 10 min and ﬁltered through meshes with a pore size of 70 lm. The stromal vascular fraction was then sub- jected to magnetic-activated cell sorting of CD14+ cells (Miltenyi Biotec, Paris, France) using CD14-labelled mag- netic beads and MS columns (Miltenyi, Paris, France), according to the manufacturer’s to yield ATM. The purity of CD14+ cells was assessed by ﬂow cytometry analysis. ATM were cultured for 24 h in endo- thelial cell basal medium, supplemented with 0.1% BSA, before treatment with GW1929 (600 nm) or dimethylsulfox- ide for 24 h.

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The recombinant adenoviruses AdGFP and AdPPARc were obtained by homologous recombination in Escherichia coli after insertion of the cDNAs into the pAdCMV2 vector (Q.BIOgene, Illkirch, France). Viral stocks were created as previously described [52]. Viral titers were determined by plaque assay on HEK 293 cells and deﬁned as plaque-form- ing unitsÆmL)1. For the infection experiments, primary human macrophages were seeded in six-well Primaria plates at a density of 106 cells per well and viral particles were added at a multiplicity of infection of 100 for 12 h. Cells were subsequently incubated for 24 h with RSG (100 nm) or dimethylsulfoxide. The CD14 negative fraction was cultured in pre-adipo- cyte basal medium (Promocell, Heidelberg, Germany) for 24 h, then washed with NaCl ⁄ Pi to remove ﬂoating cells. Adherent pre-adipocytes were then cultured in pre-adipo- cyte growth medium (Promocell), according to the manu-

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In vitro translation and EMSA

Protein extraction and western blot analysis

transfected with siRNA using the transfection reagent DharmaFECT Reagent 4. Sixteen hours after transfection, cells were incubated in the presence of GW1929 (600 nm) or vehicle (dimethylsulfoxide) and harvested 24 h later.

Measure of visfatin protein secretion by ELISA

Cells were washed twice with ice-cold NaCl ⁄ Pi and harvested in ice-cold protein lysis buffer (RIPA). Cell homogenates were collected by centrifugation at 13 000 rpm at 4 (cid:2)C for 30 minutes and protein concentrations were determined using the bicinchoninic acid assay (Pierce Interchim, Rockford, IL, USA). Ten micrograms of protein lysate was separated by 10% SDS ⁄ PAGE and transferred to nitrocellulose mem- branes (Amersham, Saclay, France). Equal loading of pro- teins was veriﬁed by Ponceau red staining. Membranes were then subjected to immunodetection using rabbit polyclonal antibodies against visfatin (ab24149; Abcam, Paris, France) or against b-actin (I-19; Santacruz Biotechnology). After incubation with a secondary peroxidase-conjugated antibody (Cell Signaling Technology, Denver, MA, USA), immunore- active bands were revealed using a chemiluminescence ECL detection kit (Amersham) and the intensity of signals was subsequently analyzed by densitometry and quantiﬁed using Quantity One software.

Plasmid cloning and transient transfection experiments

PPARc and RXRa were in vitro transcribed from the pSG5–hPPARc and pSG5–hRXRa plasmids, respectively, using T7 polymerase, and subsequently translated using the transcription and translation (TNT)-coupled transcription ⁄ translation system (Promega, Madison, WI, USA). Proteins were then incubated for 10 min at room temperature in a binding buffer (10 mm Hepes, pH 7.8, 100 mm NaCl, 0.1 mm EDTA, 10% glycerol, 1 mgÆmL)1 of BSA) contain- ing 1 lg of poly(dI-dC) and 1 lg of herring sperm DNA in a total volume of 20 lL. Double-stranded oligonucleotides containing the wild-type DR1–PPRE, present at position- the human visfatin promoter, and end- 1501 ⁄ -1513 of labeled using T4 polynucleotide kinase and [32P]dATP[cP], were added as a probe to the binding reaction. For compe- tition experiments, increasing amounts (5, 10, 50, 100 and 200-fold excess) of unlabeled visfatin–PPREwt (5¢-CAAT ACAGGGCAAAGATCATGGAAG-3¢) or visfatin–PPRE- mut (5¢-CAATACAGGAAAAAGAAAATGGAAG-3¢) oli- gonucleotides were added to the mixture 10 min before the DR1–visfatin–PPREwt. The binding reaction was incubated for a further 15 min at room temperature. For supershift assays, 2 lL of monoclonal mouse anti-human PPARc IgG (Sc-7273; Santacruz Biotechnology, Heidelberg, Germany) was added to the binding reaction. DNA–protein complexes were resolved by 6% nondenaturing PAGE in 0.25 · Tris ⁄ Borate ⁄ EDTA.

Measurement of cellular NAD content

Human RM were treated with the PPARc ligand GW1929 (600 nm, or with dimethylsulfoxide, for 24 h. Supernatants were collected and extracellular visfatin concentrations were measured using a commercially available ELISA kit with a human visfatin (COOH-terminal) enzyme immunometric assay (Phoenix Pharmaceuticals, Karlsruhe, Germany), according to the manufacturer’s instructions.

(Polyplus

siRNA

The reporter plasmid (DR1–visfatin–PPREwt)6–TK–pGL3 was generated by inserting six copies of the double-strand oligonucleotides (forward, 5¢-CAATACAGGGCAAAGAT CATGGAAG-3¢; and reverse, 5¢-CTTCCATGATCTTTG CCCTGTATTG-3) into the pTK–pGL3 plasmid. Primary human macrophages were transfected overnight in RPMI containing 10% human serum with reporter plasmids and expression vectors (pSG5–empty or pSG5–hPPARc) using jetPEI transfection, France). b-galactosidase expression vectors were used as an internal control of trans- fection efﬁciency. Subsequently, cells were incubated for an additional 24 h in RPMI containing 2% human serum in the presence of GW1929 (600 nm) or dimethylsulfoxide. At the end, cells were lysed, and luciferase and b-galactosidase activities were measured on cell extracts using a luciferase buffer (Promega).

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Total nicotinamide adenine dinucleotide (NADt = NAD + lysates using the NADH) levels were determined in cell NADH ⁄ NAD quantiﬁcation kit according to the manufac- turer’s instructions (Biovision research products, Mountain View, CA, USA). Brieﬂy, human RM, treated or not with FK866 (100 nm), infected or not with adenovirus (AdGFP, AdPPARc) and transfected or not with siRNA (siScrambled, siVisfatin) were treated with the PPARc ligands GW1929 (600 nm) or RSG (100 nm), or with dimethylsulfoxide, for 24 h. Cells were lysed in NAD+ extraction buffer after wash- ing three times with ice-cold NaCl ⁄ Pi. The NAD ⁄ NADH ratio was calculated as (NADt-NADH) ⁄ NADH. NAD levels were normalized to protein content. The results are expressed as a percentage, with the control unstimulated cells being expressed as 100%. All assays were performed in triplicate in at least three independent experiments. siRNA speciﬁc for human PBEF1 (Visfatin NAMPT), and nonsilencing control siRNA (siScrambled) were purchased from Dharmacon. Seven-day-old human macrophages were

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Visfatin induction by PPARc in human macrophages

Statistical analysis

Statistically signiﬁcant differences between groups were analysed using the Student’s t-test and were considered sig- niﬁcant when the P-value was £ 0.05.

8 Dahl TB, Yndestad A, Skjelland M, Oie E, Dahl A, Michelsen A, Damas JK, Tunheim SH, Ueland T, Smith C et al. (2007) Increased expression of visfatin in macrophages of human unstable carotid and coronary atherosclerosis: possible role in inﬂammation and plaque destabilization. Circulation 115, 972–980.

Acknowledgements

9 Moschen AR, Kaser A, Enrich B, Mosheimer B, Theurl M, Niederegger H & Tilg H (2007) Visfatin, an adipo- cytokine with proinﬂammatory and immunomodulating properties. J Immunol 178, 1748–1758. 10 Kendal CE & Bryant-Greenwood GD (2007) Pre-B-cell

We thank R. Dievart, B. Derudas, A. Blondy, C. Eberle, M. F. Six and Dr L. Arnalsteen for their contribution. We acknowledge grant support from the ‘Nouvelle Soci- ete´ Franc¸ aise d’Athe´ roscle´ rose’ (to T. H. Mayi) and Fondation Coeur et Arte` res. The research leading to these results has received funding from the European Community’s 7th Framework Programme (FP7 ⁄ 2007- 2013) under grant agreement no. 201608.

colony-enhancing factor (PBEF ⁄ Visfatin) gene expres- sion is modulated by NF-kappaB and AP-1 in human amniotic epithelial cells. Placenta 28, 305–314.

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