The adipokine chemerin has been implicated in cardiovascular complications associated with obesity and the metabolic syndrome. Chemerin has direct effects on the vasculature, augmenting vascular responses to contractile stimuli. As NO/cGMP signalling plays a role in vascular dysfunction associated with obesity and the metabolic syndrome, we hypothesized that chemerin induces vascular dysfunction by decreasing NO/cGMP signalling. Aortic rings from male Wistar rats (10–12 weeks of age) were incubated with chemerin (0.5 or 5 ng/ml for 1 h) or vehicle and isometric tension was recorded. Vasorelaxation in response to ACh (acetylcholine), SNP (sodium nitroprusside) and BAY 412272 [an sGC (soluble guanylate cyclase) stimulator] were decreased in chemerin-treated vessels. The NOS (NO synthase) cofactor BH4 (tetrahydrobiopterin), an O2 (superoxide anion) scavenger (tiron) and a SOD (superoxide dismutase) mimetic (tempol) abolished the effects of chemerin on ACh-induced vasodilation. eNOS (endothelial NOS) phosphorylation, determined by Western blotting, was increased in chemerin-treated vessels; however, the enzyme was mainly in the monomeric form, with decreased eNOS dimer/monomer ratio. Chemerin decreased the mRNA levels of the rate-limiting enzyme for BH4 biosynthesis GTP cyclohydrolase I. Chemerin-incubated vessels displayed decreased NO production, along with increased ROS (reactive oxygen species) generation. These effects were abrogated by BH4, tempol and L-NAME (NG-nitro-L-arginine methyl ester). sGC protein expression and cGMP levels were decreased in chemerin-incubated vessels. These results demonstrate that chemerin reduces NO production, enhances NO breakdown and also decreases NO-dependent cGMP signalling, thereby reducing vascular relaxation. Potential mechanisms mediating the effects of chemerin in the vasculature include eNOS uncoupling, increased O2 generation and reduced GC activity.

CLINICAL PERSPECTIVES

  • Current advances in the understanding of adipose tissue biology and its endocrine function have provided insight into the mechanisms involved in adiposity-related diseases. White adipose tissue secretes a number of proteins called adipokines, including chemerin, that participate in many physiological processes implicated in cardiovascular complications associated with obesity and the metabolic syndrome.

  • In the present study, we observed that chemerin reduces vascular NO/cGMP signalling and decreases vascular relaxation. Potential mechanisms mediating the vascular effects of chemerin included eNOS uncoupling, increased O2 generation and reduced NO production, which in turn are associated with decreased sGC protein activation and cGMP production.

  • Therefore chemerin may play a significant role in obesity-associated vascular dysfunction and may represent a new therapeutic target in adiposity-related diseases.

INTRODUCTION

Current advances in the understanding of adipose tissue biology and, in particular, its endocrine function, have provided insight into the mechanisms involved in adiposity-related pathologies. White adipose tissue secretes a number of proteins called adipokines [1,2] that participate in many physiological processes, including regulation of body weight and appetite, lipid and carbohydrate metabolism, reproduction, immunity and the inflammatory response, vascular function and blood pressure maintenance. Most adipokines with pro-inflammatory properties are overproduced with increasing adiposity, whereas the production of some adipokines with anti-inflammatory properties is decreased. Disturbances in secretion, function and balance of adipokines also occur in many CVDs (cardiovascular diseases) and appear to be implicated in the associated alterations of vascular function reported in these conditions [3].

Chemerin, also known as RARRES2 (retinoic acid receptor responder protein 2), is an adipocyte-secreted protein (adipokine) that has recently emerged as a regulator of several biological processes, including adipocyte development, metabolism and immune function. Chemerin is secreted as a 163 amino acid inactive peptide, pro-chemerin, that subsequently undergoes proteolysis to form active chemerin [4], which binds the Gi-protein-coupled specific receptor, CMKLR1 (chemokine-like receptor 1) or ChemR23 (chemerin receptor 23), expressed in macrophages, dendritic cells and adipocytes [5]. Chemerin receptor expression has also been reported in endothelial cells and VSMCs (vascular smooth muscle cells) [6]. Studies have associated chemerin with several obesity-related disorders, including insulin resistance, hypertriglyceridaemia, augmented blood pressure and Type 2 diabetes [7,8]. Serum and adipose tissue chemerin levels are increased in women with the metabolic syndrome [9], an effect that is reversed by weight and fat loss [8,10]. Additionally, genetic deletion of the primary receptor for chemerin, ChemR23, is associated with reduced adiposity and body mass [8].

Evidence also supports a relationship between chemerin and CVDs. In epicardial adipose tissue from patients with coronary artery disease, chemerin mRNA and protein expression is significantly higher compared with tissues from healthy subjects [11]. A positive correlation between chemerin expression in periaortic and pericoronary adipose tissue and aortic and coronary atherosclerosis has also been reported [12]. Chemerin levels have also been associated with cardiometabolic risk factors and the degree of coronary artery disease in patients with coronary artery stenosis [13]. Recently, we have shown that chemerin has direct effects in the vasculature, augmenting vascular responses to contractile stimuli via activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK1/2 pathway [14]. In addition, chemerin has been suggested to be an endogenous vasoconstrictor produced by both visceral adipose tissue and PVAT (perivascular adipose tissue) that modifies vascular tone via ChemR23 [6]. Although it has been demonstrated that chemerin is a potential risk factor for vascular injury, so far there is no evidence showing that chemerin influences vascular relaxation or the vasodilation-associated signalling pathways modified by chemerin.

NO/cGMP signalling plays a major role in the cardiovascular system, evoking smooth muscle relaxation and inhibiting platelet aggregation. Endothelium-derived NO stimulates the production of the second messenger cGMP by activation of sGC [soluble GC (guanylate cyclase)] in VSMCs. cGMP alters the properties of several target proteins. For example, it activates cGK (cGMP-dependent protein kinase I), which is also essential for both vascular smooth muscle relaxation [15] and the antiproliferative effects of NO [16]. Likewise, obesity-related diseases, conditions with increased levels of adipokines, are associated with defective vascular NO/cGMP-dependent signalling [17,18], which are characterized by reduced production or bioavailability of endothelium-derived NO. In later stages of the disease, endothelium-independent vasodilation induced by exogenous NO donors also becomes increasingly attenuated, along with alterations in the expression and activity of enzymes involved in NO-dependent signalling, including sGC and cGK [19]. On the basis of these observations, the present study was designed to test the hypothesis that the adipokine chemerin reduces NO/cGMP signalling in the vasculature contributing to decreased vascular relaxation.

MATERIALS AND METHODS

Animals

All experimental protocols were performed in accordance with the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee on Animal Use (CEUA) of the University of Sao Paulo, Ribeirao Preto, Brazil (Protocol n° 10.1.1295.53.7).

Male Wistar rats, 10–12 weeks of age (from the University of Sao Paulo, Ribeirao Preto, Brazil), were used. The rats were maintained on a 12-h light/dark cycle under controlled temperature (22±1°C) with ad libitum access to food and water. Animals were killed using CO2. Abdominal aortas were isolated for the experimental procedures described below.

Incubation procedures with chemerin

After killing, thoracic aortas were rapidly excised and cleaned from fat tissue in an ice-cold (4°C) Krebs–Henseleit-modified solution (130 mmol/l NaCl, 14.9 mmol/l NaHCO3, 4.7 mmol/l KCl, 1.18 mmol/l KH2PO4, 1.17 mmol/l MgSO4·7H2O, 5.5 mmol/l glucose, 1.56 mmol/l CaCl2·2H2O and 0.026 mmol/l EDTA) gassed with 5% CO2/95% O2 to maintain a pH of 7.4. Aortic rings (2–3 mm in length) were mounted on two stainless-steel wires in standard organ chambers for isometric tension recording, as described previously [20]. Vessels were allowed to equilibrate for about 30 min in Krebs–Henseleit solution. After the stabilization period, arterial integrity was assessed first by stimulation of vessels with 120 mmol/l KCl. After washing and a new stabilization period, endothelial function was assessed by testing the relaxant effect of ACh (acetylcho-line; 10−6 mol/l) on vessels contracted with PhE (phenylephrine; 10−6 mol/l). Aortic rings exhibiting a vasodilator response to ACh greater than 90% were considered endothelium-intact vessels. In experiments with endothelium-denuded vessels, aortic rings were subjected to rubbing of the intimal surface. Rings showing a maximum of 5% relaxation in response to ACh were considered to be without endothelium.

Arterial segments were incubated with either vehicle (PBS containing 0.1% BSA) or chemerin (0.5 and 5 ng/ml) for 1 h to verify the acute effects of the adipokine. Considering that no difference between the effects of the two concentrations of chemerin was observed, the experiments were performed with the lowest concentration of the adipokine. Previous studies have estimated that the plasma and serum concentrations of active chemerin are 50.5 and 72.7 ng/ml respectively in humans and 10.0 and 8.3 ng/ml respectively in mice [21]. Therefore the concentration used in the present study corresponds to physiological levels of chemerin.

Vascular functional studies

Cumulative concentration–response curves to ACh (10−9–3×10−5mol/l), SNP (sodium nitroprusside; 10−10–3×10−5 mol/l) and BAY 412722 (a direct GC stimulator; 10−10–3×10−5 mol/l) were constructed for aortic rings previously incubated with chemerin (0.5 ng/ml) or vehicle (PBS containing 0.1% BSA) for 1 h.

To verify the involvement of O2 (superoxide anion) generation on the effects of chemerin in ACh-induced vasodilation, cumulative concentration–effect curves to ACh were constructed for endothelium-intact aortic rings in the absence or presence of either the selective O2 scavenger tiron (10−4 mol/l) or the SOD (superoxide dismutase) mimetic tempol (10−3 mol/l), which were added 30 min before the incubation with vehicle or chemerin.

To investigate whether the effects of chemerin in ACh-induced vasodilation are mediated by increased vascular ROS (reactive oxygen species) generation through eNOS [endothelial NOS (NO synthase)] uncoupling, cumulative concentration–effect curves to ACh were constructed for endothelium-intact aortic rings in the absence or presence of the eNOS cofactor BH4 (tetrahydrobiopterin; 10−4 mol/l) 30 min before chemerin incubation. To verify the role of NADPH oxidase-dependent mechanisms in the effects of chemerin in ACh-induced vasodilation, cumulative concentration–effect curves to ACh were constructed for endothelium-intact aortic rings in the absence or presence of an NADPH oxidase inhibitor (apocynin; 3×10−5 mol/l), which was added 30 min before the incubation with vehicle or chemerin.

Finally, to evaluate the direct effects of chemerin on VSMCs, cumulative concentration–effect curves to SNP (10−10–3×10−5 mol/l) and BAY 412722 (10−10–3×10−5 mol/l) were constructed for aortic rings previously incubated with chemerin (0.5 or 5 ng/ml) or vehicle (PBS containing 0.1% BSA) for 1 h.

Western blot analysis

Endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h were subsequently frozen in liquid nitrogen and homogenized in a buffer (50 mmol/l Tris/HCl, 150 mmol/l NaCl, 1% Nonidet P40, 1 mmol/l EDTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 1 mmol/l sodium orthovanadate, 1 mmol/l PMSF and 1 mmol/l sodium fluoride). The proteins were extracted (80 μg) and separated by electrophoresis on 8% or 12% polyacrylamide gels, and transferred on to nitrocellulose membranes. Non-specific binding sites were blocked with 5% BSA in TBS containing 0.1% Tween 20 (for 1 h at 24°C). Membranes were incubated with antibodies (at the indicated dilutions) overnight at 4°C. Antibodies were used as follows: anti-eNOS (1:500 dilution; Cell Signaling Technology), anti-phospho-eNOS (1:250 dilution; Millipore), anti-GCα (1:500 dilution; Abcam), anti-GCβ (1:500 dilution; Sigma), anti-β-actin (1:3000 dilution; Abcam) and anti-PDE5 (phosphodiesterase 5) (1:500 dilution; Santa Cruz Biotechnology). For the analysis of the dimeric and monomeric forms of eNOS, low-temperature SDS-gel electrophoresis was used with tissue homogenates, as described previously [22]. Membranes were then probed with anti-eNOS antibody (1:300 dilution; Cell Signaling Technology). After incubation with secondary antibodies, signals were obtained by chemiluminescence, visualized by autoradiography and quantified densitometrically. eNOS uncoupling is represented inversely as the ratio of active eNOS dimers to inactive eNOS monomers. Results were normalized to β-actin expression and are expressed as units relative to the control.

ROS measurements in isolated aorta

ROS generation in isolated aorta was assessed by DHE (dihydroethidium), as described previously [23]. Aortas were excised, denuded of periaortic fat and incubated with chemerin (0.5 ng/ml) or vehicle for 1 h in the absence or presence of tempol (10−3 mol/l), BH4 (10−4 mol/l) or L-NAME (NG-nitro-L-arginine methyl ester) (a non-specific NOS inhibitor; 10−4 mol/l), which were added 30 min before the incubation with vehicle or chemerin. After the incubation protocols, aortic sections were embedded in OCT tissue-freezing medium (Leica Microsystems) and stored at −80°C. Fresh-frozen specimens were cross-sectioned at 10-μm thickness and placed on slides covered with poly-(L-lysine) solution. The tissue was loaded with the non-selective dye for ROS detection DHE (5 μmol/l; 30 min at 37°C), which was prepared in Hanks solution (composition: 1.6 mmol/l CaCl2, 1.0 mmol/l MgSO4, 145.0 mmol/l NaCl, 5.0 mmol/l KCl, 0.5 mmol/l NaH2PO4, 10.0 mmol/l dextrose and 10.0 mmol/l Hepes) at pH 7.4 [24]. Images were collected on a Leica DMI 4000B microscope and the results are expressed as the fold change relative to the control.

Determination of cGMP levels

Aortic rings incubated with chemerin (0.5 ng/ml) or vehicle for 1 h were subsequently stimulated with buffer (baseline group, 15 min), PhE (10−6 mol/l for 10 min; negative control)or PhE (for 10 min) SNP (10−5 mol/l for 5 min). Arteries were collected and immediately frozen in liquid nitrogen. cGMP was extracted and quantified using a cGMP enzyme immunoassay kit (Cayman Chemical), as described previously [25]. The weights of the dried pellets were used to standardize the different samples.

Evaluation of NO metabolite (nitrite and nitrate) production

Aortic rings samples were collected in tubes containing 20 mmol/l Tris/HCl buffer (pH=7.4). After centrifugation at 800 g for 5 min, the supernatant was removed and stored at −20°C before being analysed for NOx (nitrite and nitrate) content using an ozone-based chemiluminescence assay [26]. Briefly, samples were treated with a 2:1 volume of ice-cold ethanol (ethanol/sample) and centrifuged at 14000 g for 5 min. NOx was measured by injecting 25 μl of the supernatant in a glass purge vessel containing vanadium (III) in 1 M HCl, which reduces NOx to NO gas. A nitrogen stream was bubbled through the purge vessel containing vanadium (III), then through 1 M NaOH, and then into a NO analyser (Sievers Model 280 NO Analyzer), which detects NO released from NOx for chemiluminescent detection. The samples were normalized by protein levels, and all samples were run in duplicate.

Real-time PCR analysis

Expression of Gch1 (GTP cyclohydrolase I) mRNA was determined in isolated rat thoracic aorta using real-time PCR. The vessels incubated with chemerin (0.5 ng/ml) or vehicle for 1 h were homogenized in lysis buffer and total RNA was isolated with the RNeasy kit (Qiagen), according to the manufacturer's protocol. After isolation, mRNA was reverse-transcribed with MMLV (Moloney murine leukaemia virus) reverse transcriptase and random hexamers (Promega). Quantitative PCR analysis was performed with the ViiA™ 7 system (Applied Biosystems). SYBR green was used for the fluorescent detection of DNA generated during PCR. The PCR was performed in a total volume of 13 μl and 2× SYBR Green master mix (Qiagen). The following primer sequences were used: Gch1 (GenBank® accession number NM_024356.1) forward, 5′-ATTTGTGGGAAGGGTCCAT-3′; reverse, 5′-TTCCACAATCCTGGCAAGT-3′; and β-actin, forward 5′-CTAAGGCCAACCGTGAAAAG-3′; reverse 5′-GGGGTGTTGAAGGTCTCAAA-3′. Experiments were performed in duplicate of each sample the cycle threshold (Ct) values averaged. All Ct values were normalized to β-actin. Results are expressed as the means±S.E.M. of the Ct value obtained.

Aortic smooth muscle cell culture

VSMCs from the aorta were isolated and characterized as described previously [27]. Briefly, arteries were cleaned of adipose and connective tissue, and VSMCs were dissociated by digestion of arteriolar arcades with an enzymatic solution (2 mg/ml collagenase, 0.12 mg/ml elastase, 0.36 mg/ml soya-bean trypsin inhibitor and 2 mg/ml bovine serum albumin type I in Ham's F12 culture medium). Cells were incubated for 45 min at 37°C and then filtered through a 100-μm nylon mesh. The cell suspension was centrifuged at 2000 g and resuspended in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% bovine serum, 2 mmol/l glutamine, 20 mmol/l Hepes (pH 7.4) and antibiotics. VSMCs were identified by determination of α-actin expression using fluorescence microscopy. The absence of endothelial cells was confirmed by assessment of von Willebrand factor mRNA by real-time PCR (results not shown). Subconfluent cell cultures were rendered quiescent by serum deprivation for 24 h before experimentation. Low-passage cells (passages 4–8) from at least four different primary cultures were used in our experiments.

Lucigenin-enhanced chemiluminescence

The lucigenin-enhanced chemiluminescence assay was based on the method described by Chignalia et al. [27]. VSMCs from aortas were incubated with chemerin (0.5 ng/ml) or vehicle for 1 h in the absence or presence of tempol (10−3 mol/l) or apocynin (3×10−5 mol/l), which were added 30 min before the incubation with vehicle or chemerin. After stimulation, cells were washed and harvested in lysis buffer (20 mmol/l KH2PO4, 1 mmol/l EGTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mmol/l PMSF). A portion (50 μl) of the samples were added to a suspension containing 175 μl of assay buffer (50 mmol/l KH2PO4, 1 mmol/l EGTA and 150 mmol/l sucrose) and lucigenin (5×10−6 mol/l). NADPH (10−4 mol/l) was added to the suspension containing lucigenin. Luminescence was measured for 3 min by a luminometer (AutoLumat LB 953) before and after addition of NADPH. The activity measured with buffer alone was subtracted from each test reading to yield net activity, which was expressed as rlu (relative light units) of protein (% of vehicle). The protein levels were used to standardize the different samples.

Drugs

PhE, ACh, SNP, L-NAME, BAY 412272, tempol and tiron were purchased from Sigma. BH4 was from Axxora, and chemerin was purchased from R&D Systems.

Data and statistical analyses

The relaxation in response to ACh, SNP and BAY 412272 is expressed as a percentage of contraction in response to PhE. The individual concentration–response curves were fitted into a curve by non-linear regression analysis. pD2 (defined as the negative logarithm of the EC50 values) and maximal response (Emax) were compared by Student's t tests or ANOVA, where appropriate. Prism software (version 5.0) (GraphPad Software) was used to analyse these parameters, as well as to fit the sigmoid curves. Results are means±S.E.M., with n representing the number of animals used. P values less than 0.05 were considered significant.

RESULTS

Chemerin reduces the ACh vasodilator response by eNOS uncoupling

As shown in Figure 1(A), both of the concentrations of chemerin (0.5 and 5.0 ng/ml) reduced ACh-induced vasodilation (n=5, P<0.05). The impairment in ACh-induced vasodilation by chemerin was prevented by pre-incubation with BH4 (n=5, P<0.05; Figure 1B).

Effects of chemerin on endothelium-dependent relaxation in response to ACh, eNOS protein expression and Gch1 mRNA levels in rat aorta

Figure 1
Effects of chemerin on endothelium-dependent relaxation in response to ACh, eNOS protein expression and Gch1 mRNA levels in rat aorta

Endothelium-intact aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the relaxation in response to ACh in PhE-pre-contracted vessels was evaluated in the absence (A) or presence of the eNOS cofactor BH4 (10−4 mol/l) (B), which were added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. (C and D) Protein expression and analysis of the dimeric and monomeric forms of eNOS in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β-actin expression and are expressed as units relative to the vehicle. eNOS uncoupling was represented inversely as the ratio of active eNOS dimers to inactive eNOS monomers. (E) Gch1 (GTPCH I) mRNA expression. Endothelium-intact aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the Gch1 mRNA expression was assessed by real-time PCR. Results are expressed as the means±S.E.M. of the Ct value obtained. *P<0.05 compared with vehicle, and $P<0.05 compared with BH4+chemerin (n=4–5 per group)

Figure 1
Effects of chemerin on endothelium-dependent relaxation in response to ACh, eNOS protein expression and Gch1 mRNA levels in rat aorta

Endothelium-intact aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the relaxation in response to ACh in PhE-pre-contracted vessels was evaluated in the absence (A) or presence of the eNOS cofactor BH4 (10−4 mol/l) (B), which were added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. (C and D) Protein expression and analysis of the dimeric and monomeric forms of eNOS in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β-actin expression and are expressed as units relative to the vehicle. eNOS uncoupling was represented inversely as the ratio of active eNOS dimers to inactive eNOS monomers. (E) Gch1 (GTPCH I) mRNA expression. Endothelium-intact aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the Gch1 mRNA expression was assessed by real-time PCR. Results are expressed as the means±S.E.M. of the Ct value obtained. *P<0.05 compared with vehicle, and $P<0.05 compared with BH4+chemerin (n=4–5 per group)

Western blot analysis showed that chemerin increased eNOS (Ser1177) phosphorylation (n=5, P<0.05; Figure 1C). However, the eNOS enzyme was present mainly in the monomeric form, as demonstrated by the decreased eNOS dimer (functional eNOS)/monomer (non-functional eNOS) ratio in vessels incubated with chemerin (n=5; Figure 1D), indicating that this adipokine induces eNOS uncoupling.

Considering that BH4 is an essential cofactor for NOS enzymes activity [28,29] and Gch1 is the rate-limiting enzyme for BH4 biosynthesis, we determined Gch1 levels by real-time PCR. Gch1 mRNA levels were significantly decreased in aortas incubated with chemerin for 1 h (n=5–7, P<0.05; Figure 1E).

Chemerin-induced impairment of vasodilation is mediated by increased ROS generation and decreased NO production from uncoupled eNOS

We investigated whether ROS generation mediates the vascular dysfunction induced by chemerin. Both tiron (10−4 mol/l) and tempol (10−3 mol/l) abolished the effects of chemerin on ACh vasodilation (n=5, P<0.05; Figures 2A and 2B).

Involvement of ROS generation in the effects of chemerin

Figure 2
Involvement of ROS generation in the effects of chemerin

(A and B) Cumulative concentration–response curves to ACh in endothelium-intact aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. The relaxation in response to ACh was evaluated in PhE-pre-contracted vessels in the absence or presence of the tiron, an O2 anion scavenger (10−4 mol/l) (A) or tempol, a SOD mimetic (10−3 mol/l) (B), which were added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. Quantitative analysis (C) and representative fluorographs (D) of the DHE-positive nuclei in transverse sections of endothelium-intact rat aortas from Wistar rats. ROS generation was assessed in vessels incubated with chemerin (0.5 ng/ml) or vehicle for 1 h in the absence or presence of BH4 (10−4 mol/l), tempol (10−3 mol/l) or L-NAME (10−4 mol/l), which were added 30 min before the incubation with vehicle or chemerin. DHE scale bar, 50 μm. *P<0.05 compared with vehicle, #P<0.05 compared with chemerin, &P<0.05 compared with tiron+chemerin and P<0.05 compared with tempol+chemerin (n=5 per group).

Figure 2
Involvement of ROS generation in the effects of chemerin

(A and B) Cumulative concentration–response curves to ACh in endothelium-intact aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. The relaxation in response to ACh was evaluated in PhE-pre-contracted vessels in the absence or presence of the tiron, an O2 anion scavenger (10−4 mol/l) (A) or tempol, a SOD mimetic (10−3 mol/l) (B), which were added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. Quantitative analysis (C) and representative fluorographs (D) of the DHE-positive nuclei in transverse sections of endothelium-intact rat aortas from Wistar rats. ROS generation was assessed in vessels incubated with chemerin (0.5 ng/ml) or vehicle for 1 h in the absence or presence of BH4 (10−4 mol/l), tempol (10−3 mol/l) or L-NAME (10−4 mol/l), which were added 30 min before the incubation with vehicle or chemerin. DHE scale bar, 50 μm. *P<0.05 compared with vehicle, #P<0.05 compared with chemerin, &P<0.05 compared with tiron+chemerin and P<0.05 compared with tempol+chemerin (n=5 per group).

As uncoupled eNOS is a source of ROS, we assessed whether the effects of chemerin on ACh-induced vasodilation were mediated by ROS generation. Chemerin increased ROS generation when compared with vehicle-treated vessels (P<0.05; Figures 2C and 2D). In addition, as shown in Figures 2(C) and 2(D), chemerin-induced ROS generation was inhibited by L-NAME (10−4 mol/l), tempol (10−3 mol/l) and BH4 (10−4 mol/l) (n=9–11, P<0.05; Figures 2A and 2B), indicating that uncoupled eNOS mediates chemerin-induced O2 generation.

To determine whether eNOS uncoupling was associated with decreased NO production, we used an NO analyser to measure NO metabolites. Our data show that chemerin decreased NO production when compared with vessels treated only with vehicle (P<0.05; Figure 3). Additionally, as shown in Figure 3, the effects of chemerin on NOx levels were inhibited by both BH4 (10−4 mol/l) and tempol (10−3 mol/l) (n=4–6, P<0.05; Figure 3).

Effects of chemerin on NO production

Figure 3
Effects of chemerin on NO production

Measurement of NO metabolites NOx in endothelium-intact aortic rings also incubated with chemerin or vehicle for 1 h in the absence or in the presence of BH4 (10−4 mol/l) and tempol (10−3 mol/l). Bars represent the means±S.E.M. *P<0.05 compared with vehicle (n=5 per group).

Figure 3
Effects of chemerin on NO production

Measurement of NO metabolites NOx in endothelium-intact aortic rings also incubated with chemerin or vehicle for 1 h in the absence or in the presence of BH4 (10−4 mol/l) and tempol (10−3 mol/l). Bars represent the means±S.E.M. *P<0.05 compared with vehicle (n=5 per group).

Chemerin reduces NO-dependent cGMP signalling in VSMCs

Chemerin (0.5 and 5.0 ng/ml) significantly reduced the vascu-lar sensitivity to SNP (n=5, P<0.05; Figure 4A). The vascular sensitivity to the sGC stimulator BAY 412272 was also decreased in both endothelium-intact (n=5, P<0.05; Figure 4B) and endothelium-denuded (n=5, P<0.05; Figure 4C) vessels incubated with chemerin.

Effects of chemerin on endothelium-independent relaxation in response to SNP and on BAY 412272-induced relaxation in rat aorta

Figure 4
Effects of chemerin on endothelium-independent relaxation in response to SNP and on BAY 412272-induced relaxation in rat aorta

Endothelium-denuded aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the relaxation in response to SNP in PhE-pre-contracted vessels was evaluated (A). Each point represents the means±S.E.M. (B and C) Cumulative concentration–response curves to BAY 412272 in PhE-pre-contracted vessels was performed in both endothelium-intact (B) and endothelium-denuded (C) aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. Each point represents the mean±S.E.M. *P<0.05 compared with vehicle (n=5 per group)

Figure 4
Effects of chemerin on endothelium-independent relaxation in response to SNP and on BAY 412272-induced relaxation in rat aorta

Endothelium-denuded aortas from Wistar rats were incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h and the relaxation in response to SNP in PhE-pre-contracted vessels was evaluated (A). Each point represents the means±S.E.M. (B and C) Cumulative concentration–response curves to BAY 412272 in PhE-pre-contracted vessels was performed in both endothelium-intact (B) and endothelium-denuded (C) aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. Each point represents the mean±S.E.M. *P<0.05 compared with vehicle (n=5 per group)

Protein expression of both sGC subunits α (n=5, P<0.05; Figure 5A) and β (n=5, P<0.05; Figure 5B) was significantly decreased in vessels incubated with chemerin. The effects of chemerin were associated with increased PDE5 protein expression (n=4–5,P<0.05; Figure 5C) and decreased cGMP levels (n=5, P<0.05; Figure 5D) when compared with either SNP- or vehicle-stimulated vessels.

Effects of chemerin on sGC and PDE5 protein expression and cGMP levels

Figure 5
Effects of chemerin on sGC and PDE5 protein expression and cGMP levels

(AC) Protein expression in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β-actin expression and are expressed as units relative to the vehicle. Each point represents the mean±S.E.M. (D) cGMP levels in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h by ELISA. The weights of the dried pellets were used to standardize the different samples. For Western blotting, *P<0.05 compared with vehicle; for ELISA, *P<0.05 compared with baseline and #P<0.05 compared with vehicle (n=5–7 per group)

Figure 5
Effects of chemerin on sGC and PDE5 protein expression and cGMP levels

(AC) Protein expression in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h. Representative Western blots are shown in the upper panels, with quantitative analysis in the lower panels. Results were normalized to β-actin expression and are expressed as units relative to the vehicle. Each point represents the mean±S.E.M. (D) cGMP levels in endothelium-intact aortic segments incubated with chemerin (0.5 ng/ml) or vehicle for 1 h by ELISA. The weights of the dried pellets were used to standardize the different samples. For Western blotting, *P<0.05 compared with vehicle; for ELISA, *P<0.05 compared with baseline and #P<0.05 compared with vehicle (n=5–7 per group)

Chemerin increases ROS generation in VSMCs

Figure 6 shows that lucigenin-derived luminescence was significantly higher (P<0.05) in VSMCs exposed to chemerin (0.5 ng/ml, 1 h) than in cells incubated with vehicle. This effect was reversed by both tempol and apocynin (Figure 6A). The increased NADPH-oxidase-derived O2 generation was paralleled at the functional level because apocynin incubation (3×10−5 mol/l) also reversed chemerin-induced impairment in ACh-induced vasodilation (n=5, P<0.05; Figure 6B).

Chemerin increases ROS generation in VSMCs

Figure 6
Chemerin increases ROS generation in VSMCs

(A) Lucigenin chemiluminescence was assessed in VSMCs incubated with chemerin (0.5 ng/ml) or vehicle for 1 h and in the absence or presence of apocynin (3×10−5 mol/l) and tempol (10−3 mol/l), which were added 30 min before stimulation with chemerin. Luminescence was measured for 3 min before and after addition of NADPH. The activity measured with buffer alone was subtracted from each test reading to yield net activity, which was expressed as rlu of protein (% of vehicle). Bars represent the means±S.E.M. (n=9–11). (B) Cumulative concentration–response curves to ACh in endothelium intact aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. The relaxation response to ACh was evaluated in PhE-pre-contracted vessels in the presence of the NADPH oxidase inhibitor apocynin (3×10−5 mol/l), which was added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. *P<0.05 compared with vehicle, #P<0.05 compared with chemerin and †P<0.05 compared with apocynin+chemerin (n=5 per group).

Figure 6
Chemerin increases ROS generation in VSMCs

(A) Lucigenin chemiluminescence was assessed in VSMCs incubated with chemerin (0.5 ng/ml) or vehicle for 1 h and in the absence or presence of apocynin (3×10−5 mol/l) and tempol (10−3 mol/l), which were added 30 min before stimulation with chemerin. Luminescence was measured for 3 min before and after addition of NADPH. The activity measured with buffer alone was subtracted from each test reading to yield net activity, which was expressed as rlu of protein (% of vehicle). Bars represent the means±S.E.M. (n=9–11). (B) Cumulative concentration–response curves to ACh in endothelium intact aortas from Wistar rats incubated with chemerin (0.5 and 5 ng/ml) or vehicle for 1 h. The relaxation response to ACh was evaluated in PhE-pre-contracted vessels in the presence of the NADPH oxidase inhibitor apocynin (3×10−5 mol/l), which was added 30 min before the incubation with vehicle or chemerin. Each point represents the mean±S.E.M. *P<0.05 compared with vehicle, #P<0.05 compared with chemerin and †P<0.05 compared with apocynin+chemerin (n=5 per group).

DISCUSSION

The role of adipokines in vascular dysfunction associated with adiposity-related diseases has recently emerged. Adipokines induce genes that synthesize other cytokines and may also alter the production and release of several vascular mediators, which will, in turn, affect vascular function [30]. The fact that chemerin levels are elevated in several conditions that represent risk factors for CVDs, including obesity, the metabolic syndrome and hyper-tension [7,9,10,3133], where the presence of vascular dysfunc-tion has been constantly described, suggests a role for this adipo-kine as a link between adiposity-related disorders and CVDs.

Our group has recently demonstrated [14] that chemerin has direct effects in the vasculature, augmenting vascular responses to contractile stimuli via activation of the MEK/ERK1/2 pathway. Given the importance of NO production and endothelial function in vascular homoeostasis, as well as the modulatory role of chemerin in vascular tone, in the present study we examined the direct acute effects of chemerin on eNOS phosphorylation, NO production, endothelial function and cGMP signalling. Herein, we present the novel findings that chemerin impairs vascular relaxation by reducing NO/cGMP signalling. Potential mechanisms mediating the vascular effects of chemerin include decreased NO production and increased O2 generation from uncoupled eNOS, which in turn are associated with decreased sGC protein activation and cGMP production.

In the vasculature, NO generated by eNOS critically determines vascular tone, as well as vascular wall homoeostasis. Impaired endothelium-dependent vasodilation caused by a dysfunctional eNOS/NO pathway is a key early event in the development of the major risk factors for CVDs, which can be detected before overt vascular structural changes [18]. eNOS catalyses the formation of NO from L-arginine and O2 in a reaction requiring Ca2+/calmodulin, FAD, FMN, NADPH and BH4. A decrease in NO bioavailability may be caused by: (i) reduction in the expression or activity of eNOS, (ii) uncoupling of eNOS, resulting in increased O2 formation, or (iii) degradation of NO by reacting with O2 from other enzymatic sources, resulting in the formation of ONOO (peroxynitrite) [16,17]. In the present study, we provide, for the first time, direct evidence that chemerin impairs vascular NO-dependent relaxation. The paradoxical finding of the concomitant increase in eNOS phosphorylation in vessels incubated with chemerin is in accordance with previous studies showing that cardiovascular risk factors are associated with an increase, rather than a decrease, in eNOS expression [34,35]. The increased expression of eNOS in vascular diseases has been considered a consequence of the excessive production of H2O2, the dismutation product of O2, which can increase eNOS expression through transcriptional and post-transcriptional mechanisms. Accelerated degradation of NO (by its reaction with O2) can also occur in vascular diseases. NO and O2 react readily to form ONOO, which in turn leads to eNOS uncoupling and enzyme dysfunction. In fact, conditions of increased eNOS activation are associated with dysfunctional eNOS, resulting in increased O2 production [35,36]. On the basis of these observations, we can postulate that eNOS itself may be a source of O2 in the presence of chemerin.

Dimerization is required for eNOS catalytic function. In intact eNOS, the dimer catalyses flavin-mediated electron transfer from one monomer to the haem of the other monomer [37]. When sufficient substrate, L-arginine, and cofactor, BH4, are present, intact eNOS dimers couple their haem and O2 reduction to the synthesis of NO. If the flow of electrons within NOS is disturbed, the ferrous–dioxygen complex dissociates and O2, instead of NO, is generated from the oxygenase domain, a process referred as NOS uncoupling. NOS-catalysed reduction of O2 to O2 has been attributed to the failure of the enzyme to form dimers. Indeed, it has been shown that monomers of NOS and even isolated reductase domains have the capacity to reduce molecular O2 to O2 [37]. Our results clearly indicate that chemerin leads to destabilization of the eNOS dimer, impairing the catalytic function of the enzyme, as demonstrated by the decreased eNOS dimer/monomer ratio after exposure to chemerin. eNOS uncoupling has been linked to reduced BH4 availability, and BH4 supplementation generally restores eNOS-mediated NO formation and endothelial function in hypertension, hypercholesterolaemia and diabetes [38,39]. In fact, intracellular BH4 is required to promote and possibly stabilize eNOS protein in the active homodimer form [22]. Our observation that the impairment of Ach-induced vasodilation by chemerin was restored by BH4 indicates that chemerin leads to eNOS uncoupling by decreasing the BH4-dependent stabilization of the eNOS dimer.

The synthesis of BH4 involves a multistep process, where Gch1 is the rate-limiting enzyme required for the conversion of GTP into 7,8-dihydroneopterin 3′-triphosphate. As the first enzyme in the BH4 biosynthetic pathway, Gch1 is constitutively expressed in endothelial cells and is critical for the maintenance of BH4 levels and NO synthesis. Indeed, acute inhibition of Gch1 uncouples eNOS, induces endothelial dysfunction and elevates blood pressure [40]. Gch1 function is regulated at both the mRNA and protein levels. In the present study, we found that chemerin-induced eNOS uncoupling is accompanied by a reduction in Gch1 mRNA expression. Previous studies have investigated the transcriptional regulation of Gch1 by inflammatory cytokines in endothelial cells. In contrast with the findings in the present study, Werner-Felmayer et al. [41] reported that HUVECs (human umbilical vein endothelial cells) exposed to TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ) exhibited increased Gch1 enzymatic activity, along with an intracellular accumulation of BH4. Subsequent studies have demonstrated that this effect was attributable to induction of Gch1 mRNA [42,43]. However, it is important to consider that Gch1 mRNA activation by these cytokines was designed to provide additional support for iNOS (inducible NOS) enzymatic activity, as Gch1 mRNA activation was accompanied by increased iNOS gene expression. Considering that BH4 is an essential cofactor for eNOS activity and Gch1 deficiency uncouples eNOS, chemerin-mediated reduction of Gch1 mRNA might be a crucial mechanism mediating chemerin-induced eNOS uncoupling.

Several studies have provided evidence that reduced BH4 bioavailability is a consequence of oxidative stress, which may lead to excessive oxidation and depletion of BH4 [39,44]. Thus oxidation of BH4 may be a common cause of eNOS dysfunction in vascular diseases. In agreement with this hypothesis, our present findings have demonstrated that chemerin increased ROS generation, an effect that was prevented by O2 dismutation (with tempol) and NOS inhibition (with L-NAME), suggesting a specific role for eNOS in chemerin-induced O2 production. In addition, prevention of chemerin-induced decreased ACh relaxation and NO production by BH4 further confirms that eNOS uncoupling, as a consequence of BH4 deficiency, contributes to reducing NO production and is involved in vascular dysfunction induced by chemerin. The role of increased NO inactivation by O2 as an important mechanism for the impairment of endothelium-dependent relaxations in arteries exposed to chemerin was confirmed by our observations that the SOD mimetic tempol and the O2 scavenger tiron prevented the effects of chemerin on ACh-induced relaxation.

sGC is the main target for NO and accounts for the conversion of GTP into cGMP. Active sGC protein contains a ferrous haem heterodimer composed of α and β subunits, which is activated in response to NO binding to its haem moiety [45]. As such, sGC is crucially involved in the physiology of the cardiovascular system through modulation of vascular tone [46]. We have demonstrated in the present study that chemerin reduced BAY412272- and SNP-induced vasodilation, as well as sGC expression, suggesting that chemerin also impairs the ability of sGC to react with NO and, consequently, reduces NO signalling in VSMCs. These findings were supported further by the observation that chemerin reduces cGMP levels.

NO regulation of the cardiovascular system involves both cGMP-dependent and -independent mechanisms. cGMP, through the activation of cGK, causes smooth muscle relaxation via multiple mechanisms, including decreases in cytosolic Ca2+ concentration and/or Ca2+ sensitivity of contractile proteins [15]. cGMP directly interacts with a family of catabolic PDEs that control cGMP levels in both VSMCs and cardiac myocytes. The PDE family comprises 11 different primary isoenzymes (with a total of 48 isoforms) based on substrate affinity, selectivity and regulation mechanisms. The PDEs, by hydrolysing cGMP, counterbalance the activity of sGC [47]. In the present study, we have provided evidence that chemerin not only reduces NO availability and activity of the NO-sensitive sGC, but also induces PDE5 activation contributing to the desensitization of NO/cGMP signalling.

Haem oxidation of sGC, which occurs in conditions of oxidative stress such as aging [48] and hypertension [49], is considered one of the mechanisms responsible for attenuated NO/cGMP signalling [45]. Furthermore, S-nitrosation of sGC impairs its ability to be activated by NO [50]. Accordingly, our findings show that chemerin significantly increased ROS generation in aortic VSMCs, suggesting that chemerin-induced ROS generation can lead to sGC oxidation, decreasing the ability of sGC to react with NO in the vascular smooth muscle layer. Additionally, the present study demonstrates that apocynin, an NADPH oxidase inhibitor, reverted chemerin-induced ROS generation in aortic smooth muscle cells. Supporting our molecular data, the impairment of ACh-induced vasodilation in vessels incubated with this adipokine was also prevented by apocynin. It is also known that NADPH oxidases are at the centre of the dysfunction of other oxidases, including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction [51]. It is also known that activation of NADPH oxidase subunits mediates eNOS uncoupling and endothelial dysfunction in diabetes [52], and eNOS uncoupling induced by angiotensin II requires upstream activation of NOX [53]. Thus chemerin-induced ROS generation in VSMCs might contribute to further impairing endothelial NO production and bioavailability, indicating that the effects of chemerin on the endothelium may be potentiated by its harmful actions in vascular smooth muscle. More detailed studies will elucidate the exact role of NADPH oxidase on the effects of chemerin.

It is interesting to note that, whereas chemerin induced a leftward shift in the SNP concentration–response curve, as indicated by decreased pD2 values (decreased sensitivity), the adipokine caused a decrease in the maximal response to ACh (decreased efficacy). Although several possible mechanisms may account for this apparent discrepancy, it is important to emphasize that the experimental conditions for the SNP- and ACh-induced relaxation curves were different. Although endothelium intact aortic rings were used to address the effects of chemerin on ACh responses, endothelium-denuded vessels were used in the SNP protocols. SNP has been considered as a NO donor and therefore it does not need the endothelium to promote vasodilation. However, a variety of studies have reported the endothelial modulation of the relaxant responses to this agonist. Considering our important findings that the signalling pathways involved in NO synthesis by the endothelium are the primary mediators of the effects of chemerin on ACh-induced relaxation, SNP was used in denuded vessels to determine whether chemerin could also affect the response of the vascular smooth muscle to NO. Of great relevance, this experimental approach allowed us to demonstrate that chemerin-induced functional changes in the arterial wall may include the coupling between the endothelium and the smooth muscle cells. Accordingly, several studies have identified not only the presence of chemerin receptors on endothelial cells [6,54,55], but also functional responses by chemerin in these cells. Chemerin increases eNOS and Akt phosphorylation and cGMP levels in HUVECs [54,56]; endothelium removal or the addition of a L-NNA (NG-nitro-L-arginine; a NOS inhibitor) further exacerbates the effects of chemerin or chemerin-9 (chemerin receptor agonist) on vascular contractions [6,14]. Together, these data show that endothelial cells may play an important role in modulating the effects of chemerin upon the vasculature and it is possible that the lack of the modulatory effect of endothelial cells is responsible for the difference observed in our present study.

Previous studies have estimated that the plasma and serum concentrations of active chemerin are 50.5 and 72.7 ng/ml respectively in humans and 10.0 and 8.3 ng/ml respectively in mice [21]. Considering that our present study was performed in Wistar rats, also a rodent, the concentration used is close to that found in normal mice. We have determined previously the effects of other concentrations of chemerin (10 and 50 ng/ml), but since the effects produced by chemerin were only slightly enhanced, we chose to use the lowest concentration of chemerin. In mouse models of obesity and diabetes [32,33], serum chemerin levels are elevated, opening the possibility that the effects of chemerin observed in the present study will be further enhanced in these animal models.

In conclusion, the results of the present study demonstrate that chemerin reduces vascular NO/cGMP signalling and decreases vascular relaxation. Potential mechanisms mediating the vascular effects of chemerin include eNOS uncoupling, increased O2 generation and reduced NO production, which in turn is associated with decreased sGC protein activation and cGMP production. Since chemerin peptides are new vasoactive factors released by adipose tissue, they are relevant to arterial function and may represent a connection between obesity and vascular dysfunction.

We thank Dr Paulo Evora for the use of his laboratory and his technician Maria Cecilia for help with the NO analyser.

Abbreviations

     
  • ACh

    acetylcholine

  •  
  • BH4

    tetrahydrobiopterin

  •  
  • cGK

    cGMP-dependent protein kinase I

  •  
  • ChemR23

    chemerin receptor 23

  •  
  • CVD

    cardiovascular disease

  •  
  • DHE

    dihydroethidium

  •  
  • eNOS

    endothelial NOS

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • GC

    guanylate cyclase

  •  
  • Gch1

    GTP cyclohydrolase I

  •  
  • HUVEC

    human umbilical vein endothelial cell

  •  
  • iNOS

    inducible NOS

  •  
  • L-NAME

    NG-nitro-L-arginine methyl ester

  •  
  • MEK

    mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase

  •  
  • NOS

    NO synthase

  •  
  • NOx

    nitrite and nitrate

  •  
  • O2

    superoxide anion

  •  
  • ONOO

    peroxynitrite

  •  
  • PDE5

    phosphodiesterase 5

  •  
  • PhE

    phenylephrine

  •  
  • rlu

    relative light units

  •  
  • rlu

    relative light units

  •  
  • ROS

    reactive oxygen species

  •  
  • sGC

    soluble GC

  •  
  • SNP

    sodium nitroprusside

  •  
  • VSMC

    vascular smooth muscle cell

AUTHOR CONTRIBUTION

Karla Neves, Núbia Lobato, Rita Tostes and Ana Maria de Oliveira participated in the design of the study; Karla Neves, Núbia Lobato, Rhéure Lopes, Fernando Filgueira and Camila Zanotto conducted the experiments; Rita Tostes contributed new reagents or analytical tools; Karla Neves, Núbia Lobato and Rhéure Lopes performed the data analysis; and Karla Neves, Rhéure Alves, Núbia Lobato, Rita Tostes and Ana Maria de Oliveira wrote or contributed to the writing of the paper

FUNDING

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number 2010/02434-0], Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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