of April 3, 2018.This information is current as

Experimental Models of Arthritis7) Receptor, Mas, in−of the Angiotensin-(1

Anti-Inflammatory Effects of the Activation

Silva and Mauro Martins TeixeiraRobson Augusto Souza dos Santos, Ana Cristina Simões e Bader, Lirlândia Pires de Sousa, Tarcília Aparecida da Silva,Vasconcelos Costa, Thales Lages Bicalho Bretas, Michael

VivianThomáz Vieira, Daniela Sachs, Lívia Corrêa Barroso, Kátia Daniela da Silveira, Fernanda Matos Coelho, Angélica

ol.1000314http://www.jimmunol.org/content/early/2010/10/08/jimmun

published online 8 October 2010J Immunol 

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The Journal of Immunology

Anti-Inflammatory Effects of the Activation of theAngiotensin-(1–7) Receptor, Mas, in Experimental Models ofArthritis

Katia Daniela da Silveira,*,† Fernanda Matos Coelho,† Angelica Thomaz Vieira,†

Daniela Sachs,† Lıvia Correa Barroso,† Vivian Vasconcelos Costa,† Thales Lages Bicalho Bretas,†

Michael Bader,‡ Lirlandia Pires de Sousa,†,x Tarcılia Aparecida da Silva,{

Robson Augusto Souza dos Santos,* Ana Cristina Simoes e Silva,‖ and Mauro Martins Teixeira†

Activation of the renin-angiotensin (Ang) system induces inflammation via interaction between Ang II and type 1 receptor on leu-

kocytes. The relevance of the new arm of the renin-Ang system, namely Ang-converting enzyme-2/Ang-(1–7)/Mas receptor, for

inflammatory responses is not known and was investigated in this study. For this purpose, two experimental models were used:

Ag-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Male C57BL/6 wild-type or Mas2/2 mice were

subjected to AIA and treated with Ang-(1–7), the Mas agonist AVE 0991, or vehicle. AdIAwas performed in female rats that were

given AVE 0991 or vehicle. In wild-type mice, Mas protein is expressed in arthritic joints. Administration of AVE 0991 or Ang-

(1–7) decreased AIA-induced neutrophil accumulation, hypernociception, and production of TNF-a, IL-1b, and CXCL1. Histo-

pathological analysis showed significant reduction of inflammation. Mechanistically, AVE 0991 reduced leukocyte rolling and

adhesion, even when given after Ag challenge. Mas2/2 mice subjected to AIA developed slightly more pronounced inflammation,

as observed by greater neutrophil accumulation and cytokine release. Administration of AVE 0991 was without effect in Mas2/2

mice subjected to AIA. In rats, administration of AVE 0991 decreased edema, neutrophil accumulation, histopathological score,

and production of IL-1b and CXCL1 induced by AdIA. Therefore, activation of Mas receptors decreases neutrophil influx and

cytokine production and causes significant amelioration of arthritis in experimental models of arthritis in rats and mice. This

approach might represent a novel therapeutic opportunity for arthritis. The Journal of Immunology, 2010, 185: 000–000.

The renin-angiotensin (Ang) system (RAS) is classicallyconceived as a coordinated hormonal cascade that playsa major role in the control of cardiovascular, renal, and

adrenal functions, mainly through the actions of Ang II (1). How-ever, there is an increasing body of evidence showing the in-volvement of the RAS in inflammatory diseases (2, 3). Indeed,inflammatory cells have several components of the RAS (3, 4),and components of the RAS are upregulated in synovium samplesobtained from rheumatoid arthritis (RA) patients (5, 6). Moreover,

several studies have now shown the ability of Ang II to induceleukocyte migration, chemokine production, and activation of thetranscription factor NF-kB (7, 8). Hence, blockade of Ang II maybe useful in the context of inflammatory diseases, such as RA,in which a complex array of cytokines and leukocytes are in-volved (9). In this regard, few clinical studies have shown thatAng-converting enzyme (ACE) inhibitor (captopril) or AT1 re-ceptor antagonist (losartan) may be useful in the treatment of RA(10–12).In addition to Ang II, there are several other active fragments

of RAS metabolism, including Ang III, Ang IV, and Ang-(1–7)(13–15). In particular, the peptide Ang-(1–7) has been shown topresent several effects that are opposite to those observed afterAng II. Ang-(1–7) is formed by ACE2 (16, 17) and exerts itsactions via the G protein-coupled Mas receptor (18). The Masprotooncogene, first detected in vivo by tumorigenic propertiesoriginating from rearrangement of its 59 flanking region (19, 20),encodes a protein with seven hydrophobic transmembrane do-mains, considered to be an “orphan” G protein-coupled receptor(21). Several in vivo and in vitro studies have demonstrated thatthis receptor binds Ang-(1–7) and is involved in the biologicactions of this heptapeptide (18). Another important tool forstudying the interaction of Ang-(1–7) and its Mas receptor is thecompound AVE 0991 (22). This compound is a nonpeptide and anorally active Ang-(1–7) Mas receptor agonist that mimics theeffects of Ang-(1–7) in several organs, including blood vessels(23, 24), kidneys (25), and the heart (26).The ACE2–Ang-(1–7)–Mas receptor axis has been termed a

counterregulatory axis to control the ACE–Ang II–AT1 receptoraxis (15). This is because several actions of Ang-(1–7) are oppo-

*Department of Physiology and Biophysics, †Immunopharmacology, Department ofBiochemistry and Immunology, Institute of Biological Sciences, xClinical PathologyUnity, Technical College, {Department of Oral Surgery and Pathology, DentalSchool, Institute of Biological Sciences, and ‖Department of Pediatrics, Faculty ofMedicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; and‡Max Delbruck Center for Molecular Medicine, Berlin-Buch, Germany

Received for publication January 28, 2010. Accepted for publication August 28,2010.

This work was supported in part by Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior, Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico,Fundacao do Amparo a Pesquisa do Estado de Minas Gerais, and the EuropeanUnion’s Sixth Framework Programme (Innovative Chemokine-Based TherapeuticStrategies for Autoimmunity and Chronic Inflammation, Project Grant LSHB-CT-2005-518167).

Address correspondence and reprint requests to Dr. Mauro Martins Teixeira, AvenidaAntonio Carlos, 6627- Pampulha, Belo Horizonte, Minas Gerais, Brazil. E-mailaddress: mmtex@icb.ufmg.br

Abbreviations used in this paper: ACE, angiotensin-converting enzyme; AdIA,adjuvant-induced arthritis; AIA, Ag-induced arthritis; Ang, angiotensin; CT, control;IV, intravital microscopy; mBSA, methylated bovine serum albumin; MPO, myelo-peroxidase; RA, rheumatoid arthritis; RAS, renin-angiotensin system; SBP, systolicblood pressure; VE, vehicle; WT, wild-type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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site to the effects of Ang II (15). As Ang II has been shown topossess significant proinflammatory activity and is expressed inperiarticular tissues, we hypothesized that the counterregulatoryaxis may also be present in articular tissues and have anti-inflammatory properties by interfering with neutrophil migrationand cytokine production in vivo. To test our hypothesis, weevaluated Mas expression in arthritic tissues and the effects oftreatment with Ang-(1–7) or a specific Mas receptor agonist, AVE0991, in models of Ag-induced arthritis (AIA) in mice andadjuvant-induced arthritis (AIA) in rats. We also studied thephenotype of Mas-deficient (Mas2/2) mice subjected to AIA. Toconfirm that inhibitory effects of AVE 0991 were Mas receptor-dependent, the compound was given to Mas2/2 mice subjected toAIA.

Materials and MethodsAnimals

Eight- to 10-wk-old male C57BL/6 wild-type (WT) (Mas+/+) or Mas2/2

mice (20–25 g) and female Holtzman rats (140–170 g) were obtained fromthe animal facility of the Universidade Federal de Minas Gerais. Animalswere maintained under temperature-controlled conditions with an artificial12-h light/dark cycle and were allowed standard chow and water ad libi-tum. The study was approved by the local animals Ethics Committee.

AIA in mice

C57BL/6 WT or C57BL/6 Mas knockout mice (Mas2/2) were immunizedwith an intradermal injection of 100 mg methylated bovine serum albumin(mBSA; Sigma, St. Louis, MO), emulsified in 500 mg CFA (Sigma-Aldrich) at the base of the tail (day 0). Two weeks after immunization(day 14), Ag challenge was produced by intra-articular injection of 10 mgmBSA diluted in 10 ml sterile saline into the left knee joint. Control micereceived intra-articular injection of sterile saline (10 ml).

Mice were treated with an i.p. injection (200 ml) of AVE 0991 (0.6, 3.0,and 15.0 mg/kg) (Aventis Pharma Deutschland, Frankfurt, Germany), Ang-(1–7) (Bachem, Torrance, CA) (2 mg/kg), or vehicle (10 mM KOH in 0.9%NaCl) 60 min before and 6 h after Ag challenge (27). Neutrophil re-cruitment in the joint and periarticular tissues, production of TNF-a, IL-1b, and CXCL1, histopathological score, hypernociception, and leuko-cyte–endothelial cell interactions were assessed at 24 h (27). All surgicalprocedures were performed under ketamine and xylazine anesthesia (150mg/kg and 10 mg/kg, respectively) followed by euthanasia.

Rat adjuvant-induced arthritis

Female Holtzman rats were immunized with a single s.c. injection of 0.2 mlmineral oil–water emulsion (10:1, v/v) containing 400 mg dried Myco-bacterium butyricum into the dorsal root of the tail at day 0, as previouslydescribed (28). Control animals were s.c. injected with a single dose of 0.2ml mineral oil–water emulsion (10:1) without M. butyricum in the samesite. Rats were treated daily from day 10 to 16 (28, 29) with AVE 0991 (3mg/kg, orally, 0.5 ml) or vehicle (10 mM KOH in 0.9% NaCl). Peri-articular edema was evaluated daily, and tissue neutrophil infiltration,production of IL-1b and CXCL1, and histopathological analysis wereperformed at day 16 and the animals were killed under sodium thiopentalanesthesia (40 mg/kg).

Mas expression in articular tissues

Western blot analysis for the Ang-(1–7) Mas receptor was performed, asdescribed previously by Sousa et al. (30), in periarticular tissues, whichinclude connective soft tissues that surround bone and cartilage and alsothe synovial membrane, from AIA and control animals. A rabbit poly-clonal anti-Mas Ab (Novus Biologicals, Littleton, CO) was used at thedilution of 1:1000. Immunoreactive bands were visualized with an ECLdetection system (GE Healthcare, Piscataway, NJ), and levels of Mas werequantified by densitometric analysis software (LabImage, Leipzig, Ger-many). Levels were normalized to levels of b-tubulin in the same sampleand results are expressed as Mas/b-tubulin ratio in arbitrary units.

Tissue neutrophil infiltration

The extent of neutrophil accumulation in periarticular tissues was measuredby assaying myeloperoxidase (MPO) activity, as described elsewhere (27).The results in the synovial tissue were expressed as the relative number ofneutrophils.

Intra-articular neutrophil infiltration

The knee cavity of mice subjected to AIAwas washed with PBS (23 5 ml)24 h after Ag challenge. The total number of leukocytes was counted ina Neubauer chamber after staining with Turk’s solution, and differentialleukocyte counts were obtained after staining with May–Grunwald–Giemsa using standard morphologic criteria.

Tissue cytokines

Levels of TNF-a, IL-1b, or CXCL1 in the supernatant of processed per-iarticular tissues were assessed by ELISA in accordance with the proce-dures supplied by the manufacturer (R&D Systems, Minneapolis, MN).Periarticular tissues were obtained from dissection of the knees or pawsand stored at 220˚C until further analysis and processed as previouslydescribed (27, 28). Results were expressed as picograms of cytokine per100 mg of tissue.

Histological arthritis index

The knee joints of mice or paws from rats were dissected, fixed for 24 hin paraformaldehyde (pH 7.2), and decalcified with 14% EDTA (pH 7.2) for7–21 d at room temperature prior to embedding in paraffin. A single expertpathologist, blinded to the study protocol, analyzed 5 mm H&E-stainedsections using light microscopy. The severity of AIA in mice was mea-sured based on the semiquantitative score described by Williams et al.(31). Synovial hyperplasia, pannus formation, cellular exudates, and car-tilage/bone erosion were each scored from 0 (absent) to 3 (severe), whereasthe extent of synovial infiltrate was graded from 0 to 5. All parameterswere subsequently summed to give an arthritis index. The severity ofadjuvant-induced arthritis (AdIA) in rats ranged from 0 to 3 (0, no erosion;3, extensive erosion edema, cellular infiltration, and bone destruction) (28).

Hypernociception

Hypernociception, an index of pain, was measured by a previously validatedelectronic pressure-meter nociception paw test for mice (32). The averageof three measurements of intensity was obtained before and after intra-articular injections and before and after treatments. The results werereported as the change in withdrawal threshold, which was calculated bysubtracting the values obtained after treatment from the first measurement(before treatment).

Systolic blood pressure

Systolic blood pressure (SBP) was measured by the tail-cuff method usingan XBP1000 series rat tail blood pressure system (Kent Scientific, Tor-rington, CT), as described previously (33). The measurement performed24 h before challenge was considered as basal SBP. SBP was then mea-sured 1 h after challenge to compare with basal state (prechallenge andwithout treatment). The final measurements were performed 24 h aftereach treatment administration and the results were reported in mmHg.

Intravital microscopy

To evaluate the interaction between leukocytes and endothelium, a series ofintravital microscopy experiments were carried out as described by Vei-helmann et al. (34) and modified by Coelho et al. (27). Intravital mi-croscopy was performed 24 h after Ag challenge. AVE 0991 (3 mg/kg) wasgiven 30 min before the procedure. This time schedule allows for theevaluation of the direct effects of the drug on the migration process (27).Leukocyte–endothelial cell interaction was quantified by counting thenumber of rolling cells (cells per min) (i.e., the number of leukocytespassing a predefined vessel segment within 60 s) and the cells adhering tothe endothelium (cells per mm2) (i.e., the number of leukocytes attached tothe endothelial wall of a 100-mm vessel segment for at least 30 s).

Edema

Hind paw volume was used as an indicator of paw edema in rats and wasmeasured daily always at the same hour by using a hydroplethysmometer(model 7150; Ugo Basile, Comerio, Italy). Results are reported as changesin paw volume (ml).

Statistical analysis

Statistical analyses were performed by GraphPad Prism software, release 4.0(GraphPad Software, San Diego, CA). All data had normal distributionaccording to the Shaphiro test. Results are expressed as the mean 6 SEM.Differences between groups were evaluated by ANOVA, followed by a Stu-dent–Newman–Keuls test. The level of significance was set at p , 0.05.

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ResultsExpression of Mas in articular tissue

There was significant expression of Mas, as determined by West-ern blot, in the knee joint of control mice. Induction of arthritisdid not induce further increase in Mas expression in articular tis-sues (Fig. 1A).

Anti-inflammatory effect of AVE 0991 or Ang-(1–7) on murineAIA

Injection of Ag in the knee joint of immunized C57BL/6 miceinduced significant accumulation of neutrophils in periarticulartissue, as assessed by MPO assay, and in the articular cavity (Fig.1B). AVE 0991 administration decreased neutrophil accumulationin periarticular tissues in a dose-dependent manner and also de-creased neutrophil accumulation in the joint cavity (Fig. 1B). AVE0991 was more potent at inhibiting neutrophil accumulation intothe joint than into periarticular tissues. Treatment with Ang-(1–7)also decreased neutrophil accumulation into the joints and peri-articular tissues (Fig. 1B), confirming that the Ang-(1–7) and itsanalog, AVE 0991, exert similar effects in articular inflammation.Next, we examined levels of TNF-a, CXCL1, and IL-1b in

periarticular tissues of immunized C57BL/6 mice that receivedinjection of Ag in the knee joint. As seen in Fig. 1C, levels ofthese cytokines increased markedly after Ag challenge. Treatmentwith AVE 0991 or Ang-(1–7) significantly decreased levels ofTNF-a and CXCL1, but only AVE 0991 had a significant effect onlocal production of IL-1b (Fig. 1C).

Histological analysis showed that there was significant cellularinfiltration and synovial hyperplasia and discontinuity in mBSA-immunized and -challenged C57BL/6 mice (Fig. 2B) when com-pared with control animals (Fig. 2A). Administration of AVE 0991(3 mg/kg) decreased histological parameters (Fig. 2C) and re-duced the composite arthritis index (Fig. 2D).

Effect of AVE 0991 on hypernociception in murine AIA

Our previous studies have shown that there is hypernociception thatfollows tissue inflammation and neutrophil influx in the model ofAIA in mice (27). In the present study, Ag challenge of immunizedmice induced significant hypernociception at 24 h after challenge.Administration of AVE 0991 dose-dependently blocked hyper-nociception, an effect that was already maximal at 3 mg/kg (Fig.3A). Ang-(1–7) administration also significantly inhibited hyper-nociception, which accompanied joint inflammation (Fig. 3A).Ang-(1–7) may counteract several cardiovascular effects of Ang

II, including the ability of the latter to induce hypertension (35).Indeed, a few studies have shown that agonists of the Mas receptordid not cause hypotension per se but enhanced the hypotensiveeffects of some vasodilators (23, 36). Because hypotension maydecrease inflammation and hypernociception by decreasing bloodflow through tissues or by having central effects secondary to fallin blood pressure, we evaluated whether treatment with maximaldoses of AVE 0991 would affect blood pressure in C57BL/6 micesubjected to AIA. As seen in Fig. 3B, AVE 0091 had no effect onsystolic blood pressure even when given at 15 mg/kg.

FIGURE 1. Mas receptor expression and effects

of the treatment with AVE 0991 or Ang-(1–7) in

a model of AIA in C57BL/6 mice. Mas protein in

periarticular knee was detected by Western blot and

compared with b-tubulin (A). AVE 0991 (0.6, 3, or

15 mg/kg), Ang-(1–7) (2 mg/kg) or vehicle (VE; 10

mM KOH in 0.9% NaCl) were given 60 min before

and 6 h after challenge with mBSA. The relative

number of neutrophils in periarticular tissue, deter-

mined by myelperoxidase assay, and the neutrophil

number in the synovial cavity (B) were assessed 24 h

after arthritis induction with 10 mg of mBSA or in-

jection of 10 ml of sterile saline (control [CT]) into

the knee joints of immunized mice. The levels of

CXCL1, TNF-a, and IL-1b (C) in the periarticular

tissues were assessed by ELISA at 24 h after arthritis

induction, and the treatments used were: AVE 0991

(3 mg/kg), Ang-(1–7) (2 mg/kg), or VE (10 mM

KOH in 9% NaCl). Bars show the mean 6 SEM

results from eight mice per group. pp , 0.05 when

compared with VE-treated arthritic group; #p , 0.05

when compared with CT.

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AIA in mice with genetic deletion of the Ang-(1–7) Masreceptor

Because the administration of AVE 0991 or Ang-(1–7) preventedneutrophil accumulation, cytokine production, and hyperalgesia in

mice subjected to AIA and these are known Mas receptor agonists,

we next tested the role of the endogenous ACE2–Ang-(1–7)–Mas

receptor axis for arthritis development. To this end, AIA was

conducted in Mas2/2 and Mas+/+ mice. There was no basal dif-

ference in control (animals immunized with mBSA but challenged

with PBS),Mas2/2, andMas+/+ animals (data not show). In Fig. 4,

data in unchallenged animals were pooled and are shown as

control. The genetic deficiency of the Mas receptor was accom-

panied by slight worsening of some of the parameters in AIA.

There was greater accumulation of neutrophils in the knee jointand greater local production of TNF-a and CXCL1 (Fig. 4A, 4B).In contrast, there was similar increase in neutrophils in peri-articular tissues (Fig. 4A), comparable hypernociception (with-drawal threshold, control, 2.0 6 0.5 g; Mas+/+ plus mBSA, 4.9 60.8 g; Mas2/2 plus mBSA, 4.8 6 0.6 g; n = 10, p . 0.05), andno differences in histological arthritis indices were observed(Fig. 4D) .

To confirm that anti-inflammatory effects elicited by AVE 0991in AIAwas mediated by Mas receptor activation, we compared theadministration of AVE 0991 or vehicle (10 mM KOH in 9% NaCl)to Mas2/2 and Mas+/+ animals subjected to AIA. In agreementwith Fig. 1, administration of AVE 0991 (3 mg/kg) to Mas+/+ an-imals induced significant inhibition of neutrophil accumulation

FIGURE 2. Effects of the treat-

ment with AVE 0991 on arthritis

score in mice subjected to AIA.

Representative sections of the knee

joint of control (CT) C57BL/6 mice

(A), immunized and challenged mice

given vehicle (VE) (B), or mice given

AVE 0991 (3 mg/kg) (C). Samples

were obtained at 24 h after induction

of arthritis and were stained with

H&E (black bar, 100 mm). Arrows

indicate synovial membrane. Insets,

Higher magnification (white bars,

20 mm) views of the predominant

inflammatory infiltrate area. Original

magnification 3100 (panels) and

3400 (insets). Arthritis score (D)

was graded in a blinded manner, as

described in Materials and Methods.

Symbols represent results in single

animals, and the trace is medianvalue

for all animals. pp, 0.05 when com-

paredwithVE-treated arthritic group;#p, 0.05 when compared with CT.

FIGURE 3. Effects of the treatment with AVE 0991 on hypernociception and systolic blood pressure in C57BL/6 mice subjected to AIA. Hyper-

nociception (A), an index of inflammatory pain, was evaluated using an electronic pressure meter test before and 24 h after intra-articular injection of mBSA

(vehicle [VE] and AVE groups) or saline vehicle (control [CT]). AVE 0991 (0.6, 3, or 15 mg/kg), Ang-(1–7) (2 mg/kg), or vehicle (VE; 10 mM KOH in

0.9% NaCl) were given 60 min before and 6 h after challenge with mBSA. Results are presented as the change (Δ) in withdrawal threshold (in grams),

calculated by subtracting the 0 time mean measurements from the final time mean measurements (n = 10–15). Systolic blood pressure (B) was evaluated by

tail-cuff plethysmography 24 h before, 1 h after, and 24 h after arthritis induction in vehicle-treated and AVE-treated groups (n = 4). Results are the mean6SEM of four animals in each group. pp , 0.05 when compared with vehicle-treated arthritic group; #p , 0.05 when compared with control.

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into the knee joint (70% inhibition) and in surrounding tissue(60% inhibition) (data not shown). In contrast, AVE 0991 admin-istration did not have any significant effect on neutrophil influx inthe articular cavity of Mas2/2 mice (Mas2/2 mice, AIA plusvehicle, 12.8 6 2.3 3 104 neutrophils/joint; AIA plus AVE 0091,10.6 6 2.63 104 neutrophils/joint; p . 0.05, n = 4) or peri-articular tissue (control joints, 1.2 6 0.3 neutrophils/joint; AIAplus vehicle, 5.2 6 1.4 neutrophils/joint; AIA + AVE 0091, 5.4 61.7 neutrophils/joint; p , 0.05, n = 4). Administration of AVE0991 (3 mg/kg) reduced TNF-a (75% inhibition) (also see Fig. 1)concentration in the periarticular tissues of AIA Mas+/+ mice butcould not alter concentration of this cytokine in AIA Mas2/2 mice(Mas2/2 mice, TNF-a, CXCL1: AIA plus vehicle, 322 6 40 pg/100 mg of tissue; AIA plus AVE0091, 366 6 86 pg/100 mg oftissue; n = 5).

Anti-inflammatory mechanisms of AVE 0991 treatment

Treatment with AVE 0991 blocked local production of theneutrophil-active chemokine CXCL1 (see Fig. 1). Such inhibitioncould account for the local inhibition of neutrophil influx. How-ever, a few studies have shown that components of the RAS mayaffect leukocyte recruitment directly by affecting leukocyte–endothelial cell interactions (37, 38). To evaluate whether acti-vation of the Mas receptor would affect leukocyte–endotheliuminteractions in vivo, we used an intravital microscopy strategy inwhich AVE 0991 was given to mice much after immunization andchallenge but just prior to the intravital procedures (Fig. 5A).Using this protocol, we could avoid an effect of the compound onchemokine production and study its potential direct effect onleukocyte–endothelium interactions. As seen in Fig. 5B, treatmentwith AVE 0991 just prior to the intravital microscopy significantlyreduced leukocyte rolling by 38% and cell adhesion by 53% incomparison with vehicle administration.

Anti-inflammatory effect of AVE 0991 on rat AdIA

Next, we investigated whether treatment with AVE 0991 wouldhave anti-arthritic effects in a more chronic model of arthritis. Tothis end, we chose a model of AdIA in female Holtzman rats that wehave previously shown to be dependent on CXCR2-driven neu-trophil recruitment (39). Treatment was started at day 10 afterarthritis induction to avoid any effect on the immunization phaseof the disease. AdIA in rats is characterized by significant neu-trophil accumulation in affected joints, local production of

FIGURE 4. Induction of arthritis in mice with genetic deletion of the

Ang-(1–7) Mas receptor. Animals were immunized with mBSA (Mas+/+

and Mas2/2 C57BL/6 mice), and arthritis induction was induced by in-

jection of Ag (mBSA, 10 mg) or saline (control [CT], 10 ml) into the knee

joints of immunized mice. Relative number of neutrophils in periarticular

tissue and the neutrophil number in the synovial cavity (A) and levels of

TNF-a and CXCL1 in periarticular tissues (B) were evaluate 24 h after

arthritis induction. Hypernociception (C) and arthritis score (D) was

graded in a blind manner, as described in Materials and Methods. Bars

show the mean 6 SEM results of eight mice per group. In the D, symbols

represent results in single animals and the trace is median value for

all animals. pp, 0.05 when compared with vehicle-treated arthritic group;#p , 0.05 when compared with control.

FIGURE 5. Effects of the administration of the AVE 0991 on interaction

between leukocytes and endothelial cells in the synovial microvasculature

of mice subjected to AIA. AVE 0991 (3 mg/kg) or vehicle (VE; 10 mM

KOH in 9% NaCl) was given i.p. 30 min before intravital microscopy (IV),

as shown in (A). Rolling (B) and cell adhesion (C) of leukocyte to the

synovial endothelium were assessed at 24 h after injection of Ag (mBSA,

10 mg) or sterile saline (control [CT], 10 mg) into the knee joint of im-

munized mice. The flux of rolling cells was measured as the number of

rolling cells passing by a given point in the venule per minute. A leukocyte

was considered to be adherent if it remained stationary for at least 30 s, and

total leukocytes adhesion was quantified as the number of adherent cells

within a 100-mm length of venule. Bars show the mean 6 SEM results of

eight mice per group. pp , 0.05 when compared with vehicle (VE)-treated

arthritic group; #p , 0.05 when compared with CT.

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CXCL1, proinflammatory cytokines (TNF-a, IL-1b), joint edema,and significant joint destruction (Figs. 6, 7). Treatment with AVE0991 blocked all parameters measured. Indeed, there was signif-icant inhibition of neutrophil influx, as assessed by MPO (Fig. 6A)and confirmed by histology (Fig. 7A–C). The treatment amelio-rated the joint damage score as evidenced by synovia integrityand reduction of inflammatory infiltrate and absence of bone andcartilage resorption and pannus formation (Fig. 7D). Consistentwith the overall inhibition induced by the compound, levels ofCXCL1 and cytokines (TNF-a and IL-1b) were decreased intreated animals (Fig. 6B–D).

DiscussionThe present study demonstrates for the first time, to our knowl-edge, that activation of the Ang-(1–7) Mas receptor, which isexpressed in normal and arthritic joints, exerts significant anti-

inflammatory effects in two models of arthritis. In these sys-tems, Mas agonists reduced cell infiltration, local signs of inflam-mation (pain and edema), histological changes, and cytokine re-lease. Overall, genetic deletion of the receptor was associated withslight worsening of some aspects of arthritis in mice. Mechanis-tically, Mas agonists reduce local production of proinflammatorycytokines and directly decrease rolling and adhesion of leukocytesto endothelium at inflamed joints.It is well known that Ang II stimulates the production of che-

mokines and cytokines and expression of cell adhesion molecules,which contribute to the migration of inflammatory cells into sitesof tissue injury (see Ref. 2 for review). Consistent with the latterfinding, blockade of AT1, the major receptor mediating the in-flammatory actions of Ang II, reduces neutrophil recruitment andcytokine release in models of inflammation (12, 37, 38). However,to our knowledge, the expression of Mas receptor in periarticulartissues was demonstrated for the first time in the present study.Furthermore, we show that treatment with AVE 0991 reducedneutrophil accumulation, edema formation, and overall patho-logical score both in murine AIA and rat AdIA. The effects ofAVE 0991 were mimicked by treatment with Ang-(1–7) and wereabsent in Mas-deficient mice, confirming that the agonists wereindeed working via activation of the Mas receptor. Decreased in-flammation after treatment with Mas receptor agonists was ach-ieved at doses that caused no hemodynamic changes. Accordingly,the anti-fibrotic effect of Mas receptor agonists in cardiac cells ofhypertensive rats was also unrelated to blood pressure changes(36). Importantly, experiments in Mas-deficient mice showed thatarthritis tended to be more severe in the latter mice when com-pared with their WT counterparts, suggesting an endogenous roleof Mas receptor in controlling inflammatory responses. However,the phenotype of Mas-deficient mice was not major when com-pared with pharmacological administration of Mas receptor ago-nists, which were able to efficiently control articular inflammation.

FIGURE 6. Effects of the treatment with AVE 0991 in a model of AdIA

in rats. AVE 0991 (3 mg/kg) or vehicle (VE; 10 mM KOH in 0.9% NaCl)

was given orally daily from day 10 until day 16 after induction of disease.

The following parameters were determined at day 16: relative number of

neutrophils (A), chemokine CINC-1 in periarticular tissue (B), and levels

of the cytokines TNF-a (C) and IL-1b (D) in periarticular tissues. Hind

paw volume (E), an indicator of edema, was measured daily. Bars show the

mean 6 SEM results from six rats per group. pp , 0.05 when compared

with vehicle-treated arthritic group; #p , 0.05 when compared with con-

trol (CT).

FIGURE 7. Effects of the treatment with AVE 0991 on arthritis score in

rats subjected to AdIA. Representative photographs of H&E-stained sec-

tions of control (CT) (A), vehicle (VE)-treated (B), and AVE 0991-treated

rats (C) at day 16 are presented in original (scale bar, 200 mm) and higher

magnification (insets; scale bar, 20 mm). Insets in A and C show integrity

of synovial membrane and absence of inflammatory infiltrate. Inset in B

shows bone resorption and pannus formation (arrows). Original magnifi-

cation 3100 (panels) and 3400 (insets). The arthritis score (D) was

graded in a blinded manner, as described in Materials and Methods.

Symbols represent results in single animals, and the trace is median value

for all animals. pp , 0.05 when compared with vehicle-treated arthritic

group; #p , 0.05 when compared with control.

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Altogether, these findings show, to our knowledge, for the firsttime that Mas receptor agonists have substantial anti-inflammatoryactivity in experimental arthritis. However, despite the ability ofAVE 0991 and exogenous Ang-(1–7) to block arthritis efficientlyin a Mas receptor-dependent manner, there does not appear to bemajor role for endogenous activation of this receptor in providingan anti-inflammatory break in the context of arthritis.The decreased accumulation of neutrophils and joint damage

was associated with decreased local production of proinflamma-tory cytokines, including TNF-a and IL-1b, and neutrophil-activechemokines. It is well established that cytokines are responsiblefor the initiation and perpetuation of the inflammatory response inRA as well for the facilitation of neutrophil influx at sites of tissueinjury (40, 41). In this regard, TNF-a has a key role in the path-ogenesis of RA (42) and facilitates the influx of leukocytes, whichmay in turn increase the local production of TNF-a (43). Simi-larly, neutrophil-active chemokines, such as CXCL1, are impor-tant for neutrophil recruitment in animal models, which in turnfacilitate further chemokine production (40, 44). It is clear, there-fore, that proinflammatory cytokines and chemokines cooperatewith neutrophils to create a positive feedback loop that enhancesjoint inflammation and triggers joint damage. Treatment with Masreceptor agonists decreased both neutrophil influx and cytokineproduction, demonstrating that activation of the Mas receptor issufficient for stopping the positive interaction between cytokinesand leukocytes at inducing joint inflammation and damage.The decreased inflammatory response attained by treatment

with Mas receptor agonists was accompanied by amelioration ofarticular function, as seen by decreased hypernociception in micesubjected to AIA at 24 h after challenge, when hypernociceptionreaches its maximum in this model. Pain is a most importantsymptom in arthritic patients, frequently leading to loss of func-tion (45). Previous studies (27, 46) have clearly shown an essentialrole for neutrophils in mediating inflammatory-related hyper-nociception in mice subjected to AIA. Furthermore, there is muchevidence linking CXCL1, TNF-a, and IL-1b and in models ofinflammation in mice (47). Therefore, it appears that the abilityof Mas receptor agonists to decrease hypernociception in thesystem is secondary to the ability of the agonists to decrease theinfiltration of neutrophils and production of proinflammatorycytokines in the joint.Treatment with AVE 0991 greatly reduced neutrophil accu-

mulation in both models of arthritis. In addition to inhibiting neu-trophil influx due to the inhibition of local production of chemo-kines, it was possible that Mas agonists could be directly interferingwith the process of leukocyte–endothelial cell interaction, hencepreventing their influx. In support of the latter possibility, otherstudies have found that Ang II via AT1 receptor stimulatedleukocyte–endothelium interactions and increased neutrophil mi-gration in vivo (37, 38). In our study, treatment with the Masreceptor agonist AVE 0991 significantly inhibited rolling and celladhesion of leukocytes to endothelial cells in the joint microvas-culature of mice subjected to AIA. These results clearly demon-strate that Mas receptor agonists directly interfere with leukocyte–endothelium interactions, as the compound was given just prior tothe procedure and, hence, after the proinflammatory mediators hadbeen produced. Our studies cannot, however, determine whetherleukocytes or endothelial cells are the major site of the action ofMas receptor agonists. Previous studies have shown that the Masreceptor is expressed on endothelial cells (48) and certain leuko-cytes (49). Therefore, although Ang II via AT1 receptor stimulatesleukocyte–endothelium interactions and increases the neutrophilmigration (37, 38), Ang-(1–7) action on the Mas receptor couldcounteract these effects by inhibiting neutrophil rolling and cell

adhesion to microvessels. In several systems, especially in thecardiovascular and renal systems, the actions of Ang II are fre-quently opposite to those of Ang-(1–7) on the Mas receptor. Itappears that the actions of these two systems are also opposite inthe context of inflammation, and the major function of Mas re-ceptor is to dampen inflammation.Note that RA and other inflammatory diseases are considered

independent risk factors for cardiovascular diseases (50–53). Theseinflammatory diseases may enhance expression of proinflam-matory cytokines (TNF-a and IL-6), T cell activation (with anincreased Th1/Th2 cell ratio), and local expression of cell adhe-sion molecules and endothelin (52, 54, 55), factors associated withcardiovascular disease. Recent studies (56) have shown the ben-eficial effects of Ang II AT1 receptor antagonists in patients withcardiovascular and inflammatory-associated conditions. Our studyraises the possibility that, in addition to Ang II antagonism, theactivation of the ACE2–Ang-(1–7)–Mas receptor axis might be-come an alternative approach to treat inflammation in the con-text of cardiovascular disease.In conclusion, this study shows that Mas receptor agonists have

potent anti-inflammatory effects in two animal models of arthritis.Mechanistically, inhibition of leukocyte accumulation was due tolocal inhibition of cytokine production and a direct inhibition ofleukocyte–microvascular endothelium interactions. Inhibition ofinflammation was accompanied by functional improvement of thejoint, as seen by reduced joint hypernociception. Mas receptoragonists may represent a novel therapeutic opportunity for thetreatment of arthritis in humans.

DisclosuresThe authors have no financial conflicts of interest.

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