direct stimulation of angiotensin ii type 2 receptor reduces...

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European Journal of Pharmacology

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Direct stimulation of angiotensin II type 2 receptor reduces nitric oxideproduction in lipopolysaccharide treated mouse macrophagesRebecka Isakssona, Anna Casselbrantb,∗, Erik Elebringb, Mathias Hallbergc, Mats Larhedd,Lars Fändriksba Preparative Medicinal Chemistry, Department of Medicinal Chemistry, Uppsala University, BMC Box 574, SE-751 23, Uppsala, SwedenbDepartment of Gastrosurgical Research and Education, Sahlgrenska Academy, University of Gothenburg, SE-413 45, Gothenburg, Swedenc The Beijer Laboratory, Department of Pharmaceutical Biosciences, Uppsala University, BMC Box 591, SE-751 24, Uppsala, Swedend The Beijer Laboratory, Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, BMC Box 574, SE-751 23, Uppsala, Sweden

A R T I C L E I N F O

Keywords:Renin-angiotensin-aldosterone systemBioassayFunctional activityAntagonistAgonistInducible nitric oxide synthase

A B S T R A C T

The angiotensin II type 2 receptor (AT2) is upregulated after tissue damage and mediates protective functions inthe renin-angiotensin-aldosterone system (RAAS). One of these is to inhibit inducible nitric oxide synthase(iNOS) in activated macrophages. In the present study, we assessed the effect of AT2 receptor ligands on nitricoxide production in murine macrophages as a potential assay to determine the functional activity of an AT2receptor ligand. Mouse macrophage J744.2 and RAW264.7 were cultivated in lipopolysaccharide (LPS) to in-duce M1 differentiation and increase iNOS expression. Using Griess reagent and spectrophotometric analysis, thenitric oxide levels were determined, while employing Western blot and immunocytochemistry to determinebasal protein expression.

Using the first reported selective non-peptide AT2 receptor agonist, compound C21, we conclude that acti-vation of AT2 receptor reduces nitric oxide production in M1 macrophages. Furthermore, the AT2 receptorselective ligand compound C38, a regioisomer of C21, reported as a selective AT2 receptor antagonist exhibits asimilar effect on nitric oxide production. Thus, we propose C38 acts as a partial agonist in the macrophagesystem. Monitoring nitric oxide attenuation in M1 J744.1 and RAW264.7 macrophages provides a new methodfor characterizing functional activity of AT2 receptor ligands, foreseen to be valuable in future drug discoveryprograms.

1. Introduction

The octa-peptide angiotensin II (AngII) is the major effector of therenin-angiotensin-aldosterone system (RAAS). RAAS is well known forits role in blood pressure regulation and fluid-electrolyte balance, aneffect exerted by the AngII type 1 receptor (AT1) (Raizada et al., 1993).AngII also binds to the AngII type 2 receptor (AT2), identified in the late1980s (Chiu et al., 1989; Whitebread et al., 1989). In adults, the AT2receptor is mainly expressed in specific tissues such as uterus, adrenalgland, smooth muscle, heart, and kidney (De Gasparo et al., 2000; TheHuman Protein Atlas, 2018). During tissue damage the AT2 receptor isupregulated (Altarche-Xifro et al., 2009; Busche et al., 2000; Gallinatet al., 1998; Li et al., 2005; Nakajima et al., 1995; Nio et al., 1995), andit has been shown to inhibit cell proliferation, cause vasodilation, ex-hibit neuronal protective and regenerative properties, be involved inapoptosis, and modulate inflammation (Lu et al., 2004; Ruiz-Ortega

et al., 2000; Steckelings et al., 2010; Sumners et al., 2015; Suzuki et al.,2003; Uhal et al., 1998; Unger et al., 2015; Wang et al., 1998; Yamadaet al., 1996). In recent years, the AT2 receptor has emerged as a pro-mising new drug target (Foulquier et al., 2013; Padia and Carey, 2013;Unger et al., 2015).

The first selective non-peptide agonist of the AT2 receptor, com-pound C21 (Wan et al., 2004) (Fig. 1) has been extensively studied invivo (Hallberg M. et al., 2018; Larhed et al., 2016) and recently enteredphase II clinical trials for the indication idiopathic pulmonary fibrosis(Sumners et al., 2019). The AT2 receptor antagonist EMA401 (Fig. 1),has been evaluated clinically as a potential treatment for neuropathicpain (Smith et al., 2013; Rice et al., 2014). These varied clinical in-dications, related to the ligands’ functional activity, highlight the im-portance of determining the functional response of ligands binding tothe AT2 receptor.

The agonistic property of C21 was initially shown in an in vivo

https://doi.org/10.1016/j.ejphar.2019.172855Received 11 October 2019; Received in revised form 4 December 2019; Accepted 9 December 2019

∗ Corresponding author.E-mail address: [email protected] (A. Casselbrant).

European Journal of Pharmacology 868 (2020) 172855

Available online 16 December 20190014-2999/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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bicarbonate secretion assay in rat duodenum (Johansson et al., 2001;Wan et al., 2004). Subsequently, the agonistic function of C21 wasconfirmed using a neurite outgrowth assay in NG108-15 cells (Buissonet al., 1992; Gendron et al., 2003; Laflamme et al., 1996; Wan et al.,2004). Due to the time- and cost-consuming nature of both these assaysonly a few compounds have since been evaluated (Hallberg M. et al.,2017). The AT2 receptor selective compound C38 (Murugaiah et al.,2012) (Fig. 1) exerted antagonistic properties in the neurite outgrowthassay. In contrast to C21, that stimulated the mitogen activated proteinkinases p42/p44mapk, C38 inhibited the AngII activation of p42/p44mapk

and blocked AngII-induced neurite outgrowth. Thus, C38 acted similarto the established AT2 receptor antagonist PD123,319 in this model(Blankley et al., 1991) (Fig. 1), but at a lower dose (Guimond et al.,2013; Murugaiah et al., 2012).

The focus of this article was to identify a bioassay with potential toallow a higher throughput of AT2 receptor ligands with agonistic proper-ties. The protective function of AT2 receptor has been well established(Unger et al., 2015) and its anti-inflammatory effect have been in-vestigated by several groups. Monocytes diffuse into damaged tissue anddifferentiate to form macrophages that, depending on the micro-environ-mental stimuli, will polarize to either phenotype M1 or M2 (Wynn et al.,2013). The former phenotype secretes inflammatory cytokines and pro-duce free radicals (e.g. nitric oxide (NO)) by the inducible NO synthase(iNOS), and the latter anti-inflammatory cytokines. However, direct sti-mulation of AT2 receptor in lipopolysaccharide (LPS)-triggered monocyteshas been shown to inhibit nuclear factor kappaβ (NF-κβ) and attenuatecytokines interleukin (IL)-6 and IL-10 as well as tumor necrosis factor-α(TNF-α) (Menk et al., 2015; Rompe et al., 2010). Thus, we hypothesizedthat a decreased in NO-production via the iNOS pathway in macrophagesindicates agonistic properties of AT2 receptor ligands (Fig. 2). In aerobesolution NO is quickly converted into nitrite (Ignarro et al., 1993), whichsimplifies the bioassay for evaluate functional activity of AT2 receptor li-gands in an early drug discovery program.

2. Materials and methods

2.1. Cell culture

Mouse macrophage cell line J774.2 and RAW264.7 was purchasedfrom SigmaAldrich (Stockholm, Sweden; cat. no. 85011428 and91062702) and the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal bovine serum, 1% non-essential amino acids,and 100 IU/ml penicillin-streptomycin. All cell culture products werepurchased from Life Technologies Invitrogen AB, Lidingö, Sweden.Cells were grown in a humidified atmosphere at 5% CO2 and 37 °C.

2.2. Treatment and stimulation experiments

The macrophages were grown to a confluence near but below 95%,after which the cells were scraped loose and seeded onto 24-well cell

culture plates at a density of 0.5 × 106 cells/well. Before initiating theexperiments, the macrophages were validated with LPS. When the cellshad grown for 30 h, they were triggered to differentiate into M1 mac-rophages with 0–100 ng/ml of LPS (SigmaAldrich) and 0.2 mM L-ar-ginine (SigmaAldrich) during 16 h. The nitrite concentration was de-termined in the supernatant using the Griess reagent described below.

Selected compounds were subsequently evaluated over 16 h withsimultaneous LPS-activation to M1 differentiation. Time-matched ve-hicle-treated samples served as control. Experiments were performed intriplicates, and each experiment was repeated twice or more. Thismeans that the minimum number of analyses for each substance isn ≥ 6. After 16 h-treatments, the cell count and the viability were es-timated using trypan blue staining and a TC20™ Automated CellCounter (BioRad Laboratories, Hercules CA, USA).

2.3. Quantification of nitrite release

The nitrite (NO2−) concentration in the supernatant was measuredas an indicator of NO production using Griess reagent (SigmaAldrich).After treating the cells according to the outlined protocols, 50 μl of thesupernatant was mixed with 50 μl Griess reagent in a 96-well plate. A 6-

Fig. 1. The selective AT2 receptor li-gands used: the agonist C21 (Wan et al.,2004), C38 reported as an AT2 receptorantagonist (Murugaiah et al., 2012),antagonist PD123,319, and antagonistPD126,055 (EMA400) (Blankley et al.,1991; VanAtten et al., 1993). a Testedin HEK293 cells (Isaksson et al., 2019);b Tested in rat adrenal gland assay(Blankley et al., 1991).

Fig. 2. Proposed description of the LPS activation in relation to AT1 and AT2receptors, and the suggested antagonist activity in the system. LPS binding toToll-like receptor 4 (TLR4) will result in activation of NF-κβ, which in turnupregulates iNOS and several inflammatory cytokines (not shown). iNOS willconvert L-arginine to L-citrulline and form nitric oxide (NO). A selective agonistof AT2 receptor will result in inhibition of NF-κβ and in turn, a negativemodulation of NO should be observed.

R. Isaksson, et al. European Journal of Pharmacology 868 (2020) 172855

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point sodium nitrate standard curve for correlation of nitrite levels wasprepared with concentration ranging from 1.25 to 100 μM. The absor-bance was measured at λ 540 nm using a microplate reader (Epoch,BioTek, Winooski VT, USA). All samples were run as triplicates.

2.4. Western blot analysis

After treatment, the semi-adherent J774.2 and RAW264.7 cells werescraped into 75 μl lysis buffer in protein kinase blocking solution [1%Triton X-100 and 1 M ethylenediaminetetraacetic acid (EDTA) indeionized water] containing a protein kinase inhibitor buffer [10 mMpotassium phosphate buffer (pH 6.8), 10 mM 3-[(3-cholamidopropyl)-dimetylammonio]-1-propande sulphonate (CHAPS), and protease in-hibitor cocktail tablet Complete]. After extraction, the protein levelswere determined using the Bradford method. The protein concentrationwas used as a measure of cell viability (i. e. the number of cells cor-respond to a certain amount of protein regardless of treatment). Theproteins were separated with NuPage 10% Bis-Tris gel electrophoresisusing MOPS SDS buffer (Invitrogen AB). The proteins were transferredto a polyvinyldifluoride transfer membrane (Hybond, 0.45 μm,RPN303F, Amersham Buckinghamshire, UK) using the iBlot dry blot-ting system (Invitrogen AB). The presence of AT1 and AT2 receptors,iNOS, NF-κβ, and GAPDH was determined using antibodies for AT1receptor (rabbit polyclonal Ab; SantaCruz Biotechnology, Dallas TX,USA; cat. no. SC-1173), AT2 receptor (rabbit monoclonal Ab; Abcam,Cambridge, UK; cat. no. ab92445), iNOS (rabbit polyclonal Ab;SigmaAldrich; cat. no. ABN26), NF-κβ (rabbit monoclonal Ab, Abgent,Suzhou, China; cat. no. AJ1537a, AJ1535a), and GAPDH (rabbit poly-clonal Ab; Novus Biologicals, Abingdon Oxfordshire, UK; cat. no.NB100-56875). A HRP-conjugated secondary antibody (Cell SignalingTechnology, cat. no. 7074) was applied for 1 h at room temperature andvisualization was carried out using the WesternBright Quantum re-agents (K-12042, Advansta Corporation, Menlo Park CA, USA). Thesignal intensities of specific bands were detected and analyzed using aChemidox XRS cooled charge-couple device camera and the QuantityOne software (BioRad Laboratories, Hercules CA, USA).

2.5. Immunocytochemistry

To visualize the presence of AT1 and AT2 receptors in the macro-phages, the cells were seeded onto 8-well chamber slides (BioRadLaboratories, Solna, Sweden) at a density of 0.1 × 106 cells/well. Thecells were allowed to grow for 24 h after which they were gently wa-shed with medium and treated with 100 ng/ml LPS and 0.2 mM L-arginine for 16 h. The cells were fixed with phosphate-buffered saline(PBS) containing 4% formaldehyde for 20 min. The fixed cells were firstpermeabilized for 10 min using 0.3% Triton X-100 in PBS, and subse-quently unspecific binding of antibodies was blocked by incubating thecells for 1.5 h with 5% goat serum (Invitrogen, Rockford IL, USA; cat.no. 31872) in PBS with 0.1% Triton X-100. The cells were incubatedwith primary antibodies AT1 receptor (rabbit polyclonal Ab, SantaCruzBiotechnology) or AT2 receptor (rabbit monoclonal Ab, Abcam) or 5%goat serum (as negative control) overnight at 4 °C. The incubation withsecondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen;cat. no. A11008) was performed at room temperature for 1.5 h. Thenucleus was stained for 10 min using Hoechst nucleus stain(SigmaAldrich). Between each step outlined above, the cells were wa-shed with 0.1% Triton X-100 in PBS. The slides were sealed with anti-fade reagent ProLong Gold (Invitrogen) and analyzed at 40× magni-fication using a fluorescence microscope (Leica Microsystems, Wetzlar,Germany) equipped with DAPI and FITC filters.

2.6. Compounds evaluated

To evaluate the proposed assay, four suitable selective AT2 receptorligands were used (Fig. 1). Using the confirmed AT2 receptor agonist

C21 (Syntagon, Södertälje, Sweden) the effect of AT2 receptor activa-tion on nitric oxide levels in cells were studied with or without theestablished prototype antagonist PD123,319 (SigmaAldrich) or the AT2receptor antagonist EMA400 (AstaTech Inc., Bristol PA, USA). Theclinically evaluated EM401 is the S-enantiomer of EMA400. To confirmAT2 receptor mediated the observed effect of C21, the cells were treatedwith or without the selective AT1 receptor antagonist Losartan (Sig-maAldrich) or compound A779 (SigmaAldrich). A779 is an antagonistto the Ang (1–7)-ligand sensitive Mas receptor. In addition, the pro-posed AT2 receptor antagonist C38 was evaluated. Ligand C38 wassynthesized according to previously published methods (Murugaiahet al., 2012; Wannberg et al., 2018) and evaluated with or without theprototype antagonist PD123,319 and antagonist EMA400.

2.7. Statistical analysis

Vertical lines in figures indicate standard error of mean (S.E.M.),and data is described in text as “mean± S.E.M.“. Ordinary one-wayvariance analysis ANOVA and two-tailed Student's t-test was used forcomparing group mean, as indicated in figures. Statistical analysis wasperformed using the GraphPad Prism software (GraphPad Software, SanDiego CA, USA), and statistical significance was assumed at P < 0.05.

3. Results

3.1. Cell validation and experimental set-up

The J774.2 and RAW264.7 macrophages were validated for nitriteproduction using LPS. Cells in a passage between 5 and 25 were con-firmed to react dose-dependently to 16 h LPS stimuli (Fig. 3A and B),producing stable and repetitive nitrite levels. The calculated cellnumber, the cell viability, and the protein concentration in the wellsremained constant after varied LPS treatment (Supplementary Table 1).Cells with passages below 5 and above 25 had a lower nitrite responseafter stimulation (see Supplementary Fig. 1). Fig. 3C shows the mac-rophages at the end of the experiment, after LPS pretreatment for 16 h,nearing complete confluence.

3.2. Expression of AT1, AT2 receptors, iNOS, and NF-κβ

The basal expression of important proteins was determined. BothJ774.2 and RAW264.7 macrophages expressed all the desired proteins(AT1, AT2 receptors, iNOS, and NF-κβ, Fig. 3D). Using im-munocytochemistry, the presence of AT1 and AT2 receptors after LPSstimulation was visualized (Fig. 3E). Although the protein levels werenot measured, a notable difference is observed with a higher detectionof AT1 compared to AT2 receptor. This is likely due to the efficacy of theantibody used, although a different basal expression of these two pro-teins could also account for the difference. Key proteins after treatmentwere evaluated by Western blot in relation to LPS-treated control. Theexpression of AT2 receptor was significantly up-regulated while iNOSexpression was significantly down-regulated after LPS and C21-treat-ment. None of these receptors were changed in the macrophages afteraddition of PD123,319, suggesting an AT2 receptor involvement. TheAT1 receptor expression was stably independent of treatment (Fig. 3F).

3.3. Effect of C21 on the nitric oxide production

Using the selective AT2 receptor agonist C21 the effect of AT2 re-ceptor activation on NO production in J774.2 macrophages could bestudied. Comparing the measured nitrite levels to a time-control oneach plate, set to 100%, enabled comparison between experiments.Treating the cells with 50 ng/ml LPS and C21 at doses 1, 10, and100 μM resulted in a linear dose-dependent attenuation of nitrite(Fig. 4A). At 1 μM of C21 the nitrite levels were reduced to 79 ± 2%,10 μM C21 reduced to 48 ± 2% and 100 μM almost completely


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Fig. 3. Dose-dependent effect of LPS on nitrite levels (indirect measure of NO release) in J774.2 (A) and RAW264.7 (B) macrophages stimulated for 16 h. RAW264.7macrophages nearing confluence were observed through contrast microscope at 10 × magnification (C). Immuno-blot analysis of basal expression of iNOS, AT1 andAT2 receptors, and NF-κβ, in untreated and LPS-treated (50 ng/ml) J774.2 and RAW264.7 macrophages (D), detection of GAPDH served as a loading control.Presence of AT1 or AT2 receptors visualized in LPS-treated RAW264.7 macrophages using immunocytochemistry (E). Expression analysis of AT1 and AT2 receptors,and iNOS in LPS-treated (50 ng/ml), in LPS and C21-treated (10 μM), in LPS, C21 and PD123,319-treated (10 μM) macrophages (F). Data from five independentexperiments were normalized to LPS-treated (set to 100%). *P < 0.05; data is presented as mean ± S.E.M.

Fig. 4. The effect of AT2 receptor agonist C21 (1–100 μM) on nitrite as compared to control. The J774.2 macrophages (A) and the RAW264.7 macrophages (B) wereincubated with 50 ng/ml LPS and C21 at varying doses for 16 h after which the nitrite levels were measured. The effect of C21 1 μM and 10 μM could be blocked bytreatment with 10 μM PD123,319 (C) or 10 μM EMA400 (D) in J774.2 cells. The effect of C21 1 μM could not be blocked by simultaneous treatment with neither theAT1 receptor antagonist Losartan receptor nor Mas antagonist A779 in J774.2 cells (E). ***P < 0.001; **P < 0.01; *P < 0.05; ns = no significance (P > 0.05);data is presented as mean ± S.E.M., two-tailed Student's t-test.


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blocked the nitrite level to 10 ± 1%. A similar reductive effect on thenitrite levels was observed when treating RAW264.7 macrophages withvarying doses of C21 (Fig. 4B). The effect of C21 in J774.2 cells couldbe significantly blocked by 10 μM PD123,319 at the two lowest dosestested (Fig. 4C). Simultaneous treatment with 10 μM EMA400 inJ774.2 cells also had the ability to block the nitrite level at the twolowest concentrations of C21 (Fig. 4D). To confirm the effect of C21 wasnot linked to AT1 receptor or Mas receptor, the J774.2 macrophageswere treated with 10 μM of AT1 receptor antagonist Losartan or 10 μMof Mas receptor antagonist A779 in the presence of C21 1 μM. Neitherpretreatment with Losartan nor A779 could prevent the C21-triggeredAT2 receptor derived attenuation of nitrite (Fig. 4E). Despite majorchanges in NO-production, the calculated cell number, the cell viability,and the protein concentration in the wells was the same after varioustreatments (Supplementary Table 1).

3.4. Effect of C38 on the nitric oxide production

The proposed AT2 receptor antagonist C38 was investigated next(Fig. 5A). Surprisingly, treating the cells with C38 reduced the nitritelevels, similar to the effect of C21, indicating an agonistic function ofthe ligand in this assay. At the lowest concentration of C38 (1 μM) thenitrate levels were reduced to 69 ± 5%, 10 μM of C38 reduced nitritelevels to 43 ± 4%, and 100 μM brought the levels to 14 ± 1%.Treating RAW264.7 cells with C38 resulted in a similar dose-dependentreduction of nitrite (Fig. 5B). The effect of C38 in J774.2 cells could besignificantly blocked by both PD123,319 and EMA400 (10 μM of both)at the two lowest doses tested (Fig. 5C and D respectively). Despite thelarge shifts in NO-production, the calculated cell number, the cell via-bility, and the protein concentration in the wells was the same aftervarious treatments (Supplementary Table 1).

4. Discussion

Stimulating macrophages with LPS is known to result in NO releaseand production of pro-inflammatory cytokines. This effect results fromLPS activation of toll-like receptor 4 (TRL4), causing stimulation of NF-κβ that in turn is linked to regulation of inflammatory cytokines, as wellas TNF-α. It is further well established that NF-κβ regulate cell ex-pression of iNOS, which converts L-arginine to L-citrulline while pro-ducing NO. Direct stimulation of AT2 receptor has been shown to in-hibit NF-κβ and attenuate IL-6, IL-10, and TNF-α, while direct AT1receptor stimulation has displayed the opposite effect and upregulatedthe same proteins (Dhande et al., 2015; Guo et al., 2011; Menk et al.,2015; Rompe et al., 2010).

The present study, performed in J774.2 and RAW264.7 mousemacrophages, revealed that direct AT2 receptor stimulation with theselective agonist C21 decreases NO release in LPS-activated cells.Western blot analysis confirmed that direct AT2 receptor stimulationwith C21 increased the AT2 receptor levels. The simultaneously de-creased iNOS expression may, at least in part, explain the reduced NOoutput. The present study further revealed that the proposed AT2 re-ceptor antagonist C38 display a similar attenuating effect on NO re-lease, indicating this ligand acts as an agonist in this assay.

This contrasts to the C21 activating AT2 receptor causing vasodi-lating in human aortic endothelial cells (Peluso et al., 2018), and itsanti-fibrotic effect in pulmonary fibrosis, both mechanisms via eNOSresulting in NO release (Sumners et al., 2019). While the eNOS (via Aktsignaling) and iNOS (via protein phosphatases inhibiting NF-κβ) path-ways are different, it is interesting that the selective AT2 receptoragonist C21 can have such opposing effects on NO release in differentcells (Rompe et al., 2010). However, the precise cellular mechanismunderlaying the anti-inflammatory effects of AT2 receptor are not fullyunderstood. Using NO release in J774.2 and RAW264.7 macrophages,

Fig. 5. The effect of AT2 receptor ligand C38 (1–100 μM) on nitrite as compared to control. The J774.2 macrophages (A) and RAW264.7 macrophages (B) wereincubated with 50 ng/ml LPS and C38 at varying doses for 16 h after which the nitrite levels were measured. The effect of C38 1 μM and 10 μM could be blocked bysimultaneous treatment with 10 μM PD123,319 (C) or 10 μM EMA400 (D) in J774.2 cells, confirming the agonistic effect. **P < 0.01; *P < 0.05; data is presentedas mean ± S.E.M., two-tailed Student's t-test.


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measured with Griess reagent and spectrophotometric analysis, to testAT2 receptor ligands functional activity presents as a feasible biologicalassay, allowing a higher throughput of ligands than those previouslypresented.

The initial experiments were designed to confirm that the macro-phages had differentiated to the M1 phenotype, produced NO, and thatthe cells in this phenotypic state express desired proteins (i.e. AT1 andAT2 receptors, NF-κβ, and iNOS). Adding LPS to the medium resulted inphenotypic differentiation, giving dose-dependent NO production, andthe macrophages expressed the proteins needed to perform the sub-sequent experiments. Cells in passage 5–25 were identified as pheno-typically stable, a trend that was also observed by Taciak et al. (2018).The doses applied herein are in the micro-molar range, and althoughloss of viability could explain the ability of C21 and C38 to attenuatethe NO production, the calculated cell number, the cell viability mea-surements, as well as the stable protein concentrations seen acrossprotocols make this an unlikely explanation.

Treating the cells simultaneously with selective agonist C21 and LPSto trigger M1 phenotypic differentiation produced linear dose-responseattenuation of NO. The linearity in dose-response confirm that whileLPS continuously activate TLR4 and NF-κβ will be continuously ex-pressed, which in turn results in AT1 and AT2 receptors remaining at (ornear) the cell surface. The high attenuation at the highest doses of C21(100 μM) is likely related to the surface (or near surface) expression ofAT2 receptor resulting from the constant signaling from TLR4 to NF-κβ.Blocking the effect of C21 with simultaneous treatment with 10 μM ofPD123,319 was successful at the two lowest doses of C21 (1 and 10 μM)in J774.2 cells. The reported racemic antagonist EMA400 (10 μM),structurally related to PD123,319, could also block the effect of C21.This confirms the antagonistic character of this ligand in the J774.2macrophages. The observed effect was confirmed to derive from AT2receptor activation as treatment with an AT1 receptor or Mas blockerdid not affect the reduced NO expression.

The proposed AT2 receptor antagonist C38 displayed similar, ago-nistically linked, attenuation of NO as agonist C21. The agonisticcharacter of C38 in this assay was confirmed with simultaneous treat-ment with 10 μM PD123,319 or EMA400 that resulted in a completerecovery of NO release for the two lower doses in J774.2 macrophages.It is possible that agonistic properties of C38 could not be discerned inthe neurite outgrowth assay used previously due to assay limitationsand thus, C38 seemingly displayed antagonistic properties.Anotherpossible explanation could be different signaling pathways in the twoassays (neurite outgrowth cf. nitrite production), resulting in variedfunctional activity of the same ligand between the two.

While several authors conclude that activating AT2 receptor willgive an anti-inflammatory response (Dhande et al., 2015; Menk et al.,2015; Rompe et al., 2010; Sampson et al., 2016), in correlation with ourpresented findings, it should be noted that contrasting findings haverecently been presented. C21 was recently reported to be without effecton aortic aneurysm in a mouse model of Marfan Syndrome (Verbruggheet al., 2018). Moreover, in 2018 Shepherd et al. concluded that AT2receptor activation in macrophages trigger production of reactiveoxygen/nitrogen species (ROS/RNS, i.e. NO), in contrast with previousresearch and data presented in this article. Shepard et al. (2018) in-vestigated the effect of the clinical candidate AT2 receptor antagonistEMA401, the S-enantiomer of EMA400 (Blankley et al., 1991; VanAttenet al., 1993), and found its effect to be mediated via macrophages in-filtrating the injured nerves, and not a direct interaction of EMA401with AT2 receptor in the damaged nerves (Shepherda et al., 2018;Shepherdb et al., 2018). Shephardab et al. (2018) found that increasedlevels of ROS/RNS cause cysteine modification of the transient receptorpotential ankyrin 1 (TRPA1) channel in the dorsal-root ganglia (DRG)sensory neurons, resulting in increased hypersensitivity. Blocking AT2receptor activation reduced ROS/RNS and in turn reduced hypersensi-tivity (Shepharda et al., 2018). This is in contrast with our findings thatAT2 receptor activation in macrophages decreases the levels of NO.

Racemic EMA401 (EMA400) does exhibit antagonistic effects in ourmacrophage assay, resulting in maintained NO production whenblocking AT2 receptor activation. We acknowledge that the general-izability of in vitro in relation to in vivo data is highly limited. Still,monitoring NO-liberation from macrophages in vitro presents as asimple and useful bioassay, allowing rapid throughput of interestingligands.

4.1. Conclusion

From the studies described herein, we conclude that AT2 receptoractivation can attenuate iNOS-derived NO release in M1 phenotypicJ774.2 and RAW264.7 macrophages, and thus NO release could bemeasured as an indicator of an AT2 receptor ligands functional activity.The agonistic character of phase II clinical candidate C21 was con-firmed. The functional characteristics of the structurally related ligandC38 was previously reported as antagonistic however, when applyingC38 in the macrophage assay the ligand acts as a partial agonist. Theidentified protocol enables a higher throughput than previously re-ported assays for functional determination of ligands for AT2 receptor.

Declaration of competing interest

None.

Acknowledgements

The authors thank Christina Ek for practical support with datacollection. The authors thank Kjell and Märta Beijer Foundation,Sahlgrenska University hospital, and H.F. Sederholm Foundation forfinancial support.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejphar.2019.172855.

Authorship of the paper

LF-Conceptualization; AC, EE, LF-Methodology and validation; RI,AC, EE- Investigation; RI, MH, ML-Resources RI, AC-Writing-originaldraft; EE, MH, ML, LF-Writing-review & editing.

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Direct stimulation of angiotensin II type 2 receptor reduces nitric oxide production in lipopolysaccharide treated mouse macrophagesIntroductionMaterials and methodsCell cultureTreatment and stimulation experimentsQuantification of nitrite releaseWestern blot analysisImmunocytochemistryCompounds evaluatedStatistical analysis

ResultsCell validation and experimental set-upExpression of AT1, AT2 receptors, iNOS, and NF-κβEffect of C21 on the nitric oxide productionEffect of C38 on the nitric oxide production

DiscussionConclusion

mk:H1_17AcknowledgementsSupplementary dataAuthorship of the paperReferences

direct stimulation of angiotensin ii type 2 receptor reduces...

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