International Immunopharmacology 19 (2014) 308–316

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International Immunopharmacology

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Antibodies against the second extracellular loop of β1-adrenergicreceptors induce endothelial dysfunction in conductance and resistancearteries of the Wistar rat

M.A. Abdelkrim a,b, D. Leonetti a,d, E. Montaudon a,b, G. Chatagnon a,c, M. Gogny a,b, J.-C. Desfontis a,b,J. Noireaud a,d, M.Y. Mallem a,b,⁎a L'UNAM Université Nantes Angers Le Mans, Franceb Oniris, “UPSP 5304 de physiopathologie animale et de pharmacologie fonctionnelle”, Atlanpole-La Chantrerie, BP 40706, Nantes F-44307, Francec Oniris, “Biotechnologie Pathologie Reproduction/RSB”, Atlanpole-La Chantrerie, BP 40706, Nantes F-44307, Franced Inserm UMR 1063, IBS-IRIS, rue des Capucins, F-49100 Angers, France

⁎ Corresponding author at: “UPSP 5304 de physiopatholofonctionnelle” Atlanpole-La Chantrerie, BP 40706, Nantes F-77 85; fax: +33 240 68 77 42.

E-mail address: yassine.mallem@oniris-nantes.fr (M.Y

1567-5769/$ – see front matter © 2014 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.intimp.2014.01.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form 13 December 2013Accepted 31 January 2014Available online 13 February 2014

Keywords:AutoantibodyImmunizationβ-AdrenoceptorEndothelium dysfunctionNitric oxideHeart failure

Autoantibodies against β1-adrenoceptors (β1-ARs) have been detected in the serum of patients with variouscardiac diseases; however, the pathological impact of these autoantibodies (β1-AABs) has only been evaluatedin cardiac tissue. The purpose of the present study was to evaluate whether β1-AABs have deleterious effectson vascular reactivity in rats.An enzyme-linked immunosorbent assay was used to detect β1-AABs in sera from immunized rats over a period of1–3 months using the peptidic sequence of the second extracellular loop of human β1-AR. Functional studies wereperformed in thoracic aortic (TA) and small mesenteric artery (SMA) rings from immunized rats. Following pre-contraction with phenylephrine (0.3 μM and 3 μM for the TA and SMA respectively), cumulative concentration–response curves (CCRCs) to various β-AR agonists (isoproterenol, dobutamine, salbutamol, SR 58611A), acetylcho-line, A23187, and sodium nitroprusside (SNP) were then plotted.The relaxations induced by dobutamine, SR 58611A, and acetylcholine were significantly impaired, butsalbutamol-induced relaxationswere not affected, in both vessels from immunized rats. A significant impairmentof isoproterenol-induced relaxation was only observed in SMA. CCRCs to SNP were not modified in either of thevessels. A23187-induced relaxation was impaired in immunized rats. Following pretreatment with L-arginine,vasorelaxation to acetylcholine and SR 58611A was restored in immunized rats.This studydemonstrates that immunization against the second extracellular loop ofβ1-ARs has a deleterious impacton vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signalingpathways.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Circulating autoantibodies directed against the 2nd extracellularloop of the β1-adrenoceptor (β1-AR) and displaying agonistic activityat cardiac β1-ARs have been detected in the serum of patients withcardiac disease. The diseases in question include idiopathic dilatedcardiomyopathy [1], congestive heart failure [2], chronic Chagas heartdisease [3], Graves' hyperthyroidism [4], and to a lesser extent ischemiccardiomyopathy [5]. β1-Autoantibodies (β1-AABs) enhance cardiac cellshortening, prolong action potential duration, and increase L-type calci-um current amplitude in rat ventricular myocytes; these effects havebeen shown to be mediated via β1-ARs [6,7]. β1-AABs induce their

gie animale et de pharmacologie44307, France. Tel.: +33 240 68

. Mallem).

ghts reserved.

agonist-like effect via the β1-AR/adenylate cyclase/cAMP-dependent pro-tein kinase A cascade [8,9]. Sustainedβ1-AR activation via stimulatory β1-AABs, might have a pathogenic action by initiating the disease process orby contributing to the progression of myocardial contractile malfunction[10–13]. However, this is not sufficient to explain heart failure, becausecardiac performance is greatly dependent on the interaction betweenthe left ventricle and the arterial system [14], and any increase in post-load would contribute to cardiac failure.

CardiacAABs fromthe immunoglobulinG (IgG) family, canbe extract-ed using immunoadsorption; several clinical studies performed in pa-tients with dilated cardiomyopathy have demonstrated hemodynamicimprovement following immunoadsorption (and IgG substitution)[15–18]. In a study of nine patients with severe dilated cardiomyopathy,whowerepositive forβ1-AAB, substantial hemodynamic changes, includ-ing reducedmean arterial and pulmonary arterial pressure, and systemicvascular resistance, were found following immunoadsorption [19], butthe specific impact of β1-AAB removal is difficult to quantify.

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It has been clearly shown that the occurrence, frequency, and titer ofβ1-AABs were increased in the sera of spontaneously hypertensive rats(SHR), a model of human essential arterial hypertension, and in exper-imental renovascular hypertension in rats [20,21]. However, to the bestof our knowledge, there are no studies into the effect of β1-AAB onvascular reactivity.

The aim of this study was to analyze the functional impact of circu-lating β1-AAB in vessels taken from rats immunized against the 2ndextracellular loop of β1-AR. Very recently, Flacco et al. [22] discoveredthat the specific β-AR subtype contribution and signaling pathwaysinvolved in the vasodilator response differ between territories. Theirfindings strongly indicate that β-AR have different physiological rolesin the regulation of conductance versus resistance vessels. The presentstudy therefore sought to analyze the effects of β1-AAB in rat thoracicaortic (TA, conductance vessel) and rat small mesenteric (SMA, resis-tance vessel) arteries.

2. Materials and methods

2.1. Animals

Eleven week-old male Wistar rats (370 to 400 g) purchased fromCER Janvier (Le Genest St Isle, France) were used for these experiments.They were housed under controlled conditions of temperature (22 °C)and humidity (50%) and allowed free access to standard chow anddrinking water. After arrival, all rats were acclimatized for at least oneweek before any experiments. All experimental procedures were con-ducted in compliance with local guidelines for the care and use of labo-ratory animals (authorizations B44266, C4983, and D44271). The studywas also in compliance with the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health (NIH Publica-tion, Eighth edition, 2010).

2.2. Peptide preparation, immunization and production of antibodies

A peptide (residues 197 to 222: H–W–W–R–A–E–S–D–E–A–R–R–C–Y–N–D–P–K–C–C–D–FV–T–N–R) corresponding to the sequence of thesecond extracellular loop of human β1-AR was commercially synthe-sized by GeneCust (Dudelange, Luxembourg). Rats were randomly di-vided into 2 groups. Rats from the first group were immunized bysubcutaneous injection of the peptide (1 mg) dissolved in 1 ml of a so-lution containing Na2CO3 (0.1M) and β-mercaptoethanol 1%, conjugat-ed with Freund's adjuvant (V/V) once a month for 3 months. Rats fromthe second groupwere used as a control and received only saline conju-gated with Freund's adjuvant and purified immunoglobulin G (IgG)taken from non-immunized rats. In both groups, IgG fractions werepurified from sera using the Proteus Protein G kit (AbD SerotecMorphoSys, Kidlington, UK), in accordance with the manufacturer's in-structions, and eluted with PBS (pH 7.4). Stock concentrations of5–15 mg/ml were obtained as determined by the bicinchoninic acidprotein assay (Pierce Protein Research Products®, Thermo FischerScientific, Rockford, USA), and stored at−20 °C.

2.3. Enzyme-linked immunosorbent assay

Peptide (10 μg/ml in BIC buffer: 0.1 M Na2CO3, 0.1 M NaHCO3,100 ml distilled water QSP, pH 9.6) was used to coat individual wellsof a 96-well microtiter plate (polysorp NUNC, Denmark). Rat sera(100 μl), diluted 1/100 (v/v) in PBS–Tween 80–NaCl 0.5 M, wereadded to the coated plates and incubated for 1 h at 37 °C. After 3washeswith PBS–Tween 80 (pH 7.2), 100 μl of donkey anti-rat IgG antibodyconjugated with horseradish peroxidase (712-035-153, JacksonImmunoResearch, USA), diluted 1/10,000 in PBS – Tween 80 – NaCl0.5 M, was allowed to react for 1 h at 37 °C. After 3 washes with PBS–Tween 80, the bound antibodies were detected using 100 μl of 3,3′,5,5′-tetramethylbenzidine (T4444, Sigma-Aldrich). After 20 min, the reaction

was stopped by the addition of 50 μl of sulfuric acid 0.1 M, and the absor-bance at 450 nm was determined using an enzyme-linked immunosor-bent assay reader (Titertek Multiskan® MC, Finland). Specific IgG weretitrated as early as 1 month after starting immunization; IgG titrationwas maximal at 3 months.

2.4. Functional characterization of IgG containing β1-AAB

Normal adult rat ventricular cardiomyocytes were isolated as de-scribedpreviously [23]. The ratswere anesthetizedwithpentobarbitone(Ceva Santé animal, Libourne, France; 60 mg/kg IV) and heparinized(Heparine Choay®, Sanofi Aventis, Paris, France; 2500 IU/kg IV). Theheart was excised and mounted on a Langendorff perfusion apparatus;digestion was performed for 20 min by perfusion with 1 mg/mlcollagenase type II and 0.04 mg/ml protease type XIV (30 μM CaCl2).The atria were then excised and the left and right ventricles separated.Cardiomyocytes were freed by four rounds of mincing and gentle man-ual stirring, before being filtered through sterile nylon gauze (200 μmnylon mesh, Sephar AG, Switzerland) and progressively exposed to in-creasing Ca2+ concentrations in Tyrode solution. The cardiomyocytesin the final suspension were plated onto poly-L-lysine (100 μg/ml)-coated 35 mm culture dishes for 3 h before starting experiments.All substances used for cardiomyocyte isolation were obtained fromSigma-Aldrich except for type II collagenase (Worthington, Lakewood,NJ, USA).

Culture dishes were placed in a custom-made, thermostat regulatedchamber (37± 0.2 °C; TC-344B, Phymep, Paris, France), on amicroscopestage.Myocyteswere super-perfused at a flow rate of 3–4mlmin−1with100% O2 aerated Tyrode solution and electrically stimulated at 1 Hz atabout twice threshold using platinum electrodes. Shortening ofcardiomyocytes was recorded using a video-imaging system (Coy-ote Bay Instruments, Manchester, NH, USA) with a CCD camera, JAI-M30 (120 images/s). On- and off-line analysis was performed usingMatrox Inspector software (Coyote Bay Instruments). Cells selected fordata analysis were rod-shaped with clear striations and a stable diastoliclength at baseline.

Since stimulation ofβ1-ARsmodulates cardiac contractility, wemea-sured the effects of β1-AAB on electrically stimulated cell shortening. Astable baseline twitch amplitude was generally obtained after 10 minof super-perfusion with normal Tyrode solution. When applying IgGcontaining β1-AAB, we observed a dose-dependent increase in cellshortening, proving their ability to stimulate β1-ARs (data not shown).Conversely, IgG obtained from non-immunized animals had no effecton cardiac cell responses (for more complete results, see [23]).

2.5. Contraction–relaxation studies

The rats were anesthetized with pentobarbital (Ceva, Libourne,France; 60 mg/kg body weight intravenous injection (IV)) and eutha-nized by exsanguination. The thoracic aorta (TA) andmesenteric arcadewere removed and placed in ice-cold Krebs solution composed of(mM): NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 20;EDTA, 0.016; D-glucose, 11.1 and CaCl2, 2.5 (pH 7.4).

TA and branches of SMA were carefully removed and cleaned of fatand connective tissue. Segments of the vessels were cut into rings(2–3 mm long for TA and 1.6–2.0 mm long for SMA). TA rings weresuspended on stainless-steel wires in a 5 ml organ bath containingKrebs solution. The bath was maintained at 37 ± 0.5 °C and the Krebssolution was continuously oxygenated with a 95% O2, and 5% CO2 gasmixture. SMA rings were mounted on an isometric myograph (DMT(DanishMyo Technology A/S, Aarhus, Denmark)), filledwith Tyrode so-lution composed of (mM): NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4,1.2; NaHCO3, 20; HEPES, 10; EDTA, 0.016; D-glucose, 11.1; and CaCl2,2.5 (pH 7.4), maintained at 37 °C and continuously gassed with 21%O2, 1% CO2, and 78% N2. TA rings were progressively stretched to a rest-ing tension of 2 g. SMA rings were progressively stretched at 2 mN, as

Fig. 1. Curves illustrating the evolution of the concentration of antibodies againstanti-β1-adrenergic receptors (β1-ARs) determined by ELISA, in sera of controlrats (adjuvant-treated) (squares, n = 10) or of rats immunized with a peptide cor-responding to the second extracellular loop of the human β1-AR (circles, n = 12). Anti-body titers are defined by optical density values. Values are represented as mean ±SEM, ***P b 0.0001 compared to control group.

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described previously [24,25]. Isometric tension was recorded using aforce displacement transducer (EMKA Technologies, Paris, France forTA and Danish Myo Technology A/S, Aarhus N, Denmark for SMA) anddisplayed on a computer (Acqknowledge 3.9, MP150 BIOPAC system,Cerom, France for TA and ADinstruments, Labchart, Colorado Springs,USA for SMA). After equilibration for 1 h with the physiological solu-tions being changed every 10 min, TA and SMA rings were contractedtwice with 80 mM KCl. Endothelial function was verified by the obser-vation of at least 60% relaxation to acetylcholine (a muscarinic receptoragonist, 1 μM) in both TA and SMA rings (pre-contracted with phenyl-ephrine, a selective α1-adrenoceptor agonist; 0.3 μM and 3 μM). Afterwashing for 30 min, TA and SMA rings were contracted again withphenylephrine (0.1–1 μM and 2–3 μM respectively) to produce about80% of the maximum response obtained with KCl 80 mM.

Once contraction had reached a plateau, cumulative concentration–response curves (CCRCs) were constructed in response to the followingdrugs: isoproterenol (a non-selective β-AR agonist, 0.001–100 μM),dobutamine (a β1-AR agonist, 0.0001–100 μM), salbutamol (a β2-ARagonist, 0.001–300 μM), SR 58611A (a β3-AR agonist, 0.3–100 μM for TAand 0.001–100 μM for SMA), acetylcholine (0.001–100 μM), A23187 (acalcium ionophore, 0.001–0.3 μM), and sodium nitroprusside (a nitricoxide donor, 0.001–100 μM). The relaxation produced by each concentra-tion of each drug was measured after a steady state was reached. Valueswere expressed as the percentage change in the maximum tension ofthe rings after addition of KCl. Some TA rings were also equilibratedfor 60 min in Krebs solution containing L-arginine (a NO precursor,1 mmol/l) before constructing CCRCs to acetylcholine, SR 58611A, andphenylephrine.

2.6. Drugs

Pentobarbital solution was purchased from CEVA santé animale(France), (−)-isoproterenol hydrochloride, dobutamine hydrochloride,salbutamol, A23187, L-arginine, phenylephrine hydrochloride, acetylcho-line chloride, and SNP were obtained from Sigma-Aldrich (France). SR58611A [(RS)-N-[(25)-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydro-napht-2-yl]-(2R)-2(3-chlorophenyl)-2 hydroethanamine hydrochloride]was a generous gift from Sanofi Synthelabo Recherche (France). Aqueoussolutions of SNP were made up just prior to use and kept in the dark.All drugs were prepared as stock solutions in distilled water, with theexception of SR 58611A and A23187, which were dissolved indimethylsulphoxide (DMSO, Sigma-Aldrich). The final concentrationof the solvents in the organ bath was less than 0.1% vv−1 and wasused as a control for the effect of the active drugs.

2.7. Statistical analysis

Results were expressed as amean± SEM of n experiments, where nis the number of rat or porcine hearts. The differences between theELISA titers of the data groups were compared using the unpairedStudent's t-test with Prism ® software V.5. Relaxation was expressedas the percentage relaxation of the phenylephrine-induced contraction.Different CCRCs and their parameters (maximum effect: Emax and thenegative logarithmof the concentration producing 50%of themaximumeffect or pD2) were compared [26] using non-linear mixed effects [27]and the CCRC incomplete model using the two-way ANOVA withrepeated measures, completed where appropriate with a Bonferroni-test (PRISM®). A value of P b 0.05 was considered to be statisticallysignificant.

3. Results

3.1. Antibody production

Onemonth after the first injection,we observed a high production ofspecific antibodies in the serum of rats immunized with β1-AR antigen

peptide. After a second and a third injection, antibody titers remainedhigh and stable compared with the low level of antibody productionmeasured in the control rats who had been treated with adjuvant (opti-cal density at 3 months: 1.61 ± 0.13 and 0.07± 0.02 for immunized andcontrol rats respectively, P b 0.0001 compared to control group) (Fig. 1).

3.2. Vasoconstrictive effect ofα1-adrenoceptor agonists in TA and SMA fromimmunized rats

To evaluate the effects of immunization on vasoconstriction viaα1-ARin TA and SMA rings, CCRCs to phenylephrine (0.001 to 300 μM) wereconstructed. The maximum contraction induced by phenylephrinein TA from immunized rats was not significantly reduced (Emax =5.23 ± 0.35 g (n = 8) vs 5.21 ± 0.21 g (n = 13), for immunizedand control rats respectively) and pD2 values were not modified(6.88±0.13 (n=8) vs6.98±0.08 (n=13) for immunized and control(adjuvant-treated) rats respectively) (Fig. 2A). Similarly, the effect ofphenylephrine in SMA was not modified in immunized rats (Emax =9.95 ± 0.78 mN and pD2 = 6.26 ± 0.02 (n = 5) vs Emax = 8.81 ±0.53 mN and pD2 = 6.04 ± 0.04 (n = 5) for immunized and controlrats respectively) (Fig. 2B).

3.3. Vasorelaxant effects of β-AR agonists in TA and SMA from immunizedrats

The relaxant effects of dobutamine (0.0001–100 μM) and SR 58611A(0.01–100 μM and 0.001–100 μM for TA and SMA respectively) in TAand SMA rings from immunized rats,were significantly reduced in com-parison with relaxations obtained in vessels from control rats (Fig. 3A,Band Fig. 4A,B). With isoproterenol (0.001–10 μM), there was a generaltrend towards inhibition in TA, but relaxation was only significantlyinhibited in SMA from immunized rats (Fig. 5A,B). The relaxant effectsof salbutamol (0.001–300 μM) were not altered in TA and SMA ringsfrom immunized rats (Fig. 6A,B).

Various agonists of β-AR subtypes were also tested; the pD2 valueswere not changed in TA rings from immunized rats. However, thepercentage of maximum relaxations induced by dobutamine and SR

Fig. 2. Immunizationwith a peptide corresponding to the secondextracellular loop of humanβ1-adrenergic receptors (ARs), does not affect concentration-dependent contractions inducedby phenylephrine (a α1-AR agonist) in A, thoracic aorta (TA) and B, small mesenteric artery (SMA) rings of rats. Cumulative concentration–response curves were constructed to phenyl-ephrine (0.001–300 μM for TA and 0.001–100 μM for SMA). Each point is themean of n experiments obtained from n rats and vertical lines show the SEM.When no error bar is shown, theerror is smaller than the symbol.

311M.A. Abdelkrim et al. / International Immunopharmacology 19 (2014) 308–316

58611A were significantly decreased in TA from immunized rats. Con-versely, both the percentage of maximum relaxations and the pD2

values of CCRCs constructed with dobutamine were reduced by immu-nization in SMA. In SMA rings from immunized rats, the pD2 values of SR58611Awere significantly reduced, but not the percentage ofmaximumrelaxation. In SMA, there was a significant reduction of both the percent-age of maximum relaxations and pD2 values. Table 1 displays the pD2

values and percentage of maximum relaxations induced by the variousβ-AR subtype agonists, obtained in TA and SMA in each group of rats.

To determinewhether a general defect in endothelium-independentrelaxation was involved in the impairments observed in TA and SMAfrom immunized rats, CCRCs to SNP (0.001–100 μM) were constructedand compared with those obtained in rats from the control group. Inboth types of vessels, no detectable difference was found between thetwo groups of rats (Fig. 7A,B). The pD2 values and the percentage ofmaximum relaxation are also displayed in Table 1.

Fig. 3. Immunizationwith a peptide corresponding to the second extracellular loop of human β1

butamine (a selectiveβ1-AR agonist) in A, thoracic aorta (TA) andB, smallmesenteric artery (SMfor TA and 0.0003–100 μM for SMA) were constructed after pre-contraction to phenylephrine.SEM. When no error bar is shown, the error is smaller than the symbol. **P b 0.002 for TA and

3.4. Endothelium-dependent impairment of vasorelaxation in immunizedrats

In both groups of rats, acetylcholine (0.001–100 μM) producedconcentration-dependent relaxations in intact TA and SMA rings pre-contractedwith phenylephrine (Fig. 8A,B). Both the percentage of max-imum relaxations and pD2 values were significantly reduced in SMArings. In TA rings, only the percentage of maximum relaxation wasreduced in immunized rats compared to controls (Table 2).

The endothelium-dependent relaxation produced by A23187 (a cal-cium ionophore used as an activator of NO synthases (NOS), 0.001–0.3 μM)was reduced in TA rings from immunized rats compared to con-trol rats (Fig. 9). The percentage of maximum relaxations were 81.94±1.43 (n = 10) and 71.15 ± 3.58 (n = 9, P b 0.001) for TA from controland immunized rats respectively. There was no significant difference inpD2 values (7.81 ± 0.06 vs 7.58 ± 0.10).

-adrenergic receptors (ARs), impairs concentration-dependent relaxations induced by do-A) rings of rats. Cumulative concentration–response curves to dobutamine (0.0001–10 μMEach point is the mean of n experiments obtained from n rats and vertical lines show the***P b 0.0001 for SMA compared to control (adjuvant-treated) rats.

Fig. 4. Immunization with a peptide corresponding to the second extracellular loop of human β1-adrenergic receptors (ARs), impairs concentration-dependent relaxations induced by SR58611A (a selective β3-AR agonist) in A, thoracic aorta (TA) and B, smallmesenteric artery (SMA) rings of rats. Cumulative concentration–response curves to SR 58611A (0.01–100 μM forTA and of 0.001–100 μM for SMA)were constructed after pre-contraction to phenylephrine. Each point is themean of n experiments obtained from n rats and vertical lines show the SEM.When no error bar is shown, the error is smaller than the symbol. ***P b 0.0001 for TA and ***P b 0.0002 for SMA compared to control (adjuvant-treated) rats.

312 M.A. Abdelkrim et al. / International Immunopharmacology 19 (2014) 308–316

In TA rings from both groups of rats, CCRCs to acetylcholine and SR58611Awere performed in the presence of L-arginine (1 mM) to deter-mine whether a deficiency of the precursor amino acid needed to formNO and citrulline, was involved in the impairment of acetylcholine- andSR 58611A-induced relaxations. For both agonists, a significant im-provement with a recovery of the percentage of maximum relaxationswas observed in TA rings from immunized rats following supplementa-tion with L-arginine (Fig. 10A,B and Table 3).

4. Discussion

This study is the first to demonstrate that immunization against thesecond extracellular loop of β1-ARs impairs endothelial vasorelaxationinduced by stimulation of β1/β3-ARs and muscarinic receptors, in TAand SMA of rats immunized for 3 months.

The immunization protocol used in this study, with a synthetic pep-tide corresponding to the second extracellular loop of β1-ARs, allowedus to detect antibody titers of IgG; thesewere high, maximal, and stable

Fig. 5. Immunization with a peptide corresponding to the second extracellular loop of human β1

isoproterenol (a non-selective β-AR agonist) only in B, small mesenteric artery (SMA) but not in100 μM for TA and 0.0003–3 μM for SMA) were constructed after pre-contraction to phenylephthe SEM. When no error bar is shown, the error is smaller than the symbol. **P b 0.002 for SMA

after 3 months of immunization. ELISA was used to demonstrate thatIgG containing β1-AABs were already present only one month after im-munization. This type of immunization protocol to produce IgG contain-ing β1-AABs has already been used in rats [23,28] and rabbits [29,30].This study also characterized the functional activity (in vitro) of IgG con-tainingβ1-AABs by confirming their positive inotropic effects in isolatedcardiomyocytes from adult rats (see [23]).

The rats used in this studywere 6 months old at the end of the activeimmunization protocol. In control rats (treated with Freund's adjuvantalone), the maximal vasorelaxant responses induced by isoproterenol,dobutamine, and salbutamol, were lower (ranging between 56% and78%), than those observed in 1–2 month-old rats (80–100%, see:[22,31–33]). By contrast, the same three β-AR agonists induced maxi-mal vasorelaxant responses of almost 100% in the mesenteric arteriesof control rats. The reduced maximal response with β1 and β2-ARagonists in 6 month old rats, could be explained by a reduction in thedensity and/or the affinity of β-ARs with age, which has already beenreported in the thoracic aorta [31,34]. However, the vasorelaxant

-adrenergic receptors (ARs), impairs concentration-dependent relaxations induced by (−)-A, thoracic aorta rings. Cumulative concentration–response curves to isoproterenol (0.001–rine. Each point is the mean of n experiments obtained from n rats and vertical lines showcompared to control (adjuvant-treated) rats.

Fig. 6. Immunizationwith a peptide corresponding to the second extracellular loop of humanβ1-adrenergic receptors (ARs), does not affect concentration-dependent relaxations inducedby salbutamol (a selective β2-AR agonist) in A, thoracic aorta (TA) and B, small mesenteric artery (SMA) rings of rats. Cumulative concentration–response curves to salbutamol (0.001–300 μM) were constructed after pre-contraction to phenylephrine. Each point is the mean of n experiments obtained from n rats and vertical lines show the SEM. When no error bar isshown, the error is smaller than the symbol.

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responses observed in mesenteric arteries in this study confirm that inthose vessels, the number and affinity of β-ARs were not altered inaged rats [35]. We also found that the maximum effect of the maximalvasorelaxant response induced by isoproterenol, a non-selective β-ARagonist, was lower than that obtained with salbutamol, a selective β2-AR agonist. This is probably due to the concomitant vasoconstrictiveeffect of isoproterenol induced by the highest concentrations (from30 μM) through stimulation of α1-ARs.

Surprisingly, in the immunized rats in this study, the vasorelaxationinduced by isoproterenol, a nonselective β-AR agonist, was not im-paired in TA, whereas in SMA, there was significant inhibition ofisoproterenol-induced vasorelaxation, including a reduction in the per-centage of maximum relaxations and pD2 values of constructed CCRCs.This is probably related to the predominance of the β2-AR subtype inthis vascular bed [36,37]. This could also explain why immunizationhad no effect on the response to salbutamol, a selective β2-AR agonist,in this study in both TA and SMA. The fact that isoproterenol-inducedrelaxation was impaired in SMA in immunized rats is probably due toa higher density of β1-ARs over β2-AR in those vessels [38,39].

Several findings in this study indicate that the endothelium-dependent NO/cGMP signaling pathway is impaired by immunization.

Table 1Descriptive statistics of the % of maximum relaxations (of the phenylephrine-induced contractiand SMA from control (adjuvant-treated) and immunized rats.

TA

Control Immunized

% pD2 (n) % pD2

(±) (±) (±) (±)

Isoproterenol 69.11 6.36 (9) 54.92 6.16(4.25) (0.12) (5.30) (0.11)

Dobutamine 55.93 6.48 (8) 41.78 6.53(6.14) (0.06) (1.84) (0.08)

**Salbutamol 77.55 ND (8) 69.25 ND

(2.93) (1.46)SR 58611A 86.74 4.96 (9) 64.82 4.85

(1.88) (0.04) (2.09) (0.06)***

SNP 97.65 8.79 (7) 96.70 8.51(1.23) (0.39) (1.86) (0.19)

Values are means ± (SEM). **P b 0.001 and ***P b 0.0001 denote significant differences betwe

Firstly, in TA and SMA from immunized rats, there was a significant im-pairment of the percentage maximum relaxation to acetylcholine,which interacts with endothelial muscarinic receptors releasing NOand causing vasodilatation. Secondly, the relaxant effect of dobutamine,a selective β1-AR agonist, was impaired in SMA, and in this vascular bed,activation of β1-AR is known to be mainly mediated by the release ofendothelial NO [40]. We also found that following immunization, thepercentage of maximum relaxation induced by SR 58611A, a selectiveβ3-AR agonist, was significantly reduced in TA. It is already wellknown that the endothelium-dependent NO/cGMP signaling pathwayis involved in β3-AR responses in TA [41,42].

Contrary to our findings with TA, pD2 values of CCRCs constructedwith dobutamine were reduced by immunization in SMA. This indi-cates a reduction of the efficacy of β1-AR binding to their signalingeffectors in SMA, and emphasizes regional differences in β1-AAB-in-duced modifications of vascular β1-AR sensitivity. One explanationfor the discrepancies in our results could be the inability of β1-AABs to interact directly with β1-AR in TA during the active immuni-zation process. In accordance with our hypothesis, β1-AABs havebeen reported to behave as low efficacy agonists [1] and such agonistswould be expected to interact preferentially with β1-AR in tissues

ons) and pD2 values calculated from CCRCs constructed with various β1-AR agonists in TA

SMA

Control Immunized

(n) % pD2 (n) % pD2 (n)

(±) (±) (±) (±)

(9) 99.84 8.21 (5) 90.41 7.78 (6)(1.38) (0.04) (3.29) (0.12)

**(14) 99.52 8.05 (5) 92.09 7.70 (8)

(0.34) (0.07) (1.93) (0.04)*** ***

(8) 99.68 5.79 (5) 91.5 5.98 (5)(3.11) (0.05) (1.80) (0.03)

(9) 97.14 6.00 (6) 95.98 5.64 (8)(2.82) (0.06) (0.93) (0.13)

***(6) 93.5 8.23 (5) 97.27 8.07 (5)

(1.80) (0.22) (2.67) (0.08)

en control and immunized rats. ND denotes that it could not be determined.

Fig. 7. Immunizationwith a peptide corresponding to the second extracellular loop of humanβ1-adrenergic receptors (ARs), does not affect concentration-dependent relaxations inducedby sodium nitroprusside (SNP) (a nitric oxide donor) in A, thoracic aorta (TA) and B, small mesenteric artery (SMA) rings of rats. Cumulative concentration–response curves to SNP(0.001–1 μM for TA and 0.001–100 μM for SMA) were constructed after pre-contraction to phenylephrine. Each point is the mean of n experiments obtained from n rats and verticallines show the SEM. When no error bar is shown, the error is smaller than the symbol.

Fig. 8. Immunization with a peptide corresponding to the second extracellular loop of human β1-adrenergic receptors (ARs), impairs concentration-dependent relaxations induced byacetylcholine (a muscarinic receptor agonist) in A, thoracic aorta (TA) and B, small mesenteric artery (SMA) rings of rats. Cumulative concentration–response curves to acetylcholine(0.001–30 μM for TA and 0.0003–100 μM for SMA) were constructed after pre-contraction to phenylephrine. Each point is the mean of n experiments obtained from n rats and verticallines show the SEM. When no error bar is shown, the error is smaller than the symbol. ***P b 0.0001 for TA and **P b 0.002 for SMA compared to control (adjuvant-treated) rats.

314 M.A. Abdelkrim et al. / International Immunopharmacology 19 (2014) 308–316

with high receptor density and/or coupling efficiency (pD2 values of do-butamine were actually higher in SMA than in TA). However, it shouldbe noted that the β1-AAB-induced modification of vascular β1-ARsensitivity is unlikely to be specific to β1-AR since in SMA, for CCRCs

Table 2Descriptive statistics of the % of maximum relaxations (of the phenylephrine-induced contractifrom control (adjuvant-treated) and immunized rats.

TA

Control Immunized

% pD2 (n) % pD2 (n)

(±) (±) (±) (±)

89.27 7.41 (9) 64.19 7.27 (13)(1.83) (0.02) (2.5) (0.05)

***

Values are means ± (SEM). *P b 0.05, **P b 0.001 and ***P b 0.0001 denote significant differen

constructed with SR 58611A and acetylcholine, pD2 values were alsoreduced by immunization, suggesting that the efficacy of β3-AR andmuscarinic receptor binding to their endothelium-dependent signalingpathway was also affected by immunization. It is therefore conceivable

ons) and pD2 values calculated from CCRCs constructed with acetylcholine in TA and SMA

SMA

Control Immunized

% pD2 (n) % pD2 (n)

(±) (±) (±) (±)

99.98 7.93 (5) 92.57 7.58 (11)(0.79) (0.08) (1.66) (0.10)

** *

ces between control and immunized rats.

Fig. 9. Immunization with a peptide corresponding to the second extracellular loopof human β1-adrenergic receptors (ARs), impairs concentration-dependent relax-ations induced by A23187 (a NOS activator) in thoracic aorta rings of rats. Cumulativeconcentration–response curves to A23187 (0.001–0.3 μM) were constructed after pre-contraction to phenylephrine. Each point is the mean of n experiments obtained from nrats and vertical lines show the SEM. When no error bar is shown, the error is smallerthan the symbol. **P b 0.0016 compared to control (adjuvant-treated) rats.

Table 3Descriptive statistics of the% ofmaximumrelaxations (of the phenylephrine-induced con-tractions) and pD2 values calculated from CCRCs constructed with acetylcholine and SR58611A following L-arginine pre-treatment in TA from control (adjuvant-treated) and im-munized rats.

TA

Control Immunized

% pD2 (n) % pD2 (n)

(±) (±) (±) (±)

Acetylcholine 83.91 7.37 (5) 92.36 7.5 (5)(3.94) (0.04) (1.76) (0.05)

SR 58611A 90.79 5.48 (5) 89.97 5.42 (5)(6.32) (0.23) (1.59) (0.12)

Values are means ± (SEM).

315M.A. Abdelkrim et al. / International Immunopharmacology 19 (2014) 308–316

that alterations in vascular reactivity are not related to differences inreceptor sensitivity per se but to differences in the sensitivity of mecha-nisms linked to endothelial receptors.

To determine whether the observed impairment of vasorelaxationdue to immunization was specific to endothelial tissue and did not in-volve smooth muscle cells, we tested the endothelium-independentvasorelaxant effects of SNP, a donor of NO. This study did not revealany changes in the vasorelaxant action of SNP in either TA or SMA,confirming that the dysfunction was essentially localized in the endothe-lium.We also observed significant inhibition of the vasorelaxant effect ofA23187 in TA after immunization. A23187 is a calcium ionophore, whichacts as a receptor-independent vasodilator, by activating endothelial NOS

Fig. 10. L-Arginine (a substrate for nitric oxide synthesis) restored impairment due to immunizareceptors (ARs) of relaxations induced in thoracic aorta rings of rats by A, acetylcholine (a mconcentration–response curves to acetylcholine (0.001–30 μM) and SR 58611A (0.01–100 μM) wphenylephrine. Each point is the mean of n experiments obtained from n rats and vertical lines sh

[43]. Our result indicates an impairment of NO synthesis after immuniza-tion.We thereforewent on to demonstrate that pre-treatment of TA ringsfrom immunized ratswith L-arginine, a substrate of NO synthase and thusa precursor of NO synthesis, completely restores the vasorelaxant effectsof acetylcholine and SR 58611A. The latter two agonists interact withmuscarinic receptors and β3-ARs respectively, releasing NO and causingvasorelaxation. Thus, endothelial NO-induced vasorelaxation was im-paired by immunization and clearly improved following pretreatmentwith a precursor of NO delivery, such as L-arginine.

Several studies have demonstrated the deleterious effects of autoanti-bodies against the second extracellular loop of β1-AR and their primarypathogenic role in the pathophysiology of some forms of cardiomyopathyand heart failure in humans [5,44,45] and in immunized rats [46].

5. Conclusions

This study provides strong evidence that, in addition to cardiac effects,these autoantibodies affect vascular endothelial function by inducing spe-cific β1-AR and/or nonspecific (including β3-AR and muscarinic recep-tors), depending on the target vascular bed, deleterious effects onendothelial NO-dependent signaling pathways. Li et al. [47] recently re-ported that circulating autoantibodies to β2-AR and/or muscarinic recep-tors may exacerbate hypotension in patients with orthostatic disorders.

tionwith a peptide corresponding to the second extracellular loop of humanβ1-adrenergicuscarinic receptor agonist) and B, SR 58611A (a selective β3-AR agonist). Cumulativeere constructed after 1 hour pretreatment with L-arginine (1 mM) and pre-contraction toow the SEM. When no error bar is shown, the error is smaller than the symbol.

316 M.A. Abdelkrim et al. / International Immunopharmacology 19 (2014) 308–316

Acknowledgments

The study was supported by “La Société Française d'HypertensionArtérielle” and the “Fondation Langlois”. We thank Chantal Thorin forher help concerning the statistical analysis and Carole Lenne for techni-cal assistance.

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