Original Article

Angiotensin-(1^9) reverses experimental hypertension and cardiovascular damage by inhibition of the angiotensin converting enzyme/Ang II axis Maria Paz Ocaranza a, Jackeline Moya a, Victor Barrientos b,c, Rodrigo Alzamora b,c, Daniel Hevia b,c, Cristobal Morales a,d, Melissa Pinto a, Nicola´s Escudero a, Lorena Garcı´a d, Ulises Novoa a, Pedro Ayala d, Guillermo Dı´az-Araya d, Ivan Godoy a, Mario Chiong d, Sergio Lavandero d,e, Jorge E. Jalil a, and Luis Michea b,c

Background: Little is known about the biological effects of angiotensin-(1–9), but available evidence shows that angiotensin-(1–9) has beneficial effects in preventing/ ameliorating cardiovascular remodeling. Objective: In this study, we evaluated whether angiotensin-(1–9) decreases hypertension and reverses experimental cardiovascular damage in the rat. Methods and results: Angiotensin-(1–9) (600 ng/kg per min for 2 weeks) reduced already-established hypertension in rats with early high blood pressure induced by angiotensin II infusion or renal artery clipping. Angiotensin(1–9) also improved cardiac (assessed by echocardiography) and endothelial function in smalldiameter mesenteric arteries, cardiac and aortic wall hypertrophy, fibrosis, oxidative stress, collagen and transforming growth factor type b
1 protein expression (assessed by western blot). The beneficial effect of angiotensin-(1–9) was blunted by coadministration of the angiotensin type 2(AT2) receptor blocker PD123319 (36 ng/kg per min) but not by coadministration of the Mas receptor blocker A779 (100 ng/kg per min). Angiotensin(1–9) treatment also decreased circulating levels of Ang II, angiotensin-converting enzyme activity and oxidative stress in aorta and left ventricle. Whereas, Ang-(1–9) increased endothelial nitric oxide synthase mRNA levels in aorta as well as plasma nitrate levels. Conclusion: Angiotensin-(1–9) reduces hypertension, ameliorates structural alterations (hypertrophy and fibrosis), oxidative stress in the heart and aorta and improves cardiac and endothelial function in hypertensive rats. These effects were mediated by the AT2 receptor but not by the angiotensin-(1–7)/Mas receptor axis. Keywords: angiotensin II, angiotensin-(1–9), cardiovascular remodeling, fibrosis, hypertension, hypertrophy, renin–angiotensin system Abbreviations: ACE2, angiotensin-converting enzyme 2; ACEIs , angiotensin-converting enzyme inhibitors; Ang, angiotensin; AT1R, angiotensin type 1 receptor; AT2R, angiotensin type 2 receptor; C, control rat; Cch, carbachol; EEL, external elastic lamina; eNOS, endothelial nitric oxide

synthase; FBS, fetal bovine serum; FLVW, free left ventricular wall; IEL, internal elastic lamina; IVS, interventricular septum; L-NAME, NG-nitro-L-arginine methyl ester; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; RAS, renin– angiotensin system; RT-PCR, reverse transcription polymerase chain reaction; SHRSP, stroke-prone spontaneously hypertensive rats; TGF-b1, transforming growth factor type b1; aSMA, a-smooth muscle actin

INTRODUCTION

A

ngiotensin (Ang) II plays a central role in the regulation of blood pressure (BP) as the main effector peptide of the renin–angiotensin system (RAS) [1]. Both Ang-(1–7) and Ang-(1–9) have been identified as new members of RAS [2]. Ang-(1–7) is produced from Ang I or Ang II by the angiotensin-converting enzyme 2 (ACE2). Ang-(1–7) binds to the G protein-coupled receptor Mas [3]. Ang-(1–9) is generated from Ang I by the carboxypeptidases ACE2 and cathepsin A [4,5]. Ang-(1–9) is present in plasma of healthy volunteers, patients and animals treated with ACE inhibitors or AT1 receptor blockers [6–8]. Little is known about the biological effects of Ang-(1–9), but available evidence shows beneficial effects in preventing/ameliorating cardiovascular remodeling [9–11]. We and

Journal of Hypertension 2014, 32:771–783 a Division de Enfermedades Cardiovasculares, Escuela de Medicina, Pontificia Universidad Cato´lica de Chile, Santiago, bMillennium Institute on Immunology and Immunotherapy, cInstituto de Ciencias Biomedicas, Facultad de Medicina, Santiago, d Advanced Center for Chronic Diseases & Centro Estudios Moleculares de la Celula, Facultad Ciencias Quimicas y Farmaceuticas & Facultad Medicina, Universidad de Chile, Santiago, Chile and eDepartment of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, Texas, USA

Correspondence to Marı´a Paz Ocaranza, PhD, Divisio´n de Enfermedades Cardiovasculares, Escuela de Medicina, Pontificia Universidad Cato´lica de Chile, Marcoleta 391, Santiago 8330024, Chile. Tel: +562 2354 3407; fax: +562 2632 1924; e-mail: [email protected] Received 28 April 2012 Accepted 27 November 2013 J Hypertens 32:771–783 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000094

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others have shown that Ang-(1–9) is an effective antihypertrophic peptide [9,11]. Ang-(1–9) ameliorates cardiac fibrosis and ex-vivo aortic nitric oxide bioavailability in stroke-prone spontaneously hypertensive rats [10]. Both effects were prevented by the angiotensin type 2 receptor (AT2R) blocker PD123319 [10]. These data suggest that cardiovascular effects of Ang-(1–9) could be mediated by the AT2R [10]. In this study, we evaluated whether Ang-(1–9) decreases hypertension and reverses/ameliorates cardiovascular damage in two models of experimental hypertension: rats with Ang II infusion and rats with renal artery clipping (Goldblatt model). In both models, there was an early development of Ang II-dependent hypertension. Hypertensive animals were compared with control rats and with hypertensive rats receiving Ang-(1–9) infusion. Plasma levels of Ang II, Ang-(1–9) and Ang-(1–7) were determined by high-performance liquid chromatography and radioimmunoassay. We also examined the role of the Mas receptor and/or the AT2R in mediating the effects of Ang-(1–9) using a pharmacological approach. Morphometric and biochemical studies were performed to evaluate the effects of Ang-(1–9) infusion in the heart and smalldiameter arteries from hypertensive and control animals. Cardiac function was assessed by echocardiography, and effects of Ang-(1–9) on endothelial function, oxidative stress and nitric oxide production were also evaluated. Ang-(1–9) reduced hypertension and secondary end-organ damage via the AT2R.

METHODS Hypertensive experimental models All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011), and approved by our institutional Bioethical Committee. Male Sprague–Dawley rats (150  50) were used in this study. Two different procedures for the induction of hypertension were performed: Goldblatt (two kidneys and one clipped) and Ang II infusion (osmotic minipump). Rats were housed in a temperature-controlled and humidity-controlled room maintained on a 12 : 12-h light-dark schedule, with free access to food and water. To test whether Ang-(1–9) reverted hypertension and/or tissue damage, two experimental models were used: Ang II infusion model (400 ng/kg per min, n ¼ 37) for 14 days using osmotic minipumps Alzet (Alzet 2002; Alzet, Cupertino, California, USA), implanted in the jugular vein under ketamine HCl/xylazine [35 and 7 mg/kg intraperitoneally (i.p.), respectively [11]. Control rats (C, n ¼ 14) were infused with vehicle. After 14 days of vehicle and Ang II infusion, control and Ang II rats with SBP at least 140 mmHg were randomized, and new osmotic minipumps Alzet (Alzet 2002) were implanted to obtain the following experimental subgroups: Ang II plus vehicle (Ang II, n ¼ 11); Ang II plus Ang-(1–9) [Ang II/Ang-(1–9), n ¼ 12]; Ang II plus Ang-(1– 9) and Mas receptor blocker A779 [Ang II/Ang-(1–9)/A779, n ¼ 7]; and Ang II plus Ang-(1–9) and the AT2 receptor blocker PD123319 [Ang II/Ang-(1–9)/PD123319, N ¼ 7]. 772

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The Goldblatt model (two kidneys, one clip, n ¼ 38) was established as described previously [12]. Sham-operated rats were the control group (C, n ¼ 12). Twenty-eight days after the renal surgery, control and Goldblatt rats that developed SBP at least 140 mmHg were randomized, and osmotic minipumps Alzet (Alzet 2002) were implanted to obtain the following experimental subgroups: Control rats plus vehicle (C, n ¼ 12); Goldblatt plus vehicle (Goldblatt, n ¼ 11); Goldblatt rats plus Ang-(1–9) [GB/Ang-(1–9), n ¼ 12]; Goldblatt rats plus Ang-(1–9) and A779 [GB/Ang(1–9)/A779, n ¼ 8]; and Goldblatt rats treated plus Ang-(1–9) and PD123319 [GB/Ang-(1–9)/PD123319, N ¼ 7]. In the treatment groups, either Ang-(1–9) (600 ng/kg per min), A779 (100 ng/kg per min) or PD123319 (36 ng/kg per min) were used [13]. All treatments were administered for 14 days as described [11].

Echocardiography and hemodynamic analysis SBP was measured by a noninvasive pressure device using volume pressure recording, CODA 2 (Kent Scientific Corporation, Torrington, Connecticut, USA). Measurements were obtained in conscious rats restrained in a thermal plastic chamber as described [14]. Echocardiography and hemodynamic analyses were performed as described previously [8].

Cardiac hypertrophy Cardiac hypertrophy was quantified by cardiac weight (mg), and ratios between cardiac weight and body weight (g) or tibial length (mm). Morphological and morphometric analyses were performed in transverse mid-ventricular slices (5 mm) from paraffin-embedded hearts, stained with hematoxylin and eosin and examined by light microscopy [11]. Cardiomyocyte size (area and perimeter) was determined as described by Ocaranza et al. [11].

Vascular morphometry Lumen and media cross-sectional areas were determined by the measurement of internal elastic lamina (IEL) and external elastic lamina (EEL) lengths traced manually on digitized images using Network Information ServicesElement Basic Research, Nikon Systems. Media area was obtained by subtracting the area encompassed by the IEL from the area encompassed by the EEL. Lumen area represented the area enclosed by the IEL. The media-tolumen ratio was calculated by dividing the media crosssectional area by the lumen cross-sectional area. The average media thickness (in mm) was calculated using the vessel outer diameter at the EEL and the inner diameter at the IEL (EEL diameter – IEL diameter)/2) [15].

Plasma, left ventricle and aortic wall angiotensin-converting enzyme activities Plasma, left ventricle and aorta ACE enzymatic activity was measured fluorometrically by the hydrolysis of Z-phenyl-Lhistidyl-L-leucine (Bachem Bioscience Inc., King of Prussia) as described previously [16] and was expressed in U/ml or U/mg protein, respectively (1U ¼ 1 nmol L-histidyl-Lleucine/min). Volume 32  Number 4  April 2014

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Angiotensin-(1–9) in hypertension

Circulating angiotensin levels Blood was collected from the inferior vena cava directly into a syringe containing 5 ml 4 mol/l guanidine thiocyanate. The blood was centrifuged, and the plasma was immediately extracted with Sep-Pak C18 cartridges (Waters Chromatography Division, Milford, Massachusetts, USA). The peptides were acetylated before high-performance liquid chromatography (HPLC) [17]. After reconstitution in water, each HPLC fraction was assayed in duplicate with the amino terminal-directed antibody A41, which measures acetyl-Ang II, acetyl-Ang I, acetyl-Ang-(1–7) and acetylAng-(1–9). Acetylation, HPLC and radioimmunoassay were performed as described by Ocaranza et al. [8].

Evaluation of cardiac fibrosis Formalin-fixed heart sections were dehydrated, embedded in paraffin, sectioned 5 mm thick and stained with hematoxylin–eosin and with the collagen-specific stain Sirius red F3BA in saturated aqueous picric acid (pH 2.0) for 90 min [18]. For each heart, 20 sections were examined, and collagen was quantified by computer-assisted morphometry. The automated system included an image-analysis processor based on mathematical morphology software connected to a personal computer. Each field sent to the image analyzer was transmitted by a video camera connected to a microscope and transformed into a digital image [18].

Collagen score Under light microscopy, one blind observer ranked the amount of collagen in the interventricular septum and in the free left ventricular wall from 1 (lowest amount) to 4 (highest amount) [18].

Transforming growth factor type-b1 and collagen I levels in the left ventricle and aorta wall The levels of transforming growth factor type-b1 (TGF-b1) and collagen I were assessed by western blot as previously described [19]. Each blot was quantified by scanning densitometry with the Un-Scan-It software.

Aortic wall endothelial nitric oxide synthase mRNA levels The room temperature-PCR assay was performed using the primers for endothelial nitric oxide synthase (eNOS) described previously [20].

Assessment of plasma nitrate levels Nitrate was quantified by capillary zone electrophoresis [21] using a Waters Quanta 4.000 (Waters Corporation, Milford, Massachusetts, USA). Results are expressed as mmol/l nitrate.

Vascular reactivity assays Small diameter mesenteric arteries were isolated from rats of different experimental groups at the end of the experimental period [22]. After euthanasia, the superior mesenteric artery was removed and placed in ice-cold (48C) physiological Krebs-Ringer bicarbonate, containing (mmol/l): 120 NaCl, 4.2 KCl, 1.18 KH2PO4, 1.2 MgSO4, Journal of Hypertension

1.3 CaCl2, 25 Na2CO3, 5 D-glucose and 1.2 pyruvate, equilibrated with 95% O2/5% CO2 (pH 7.4). The tissue was cleaned of all adipose and connective tissue, avoiding tissue stretching. The artery was cut into segments containing first and second order branches. Arteries were cannulated (MicroFil micropipette, WPI), perfused (12– 15 ml/min; syringe pump SP101i, WPI) and pressurized (60 mmHg, pressure transducer MLT0380/A, PowerLab 4/30, AD instruments) as previously described [22] with modified Krebs-Ringer bicarbonate (KRB) containing (in mmol/l): 120 NaCl, 4.2 KCl, 1.18 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, 25 Na2HCO3, 5 D-glucose, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 1.2 pyruvate, equilibrated with 95%O2/5%CO2 (pH 7.4, 378C, BTC 9090 temperature controller, Brainchild Electronic Ltd). Arteries were allowed a 20-min equilibration period. In the present study, 30 arteries were used (average basal diameter of 367  49 mm). Before starting the experiments, arterial viability was checked by adding phenylephrine (100 mmol/l, 2 min) to the superfusion. Endothelium integrity was tested by the subsequent addition of acetylcholine (1 mmol/l). Only arteries that showed sustained vasoconstriction after addition of phenylephrine and complete relaxation in the presence of acetylcholine were used. Thereafter, arteries were washed three times (superfusion exchange, 1-min wash), followed by a 10-min period of stabilization. Video microscopy images were acquired every 8 s using a zoom stereo-microscope (Olympus SZ61) coupled to a CCD camera (Moticam 2000; Motic China Group Co), and external artery diameter was determined for each image. The arterial images and pressure were recorded continually during the experiments. Thoracic aorta from 8 weeks old Sprague–Dawley rats was removed and placed in KRB. The tissue was cleaned and cut into rings (each 4–5 mm long; 4–6 mg weight). Extreme care was taken during preparation of the rings to avoid stretching the tissue. Standard isometric tension measurements were performed as previously described [23]. Briefly, the rings were mounted on two 30-gauge (0.25 mm) stainless steel wires; the lower one was attached to a stationary glass rod, and the upper one was attached to a MLT500/A force-displacement transducer (AD Instruments, Colorado Springs, Colorado, USA) at 378C. The transducer was connected to a PowerLab 15T analog-digital converter (AD Instruments) for continuous recording of blood vessel tension. Data were recorded using LabChart 5.0 software (AD Instruments). After a 45-min equilibration period at a tension of 1 g, the response of aortic rings was tested by a near-maximal contractions with epinephrine (1 mmol/l) followed by relaxation with carbachol (10 mmol/l). The vasorelaxant effect of Ang-(1–9) was measured in only the rings that contracted in response to epinephrine and then fully relaxed in response to carbachol. Ang-(1–9) [10 fmol/l – 10 nmol/l] was added in increasing cumulative concentrations once the response to phenylephrine stabilized. To elucidate the role of AT2R, Mas receptor and AT1R activation on the vascular reactivity assays, endothelium-intact aortic rings were preincubated with PD123319 (1 mmol/l), A779 (500 nmol/l) or losartan (1 mmol/l). The endothelium and nitric oxidedependence of Ang-(1–9) vasodilator effect was tested in www.jhypertension.com

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endothelium-denuded and NG-nitro-L-arginine methyl ester (L-NAME, 300 mmol/l) pretreated aortic rings, respectively. Sodium nitroprusside (1 mmol/l) was added at the end of the recording to assess for vascular muscle responsiveness to nitric oxide. Experiments with Ang-(1–9) and other vasoactive substances were performed after washing with KRB (4) and 20 min of equilibration.

Cardiac and vascular O2 – production and NAD(P)H oxidase activity was estimated with lucigenin-enhanced chemiluminescence as described previously [20].

gel electrophoresis. Collagen type I was detected by western blot using anticollagen (AB292; Abcam, Cambridge, Massachusetts, USA) and revealed using enhanced chemiluminescence substrate (ECL). Bands were quantified by densitometric analysis [25]. For myofibroblast differentiation, cardiac fibroblasts were incubated for 72 h with Ang II, Ang-(1–9) and Ang II þ Ang-(1–9) in DMEM F-12. Cells were disrupted, and equal amount of proteins were resolved by 12% polyacrylamide gel electrophoresis. Alpha-smooth muscle actin (a-SMA) was detected by western blot using anti-aSMA (A2547; Sigma-Aldrich Chemical Co, St Louis, Missouri, USA) and revealed using ECL. Bands were quantified by densitometric analysis [25].

Cardiac fibroblast isolation and cell culture

Statistical analysis

Measurement of O2 – production and NAD(P)H oxidase activity

Male Sprague–Dawley rats (250–300 g) were anesthetized with ketamine-xylazine (66 and 1.6 mg/kg i.p., respectively). Adult rat cardiac fibroblasts were isolated by retrograde aortic perfusion. Briefly, hearts were digested with collagenase type B solution for 1 h, and cells were centrifuged at 500 rpm for 2 min. The supernatant, mainly adult rat cardiac fibroblasts, was centrifuged at 1000 rpm for 10 min, resuspended in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM F-12) plus 15% fetal bovine serum (FBS) and then seeded in nontreated culture dishes during 3 h. Cells were washed with phosphatebuffered saline to eliminate debris and nonadherent cells [24].

Measurement of cell viability, collagen and myofibroblast differentiation Adult rat cardiac fibroblasts were used at passage one, and seeded on 60 mm cell culture dishes at density of 2  10 –4 cells/cm2 in DMEM F-12 plus 10% FBS and incubated at 378C for 3 h. To synchronize cells, they were washed and incubated in DMEM F-12 (without serum, 0% FBS) for 24 h before experimentation [25]. For cell viability, cardiac fibroblasts were incubated for 24 h with Ang II, Ang-(1–9) and Ang II þ Ang-(1–9) in DMEM F-12 5% FBS. Cells were trypsinized, and viability was assayed using trypan blue method [25]. For collagen synthesis, cardiac fibroblasts were incubated for 24 h with Ang II, Ang-(1–9) and Ang II þ Ang-(1–9) in DMEM F-12, 0% FBS and 10 ng/ml ascorbate. Cells were disrupted, and equal amount of proteins were resolved by 6% polyacrylamide

Results (mean  SEM) were compared using a single factor analysis of variance followed by the Student–Newman– Keuls test. A P  0.05 was considered statistically significant.

RESULTS Angiotensin-(1–9) decreases blood pressure in hypertensive rats To evaluate whether Ang-(1–9) reduces already-established experimental hypertension, rats were subjected to Ang II infusion or renal artery clipping (Goldblatt model). After 4 and 6 weeks of surgery, circulating Ang II levels increased significantly; by 66 and 77%, in Goldblatt and Ang II rats, respectively. Whereas, Ang-(1–9) circulating levels decreased by 47 and 42%, respectively, in both experimental models (Tables 1 and 2). Infusion with Ang-(1–9) in hypertensive rats for 2 weeks (starting on the 2nd and 4th week after procedure) significantly increased Ang-(1–9) plasma levels and decreased Ang II level by 38 and 25%, ACE activity by 12 and 22%. Ang-(1–9) infusion reduced SBP both in Ang II and Goldblatt rats, respectively (154  4 vs. 139  3 mmHg, P < 0.05 and 161  3 vs. 132  3 mmHg, P < 0.05, n ¼ 7–14 per group). However, the coinfusion of Ang-(1–9) with the AT2R antagonist PD123319 did not modify Ang II levels and ACE activity with respect to hypertensive rats treated with Ang-(1–9) (Tables 1 and 2). However, the antihypertensive effect of Ang-(1–9) was blocked by PD123319 but not by the Mas receptor blocker, A779 (Fig. 1).

TABLE 1. Circulating angiotensin II, (1–7), (1–9) and angiotensin converting enzyme activities in hypertensive rats by angiotensin II infusion

N Ang II (fmol/ml) Ang-(1–7) (fmol/ml) Ang-(1–9) (fmol/ml) Plasma ACE activity (U/ml) LV ACE activity (U/mg prot) Aorta ACE activity (U/mg prot)

C

Ang II

Ang II þ Ang -(1–9)

Ang II þ Ang (1–9)þA779

Ang II þ Ang-(1–9) þ PD

11 20.2  2.8 5.9  0.8 5.3  1.0 110  5.0 1.4  0,1 154  10

10 35.8  0.9a 6.1  0.5 3.1  0.5a 134  6.0a 2.9  0.2a 219  22a

10 22.3  1.7b 5.0  0.7 14.4  0.7a,b 111  4.0b 1.5  0.3b 165  13b

7 21.7  2.1b 7.9  0.6a,b,c 13.9  0.9a,b 100  3b 1.3  0.2,b 170  6,b

7 31.5  1.8a,c 5.2  0.4 16.9  0.4a,b,c 129  2a,c 2.5  0.3a,c 235  21a,c

Values as mean  SEM. A779, Ang-(1–7) blocker receptor; ACE, angiotensin-converting enzyme; Ang II, angiotensin II; Ang, angiotensin; N, number of animals. a P < 0.05 vs. control. b P < 0.05 vs. Ang II. c P < 0.05 vs. Ang IIþ Ang-(1–9) (after ANOVA).

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Angiotensin-(1–9) in hypertension TABLE 2. Circulating angiotensin II, (1–7), (1–9) and angiotensin converting enzyme activities in hypertensive Goldblatt rats

n Ang II (fmol/ml) Ang-(1–7) (fmol/ml) Ang-(1–9) (fmol/ml) Plasma ACE activity (U/ml) LV ACE activity (U/mg prot) Aorta ACE activity (U/mg prot)

Control GB

GB

GBþAng-(1–9)

GB þ Ang-(1–9)þA779

GB þ Ang-(1–9)þPD

11 24.3  1.6 6.1  0.4 6.4  0.8 115  2.0 1.7  0,3 167  9

12 40.4  0.5a 6.3  0.2 3.4  0.1a 134  4.0a 3.4  0.3a 245  23a

11 30.1  0.3a,b 5.9  0.1 9.2  0.8a,b 118  1.0a,b 1.9  0.2b 160  13b

7 29.4  1.1a,b 6.9  0.1a,b,c 8.7  0.3a,b 121  3.0a,b 1.6  0.4,b 171  14b

7 39.2  0.7a,c 5.8  0.4 12.2  0.7a,b,c 132  2a,c 2.9  0.3a,c 239  11a,c

Values as mean  SEM. A779, Ang-(1–7) blocker receptor; ACE angiotensin-converting enzyme; Ang, angiotensin; GB, Goldblatt; N, number of animals. a P < 0.05 vs. control. b P < 0.05 vs. GB. c P < 0.05 vs. GB þ Ang-(1–9) (after ANOVA).

Effects of angiotensin-(1–9) administration on myocardial damage Tables 3 and 4 summarize body weight, cardiac weight/ body weight ratio and left ventricular (LV) fractional shortening in Ang II, Goldblatt and control rats. As expected, in the untreated hypertensive rats, cardiac weight/body weight and cardiac weight/tibial length ratios increased at weeks 4 and 6. Echocardiographic studies of Ang II and Goldblatt rats showed significant increases in LV fraction shortening (LVFS) and LV ejection fraction (LVEF) after (a) *†

200

*† * 150

*

*

*

†

†

3

4

AngII AngII-Ang-(1-9) AngII-Ang-(1-9)-A779 AngII-Ang-(1-9)-PD Sham

(b)

SBP (mmHg)

100 0 0

1

2

*†

200

*† *

* 150

*

* †

*

* †

GB GB-Ang-(1-9) GB-Ang-(1-9)-A779 GB-Ang-(1-9)-PD Sham

100 0 0

1

2

3

4

5

6

Weeks FIGURE 1 Ang-(1–9) infusion decreased SBP of hypertensive rats. SBP in each group was determined by the CODA indirect tail-cuff method. (a) Two weeks after surgery to implant an Ang II minipump (arrow), hypertensive rats were randomized to receive vehicle (n ¼ 11), Ang-(1–9) (n ¼ 12), Ang-(1–9)þA779 (n ¼ 7) or Ang(1–9)þPD123319 (n ¼ 7). Control rats were implanted with a minipump with vehicle (C, n ¼ 14). (b), Four weeks after renal artery clipping (arrow), Goldblatt rats were randomized to receive vehicle (n ¼ 11), Ang-(1–9) (n ¼ 12), Ang-(1–9) þ A779 (n ¼ 8) or Ang-(1–9) þ PD123319 (n ¼ 7). Control rats were implanted with minipump with vehicle (S, n ¼ 12). Ang, angiotensin; GB, Goldblatt rats; A779, Ang-(1–7) blocker receptor; PD123319, AT2 receptor blocker. The values represent mean  SEM. P < 0.05 vs. control; yP < 0.05 vs. Ang II or GB rats; z P < 0.05 vs. Ang II or GB plus Ang-(1–9) or Ang-(1–9) þ 779 rats (after ANOVA).

Journal of Hypertension

4 and 6 weeks, as compared to control rats. Continuous administration of Ang-(1–9) to hypertensive rats for 2 weeks significantly blunted the increase of cardiac weight, cardiac weight/body weight and cardiac weight/ tibial length ratios induced both by Ang II administration and Goldblatt surgery. Ang-(1–9) also blunted the changes in LVEF and LVFS in both hypertensive models, with no effect in control rats. The coadministration of Ang-(1–9) with A779 did not affect the ability of Ang(1–9) to reverse cardiac hypertrophy and the increase in LVEF and LVFS observed in hypertensive rats (Tables 3 and 4). However, the AT2R antagonist PD123319 abolished the beneficial effects of Ang-(1–9) on cardiac hypertrophy. Consistently, both cardiomyocyte area and perimeter increased in Ang II-treated rats (Fig. 2a) and Goldblatt rats (Fig. 2b). The administration of Ang-(1–9) to hypertensive rats reversed the increase in cardiomyocyte area and perimeter (Fig. 2a,b). The coadministration of the Mas receptor blocker A779 with Ang-(1–9) had no effect on the Ang-(1–9)-dependent suppression of cardiomyocyte hypertrophy (Fig. 2a,b). However, the coadministration of Ang-(1–9) with PD123319 blunted the antihypertrophic effect of Ang-(1–9). Picrosirius red staining of heart sections revealed increased total collagen LV staining in hypertensive rats, as compared with control rats (Fig. 2c,d), both in the subendocardium and myocardium (Fig. 2e,f). Ang-(1–9) infusion for 2 weeks reduced collagen staining in subendocardium and myocardium. The reduction in collagen staining caused by Ang-(1–9) was resistant to A779 coadministration, but it was prevented by the coadministration of PD123319 (Fig. 2c–f). Consistent with cardiac collagen morphometry, cardiac collagen I, measured by western blot, increased significantly in hypertensive rats, which was reversed by Ang-(1–9) (Fig. 2g). The beneficial effect of Ang-(1–9) was blunted by coadministration with PD123319 but was not modified by coadministration with A779 (Fig. 2g). Using cultured adult rat cardiac fibroblasts, we observed that Ang-(1–9) reduced Ang II-induced fibroblast proliferation (Fig. 3a), and also reduced collagen content by 64% (Fig. 3b). In order to test whether Ang-(1–9) affects the myofibroblast axis, we evaluated the effect of Ang-(1–9) on the differentiation of fibroblasts to myofibroblasts by assessing a-SMA levels as a marker. As showed in Fig. 3c, Ang-(1–9) had no effect on a-SMA levels. www.jhypertension.com

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Ocaranza et al. TABLE 3. Effects of angiotensin-(1-9) administered for 2 weeks with or without A779 and PD123319 on echocardiographic indices of left ventricular size and function in hypertensive rats by angiotensin II infusion

N BW (g) CW (mg) RCW (mg CW/BW) CW/TL (mg/cm) LVFS (%) LVEF (%)

C

Ang II

Ang II þ Ang-(1–9)

Ang II þ Ang-(1–9) þ A779

Ang II þ Ang-(1–9) þ PD

14 318  8 990  10 321  7 25.9  0.4 31  2 53  2

11 305  1 2 1250  10a 425  10a 30.4  0.4a 37  3a 58  2a

12 319  5 990  20b 378  16b 26.5  0.7b 30  1b 50  1b

7 317  17 950  50b 357  4b 26.9  1.1b 29  3b 49  3b

7 312  14 1200  40a,c 406  14a,c 31.3  0.9a,c 38  1a,c 60  3a,c

Values as mean  SEM. A779, Ang-(1-7) blocker receptor; Ang II, angiotensin II; Ang-(1-9), angiotensin-(1-9); BW, body weight; C, control; CW, cardiac weight; LVEF, left ventricular ejection fraction; LVFS, left ventricular fraction shortening; PD121933, AT2 receptor blocker; TL, tibia length. a P < 0.05 vs. C. b P < 0.05 vs. Ang II. c P < 0.05 vs. Ang II þ Ang-(1–9) and Ang II þ Ang-(1–9) þ A779 (after ANOVA).

Effects of angiotensin-(1–9) on vasculature of hypertensive rats Aortic wall dimensions after the treatment of Ang II rats with Ang-(1–9), Mas and AT2R blockers are displayed in Fig. 4a and b. Tunica media of hypertensive rats was thicker than tunica media of control rats. Ang-(1–9) reversed (P < 0.05) the thickening of the thoracic aortas of hypertensive rats. The effect of Ang-(1–9) on vascular morphometry was not reversed by A779, but PD123319 coinfusion blocked the effect of Ang-(1–9) on the tunica media of hypertensive rats. The collagen I protein content of the aortic wall increased in hypertensive rats vs. control rats (Fig. 4c). The treatment of hypertensive rats with Ang-(1–9) reversed the increase in collagen I abundance observed in hypertensive rats. The effect of Ang-(1–9) was not modified by A779, but was blocked by coinfusion with PD123319. The abundance of TGF-b1 protein in aortic wall of Goldblatt rats increased as compared to control rats. Ang-(1–9) treatment prevented the upregulation of TGF-b1 in an A779-insensitive/PD123319-sensitive manner (Fig. 4c). We evaluated endothelial function in small-diameter mesenteric arteries of Ang II hypertensive rats with/without Ang-(1–9) treatment. Arteries were perfused-pressurized and tested ex vivo. Only arteries that showed sustained vasoconstriction after addition of phenylephrine (Phe) and complete relaxation in the presence of acetylcholine (Ach) were used. Arteries from Ang II-treated rats showed reduced vasodilation in response to Ach (Fig. 4d). However, Ang-(1–9) coinfusion in addition to Ang II prevented

the lower vasodilation in response to Ach. The beneficial effect of Ang-(1–9) observed in Ang II-treated rats was blocked by coinfusion with PD123319 but not by A779 (Fig. 4d). The administration of Ang-(1–9) to nonhypertensive rats did not modify vasodilation in response to Ach. The analyses of aortic eNOS mRNA levels showed that Ang-(1–9) significantly increased the eNOS mRNA levels Goldblatt rat aorta by three-fold. This effect was blunted by PD123319 but not by A779 (Fig. 5a). Consistently, plasma nitrate levels in Goldblatt rats were 40% lower compared those from control rats (P < 0.05). Ang-(1–9) infusion to Goldblatt rats increased plasma nitrate levels to levels observed in control rats, and PD123319 but not A779 blocked this effect (Fig. 5a). Cardiac and aortic O2 – production and NADPH oxidase activity were significantly increased in the myocardium (46 and 30%, respectively, Fig. 5b) and in the aorta (180 and 133%, respectively, Fig. 5c) in Ang II-treated rats compared with sham rats (P < 0.01). Ang-(1–9) produced a significant reduction in cardiac and aortic O2
production and NADPH oxidase activity (P < 0.05) compared with Ang II-treated rats. These effects were not modified by the Mas receptor blocker but were abolished in the presence of PD123319.

Angiotensin-(1–9) produces an angiotensin type 2 receptor-dependent vasodilatory effect in rat aortic rings To test the potential direct vasodilatory effect of Ang-(1–9), thoracic aortic rings from Sprague–Dawley rats with intact

TABLE 4. Effects of angiotensin-(1–9) administered for 2 weeks with or without A779 and PD123319 on echocardiographic indices of left ventricular size and function in hypertensive rats by pressure overload

N BW (g) CW (mg) RCW (mg CW/BW) CW/TL (mg/cm) LVFS (%) LVEF (%)

Control

GB

GB þ Ang-(1–9)

GB þAng-(1–9) þA779

GB þ Ang-(1–9) þPD123319

12 250  7 680  10 330  20 203  9 28  1 49  1

11 248  14 1010  30a 410  10a 255  10a 34  1a 56  1a

12 238  6 840  40a,b 350  12b 225  8b 25  1b 47  2b

8 245  21 830  10a,b 340  10b 216  8b 26  3b 45  2b

7 230  9 981  19a 398  23a,c 244  13a,c 31  2a,c 52  3a,c

Values as mean  SEM. Control rats went sham surgery. GB, Goldblatt rats; PD121933, AT2 receptor blocker; BW, body weight; CW, cardiac weight; TL, tibia length; LVFS, left ventricular fraction shortening; LVEF, left ventricular ejection fraction; A779, Ang-(1–7) blocker receptor; Ang-(1–9), angiotensin-(1–9). a P < 0.05 vs. control. b P < 0.05 vs. GB. c P < 0.05 vs. GB þ Ang-(1–9) and GB þ Ang-(1–9) þ A779 (after ANOVA).

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Angiotensin-(1–9) in hypertension (a)

(c)

Ang II +

*‡

*

250

*† ‡ 200

*† 150

0

70

Ang II-PD +

* †

Sham –

*† ‡

*† ‡

60

Ang-(1-9)

50 40

0 Sham Ang II Ang II Ang II Ang II A779 PD – – + + +

Ang-(1-9)

Ang II-A779 +

Sham Ang II Ang II Ang II Ang II A779 PD – – + + +

Ang-(1-9)

(b)

Ang II – Total collagen volumetric fraction (%)

Cardiomyocyte area (µm2)

Ang II –

Cardiomyocyte perimeter (µm)

Sham –

Ang-(1-9)

Ang II +

4

Ang II-A779 +

* †

†

*‡

Ang II A779 +

Ang II PD +

Ang II-PD +

2

0

Sham

Ang II

Ang II

–

–

+

Ang-(1-9)

(d)

GB –

GB +

*† ‡ 250

*

200

*†

*† ‡

150

0 Sham Ang-(1-9)

GB

–

–

GB +

GB A779 +

GB PD +

GB-A779 +

GB-PD +

Ang-(1-9)

70

*† ‡

*

60

*

50

†

*

†

40

0 Sham

Ang-(1-9)

GB

–

GB

–

+

GB A779 +

GB PD +

*‡

* †

†

GB A779 +

4

0

Sham

GB

GB

–

–

+

GB PD +

Collagen I

*‡

1

0

†

2.5

† Collagen I level (fold)

Myocardial * † †

GB-PD +

β-actin

0

Ang-(1-9)

*‡

*

†

1

2

5.0

Collagen volumetric fraction (%)

Collagen volumetric fraction (%)

†

8

GB-A779 +

(g)

*‡

*

GB +

Subendocardial

Subendocardial 2

GB –

Ang-(1-9)

(f)

(e)

Sham – Total collagen volumetric fraction (%)

Sham –

Cardiomyocyte perimeter (µm)

Cardiomyocyte area (µm2)

Ang-(1-9)

0

Myocardial 5.0

*‡

*

4

* 2

*‡ † †

0

Ang-(1-9)

Sham

Ang II

Ang II

–

–

+

Ang II A779 +

Ang II PD +

†

2.5

†

0 Sham Ang II Ang II Ang II Ang II A779 PD – – + + +

Ang-(1-9)

Sham

GB

GB

–

–

+

GB A779 +

GB PD +

FIGURE 2 Ang-(1–9) attenuates cardiac remodeling secondary to experimental hypertension. Hypertensive rats received vehicle, Ang-(1–9), A779 or PD123319 for the last 14 days of the experiment, as described in Figure 1. (a) and (b), Cardiomyocyte area and perimeter of left ventricular tissue sections stained with hematoxylin and eosin (400, scale bar ¼ 50 mm) of hypertensive rats induced by Ang II infusion or GB, respectively. (c) and (d), Interstitial collagen volumetric fraction of left ventricular tissue sections stained with Picrosirius red (200, bar ¼ 50 mm) of hypertensive rats induced by Ang II or GB, respectively. Subendocardial and myocardial collagen volumetric fraction (ICVF) in hypertensive rats by Ang II (e) or GB (f). Collagen type I content in the left ventricle of hypertensive rats by Ang II infusion (g). Ang, angiotensin; GB, Goldblatt rats. Bars represent mean  SEM (7–12 rats, n ¼ 300 cells). P < 0.001 vs. control, yP < 0.001 vs. Ang II or GB, zP < 0.001 vs. Ang II or GB plus Ang-(1–9) or Ang II or GB plus Ang-(1–9) þ A779 rats (after ANOVA).

endothelium were precontracted with epinephrine. The relaxation of the established muscle tone was measured with cumulative additions of Ang-(1–9) (Fig. 6a). Ang-(1– 9) displayed a significant vasodilatory effect at picomolar Journal of Hypertension

concentrations with a maximal effect observed at a concentration of 1 nmol/l. The EC50 value for the vasorelaxant effect of Ang-(1–9) in aortic rings was 22.2  2.0 pmol/l with a maximal effect of 33  3% of relaxation compared www.jhypertension.com

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Ocaranza et al. (a)

(b)

(c) 8

Collagen level (fold)

†

1

3

6

α-SMA level (fold)

2

Cell number (fold)

*

*

4

†

2

1

2

0

0

0

–

+

+

Ang II

–

+

–

+ Ang II

–

+

–

+

Ang II

–

–

+

Ang-(1-9)

–

–

+

+ Ang-(1-9)

–

–

+

+

Ang-(1-9)

FIGURE 3 Analyses of the effect of Ang-(1–9) on fibroblast proliferation in vitro. Cultured cardiac fibroblast isolated from adult rat heart were treated with Ang II or Ang II þ Ang-(1–9). At 24–48 h cells were trypsinized and counted using trypan blue staining. (a), Fibroblast proliferation. (b) Collagen I expression in fibroblasts measured by western blot. (c) Differentiation of fibroblasts to myofibroblasts by assessing a-SMA levels by western blot. Mean  SEM. P < 0.05 vs. control, yP < 0.05 vs. Ang II (after ANOVA).

with control (R2 ¼ 0.65, Fig. 6a). The Ang-(1–9)-induced vasodilation was fully blocked by PD123319 but not by A779 and losartan (Fig. 6b and c), and it was completely abolished by endothelium ablation as well as by L-NAME (Fig. 6d and e). (a)

DISCUSSION In the present study, we tested the effects of Ang-(1–9) in two models of experimental hypertension, Ang II infusion and Goldblatt rats. To assess if Ang-(1–9) is an (d)

(c)

Collagen I β-actin

+

–

+

*

120

Ang II-A779 Ang II-PD

†

†

0

Sham –

GB –

GB +

GB-A779 +

+

6

*

60

Ang-(1-9)

Ang-(1-9)

*† ‡

4

*† 2

0

Sham Ang II Ang II Ang II Ang II A779 PD – + – + +

4

TGF-β level (fold)

(b)

*

GB +

Media thickness (µm)

GB –

GB-A779 +

120

*

*†

†

*

Sham

GB

GB

–

+

–

GB A779 +

GB PD +

GB-PD +

Control Ang II

40

Ang II + Ang-(1-9)

80

120 ‡

0

‡

Ang II + Ang-(1-9)

‡

Ang II + Ang-(1-9) + A779 Ang II + Ang-(1-9) + PD

40

80

†

10

8

6

4

Acetylcholine (-log [M]) Treatment Sham GB – –

Sham –

*‡

120 †

2

0

Ang-(1-9)

*‡

0

*

Dilation (% vs Phe contraction)

–

Media thickness (µm)

Ang-(1-9)

Ang II

Ang II

Collagen level (fold)

Sham

Dilation (% vs Phe contraction)

TGF-β

GB GB-A779 + +

EC50 (µmol/l)

Control Ang II Ang II + Ang-(1-9) Ang II + Ang-(1-9) + A779 Ang II + Ang-(1-9) + PD

0.011 0.066 0.001 0.025 1.003

60

0

Ang-(1-9)

FIGURE 4 Ang-(1–9) attenuates vascular remodeling and improves endothelial dysfunction secondary to experimental hypertension. Hypertensive rats received vehicle, Ang-(1–9), A779 or PD123319 for the last 14 days of the experiment, as described in Figure 1. (a) and (b), Media thickness of aortic tissue sections stained with hematoxylin and eosin of hypertensive rats induced by Ang II and GB, respectively. Magnification bars, in the upper panel bar ¼ 1 mm and the lower panel the bar ¼ 25 mm. (c), TGFb1 and collagen type I in the aortic wall of GB rats, measured by western blot. (d), Endothelial function of hypertensive rats by Ang II infusion were randomized to receive vehicle, Ang-(1–9) or A779 for the last 14 days of the experiment. Small-diameter mesenteric arteries were isolated, perfused and pressurized (60 mmHg) for contractility studies. Maximal contraction was induced by phenylephrine (Phe), and the vasodilation in response to increasing dose of acetylcholine (Ach) was determined by video-microscopy. The graph depicts the dose-response curve to Ach (mean  SEM) for each experimental group. P < 0.001 vs. control, Ang II plus Ang-(1–9), Ang II plus Ang-(1–9) and plus A779 (after ANOVA). Graph bars represent mean  SEM of media thickness in aortic sections from 7–12 animals in each group.  P < 0.001 vs. control, yP < 0.001 vs. Ang II or GB, zP < 0.001 vs. Ang II or GB plus Ang-(1–9) or Ang II or GB plus Ang-(1–9) þ A779 rats (after ANOVA). GB, Goldblatt rats; TGFb1, transforming growth factor b1.

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Angiotensin-(1–9) in hypertension (b)

(a)

(c) 1600

eNOS

4

†

600

†

†

400

200

‡

*

1200

†

800

†

400

‡

2

0

0

25

18

*‡

*

0

†

†

‡

* 2

†

15

†

RLUx 10–3

†

‡

*

20

4

Plasma nitrate (µmol/l)

†

RLUx 10–3

eNOS mRNA level (fold)

18S

*‡

*

RLU/sec/mg protein

RLU/sec/mg protein

800

10

12

†

†

+ + – –

+ + + –

6

5 0

0

Ang-(1-9)

Sham

GB

GB

–

–

+

GB A779 +

GB PD +

0 – – – –

+ – – –

+ + – –

+ + + –

+ + – +

Ang-II Ang-(1-9) A779 PD123319

– – – –

+ – – –

+ + – +

Ang-II Ang-(1-9) A779 PD123319

FIGURE 5 Effect of Ang-(1–9) in aorta eNOS mRNA, plasma nitrates levels and oxidative stress in left ventricle and aorta of hypertensive rats. Hypertensive rats received vehicle, Ang-(1–9), A779 or PD123319 for the last 14 days of the experiment, as described in Figure 1. (a), Aorta eNOS mRNA levels and plasma nitrate levels of GB rats. (b), NADPH oxidase activity (upper) and superoxide production (lower) in the left ventricle of hypertensive rats by Ang II infusion. (c), NADPH oxidase activity (upper) and superoxide production (lower) in the aorta of hypertensive rats by Ang II infusion. Results are presented as mean  SEM (8–12 rats per group). P < 0.001 vs. control, y P < 0.001 vs. Ang II or GB, zP < 0.001 vs. Ang II or GB plus Ang-(1–9) or GB plus Ang-(1–9)þ A779 rats (after ANOVA). eNOS, endothelial nitric oxide synthase; GB, Goldblatt rats; RLU, relative light units.

antihypertensive peptide able to ameliorate/reverse endorgan damage, we choose to start Ang-(1–9) infusion after hypertension was already established. We observed that Ang-(1–9) infusion: decreased SBP, reversed cardiovascular remodeling, improved the cardiovascular function of hypertensive animals and decreased the levels of Ang II, cardiovascular oxidative stress and ACE activity. Moreover, we evidenced dose-dependent vasodilation in response to Ang-(1–9) in isolated precontracted aortic rings. All these effects were dependent on AT2R activity. Our data showed that the continuous infusion of Ang-(1–9) decreased SBP in animals that were already hypertensive for several weeks. Contrasting with these results, a recent study did not find any antihypertensive effect of Ang-(1–9) infusion in stroke-prone spontaneously hypertensive rats (SHRSP) [10]. This difference could be the result of a higher dosage used in our study (600 ng/kg per min) as compared to the dosage used in SHRSP (100 ng/kg per min) [10]. In previous studies in deoxycorticosterone acetate salt hypertensive rats and normotensive control animals, we observed that the Rho kinase inhibitor fasudil reduced BP and increased Ang-(1–9) plasma levels, without modifying Ang-(1–7) levels [16]. Thus, the findings of the present study are consistent with the previous report. The low levels of Ang-(1–9) in Goldblatt and Ang II-infused rats and the antihypertensive effect of Ang-(1–9) observed in the present study suggest that Ang-(1–9) may be implicated in long-term BP regulation and could be a relevant target for the treatment of hypertension. Previous studies have shown that AT2R activation causes vasodilation of precontracted isolated arteries [26], results in bradykinin release [27] and increases the production of Journal of Hypertension

nitric oxide and cyclic guanosine monophosphate (cGMP) through bradykinin-dependent or independent pathways [28]. We observed that the antihypertensive effect of Ang-(1–9) was blocked by the AT2R-antagonist, both in Goldblatt and Ang II-infused rats, whereas the Mas receptor blocker did not block the antihypertensive action of Ang-(1–9). To test if Ang-(1–9) has a direct effect on AT2R, we used aortic rings precontracted with epinephrine. We observed that Ang-(1–9) caused dose-dependent vasodilation with an EC50 of 22.2 pmol/l, a concentration similar to the Ang-(1–9) levels we found in plasma of hypertensive rats infused with Ang-(1–9). Moreover, vasodilation induced by Ang-(1–9) was prevented by the AT2R pharmacological blocker and was resistant to the Mas blocker, supporting the hypothesis of Ang-(1–9) action via AT2R. Our studies also show that Ang-(1–9)-induced vasodilation was prevented by endothelium ablation or eNOS blockade with L-NAME. All these results suggest that SBP reduction caused by the infusion of Ang-(1–9) in the present study is mediated by the AT2R activation and may involve the endothelial nitric oxide production. A recent study with human embryonic kidney-293 cells stably transfected with either AT1R or AT2R confirmed previous reports that Ang II has high-binding affinity for AT2R (5.2  10
10 mol/l) [29–34], whereas Ang-(1–7) has lower affinity for AT2R (2.5  10
7 mol/l, respectively) [29]. The experiments in rats of the present study showed plasma Ang-(1–7) levels in the range 5–7  10 –12 mol/l. In addition, the studies with isolated aortic rings showed that Ang-(1–9) has a dose-dependent vasodilatory action with an EC50 ¼ 2.2  10 –11 mol/l, an effect that was not affected by the Mas receptor blocker. These observations www.jhypertension.com

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Ocaranza et al.

% of vasodilation

(a)

(b)

50

** **

40

*

30

1g

20

5 min

10

–14

–12

–10

Epinephrine Ang-(1-9) Cch PD123319

Epinephrine Ang-(1-9) Cch

0 –8

Log [Ang-(1-9)] M (c)

Ang-(1-9) Ang-(1-9) + PD123319 Ang-(1-9) + A779 Ang-(1-9) + Losartan 1g

% of vasodilation

50

5 min

40 30

Epinephrine Ang-(1-9) Cch A779

20 10

*

0

(d)

Epinephrine Ang-(1-9) Cch Losartan

(e)

1g

SNP

SNP

Ang-(1-9) Ang-(1-9) – endothelium Ang-(1-9) + L–NAME

5 min

Epinephrine Ang-(1-9) Cch

Epinephrine Ang-(1-9) Cch L-NAME

% of vasodilation

50 40 30 20 10

*

*

0

FIGURE 6 Vasodilatory effect of Ang-(1–9) in rat aortic rings. (a) Effect of cumulative concentrations of Ang-(1–9) (10 fmol/l – 10 nmol/l) in endothelium-intact aortic rings precontracted with epinephrine (1 mmol/l). Ang-(1–9) was added when vascular contractions induced by epinephrine reached a plateau. Carbachol (Cch, 10 mmol/l) was added at the end of the recording to assess the endothelial function. Data are expressed as percentage of vasodilation and shown as mean  SEM (n ¼ 5), P < 0.01 and  P < 0.001 vs. Ang-(1–9) 10 fmol/l post-ANOVA. (b) Tracing illustrating of precontracted aortic rings with epinephrine 1 mmol/l), pretreated with vehicle (n ¼ 4), the AT2R antagonist PD123319 (1 mmol/l, n ¼ 4), the Mas receptor antagonist A779 (500 nmol/l, n ¼ 4) and the AT1R antagonist, losartan (1 mmol/l, n ¼ 6) for 10 min. Ang-(1–9) (1 nmol/l) was added to elicit relaxation when vascular contractions induced by epinephrine reached a plateau in endothelium-intact aortic rings. (c) Graph summarizing the effect of PD123319, A779 and losartan on the vasodilatory effect of Ang-(1–9). Data are expressed as percentage of vasodilation (mean  SEM), P < 0.01 vs. Ang-(1–9). (d) The endothelium and nitric oxide-dependence of Ang-(1–9) vasodilatory effect was tested in endothelium-denuded (n ¼ 4) and L-NAME (300 mmol/l, n ¼ 3) pretreated aortic rings respectively. Sodium nitroprusside (SNP; 1 mmol/l) was added at the end of the recording to assess for vascular muscle responsiveness to nitric oxide. The graph shows the effect of endothelium ablation and endothelial nitric oxide synthase inhibition on the vasodilatory effect of Ang-(1–9) in aortic rings. Relaxation is expressed as a percentage of the maximal contraction (mean  SEM), P < 0.01vs Ang-(1–9).

suggest that the beneficial actions of Ang-(1–9) infusion in BP and cardiovascular remodeling are not mediated by the Mas receptor. Flores et al. [9] recently demonstrated that Ang-(1–9) binds to the AT2R (pKi ¼ 6.3  0.1) in HeLa cells exogenously expressing the receptors via adenovirusmediated gene transfer. Thus, new studies are needed to confirm binding affinity and selectivity of AT1R, AT2R and Mas receptor for Ang-(1–9). AT2R is the predominant Ang II receptor subtype in the fetal and early postnatal period, and the absence of AT2R negatively affect cardiac and renal maturation and growth [35–37]. Thus, studies with tissuespecific and inducible AT2R knockout in mice will help to 780

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clarify the role of AT2R in Ang-(1–9) beneficial actions on BP and cardiovascular remodeling. The influence that AT2R activation may have on BP in vivo is controversial. Studies using AT2R-knockout mice show elevated SBP and enhanced sensitivity to vasopressor effects of Ang II [38]. Conversely, lentiviral expression of the AT2R in spontaneously hypertensive rat hearts did not affect BP or cardiac hypertrophy compared with wild-type rats [39]. However, a subsequent study in rats with AT2R adenoviral transfection has shown lower BP values than control rats [40]. Overexpression of AT2R in vascular smooth muscle cells of transgenic mice did not alter SBP Volume 32  Number 4  April 2014

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Angiotensin-(1–9) in hypertension

but markedly impaired Ang II-induced vasopressor activity [41]. Selective stimulation of AT2R by CGP42112 in Sprague–Dawley rats [42] and by compound 21 in spontaneously hypertensive rats (SHRs) [43] decreased SBP. The lowering of SBP using AT2R agonist treatment was potentiated by concomitant AT1R blockade [42,43]. The results from Ang-(1–9) infused rats and aortic rings indicate that the selective activation of AT2R may contribute to the antihypertensive action of Ang-(1–9) infusion in vivo we observed in Ang II and Goldblatt rats. However, further studies are needed to evaluate the potential beneficial effect of Ang-(1–9) in controlling BP with and without additional AT1R blockade. According to our data, one contributing factor to Ang-(1–9)-dependent BP reduction could be a reduction of total peripheral resistance. In the kidney, the AT2R is expressed in the vasculature and the proximal tubules [44], and several studies have demonstrated that AT2R activation enhances natriuresis [44–46]. Thus, a second mechanism implicated in the antihypertensive action of Ang-(1–9) would be increased natriuresis due to renal AT2R agonism and enhanced nitric oxide-cGMP cascade in the kidney. Some studies have suggested that Ang-(1–9) may be an endogenous ACEI. Donoghue et al. [2] proposed that Ang(1–9) is a competitive ACEI because it is itself an ACE substrate. In the present study, the infusion of Ang-(1–9) significantly decreased ACE activity and circulating Ang II levels in hypertensive rats. Previous observations also showed that the chronic administration of Ang-(1–9) to myocardial infarcted rats by osmotic minipumps vs. vehicle for 2 weeks decreased plasma Ang II levels, inhibited ACE activity and also prevented cardiomyocyte hypertrophy [11]. Thus, our results indicate that the beneficial effects of Ang-(1–9) may also implicate the downregulation of ACE activity. Interestingly, we observed that the coadministration of Ang-(1–9) and PD123319 blocked the suppressing effects of Ang-(1–9) on circulating Ang II and ACE activity, suggesting that the downregulation of ACE activity may be a consequence of AT2R activity. The results of the present study show that Ang-(1–9) infusion reversed cardiovascular remodeling and improved the cardiovascular function of hypertensive animals. These effects could result from the antihypertensive action of Ang(1–9) and the beneficial effects of the peptide in several tissues. The functional studies in resistance arteries ex vivo showed that Ang-(1–9) preserved endothelium-dependent relaxation induced by Ach in Ang II rats. Ang-(1–9) also increased aortic eNOS mRNA levels, an effect associated with higher nitrate plasma levels. These effects of Ang-(1– 9) were blocked by PD123319, showing that Ang-(1–9) increased nitric oxide bioavailability by a mechanism mediated by the AT2R. In agreement with our results, in aortic rings of SHRSP that received chronic Ang-(1–9)– infusion, eNOS blockade significantly increased contractile response to Phe. However, eNOS blockade did not alter contractile response to Phe of aortic rings from vehicleinfused SHRSP [10]. These results suggest that Ang-(1–9) increased nitric oxide availability in SHRSP aorta [10]. In addition to nitric oxide, other vasodilators may be implicated. The incubation of Chinese hamster ovary cells with Ang-(1–9) potentiated the release of arachidonic acid Journal of Hypertension

[Hyp3Tyr(Me)8]bradykinin and resensitized the B2 receptor desensitized by bradykinin [4]. Also, Ang-(1–9) was significantly more active than Ang-(1–7), enhancing the effect of an ACE-resistant bradykinin analogue on the B2 receptor, and augmented arachidonic acid and nitric oxide release by kinin in human pulmonary endothelial cells [47]. In the current study, Ang-(1–9) reversed LV and cardiomyocyte hypertrophy in both hypertensive models through the AT2R and not by Ang-(1–7). Previous observations also showed that Ang-(1–9) prevented cardiomyocyte hypertrophy both in vivo and in vitro [11]. The antihypertrophic effect of Ang-(1–9) was not mediated by the Mas receptor and was not associated with an increase of Ang-(1–7) plasma levels [11]. By using radioligand binding assays in HeLa cells that expressed AT1 or AT2R, Flores et al. [9] reported that that in the H9c2 cell line and adult rabbit cardiomyocytes, Ang-(1–9) prevented cardiomyocyte hypertrophy induced by Ang II or vasopressin which was prevented by PD123319, implicating the AT2R [9]. Thus, the reversion of cardiac hypertrophy in our Ang II and Goldblatt rats may reflect both direct AT2R stimulation due to Ang-(1–9) and the beneficial effect of afterload reduction by reducing hypertension. The role of AT2R activation in modulating cardiomyocyte hypertrophy is contentious. In the SHRs, cardiac AT2R overexpression attenuated cardiac hypertrophy despite elevated SBP [39]. However, in cultured cardiomyocytes overexpressing both the AT1R and AT2R, Ang II stimulation promoted hypertrophy, and AT2R-mediated enhanced basal hypertrophy was maintained and was additive to that of the AT1R [48]. In addition to cardiac hypertrophy reduction, Ang-(1–9) decreased cardiac fibrosis, vascular TGFb1 expression induced by hypertension and collagen I upregulation in both cardiac and vascular tissues. These effects were blocked here by pharmacological inhibition of the AT2R. Our results are also consistent with the previous data on cardiovascular protective mechanisms mediated by AT2R stimulation [9]. In agreement with our current findings, Ang(1–9) infusion reduced cardiac fibrosis and collagen I expression in SHRSP [10]. This antifibrotic action of Ang(1–9) in SHRSP was blocked by PD123319 coinfusion, consistent with the role of AT2R stimulation [10]. Ang(1–9) also inhibited fibroblast proliferation in vitro in a PD123319-sensitive manner [10]. These results show that AT2R activation by Ang-(1–9) has an important antifibrotic action that may implicate a direct effect on fibroblasts. This evidence suggests a potential approach for hypertensive cardiovascular complications, through antihypertrophic and antifibrotic effects of chronic AT2R stimulation by Ang-(1–9). Whether the antiremodeling effects of Ang-(1–9) here observed were due to direct activation of the AT2R by Ang-(1–9) or secondary to its antihypertensive effect is hard to analyze. Our current data in both hypertensive experimental models show cardiovascular protection due to Ang-(1–9) in parallel to BP reduction. Therefore, we cannot rule out a protective effect of this peptide as a consequence of its BP reduction properties in hypertension. In this regard, it would be interesting to evaluate the effect of lower Ang-(1–9) doses (without antihypertensive effect) on hypertensive cardiovascular remodeling. www.jhypertension.com

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Ocaranza et al.

Interestingly, in rats with significant cardiac remodeling but without hypertension (with experimental myocardial infarction) randomized to receive the ACEI enalapril or the Ang II receptor blocker candesartan for 8 weeks, both treatments prevented LVH and increased plasma Ang-(1– 9) levels by several folds. Remarkably, Ang-(1–9) levels correlated negatively with different LVH markers with or without adjustment for BP reduction [11]. This effect was specific as neither Ang-(1–7), Ang II nor bradykinins were correlated with LVH. Apart from, in in-vitro experiments with cardiomyocytes incubated with norepinephrine or with insulin growth factor type 1, Ang-(1–9) also prevented hypertrophy, and this effect was not modified by the coincubation with Ang-(1–9) and A779 [11]. Together, these previous observations strongly suggest that a direct antiremodeling effect of Ang-(1–9) – through AT2R activation – in our current hypertensive experimental models is also possible beyond those effects due to BP reduction of Ang-(1–9). In conclusion, Ang-(1–9) has a vasorelaxant effect in arteries that depends on AT2R activity, the endothelium and nitric oxide production. Chronic Ang-(1–9) administration to hypertensive rats reduces SBP, improves cardiac and endothelial function and ameliorates cardiovascular remodeling and oxidative stress. Our study shows that cardiovascular Ang-(1–9) beneficial effects are not dependent on Mas receptor stimulation. These results show for the first time that Ang-(1–9) exerts opposite effects to those of Ang II, and suggest that a relative reduction of Ang-(1–9) levels as compared to Ang II may be implicated in hypertension.

ACKNOWLEDGEMENTS This work was supported by Fondecyt 1100874 to M.P.O, Fondef D11/1122 to M.P.O., J.E.J., M.C., S.L., L.M. Millennium Institute on Immunology and Immunotherapy (no. P09/016-F) to L.M. and ANILLO ACT 1111 and FONDAP 15130011 to S.L., L.G., M.C. C.M. holds a PhD fellowship from CONICYT, Chile.

Conflicts of interest There are no conflicts of interest.

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Reviewer’s Summary Evaluation

Referee 2

Referee 1 The roles of Ang(1–9) and AT2 receptors are better understood by the study. The results are important suggesting that AT2 receptors bind Ang(1–9) that activates an important pathway counteracting the deleterious effects of angiotensin II, a pathway that is therefore protective of the vasculature. The role of AT2 receptors remains far from being elucidated but this study confirms the importance of endothelial AT2 receptors and of Ang(1–9).

Journal of Hypertension

Strengths: The study advances our understanding of the impact that Ang1–9 has on cardiovascular damage during hypertension. The data distinguishes between the role of the AT2 and Mas receptors to mediate the effects of Ang1–9. Limitations: The major limitation of the study is that the experimental design cannot separate potential blood pressure dependent and independent effects of Ang1–9.

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