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Research Article

Reduced isoproterenol-induced renin-angiotensin changes andextracellular matrix deposition in hearts of TGR(A1–7)3292 ratsAna Paula Nadu, PhDa, Anderson J. Ferreira, PhDb, Timothy L. Reudelhuber, PhDc,

Michael Bader, PhDd, and Robson A. S. Santos, MDa,*aDepartments of Physiology and Biophysics and

bMorphology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil;cLaboratory of Molecular Biochemistry of Hypertension, Clinical Research Institute of Montreal, Quebec, Canada; and

dMax-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany

Manuscript received February 24, 2008 and accepted April 17, 2008

bstract

e investigated the expression of specific extracellular matrix (ECM) proteins in cardiac hypertrophy induced by isoproterenol inGR(A1–7)3292 rats. Additionally, changes in circulating and tissue renin-angiotensin system (RAS) were analyzed. Left ventricles

LV) were used for quantification of collagen type I, III, and fibronectin using immunofluorescence-labeling techniques. AngiotensinAng) II levels were measured by radioimmunoassay. Expression of RAS components was assessed by semi-quantitative poly-erase chain reaction (PCR) or real-time PCR. Isoproterenol treatment induced an increase in the expression of collagen I, III, andbronectin in normal rats. Collagen I and fibronectin expression were decreased in TGR(A1–7)3292 at basal conditions and bothroteins increased by isoproterenol treatment; however, the levels achieved were still significantly lower than those observed inreated normal rats. The increase in collagen III observed in normal rats was completely blunted in TGR(A1–7)3292. Moreover,GR(A1–7)3292 presented lower Ang II levels and angiotensinogen expression and a higher angiotensin-converting enzyme 2

ACE2) expression in LV. Isoproterenol treatment increased cardiac Ang II concentration only in normal rats, which was associatedith an increase in ACE2 and a decrease in Mas expression. These observations suggest that Ang-(1–7) specifically modulates the

xpression of RAS components and ECM proteins in LV. J Am Soc Hypertens 2008;2(5): 341–348. © 2008 American Society ofypertension. All rights reserved.

Journal of the American Society of Hypertension 2(5) (2008) 341–348

eywords: Mas receptor; ACE2; collagen; hypertrophy.

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Angiotensin (Ang) II, the main end-product of the renin-ngiotensin system (RAS) cascade, stimulates the biosyn-hesis of cardiac extracellular matrix (ECM) proteins, lead-ng to interstitial and perivascular fibrosis.1,2 On the otherand, Ang-(1–7), a bioactive component of the RAS, is

This work was supported by CNPq-PRONEX (Conselho Na-ional de Desenvolvimento Científico e Tecnológico), FAPEMIG-RONEX (Fundação de Amparo à Pesquisa de Minas Gerais), andAPES (Coordenação de Aperfeiçoamento de Pessoal de Níveluperior).

Conflict of interest: none.*Corresponding author: Robson A. S. Santos, MD, Departa-

ento de Fisiologia e Biofísica, Av. Antônio Carlos, 6627 – ICBUFMG, 31.270-901 – Belo Horizonte, MG, Brazil. Tel: (55-31)

499 2957; fax: (55-31) 3499 2924.

tE-mail: marrob@ciclope.Lcc.ufmg.br

933-1711/08/$ – see front matter © 2008 American Society of Hyperteoi:10.1016/j.jash.2008.04.012

elieved to be an antifibrotic peptide.3,4 Therefore, themount of ECM proteins in the heart could also depend ofhe balance between Ang II and Ang-(1–7) levels.

Many studies have indicated that the heart is an im-ortant target for Ang-(1–7) actions.5–7 Among its ben-ficial effects in the heart, the antifibrotic/antitrophicffect is believed to be one of the most important car-ioprotective actions of this peptide. The antiprolifera-ive and antitrophic properties of the Ang-(1–7) could behe mechanism by which this peptide improves the car-iac function and protects the heart against remodel-ng.5,7–9 Ang-(1–7) reduces the growth of cardiomyo-ytes,10 acting through the Mas receptor11 and inhibitsollagen synthesis in cardiac fibroblasts.12 Hearts fromas-deficient mice exhibits a marked change in ECM

rotein expression to a pro-fibrotic profile and importantardiac dysfunction.3,13 In addition, it has been reported

hat Ang-(1–7) prevents cardiac fibrosis elicited by either

nsion. All rights reserved.

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342 A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

eoxycorticosterone acetate (DOCA)-salt treatment orng II infusion independent of blood pressure (BP)

hanges.6,14

We have found that a 2.5-fold increase in plasma Ang-1–7) levels in transgenic rats TGR(A1–7)3292 (TG) har-oring an Ang-(1–7)-producing fusion protein leads to aess pronounced cardiac hypertrophy induced by isoproter-nol, increase in dP/dt, reduction in the duration of reper-usion arrhythmias, and an improvement in the postisch-mic cardiac function in isolated perfused hearts.15 Also,his TG line presents an increased stroke volume and aecreased total peripheral resistance.16 The aim of the cur-ent study was to investigate the effects of isoproterenol onhe expression of specific ECM proteins in hearts fromGR(A1–7)3292 and determine whether the changes inCM expression are associated with alterations in cardiacAS components.

ethods

nimals

Male Sprague-Dawley (SD) and TGR(A1–7)3292 ratseighting 300 to 350 g were obtained from transgenic

nimal facilities at the Laboratory of Hypertension, Federalniversity of Minas Gerais, Brazil. All experimental pro-

ocols were performed in accordance with the guidelines forhe humane use of laboratory animals of our Institute andpproved by local authorities.

eart Hypertrophy

Heart hypertrophy was induced in SD and TG rats byaily injection of isoproterenol (2 mg/kg intraperitoneally, 7ays) and control groups (SD and TG) received injections ofehicle (0.9% NaCl 0.1 mL/100 g intraperitoneally, 7 days).t the end of the 7-day period, the rats were decapitated and

he hearts removed. The heart chambers were dissected andhe left ventricles (LV) were immediately frozen or fixed forCM protein analysis.

mmunohistochemical Analysis

The LVs of SD and TG rats treated or not with isopro-erenol (n � 3 for each group) were harvested, washed inhosphate-buffered saline to remove excess blood, andryofixed in a –80° C solution of 80% methanol and 20%imethyl sulfoxide for 7 days. Immunofluorescence labelingnd quantitative confocal microscopy were used to investi-ate the distribution and quantity of collagen types I, III,nd fibronectin according to Santos et al.3 Nuclei wereabeled with 4=6-diamidino-2-phenylindole dihydrochlorideDAPI) cat. #D1306, Molecular Probes (Carlsbad, CA).

ng II Measurements

After decapitation, the LVs of rats treated or not with

soproterenol (n � 6–9) were collected and frozen imme- C

iately. The organs were homogenized with 0.045 N HCl inthanol (10 mL/g of tissue) containing 0.9 �mol/L p-hy-roxymercuribenzoate, 131.5 �mol/L of 1,10-phenanthro-ine, 0.9 �mol/L phenylmethylsulfonyl fluoride, 1.75mol/L pepstatin A, 0.032% Ethylenediamine Tetraaceticcid (EDTA), and 0.0043% protease-free bovine serum

lbumin. In addition, blood samples for Ang II measure-ents were collected in polypropylene tubes containing 1mol/L p-hydroxymercuribenzoate, 30 mmol/L of 1,10-

henanthroline, 1 mmol/L phenylmethylsulfonyl fluoride, 1mol/L pepstatin A, and 7.5% EDTA. After extraction of

eptides using Bond-Elut phenylsilica cartridges (Varian,nc., Palo Alto, CA) from tissue homogenates and plasma,ollected as described above, Ang II levels were measuredy radioimmunoassay according to Botelho et al.17 Proteinoncentration in the crude homogenates was determined byhe Bradford method.18

emiquantitative PCR

LVs obtained from rats that received vehicle or isoprotere-ol (n � 3–4) were homogenized, and the total RNA wassolated with TRIzol reagent (Invitrogen Life Technologies,arlsbad, CA) according to the manufacturer’s protocol. RNA

amples (1 �g) were treated with DNAse to eliminate genomicNA present in the samples. Total RNA treated with DNAseas reverse transcribed using random primers. The single-

tranded cDNA was amplified by polymerase chain reactionPCR) using 33 cycles for angiotensin-converting enzyme 2ACE2) under the following conditions: 30 seconds, 95° C; 30econds, 60° C; 60 seconds, 72° C; and 35 cycles for angio-ensinogen under the following conditions: 30 seconds, 95° C;0 seconds, 56° C; 60 seconds, 72° C. cDNA was amplifiedsing the following set of primers: ACE2, forward 5=-AAAGTTCACTTGCTTCTTGG-3= and reverse 5=-TCAT-TAAATGGTGCTCATGG-3=; angiotensinogen, forward 5=-CACCCCTGCTACAGTCCAC-3= and reverse 5=-ACCC-CTCTAGTGGCAAGTT-3=. The same samples were em-loyed for Glyceraldehyde 3-phosphate dehydrogenaseGAPDH) cDNA amplification using the forward primer 5=-TGTTGCCATCAACGACCCC-3= and the reverse primer=-ATGAGCCCTTCCACAATGCC-3= to ensure that equalmounts of RNA samples were reversely transcribed. Theesults were reported as the ratio between ACE2 or angio-ensinogen and GAPDH values.

eal-Time PCR

Real-time PCR was used to assess Mas receptor ex-ression (n � 4 –5) using the forward primer 5=-TGAC-ATTGAACAGATTGCCA-3= and the reverse primer=-TGTAGTTTGTGACGGCTGGTG-3=. The same sam-les were employed for GAPDH cDNA amplificationsing the forward primer 5=-ATGTTCCAGTATGACTC-ACTCACG-3= and the reverse primer 5=-GAAGACAC-

AGTAGACTCCACGACA-3= to ensure that equal

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343A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

mounts of RNA were reversely transcribed. The resultsere reported as the ratio of Mas to GAPDH.

tatistical Analysis

All data are reported as means � SEM. Statistical sig-ificance was estimated using one-way analysis of varianceollowed by Newman-Keuls posttest or Student’s t test. A Palue � .05 was considered significant.

esults

Under basal conditions, TG rats presented a lower ex-ression of collagen type I and fibronectin (Figures 1 and 2),ut not of collagen type III in LVs (Figure 3). Isoproterenolreatment induced an increase in the expression of the col-agen type I, III, and fibronectin in SD rats. However, in TGats, only the expression of collagen type I and fibronectinere increased after isoproterenol treatment and the levels

f expression achieved were still significantly lower than v

hose observed in treated SD rats (collagen type I: 18.5 �.9 vs. 22.8 � 1.9 arbitrary units in SD, Figure 1; fibronec-in: 11.6 � 0.9 vs. 19.6 � 1.4 arbitrary units in SD, Figure). Interestingly, the increase in the expression of collagenype III observed in isoproterenol-treated SD rats was com-letely blunted in TG rats (TG: 13.60 � 1.07 vs. 13.64 �.04 arbitrary units in isoproterenol-treated rats; SD: 12.95

0.87 vs. 20.85 � 0.97 arbitrary units in isoproterenol-reated rats; Figure 3).

Because Ang II is closely involved in the regulation ofhe ECM proteins, its levels were evaluated in hearts andlasma of SD and TG rats at basal conditions and aftersoproterenol treatment. As shown in Table 1, cardiacng II levels were significantly decreased in TG rats atasal conditions and after isoproterenol treatment. Inddition, the increase in Ang II concentration induced bysoproterenol treatment observed in hearts from SD rats1.23 � 0.06 vs. 1.06 � 0.07 pg/mg protein in vehicle-reated SD rats), was not present in TG rats (0.83 � 0.10

Figure 1. Immunofluorescent localization of collagen type Iin LVs. Low-magnification images reveal the overall level ofimmunostaining in (A) SD vehicle, (B) SD ISO, (C)TGR(A1–7)3292 (TG) vehicle, and (D) TG ISO groups. (E)Negative control was obtained when the primary antibodywas omitted from the incubation procedure. Original mag-nification, 40�. (F) Quantification of collagen type I in LVs.Data are shown as means � SEM. A.U., arbitrary unit; ISO,isoproterenol; LV, left ventricle; SD, Sprague-Dawley. *P �.05. **P � .01 (one-way analysis of variance followed byNewman-Keuls posttest).

s. 0.77 � 0.04 pg/mg protein in vehicle-treated TG rats).

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344 A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

n contrast, plasma Ang II levels were augmented in TGats at basal conditions and after isoproterenol treatment.

oreover, isoproterenol administration did not changehe plasma Ang II concentration in SD and TG rats.

To evaluate possible mechanisms underlying the selec-ive change in cardiac Ang II levels observed in TG rats, wenalyzed the expression of ACE2 and angiotensinogen.CE2 expression was significantly increased in LVs of TG

ats at basal conditions (0.151 � 0.033 vs. 0.068 � 0.006 inD rats), but not after isoproterenol treatment, which in-uced an increase in ACE2 expression only in SD ratsTable 2). Hearts from TG rats also presented a lowerxpression of angiotensinogen before and after isoprotere-ol treatment. No significant changes in angiotensinogenxpression were observed after isoproterenol treatment inoth strains (Table 2). The expression of Mas receptorRNA was similar in SD and TG rats under basal condi-

ions. Isoproterenol treatment induced a significant decreasen the expression of Mas in SD rats (0.140 � 0.067 vs..541 � 0.140 in vehicle-treated rats), but not in TG rats

Figure 2. Immunofluorescent localization of fibronectin inLVs. Low-magnification images reveal the overall level ofimmunostaining in (A) SD vehicle, (B) SD ISO, (C)TGR(A1–7)3292 (TG) vehicle, and (D) TG ISO groups. (E)Negative control was obtained when the primary antibodywas omitted from the incubation procedure. Blue stainingcorresponds to nuclei labeled with 4=6-DAPI. Original mag-nification, 40�. (F) Quantification of fibronectin in LVs.Data are shown as means � SEM. A.U., arbitrary unit;DAPI, diamidino-2-phenylindole dihydrochloride; ISO, iso-proterenol; LV, left ventricle; SD, Sprague-Dawley. *P �.05. ***P � .001 (one-way analysis of variance followed byNewman-Keuls posttest).

Table 2). t

iscussion

In the current study, using a transgenic animal model,hich presents a suitable increase of 2.5-fold in the plasmang-(1–7) levels, we demonstrated that Ang-(1–7) plays aifferential control in the amount of specific ECM proteins,ontributing to the antifibrotic effect of this peptide. Theseng-(1–7) actions were further evidenced when the ratsere challenged with isoproterenol, a well-known �-adren-

rgic agonist able to induce cardiac remodeling. In addition,GR(A1–7)3292 rats showed a significant reduction in theardiac Ang II levels even after isoproterenol treatment,uggesting that Ang-(1–7) might exerts its antifibrotic ef-ects also through its regulating effect on the cardiac Ang IIevels, since this peptide is believed to induce accumulationf ECM proteins in the heart.Many studies have proposed that the cardioprotective

xis of the RAS composed by ACE2, Ang-(1–7), and Maslays antiproliferative effects in the heart.3,6,10,12,14,19,20

ng-(1–7) binds to a specific site distinct from Ang II

ype 1 and type 2 receptors in adult rat cardiac fibroblasts

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345A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

o inhibit the collagen synthesis.12 Interestingly, Ang-1–7) infusion prevents collagen deposition in DOCA-alt animals14 and in Ang II-infused rats (6) independentf changes in BP. In addition, the compound AVE 0991,synthetic Ang-(1–7) analogue, decreases the ECM pro-

ein deposition induced by isoproterenol treatment with-ut any changes in BP.21 Because Mas-deficient miceresent a marked change in the ECM protein expressiono a pro-fibrotic pattern,3 these findings indicate thatng-(1–7) acts directly via Mas present in cardiac fibro-lasts to exert its antifibrotic effects. Our present data aren accordance with these previous studies, because theardiac ECM proteins in TG rats, which are normoten-ive,15 were finely regulated by Ang-(1–7). The directffect of Ang-(1–7) on the fibroblast functions12 is cor-oborated by our findings, after a reduction of some ECMroteins was also observed in untreated TG rats that didot show any cardiac deleterious consequences caused bysoproterenol treatment. It is important to note that, de-endent of the experimental condition (treated or not withsoproterenol), Ang-(1–7) selectively regulated the me-

abolism of each ECM protein analyzed. A similar find- r

ng was also observed in previous studies.3,21 It remainso be established if Ang-(1–7) influences the synthesis oregradation of ECM proteins or both. In this aspect, theffects of Ang-(1–7) on the matrix metalloproteinasesetabolism should be considered in future studies. In

act, Pan et al22 have found that Ang-(1–7) and Ang IIave opposite effects on the regulation of matrix metal-oproteinases and tissue inhibitors of matrix metallopro-einases transcription in primary cultures of fibroblastsnd cardiomyocytes.22

In addition to its antifibrotic effect, Ang-(1–7) has beenlso described as an antitrophic peptide in the heart.7,10,12

allant et al10 have reported that Ang-(1–7) directly inhibitshe growth of cultured cardiomyocytes through activation of

as. Moreover, it has been demonstrated that the hypertro-hic effects of conditioned medium from Ang II–treateddult rat cardiac fibroblasts are attenuated in cardiomyo-ytes pretreated with Ang-(1–7).12 The antitrophic effect ofng-(1–7) was also viewed in our model, because in arevious study it was observed that the cardiac hypertrophicffect of isoproterenol was attenuated in TGR(A1–7)3292

Figure 3. Immunofluorescent localization of collagen typeIII in LVs. Low-magnification images reveal the overall levelof immunostaining in (A) SD vehicle, (B) SD ISO, (C)TGR(A1–7)3292 (TG) vehicle, and (D) TG ISO groups. (E)Negative control was obtained when the primary antibodywas omitted from the incubation procedure. Blue stainingcorresponds to nuclei labeled with 4=6-DAPI. Original mag-nification, 40�. (F) Quantification of collagen type III inLVs. Data are shown as means � SEM. A.U., arbitrary unit;DAPI, diamidino-2-phenylindole dihydrochloride; ISO, iso-proterenol; LV, left ventricle; SD, Sprague-Dawley. ***P �.001 (one-way analysis of variance followed by Newman-Keuls posttest).

ats.15 These data suggest that, besides its role in regulating

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346 A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

he ECM protein balance in the heart, Ang-(1–7) is alsomportantly involved in the maintenance of the myocytestructure.

It is well-established that Ang II increases the cardiacCM proteins content acting through AT1 receptors (1). On

he other hand, it has been reported that chronic infusion ofng-(1–7) in Wistar rats significantly decreases the Ang II

evels in the heart.23 In the present study, the antifibroticffect observed in TG rats was accompanied by a significantecrease in LV Ang II levels at basal conditions and aftersoproterenol treatment. Therefore, Ang-(1–7) could alsoegulate the ECM proteins content by decreasing the Ang IIoncentration in the heart. Also, isoproterenol treatmentnduced an increase in Ang II concentration in hearts fromD rats, but not in TG rats, further suggesting that thentifibrotic effect observed in TG rats involves a downregu-ation of cardiac Ang II. The regulatory action of Ang-(1–7)n the Ang II levels is apparently heart-specific, because noignificant alterations in the Ang II levels of other organs suchs lung, kidney, adrenal glands, and testis were observed in TGats (Ferreira AJ, Nadu AP, Reudelhuber TL, Bader M, SantosAS, unpublished data). A similar finding was also reportedy Mendes et al.23 Alternatively, Iwata et al12 have shown thatretreatment of adult rat cardiac fibroblasts with Ang-(1–7)nhibits the Ang II–induced increases in collagen synthesis.his possibility as well as a direct effect of Ang-(1–7) inbroblasts is particularly likely, considering that increases of

ocal Ang II did not change heart remodeling.24

The higher plasma Ang II levels observed in TG rats wasot fully investigated in the current study; however, lungCE expression and activity, plasma ACE activity, andlasma renin activity are not the cause of this effect, sinceo significant differences were observed between SD andG rats (Ferreira AJ, Nadu AP, Reudelhuber TL, Bader M,antos RAS, unpublished data).To identify possible mechanisms underlying the regula-

ion of the Ang II levels in the heart, we examined the

able 1eft ventricle and plasma angiotensin II levels in Sprague-awley and TGR(A1–7)3292 rats treated or not with

soproterenol

eft ventricle Angiotensin II (pg/mg protein)Vehicle ISO

D 1.06 � 0.07 (n � 6) 1.23 � 0.06* (n � 7)G 0.77 � 0.04†† (n � 6) 0.83 � 0.10†† (n � 7)lasma Angiotensin II (pg/mL)

Vehicle ISOD 76.69 � 10.14 (n � 8) 72.94 � 9.40 (n � 7)G 112.0 � 12.37 (n � 8)† 118.0 � 14.06 (n � 9)†

ISO, isoproterenol; SD, Sprague-Dawley; TG, TGR(A1–7)3292.The data are expressed as mean � SEM.* P � .05 when compared with vehicle-treated SD animals and† P � .05;†† P � .01 when compared with the corresponding SD control

Mats (Student’s t test).

xpression of some RAS components in SD and TG rats.ecause angiotensinogen is the precursor of the angiotensineptides, changes in its expression could explain the down-egulation of Ang II in hearts of TGR(A1–7)3292 rats. Inact, TG rats presented a lower expression of angiotensino-en mRNA in both basal and isoproterenol conditions. Also,e found that the expression of ACE2 is augmented in theV of TG rats compared with SD rats at basal conditions.ecause ACE2 is an Ang II–degrading enzyme, it couldontribute to the lower Ang II concentration in the hearts ofG rats. However, no significant changes in ACE2 expres-ion was observed in TG rats treated with isoproterenol.dditionally, isoproterenol treatment induced an increase ofCE2 expression only in SD rats. These findings are in

greement with previous reports showing that ACE2 iselectively regulated according to the pathologic stimulusnd to the duration of the injury.25,26 Furthermore, becauseCE2 is the main Ang-(1–7)–forming enzyme, an increased

ocal production of this peptide could be the cause of theownregulation of its receptor observed in SD rats treatedith isoproterenol. Nevertheless, changes in the expressionf Mas are not involved in the regulation of the Ang IIevels in TG rats, after its expression was similar to thatbserved in SD rats.

onclusion

A growing body of evidence suggests that the heart is anmportant target for the actions of the ACE2-Ang-(1–7)-

able 2ffects of isoproterenol treatment on angiotensinogen, ACE2nd Mas expression in left ventricles of Sprague-Dawley andGR(A1–7)3292 rats

CE2 expressionVehicle ISO

D 0.068 � 0.006 (n � 4) 0.142 � 0.013** (n � 4)G 0.151 � 0.033 (n � 4)† 0.159 � 0.035 (n � 4)ngiotensinogen expression

Vehicle ISOD 0.046 � 0.003 (n � 3) 0.045 � 0.001 (n � 3)G 0.031 � 0.001†† (n � 3) 0.032 � 0.005† (n � 3)as receptor expression

Vehicle ISOD 0.541 � 0.140 (n � 4) 0.140 � 0.067* (n � 4)G 0.483 � 0.140 (n � 5) 0.479 � 0.161 (n � 5)

ACE2, angiotensin-converting enzyme 2; ISO, isoproterenol;D, Sprague-Dawley; TG, TGR(A1–7)3292.The data are expressed as mean � SEM.* P � .05, ** P � .01 when compared with vehicle-treated SD

nimals and† P � .05,†† P � .01 when compared with the corresponding SD control

ats (Student’s t test). The results are reported as the ratio betweenCE2, angiotensinogen, or Mas and GAPDH values.

as axis. Among its cardiac effects, the antifibrotic/antitro-

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347A.P. Nadu et al. / Journal of the American Society of Hypertension 2(5) (2008) 341–348

hic action is increasingly considered to play an importantole in the homeostasis of the heart. ACE2 overexpressionn the heart (20), Ang-(1–7) infusion,6,14 and deletion of

as receptor3 have validated this hypothesis. In the presenttudy, using a transgenic model, we provided further evi-ence for a key role of Ang-(1–7) in the modulation of thexpression of ECM proteins in the heart. Further studies inas�/� and ACE2�/� animals would be important to fur-

her pursuing the role of Ang-(1–7)/Mas/ACE2 in hearttructure and function.

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