Role of AT2 Receptors in the Cardioprotective Effect of AT1

Antagonists in MiceJiang Xu, Oscar A. Carretero, Yun-He Liu, Edward G. Shesely, Fang Yang,

Alissa Kapke, Xiao-Ping Yang

Abstract—Angiotensin II (Ang II) acts mainly on two receptor subtypes: AT1 and AT2. Most of the known biologicalactions of Ang II are mediated by AT1 receptors; however, the role of AT2 receptors remains unclear. We tested thehypothesis that the cardioprotective effects of AT1 receptor antagonists (AT1-ant) after myocardial infarction (MI) arepartially mediated by activation of AT2 receptors; thus in AT2 receptor gene knockout mice (AT22/Y), the effect ofAT1-ant will be diminished or absent. MI was induced by ligating the left anterior descending coronary artery. Fourweeks later, AT22/Y and their wild-type littermates (AT21/Y) were started on vehicle, AT1-ant (valsartan, 50 mg/kgper day), or ACE inhibitor (enalapril, 20 mg/kg per day) for 20 weeks. Basal blood pressure and cardiac function as wellas remodeling after MI did not differ between AT21/Y and AT22/Y. AT1-ant increased ejection fraction and cardiacoutput and decreased left ventricular diastolic dimension, myocyte cross-sectional area, and interstitial collagendeposition in AT21/Y, and these effects were significantly diminished in AT22/Y. ACE inhibitors improved cardiacfunction and remodeling similarly in both strains. We concluded that (1) activation of AT2 during AT1 blockade playsan important role in the therapeutic effect of AT1-ant and (2) the AT2 receptor may not play an important role inregulation of cardiac function, either under basal conditions after MI remodeling or in the therapeutic effect of ACEinhibitors. (Hypertension. 2002;40:●●●-●●●.)

Key Words: receptors, angiotensinn angiotensin antagonistn myocardial infarctionn heart failuren mice

Angiotensin II (Ang II), the principal effector peptide inthe renin-angiotensin system (RAS), binds to two dis-

tinct receptor subtypes, AT1 and AT2.1,2 Most well-knownactions of Ang II in the cardiovascular system are mediatedby the AT1 receptor,3,4 whereas little is known about thefunction of the AT2 receptor.5 It has been proposed thatblockade of the AT1 receptor increases Ang II, which acti-vates the AT2 receptor and releases kinins and nitric oxide(NO), leading to cardioprotection.2,6–8We previously showedthat in a rat model of heart failure (HF) induced by myocar-dial infarction (MI), AT1-ant had a cardioprotective effectsimilar to an ACE inhibitor (ACEi), which was partiallyblocked by an AT2-ant or B2 kinin receptor antagonist(B2-ant).2 We also found that in B2 or endothelial NOsynthase (eNOS) knockout mice, the effect of AT1-ant wasdiminished compared with their wild-type controls,7,9 sug-gesting a kinin/NO-mediated mechanism. Other studies haveshown that activation of the AT2 receptor counterbalances theeffect of the AT1 receptor, thereby inhibiting cell growth,proliferation, and hypertrophy.10–13 In cultured bovine aorticendothelial cells14 or aortas from mice overexpressing theAT2 receptor gene,15 Ang II was found to increase the release

of cGMP; this effect was further enhanced by an AT1-ant butmarkedly blocked by an AT2-ant, B2-ant or NO synthesisinhibitor, suggesting that Ang II–stimulated release of cGMPis mediated by kinins and NO through activation of the AT2

receptor.The role of the AT2 receptor in regulation of blood pressure

(BP) and cardiac function under physiological conditions, orin the pathophysiology of cardiac and vascular remodeling, isnot fully understood. It has been reported that disruption ofthe AT2 receptor in mice (AT22/Y) increases systolic BP andleads to hypersensitivity to Ang II or susceptibility to DOCA-salt hypertension.5,16,17 Wu et al18,19 recently showed thatcoronary arterial thickening and perivascular fibrosis inducedby aortic banding or cuff-induced neointima formation andinflammation were exaggerated and the response to AT1-antdiminished in AT2 receptor knockout mice. On the otherhand, Senbonmatsu et al20 and Mifune et al21 reported thattargeted deletion of AT2 receptors prevented left ventricularhypertrophy induced by pressure overload, whereas stimula-tion of AT2 increased collagen synthesis.

To clarify the role of AT2 receptors in the therapeutic effectof AT1-ant and in regulation of cardiac function and remod-

Received April 9, 2002; first decision April 29, 2002; revision accepted June 26, 2002.From the Hypertension and Vascular Research Division (J.X., O.A.C., Y.-H.L., E.G.S., F.Y., A.K., X.-P.Y), Department of Internal Medicine, and the

Division of Biostatistics and Research Epidemiology (A.K.), Henry Ford Hospital, Detroit, Mich; and Wuhan University School of Medicine (J.X.),Wuhan, People’s Republic of China.

Correspondence to Xiao-Ping Yang, MD, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI48202–2689. E-mail xpyang1@hfhs.org

© 2002 American Heart Association, Inc.

Hypertensionis available at http://www.hypertensionaha.org DOI: 10.1161/01.HYP.0000029095.23198.AD

1

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

eling after MI, we used AT22/Y mice to study whether lackof AT2 receptors (1) diminishes the cardioprotective effects ofAT1-ant and ACEi and (2) affects cardiac hemodynamics andfunction as well as morphology and histology, either underbasal conditions or after MI.

Methods

AnimalsAT22/Y mice were originally developed on a hybrid geneticbackground, then back-crossed to inbred strain C57BL/6J by ourMutant Mouse Core until congenic status was reached (101back-cross generations). Since the AT2 receptor gene is X-linked, maleknockouts were obtained as homozygous offspring (2/Y) of ho-mozygous dams (2/2). Wild-type littermates (AT21/Y) were usedas controls. Animals were housed in an air-conditioned room with a12-hour light/dark cycle and given standard chow with free access totap water. This study was approved by the Henry Ford HospitalInstitutional Animal Care and Use Committee (IACUC).

Induction of Myocardial InfarctionMale mice 10 to 12 weeks old (22 to 25 g) were anesthetized withsodium pentobarbital (50 mg/kg IP), intubated, and ventilated withroom air by a positive-pressure respirator (Harvard 680). MI wascreated as described previously.7,9

Systolic Blood Pressure and EchocardiographySystolic BP (SBP) was measured with a noninvasive computerizedtail-cuff system (BP-2000, Visitech Systems).22 Cardiac geometryand function were evaluated in awake mice, using a Dopplerechocardiographic system equipped with a 15-MHz linear transducer(Acuson c256), as described previously.7,23 All primary measure-ments, including LV wall thickness and diastolic dimensions(LVDd), were traced manually and digitized by goal-directed,diagnostically driven software installed within the echocardiograph,and ejection fraction (EF), cardiac output (CO), and mass werederived from these data.

Plasma Renin ConcentrationAt the end of the experiment, mice were lightly anesthetized withether, and blood was collected in a microhematocrit tube bypuncturing the retro-orbital plexus. Plasma renin concentration(PRC) was determined by Skinner’s method.24

Histopathological StudyHearts were stopped at diastole by intraventricular injection of 15%KCl. The LV was sectioned transversely into 3 slices and rapidlyfrozen in isopentane precooled in liquid nitrogen, then stored at270°C. Infarct size was determined by Gomori trichrome stainingand expressed as the ratio of the infarcted portion to total LVcircumference.7 For myocyte cross-sectional area and interstitialcollagen fraction, 6-mm sections from each slice were double-stainedwith (a) fluorescein-labeled peanut agglutinin to delineate the myo-cyte cross-sectional area (MCSA) and interstitial space and (b)rhodamine-labeledGriffonia simplicifolia lectin I to show the cap-illaries.2 MCSA was measured by computer-based planimetry (Jan-del). ICF was calculated as percentage of total surface area occupiedby the interstitial space minus percentage of total surface areaoccupied by the capillaries.

Experimental ProtocolsDoses of AT1-ant (valsartan, 10, 20, 40, and 60 mg/kg per day;Novartis) and ACEi (enalapril, 5, 10, and 20 mg/kg per day; Merck)were tested for their inhibitory effect on mean blood pressure (MBP)response to Ang II or Ang I (12.5, 25, 50, and 100mg/mouse).AT1-ant and ACEi were administered in drinking water for 4 weeks.We found that valsartan at 40 mg/kg per day blocked 70% of thevasopressor effect of exogenous Ang II, and enalapril at 20 mg/kgper day blocked 67% of the vasopressor effect of Ang I. Therefore,50 mg valsartan and 20 mg enalapril were chosen for the presentstudy. Four weeks after MI or sham MI, each strain was divided into(1) sham MI; (2) MI1vehicle (tap water); (3) MI1AT1-ant; and (4)MI1ACEi, with treatment continuing for 20 weeks.

Data AnalysisData are expressed as mean6SEM. Mortality rates were comparedby means of a Cox proportional hazards model. ANOVA withrepeated measures was used to compare changes (after treatment)from week 4 (before treatment) within and between strains in SBPand echocardiographic parameters. When significant group or straininteractions were observed over time, Student’st test was used tocompare the prespecified group (or strain) at all time points. A pairedt test was used to compare the difference between the fourth weekand the average from 8 through 24 weeks between strains and withintreatment groups. For heart and lung weight, infarct size, PRC, andhistopathological data, Wilcoxon’s 2-sample test was used. Whenmultiple comparisons were performed, Hochberg’s method was usedto adjust the alpha level of significance.25

Effect of AT1-ant and ACEi on Heart, Lung, and Liver Weight and PRC in AT21/Y and AT22/Y Mice

Parameters

AT21/Y AT22/Y

Sham MI (n58)

MI

Sham MI (n58)

MI

Vehicle(n511)

AT1-ant(n511)

ACEi(n510)

Vehicle(n510)

AT1-ant(n510)

ACEi(n59)

BW, g 30.860.9 31.260.7 31.360.5 30.860.6 30.560.8 29.860.9 31.160.8 31.860.7

Atria, mg 9.761 22.161.8*** 16.861.7† 17.161.8† 11.361.2 2162.8** 18.361.5 1662.2

RV, mg 23.461.8 40.264*** 31.261.8 32.563.6 22.761.1 39.565.4*** 33.861.9 29.862.2

LV, mg/10 g 31.661.9 49.561.9*** 40.261.6‡ 42.361.9† 29.460.9 48.165.2** 45.361.8 41.866.2

Lungs, mg/10 g 57.263 58.763.8 51.861.7 55.863.7 52.862.9 65.168 56.162.1 54.962.2

Liver, mg/10 g 47068 440614 454612 467615 486616 458630 469612 450614

PRC, mg AI(mL/h) 0.860.3 1.360.2* 5.961.3‡ 14.968.3‡ 1.360.6 2.961.3 4.2261.2 20.769.2

IS, % z z z 41.562.2 39.561.7 37.462.8 z z z 38.463.1 36.562 37.462

Values are mean6SE. BW indicates body weight; RV, right ventricular weight; LV, left ventricular weight corrected for BW; PRC, plasma renin concentration; IS,infarct size.

*P,0.05 vs sham; **P,0.01 vs sham; ***P,0.001 vs sham; †P,0.05 vs vehicle; ‡P,0.01 vs vehicle.

2 Hypertension September 2002

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

ResultsEarly and Late MortalityAT22/Y appeared to have higher mortality rates duringsurgery than AT21/Y; however, it did not reach statisticalsignificance (27.9% versus 14.1%). During the first 4 weeksof MI, mortality rates were similar between strains. In theAT1-ant group, 2 mice died during treatment (16.7%),whereas none of the AT21/Y mice died. In vehicle- andACEi-treated mice, mortality rates were not statisticallydifferent between AT22/Y and AT21/Y (28.6% versus26.7% with vehicle; 0 versus 9.1% with ACEi).

Body, Heart, Lung and Liver Weight, PlasmaRenin Concentration, and Infarct SizeThere was no significant difference in any of these parametersbetween strains in the sham-ligated groups. In theMI1vehicle groups, heart weight increased similarly in bothstrains. AT1-ant and ACEi reduced heart weight significantly

in AT21/Y but not AT22/Y. There was no significant changein lung or liver weight after MI. PRC was significantlyincreased after MI in AT21/Y and tended to increase inAT22/Y, but this was not statistically significant. ACEiincreased PRC 10-fold in AT21/Y and 7-fold in AT22/Y.AT1-ant significantly increased PRC in AT21/Y but notAT22/Y. Infarct size was similar in all groups (Table).

Systolic Blood Pressure and Heart RateBasal SBP and HR were similar between strains in all groups.After MI, SBP in the MI1vehicle group was lower than shamand tended to decrease further with ACEi or AT1-ant in bothstrains (Figure 1, top). There was a slight increase in HR afterMI, but it was not statistically significant. Drug treatment hadno effect on HR (Figure 1, bottom).

Cardiac Function and RemodelingThere was no difference between sham-ligated AT21/Y andAT22/Y with regard to EF, CO, LVDd, or mass. After MI,EF and CO decreased, whereas LVDd and mass increasedsignificantly by 1 month and progressed similarly over timein both strains (Figure 2). AT1-ant (20 weeks’ treatment)significantly increased EF by 69614% and CO by 37611%and reduced LVDd by 1463% and mass by 1666% inAT21/Y, and these effects were diminished in AT22/Y (EF:1667%, CO:2966%; LVDd:20.662 and mass:1565%)(Figures 3 through 5). ACEi increased EF and CO anddecreased LVDd and mass similarly in both strains.

Myocyte Size and Interstitial Collagen FractionMCSA and ICF were similar among sham-ligated mice. AfterMI, MCSA and ICF increased similarly in vehicle-treatedgroups from both strains. AT1-ant significantly decreasedMCSA and ICF in AT21/Y, and this effect was attenuated inAT22/Y. The effect of ACEi on these parameters was similarin both strains (Figures 6 and 7).

DiscussionWe found that although SBP, LV performance, and cardiacmorphology/histology were similar between AT21/Y and

Figure 1. Effect of AT1-ant and ACEi on SBP and HR in AT22/Yand AT21/Y after MI; 8 to 24 weeks, average SBP from week 8to week 24.

Figure 2. Comparison of cardiac functionand remodeling between AT22/Y andAT21/Y mice with MI or sham MI. B, beforeMI; EF, ejection fraction; CO, cardiac outputcorrected for body weight (BW); LVDd: leftventricular diastolic dimension.

Xu et al Heart Failure in AT 2 Receptor Knockout Mice 3

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

AT22/Y, both under normal conditions and during thedevelopment of CHF, the beneficial cardiac effects of AT1-ant were significantly diminished in AT22/Y mice, suggest-ing that the cardioprotective effect of AT1-ant is mediated atleast in part via the AT2 receptor. The response to ACEi wassimilar between AT21/Y and AT22/Y.

The RAS plays an important role in cardiovascular, elec-trolyte, and fluid homeostasis through its effector Ang II. AngII can also be a pathological factor in cardiac hypertrophy,

fibrosis, and CHF. The biological effects of Ang II aremediated by at least two known subtypes, AT1 and AT2.1,26

Most known biological actions of Ang II, such as vasocon-striction, cellular proliferation, and matrix deposition, areattributable to the AT1 receptor, whereas the physiologicaland pathophysiological functions of the AT2 receptor remaincontroversial. Studies have shown that activation of AT2

inhibits cell growth and proliferation, promotes cell differen-tiation and counterbalances the effect of AT1.10–13 Overex-

Figure 3. Representative 2D M-modeechocardiograms showing LV dilatation(DD, diastolic dimension) and reducedinterventricular septum (IS) and posteriorwall (PW) motion in mice with HF as wellas diminished response to AT1-ant inAT22/Y mice. Sham indicates sham cor-onary ligation.

Figure 4. Effect of AT1-ant and ACEi on EFin AT21/Y and AT22/Y with MI. Curvedgraphs show time and group effect; bargraph shows average percentage increasefrom before treatment (week 4) to aftertreatment (8 to 24 weeks). B, Before MI.

4 Hypertension September 2002

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

pression of AT2 attenuated the vasopressor response toexogenous Ang II,27 whereas deletion of the AT2 receptor(AT22/Y) increased SBP and led to hypersensitivity to AngII or susceptibility to DOCA-salt hypertension.10,16However,Hein et al17 showed that basal SBP was no different betweenAT21/Y and AT22/Y mice, although AT22/Y had increasedsensitivity to Ang II. More recently, Senbonmatsu et al20 andMifune et al21 showed that lack of AT2 receptors preventedthe development of LV hypertrophy, whereas stimulation of

AT2 increased collagen synthesis. In the present study, weobserved no difference between AT21/Y and AT22/Y withregard to SBP, cardiac function, chamber dimensions, colla-gen deposition or myocyte size, either under basal conditionsor after MI, suggesting that the AT2 receptor is not importantfor regulation of cardiac hemodynamics and function or thepathophysiology of cardiac remodeling after MI. However,we cannot exclude the possibility that we did not observe adifference in cardiac remodeling between AT21/Y andAT22/Y due to the fact that the infarcts in our study were toolarge (35% to 45% of the LV) and injury too severe, so thatthe compensatory capacity of the residual noninfarcted myo-cardium reached a maximum and no further functional and/orhistopathological difference could be detected betweenstrains.

Figure 5. Effect of AT1-ant and ACEi on LV diastol-ic dimension (LVDd) in AT21/Y and AT22/Y withMI. Curved graphs show time and group effect; bargraph shows average percentage decrease frombefore treatment (week 4) to after treatment (8 to24 weeks). B, Before MI.

Figure 6. Representative slides showing increased MCSA andinterstitial collagen deposition (green staining) in mice with heartfailure induced by MI. AT1-ant and ACEi reduced MCSA andcollagen deposition; effect of AT1-ant was diminished in AT22/Ymice.

Figure 7. Effect of AT1-ant and ACEi on MCSA and ICF inAT21/Y and AT22/Y after MI.

Xu et al Heart Failure in AT 2 Receptor Knockout Mice 5

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Despite the fact that cardiac hemodynamics and pheno-type in AT22/Y mice were similar to their wild-typecontrols, we found that AT22/Y mice with MI exhibited adiminished response to the therapeutic effect of AT1-ant,suggesting that the AT2 receptor is an important compo-nent in the cardioprotective effect of AT1-ant. This agreeswith our previous finding that in rats with MI, AT1-ant hada cardioprotective effect similar to ACEi, while part of theeffect of AT1-ant was blocked by an AT2 receptor antag-onist (AT2-ant), which by itself had no effect on LVfunction or remodeling.2 Since lack of AT2 receptors inmice did not aggravate cardiac dysfunction and remodel-ing, our data may suggest that the AT2 receptor only exertsa cardioprotective action when the AT1 receptor is blocked.Inhibition of AT1 may stimulate renin release, in turnincreasing circulating Ang II levels; increased Ang II bindsto the AT2 receptor and thereby activates AT2-mediatedaction, such as inhibiting myocyte hypertrophy and/orfibroblast proliferation, leading to cardioprotection.7,8,10

The mechanism(s) responsible for the action of the AT2

receptor remains unclear. Masaki et al27 reported that over-expression of AT2 in mice with HF significantly inhibitedAng II–induced mitogen-activated protein kinase activationin fibroblasts, inhibiting collagen synthesis. Activation ofAT2 during blockade of AT1 may also stimulate the release ofautacoids such as PGE2 and NO, either directly and/or viastimulation of kinins.2,14,15,27 Using cultured bovine aorticendothelial cells, Seyedi et al14 found that Ang II induced a 6-to 7-fold increase in cGMP release; this effect was abolishedby a kinin antagonist and a NO synthesis inhibitor, markedlyinhibited by an AT2-ant, but only marginally inhibited by anAT1-ant. In aortas from mice with overexpression of the AT2

receptor gene, Ang II caused a significant increase in cGMP,which was further enhanced by an AT1-ant but blocked by anAT2-ant, B2 kinin antagonist, or NOS inhibitor.15 Theseobservations may suggest that both kinins and NO areinvolved in the AT2 signaling cascade, which mediates theaction of the AT2 receptor. Tsutsumi et al15 also reported thatmice overexpressing the AT2 receptor had increased kinino-genase activity, which may be responsible for Ang II-stimulated kinin release. Furthermore, we recently demon-strated that the therapeutic effect of AT1-ant on cardiacfunction and remodeling post-MI was diminished in B2 kininreceptor knockout mice (B22/2)7 or endothelial NOS knock-out mice,9 which may provide further evidence that kininsand endothelium-derived NO play an important role in thebeneficial cardiac effect of AT1-ant. The increase in kininrelease produced by activation of AT2 may also be partiallydue to inhibition of ACE activity. It has been shown that AT2

receptors may have a inhibitory effect on ACE activity, sinceAT22/Y mice had increased ACE activity and exhibited adecreased vasodepressor response to bradykinin.4 Taken to-gether, these results strongly suggest that production of kininsand NO by activation of AT2 should be considered a potentialcomplementary or mediator pathway during AT1 receptorblockade.

Inhibition of ACE decreases formation of Ang II anddegradation of BK and secondarily stimulates release of NOand PGs.2,28,29Inhibition of ACE may also increase Ang 1–7

by accelerating its formation (due to increased Ang I, whichis cleaved to Ang 1–7 by endopeptidases) and decreasing itsdegradation (Ang 1–7 is degraded to Ang 1–5 by ACE).30,31

It has been suggested that Ang 1–7 is an endogenouscompetitive inhibitor of Ang II and is able to stimulate releaseof kinins, PGs, and NO through non-AT1 and non-AT2

receptors.31,32 Therefore, if part of the effect of ACEi ismediated by increased Ang 1–7, lack of the AT2 receptor mayhave no impact on the effect of ACEi. Indeed, we found thatthe beneficial cardiac effect of ACEi was preserved inAT22/Y mice, suggesting that the AT2 receptor is notinvolved in the action of ACEi.

It is well known that ACE inhibition stimulates reninrelease, as we found in our study. Theoretically, blockade ofAT1 should also increase renin release due to a feedbackmechanism. However, we only saw a slight increase in PRCafter AT1-ant treatment in both AT21/Y and AT22/Y mice.Since we have confirmed that the dose of AT1-ant we usedblocked about 70% of exogenous Ang II–induced vasocon-striction, similar to the effect of ACEi on exogenous Ang I,we assume this dose is sufficient to block the action ofendogenous Ang II. At the present time, we do not have agood explanation as to why antagonism of AT1 did notincrease PRC. It is possible that renin release is mediated bymechanisms beyond the AT1 receptor, which need to beinvestigated further.

PerspectiveThe primary findings of the present study are (1) under basalconditions, cardiac hemodynamic, functional, and histologi-cal phenotypes are similar between AT21/Y and AT22/Ymice; (2) after MI, progression of cardiac dysfunction andremodeling is also similar between the two strains; and (3)blockade of the AT1 receptor improves cardiac function andregresses remodeling after MI, and this effect of AT1-ant isattenuated in AT22/Y mice, whereas the effect of ACEi ispreserved. Our data suggest that the AT2 receptor does notplay an essential role in regulation of cardiac function andmorphology, either under normal conditions or during thedevelopment of HF; however, activation of AT2 plays asignificant role in the therapeutic effect of AT1-ant.

AcknowledgmentsThis work was supported by National Institutes of Health grantHL-28982 and a Novartis Pharmaceuticals research grant.

References1. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau

VJ. Expression cloning of type 2 angiotensin II receptor reveals a uniqueclass of seven-transmembrane receptors.J Biol Chem. 1993;268:24539–24542.

2. Liu Y-H, Yang X-P, Sharov VG, Nass O, Sabbah HN, Peterson E,Carretero OA. Effects of angiotensin-converting enzyme inhibitors andangiotensin II type 1 receptor antagonists in rats with heart failure: role ofkinins and angiotensin II type 2 receptors.J Clin Invest. 1997;99:1926–1935.

3. Dzau VJ. Tissue renin-angiotensin system in myocardial hypertrophy andfailure. Arch Intern Med. 1993;153:937–942.

4. Hunley TE, Tamura M, Stoneking BJ, Nishimura H, Ichiki T, Inagami T,Kon V. The angiotensin type II receptor tonically inhibits angiotensin-converting enzyme in AT2 null mutant mice.Kidney Int. 2000;57:570–577.

6 Hypertension September 2002

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

5. Siragy HM, Inagami T, Ichiki T, Carey RM. Sustained hypersensitivity toangiotensin II and its mechanism in mice lacking the subtype-2 (AT2)angiotensin receptor.Proc Natl Acad Sci U S A. 1999;96:6506–6510.

6. Campbell DJ, Kladis A, Valentijn AJ. Effects of losartan on angiotensinand bradykinin peptides and angiotensin-converting enzyme.J Car-diovasc Pharmacol. 1995;26:233–240.

7. Yang X-P, Liu Y-H, Mehta D, Cavasin MA, Shesely E, Xu J, Liu F,Carretero OA. Diminished cardioprotective response to inhibition ofangiotensin-converting enzyme and angiotensin II type 1 receptor in B2

kinin receptor gene knockout mice.Circ Res. 2001;88:1072–1079.8. Horiuchi M, Akishita M, Dzau VJ. Recent progress in angiotensin II type

2 receptor research in the cardiovascular system.Hypertension. 1999;33:613–621.

9. Liu Y-H, Xu J, Yang X-P, Yang F, Shesely E, Carretero OA. Effect ofACE inhibitors and angiotensin II type 1 receptor antagonists on endo-thelial NO synthase knockout mice with heart failure.Hypertension.2001;39:375–381.

10. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A,Niimura F, Ichikawa I, Hogan BLM, Inagami T. Effects on bloodpressure and exploratory behaviour of mice lacking angiotensin II type-2receptor [letter].Nature. 1995;377:748–750.

11. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. Theangiotensin AT2-receptor mediates inhibition of cell proliferation in cor-onary endothelial cells.J Clin Invest. 1995;95:651–657.

12. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediatesprogrammed cell death.Proc Natl Acad Sci U S A. 1996;93:156–160.

13. Siragy HM, Senbonmatsu T, Ichiki T, Inagami T, Carey RM. Increasedrenal vasodilator prostanoids prevent hypertension in mice lacking theangiotensin subtype-2 receptor.J Clin Invest. 1999;104:181–188.

14. Seyedi N, Xu X, Nasjletti A, Hintze TH. Coronary kinin generationmediates nitric oxide release after angiotensin receptor stimulation.Hypertension. 1995;26:164–170.

15. Tsutsumi Y, Matsubara H, Masaki H, Kurihara H, Murasawa S, Takai S,Miyazaki M, Nozawa Y, Ozono R, Nakagawa K, Miwa T, Kawada N,Mori Y, Shibasaki Y, Tanaka Y, Fujiyama S, Koyama Y, Fujiyama A,Takahashi H, Iwasaka T. Angiotensin II type 2 receptor overexpressionactivates the vascular kinin system and causes vasodilation.J Clin Invest.1999;104:925–935.

16. Tanaka M, Tsuchida S, Imai T, Fujii N, Miyazaki H, Ichiki T, Naruse M,Inagami T. Vascular response to angiotensin II is exaggerated through anupregulation of AT1 receptor in AT2 knockout mice.Biochem BiophysRes Commun. 1999;258:194–198.

17. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural andcardiovascular effects of disrupting the angiotensin II type-2 receptorgene in mice [letter].Nature. 1995;377:744–747.

18. Wu L, Iwai M, Nakagami H, Chen R, Suzuki J, Akishita M, de GasparoM, Horiuchi M. Effect of angiotensin II type 1 receptor blockade oncardiac remodeling in angiotensin II type 2 receptor null mice.Arte-rioscler Thromb Vasc Biol. 2002;22:49–54.

19. Wu L, Iwai M, Nakagami H, Li Z, Chen R, Suzuki J, Akishita M, deGasparo M, Horiuchi M. Roles of angiotensin II type 2 receptor stimu-lation associated with selective angiotensin II type 1 receptor blockadewith valsartan in the improvement of inflammation-induced vascularinjury. Circulation. 2001;104:2716–2721.

20. Senbonmatsu T, Ichihara S, Price E Jr, Gaffney FA, Inagami T. Evidencefor angiotensin II type 2 receptor-mediated cardiac myocyte enlargementduring in vivo pressure overload.J Clin Invest. 2000;106:R25–R29.

21. Mifune M, Sasamura H, Shimizu-Hirota R, Miyazaki H, Saruta T. An-giotensin II type 2 receptors stimulate collagen synthesis in culturedvascular smooth muscle cells.Hypertension. 2000;36:845–850.

22. Alfie ME, Sigmon DH, Pomposiello SI, Carretero OA. Effect of high saltintake in mutant mice lacking bradykinin-B2 receptors.Hypertension.1997;29:483–487.

23. Yang X-P, Liu Y-H, Rhaleb N-E, Kurihara N, Kim HE, Carretero OA.Echocardiographic assessment of cardiac function in conscious and anes-thetized mice.Am J Physiol. 1999;277:H1967–H1974.

24. Skinner SL, Dunn JR, Mazzetti J, Campbell DJ, Fidge NH. Purification,properties and kinetics of sheep and human renin substrates.Aust J ExpBiol Med Sci. 1975;53:77–88.

25. Hochberg Y, Benjamini Y. More powerful procedures for multiple sig-nificance testing.Stat Med. 1990;9:811–818.

26. Harada K, Sugaya T, Murakami K, Yazaki Y, Komuro I. Angiotensin IItype 1A receptor knockout mice display less left ventricular remodelingand improved survival after myocardial infarction.Circulation. 1999;100:2093–2099.

27. Masaki H, Kurihara T, Yamaki A, Inomata N, Nozawa Y, Mori Y,Murasawa S, Kizima K, Maruyama K, Horiuchi M, Dzau VJ, TakahashiH, Iwasaka T, Inada M, Matsubara H. Cardiac-specific overexpression ofangiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects.J Clin Invest. 1998;101:527–535.

28. Carretero OA, Scicli AG. The kallikrein-kinin system as a regulator ofcardiovascular and renal function. In: Laragh JH, Brenner BM, eds.Hypertension: Physiology, Diagnosis, and Management. 2nd ed. NewYork, NY: Raven Press; 1995:983–999.

29. Pitt B, Segal R, Martinez FA, Meurers G, Cowley AJ, Thomas I,Deedwania PC, Ney DE, Snavely DB, Chang PI, on behalf of the ELITEStudy Investigators. Randomised trial of losartan versus captopril inpatients over 65 with heart failure (Evaluation of Losartan in the ElderlyStudy, ELITE).Lancet. 1997;349:747–752.

30. Chappell MC, Gomez MN, Pirro NT, Ferrario CM. Release of angioten-sin-(1–7) from the rat hindlimb: influence of angiotensin-convertingenzyme inhibition.Hypertension. 2000;35:348–352.

31. Tallant EA, Lu X, Weiss RB, Chappell MC, Ferrario CM. Bovine aorticendothelial cells contain an angiotensin-(1–7) receptor.Hypertension.1997;29:388–393.

32. Ferrario CM, Chappell MC, Tallant EA, Brosnihan KB, Diz DI. Coun-terregulatory actions of angiotensin-(1–7).Hypertension. 1997;30:535–541.

Xu et al Heart Failure in AT 2 Receptor Knockout Mice 7

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from

Xiao-Ping YangJiang Xu, Oscar A. Carretero, Yun-He Liu, Edward G. Shesely, Fang Yang, Alissa Kapke and

Antagonists in Mice1 Receptors in the Cardioprotective Effect of AT2Role of AT

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2002 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension published online July 29, 2002;Hypertension. 

http://hyper.ahajournals.org/content/early/2002/07/29/01.HYP.0000029095.23198.AD.citationWorld Wide Web at:

The online version of this article, along with updated information and services, is located on the

  http://hyper.ahajournals.org//subscriptions/

is online at: Hypertension Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer this process is available in the

click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,

can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on July 14, 2018http://hyper.ahajournals.org/

Dow

nloaded from