Angiotensin-(1–7) and angiotensin-(1–9): function in cardiac and vascular remodelling

Clinical Science (2014) 126, 815–827 (Printed in Great Britain) doi: 10.1042/CS20130436

Angiotensin-(1–7) and angiotensin-(1–9): functionin cardiac and vascular remodellingClare A. McKINNEY∗, Caroline FATTAH∗, Christopher M. LOUGHREY∗, Graeme MILLIGAN† andStuart A. NICKLIN∗

∗Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, U.K.†Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, U.K.

AbstractThe RAS (renin–angiotensin system) is integral to cardiovascular physiology; however, dysregulation of this systemlargely contributes to the pathophysiology of CVD (cardiovascular disease). It is well established that AngII(angiotensin II), the main effector of the RAS, engages the AT1R (angiotensin type 1 receptor) and promotes cellgrowth, proliferation, migration and oxidative stress, all processes which contribute to remodelling of the heart andvasculature, ultimately leading to the development and progression of various CVDs, including heart failure andatherosclerosis. The counter-regulatory axis of the RAS, which is centred on the actions of ACE2(angiotensin-converting enzyme 2) and the resultant production of Ang-(1–7) [angiotensin-(1–7)] from AngII,antagonizes the actions of AngII via the receptor Mas, thereby providing a protective role in CVD. More recently,another ACE2 metabolite, Ang-(1–9) [angiotensin-(1–9)], has been reported to be a biologically active peptide withinthe counter-regulatory axis of the RAS. The present review will discuss the role of the counter-regulatory RASpeptides Ang-(1–7) and Ang-(1–9) in the cardiovascular system, with a focus on their effects in remodelling of theheart and vasculature.

Key words: angiotensin, cardiovascular system, remodelling, renin–angiotensin system (RAS)

INTRODUCTION

The renin–angiotensin systemThe RAS (renin–angiotensin system) is a key component of car-diovascular physiology, playing an integral role in the regulationof vascular tone, blood pressure and electrolyte balance (Fig-ure 1). However, dysregulation of the RAS largely contributes tothe pathogenesis of various CVDs (cardiovascular diseases). Theextent to which chronic dysregulation of the RAS contributesto CVD is demonstrated by the fact that inhibitors of this sys-tem, including ACE (angiotensin-converting enzyme) inhibitorsand AT1R (angiotensin type 1 receptor) antagonists are amongthe most effective treatments [1]. ACE, which is most highlyexpressed in the vascular endothelium, lung, kidneys and in-testine, converts AngI (angiotensin I) into AngII (angiotensinII), the main effector of this system [2–4]. AngII is integrally

Abbreviations: ACE, angiotensin-converting enzyme; AF, atrial fibrillation; Ang-(1–7) etc., angiotensin-(1–7) etc.; AngI etc., angiotensin I etc.; ApoE, apolipoprotein E; AT1R, angiotensintype 1 receptor; AT2R, angiotensin type 2 receptor; BK2R, bradykinin type 2 receptor; CVD, cardiovascular disease; ECC, excitation–contraction coupling; EPC, endothelial progenitorcell; ERK, extracellular-signal-regulated kinase; GPCR, G-protein-coupled receptor; LAD, left anterior descending coronary artery; α-MHC, α-myosin heavy chain; L-NAME,NG-nitro-L-arginine methyl ester; LV, left ventricular; MAPK, mitogen-activated protein kinase; MI, myocardial infarction; MrgD, Mas-related gene D; NOS, NO synthase; eNOS, endothelialNOS; nNOS, neuronal NOS; NOX4, NADPH oxidase 4; RAS, renin–angiotensin system; rhACE2, recombinant human ACE2; ROCK, RhoA/Rho-associated kinase; ROS, reactive oxygenspecies; SHR, spontaneously hypertensive rat; SHRSP, spontaneously hypertensive stroke prone rats; VSMC, vascular smooth muscle cell.

Correspondence: Dr Stuart A. Nicklin (email [email protected]).

involved in regulation of vascular tone, blood pressure and elec-trolyte balance. The effects of AngII are mediated via interactionwith two GPCRs (G-protein-coupled receptors) the AT1R andAT2R (angiotensin type 2 receptor) respectively, which tend tohave opposing actions; however, the majority of the effects ofAngII are mediated via the AT1R (Figure 1). AngII stimulationof the AT1R results in coupling of the receptor to various hetero-trimeric G-proteins, including Gq/11, Gi/o and G12/13, resulting inmultiple signalling cascades leading to vasoconstriction, prolifer-ation, sodium retention and oxidative stress, all processes whichare largely involved in the pathogenesis of CVDs (reviewed in[5]). In contrast with the AT1R, the role and function of the AT2Ris not as clearly defined. However, it is thought to counterbalancesome actions of the AT1R by inhibiting growth and proliferation,reducing tissue remodelling and increasing vasodilation and NOproduction [6–10].

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C. A. McKinney and others

Figure 1 The RASThe RAS is a cascade of interconverted peptides that are generated mainly by the actions of ACE and ACE2. The traditionalaxis of the RAS is via ACE-mediated generation of AngII which signals mainly via the AT1R and also the AT2R. Thecounter-regulatory axis of the RAS is centred on the actions of ACE2 and production of Ang-(1–7) and Ang-(1–9) from AngIIand AngI respectively and counteracts the pathological effects of AngII at the AT1R. Ang-(1–7) exerts protective effects viaMas, whereas Ang-(1–9) has recently been reported to act via the AT2R. Cp A, cathepsin A; CxA, carboyxpeptidase A; NEP,neutral endopeptidase; POP, prolyl endopeptidase; TOP, thimet oligopeptidase.

The counter regulatory axis of therenin–angiotensin systemThe traditional view of the RAS has undergone significantchanges, largely due to the discovery of the enzyme ACE2 [11–13] (Figure 1). ACE2 is a homologue that shares approximately42 % identity with ACE and is highly expressed in a number of tis-sues, including the heart, vasculature and kidney [12,13]. Owingto structural differences ACE acts mainly as a peptidyl dipepti-dase, whereas ACE2 acts as a carboxypeptidase [11]. ACE2 func-tions in the RAS by cleaving the C-terminal residues from AngIand AngII, thereby reducing AngII levels (and hence reducing itsdeleterious effects) and producing Ang-(1–9) [angiotensin-(1–9)]and Ang-(1–7) [angiotensin-(1–7)] respectively [13]. Ang-(1–9)can be further cleaved by ACE to increase Ang-(1–7) levels [13](Figure 1).

The majority of Ang-(1–7) is produced via the actions ofACE2 and it is its main product as ACE2 has approximately 400-fold greater affinity for AngII than AngI [14]. Ang-(1–7) canalso be produced less efficiently via hydrolysis of Ang-(1–9) byACE or via the actions of alternative enzymes, including prolylendopeptidase, neutral endopeptidase or thimet oligopeptidase[13,15,16] (Figure 1). The half-life of Ang-(1–7) in the circula-tion is approximately 10 s [17,18] and circulating levels of Ang-(1–7) are reported to be 20 pg/ml [19]. Ang-(1–7) can opposethe actions of AngII in a number of tissues, mainly by inhib-iting cell growth, migration and inflammation that occurs as a

result of AngII, ultimately preventing adverse remodelling andsubsequent dysfunction of the cardiovascular system [20–23].Originally the mechanism by which Ang-(1–7) exerted its effectswas unknown, but research by Rowe et al. [24] suggested that,due to low affinity, the effects of Ang-(1–7) were unlikely to bemediated by signalling via either of the angiotensin receptors.Receptor-binding studies later showed that Ang-(1–7) could bindto the GPCR Mas and further research in Mas-deficient miceidentified Ang-(1–7) as the endogenous ligand for this receptor[25]. Moreover, despite the wealth of evidence that Ang-(1–7) isa Mas ligand, there is also more recent evidence that, althoughAng-(1–7) has relatively low affinity for the AT2R, it may alsoelicit certain biological effects via this receptor. For example, instable cell lines generated to express either the AT1R or AT2R,Ang-(1–7) was found to bind the AT2R with higher affinity thanthe AT1R [26]. The Ang-(1–7)–AT2R interaction has also beenobserved in vivo. In isolated mouse hearts exposed to the AT2Rantagonist PD123,319, Ang-(1–7) increased perfusion pressure,an effect not observed with Ang-(1–7) infusion alone and whichwas independent of both the AT1R or Mas [27]. It was alsoobserved that, in the presence of AT1R blockade, Ang-(1–7) re-duced blood pressure in both normotensive and SHRSP (spon-taneously hypertensive stroke prone rats), an effect mediated viathe AT2R [28]. This effect was preserved in aged normotens-ive rats under similar experimental conditions; however, thevasodepressor effect was via both the AT2R and Mas receptor

816 C© The Authors Journal compilation C© 2014 Biochemical Society

Role of Ang-(1–7) and Ang-(1–9) in cardiac and vascular remodelling

in the aged rats [29]. Conversely, in mice lacking the angiotensinreceptors, Ang-(1–7) reduced mean arterial pressure, suggestingthat the AT2R may not be responsible for the vasodepressor effectof Ang-(1–7) [30].

In contrast with Ang-(1–7), there is much less known aboutAng-(1–9). Ang-(1–9) can be generated from AngI by ACE2[13] or through the activity of carboxypeptidase A or cathepsinA [31,32]. Although circulating levels of Ang-(1–9) have beenreported to be approximately 2–6 fmol/ml in healthy subjects,these levels are thought to increase in pathological states [32–34]. For example, in human heart failure patients, Ang-(1–9) isformed at a rate of 1 nM/min per mg in the myocardium and alarge proportion of available AngI is rapidly converted into equallevels of Ang-(1–9) and AngII, suggesting that in pathologicalconditions the heart functions to increase levels of Ang-(1–9)[32]. However, there is currently little known about the biologicaleffects of Ang-(1–9) in the cardiovascular system. Originally itwas thought to be biologically inactive, contributing indirectly tocounter-regulate actions of AngII by competing with AngII forthe ACE active site, resulting in reduced AngII and increasedAng-(1–7) levels [13,35]. However, research has demonstratedthat Ang-(1–9) exerts direct biological effects in the cardiovas-cular system, and these effects may be via the AT2R [36]. Usingradioligand-binding assays it was demonstrated that Ang-(1–9)could bind to both the AT1R and AT2R and in cardiomyocytesAng-(1–9) mediated anti-hypertrophic effects via the AT2R, asPD123,319 (an AT2R antagonist) blocked these effects [36]. Thissuggests that, despite having approximately 100-fold lower af-finity for the AT2R than AngII, Ang-(1–9) may elicit functionaleffects via this receptor (Figures 1 and 2). Although further workis required to fully establish the signalling mechanisms elicitedby Ang-(1–9), the selective activity of this peptide at the AT2Ris possibly due to the pharmacological concept of functional se-lectivity where ligands have the ability to induce unique ligand-specific conformations that can result in differential activation ofsignalling pathways [37]. It has been shown previously that, al-though the AT1R exists in a constrained conformation, the AT2Rexists in a relaxed state [38] and it is therefore possible that theadditional histidine residue present in Ang-(1–9) may stabilizethe AT2R in a distinct conformational state, leading to the activa-tion of pathways to counter-regulate the effects of AngII and theAT1R; however, this remains to be demonstrated experimentally.Alternatively, Ang-(1–9) may be metabolized to Ang-(1–7) or an-other peptide, which may act at either the AT2R with high affinityor an alternative receptor which is also sensitive to PD123,319,as has been shown previously for the recently reported counter-regulatory RAS receptor MrgD (Mas-related gene D receptor)[39].

The counter regulatory axis of the RAS in cardiacremodellingAngII signalling via the AT1R is a key mechanism which driveschanges in cardiac structure and function, known as remodelling,post-cardiac injury in vivo which can lead to the progressive de-cline of cardiac function. Initially, remodelling within the heartis an adaptive process where changes to the myocardium whichare required for maintenance of cardiac function in response

to injury occur. However, over time this remodelling becomesmaladaptive, leading to chronic remodelling that is associatedwith development of cardiac dysfunction and increased risk ofheart failure [40]. These changes affect both cardiac structureand function and include cardiac hypertrophy, fibrosis and dila-tion, inflammation and secondary electrical remodelling, whichcauses changes in various ion channels and ECC (excitation–contraction coupling) [40–44]. Both Ang-(1–7) and Ang-(1–9)have been demonstrated to mediate a beneficial role in cardiacstructural and functional remodelling, and this evidence is de-tailed in the following sections. In addition to a direct protectiveeffect of the peptides, modulation of levels of the angiotensinpeptides by ACE2 has been shown to be beneficial. Variousstudies using ACE2-knockout mice have demonstrated that lossof this enzyme results in increased AngII levels in the heart,kidneys and plasma, associated with impaired cardiac functionand increased remodelling following MI (myocardial infarction)[45,46]. Furthermore, diabetic Akita mice deficient in ACE2 ex-pression develop cardiac myopathy, associated with increasedAngII levels and AT1R signalling [47]. These studies suggest aprotective role for ACE2 in cardiac remodelling through reducedAngII production. However, other studies have shown the oppos-ite in that overexpression of ACE2 is potentially associated witha worsened cardiac phenotype [48,49]. This phenomenon couldbe due to supraphysiological levels of expression at specific un-wanted sites and therefore further studies are required to addressthis conflicting data.

STRUCTURAL REMODELLING OF THE HEART

Effects of Ang-(1–7)Ang-(1–7) has been extensively studied with regards to its ef-fects in the heart (Figure 2). Collagen deposition in the SHR(spontaneously hypertensive rat) heart as a result of salt-inducedcardiac remodelling has been shown to be associated with de-creased levels of cardiac Ang-(1–7) and Mas, independent ofchanges in cardiac AngII levels, suggesting the loss of the pro-tective effect of Ang-(1–7) contributes to hypertension-inducedfibrosis in this model [50]. Lack of Mas was also shown to in-crease levels of collagens type I and type III and fibronectin inthe hearts of Mas-deficient mice [51]. In the rat DOCA (deoxy-corticosterone acetate)-salt model of hypertension, Ang-(1–7)delivered via mini-pump was shown to prevent myocardial andperivascular fibrosis, which was independent of effects on bloodpressure or cardiac hypertrophy [52]. Using a transgenic rat strainTG(hA1-7)L7301, which expresses an Ang-(1–7) fusion proteinin a cardiac-specific manner via the α-MHC (α-myosin heavychain) promoter, it was demonstrated that the transgenic ratswere more resistant to isoproterenol-induced cardiac stress thanwild-type controls [53]. After high-dose isoproterenol admin-istration for 7 days, analysis of collagen types I and III andfibronectin showed reduced deposition in the heart comparedwith control rats [53]. Furthermore, in the TGR(A1-7)3292 rats,higher circulating levels of Ang-(1–7) were also apparent, and therats were particularly resistant to isoproterenol-induced cardiac

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hypertrophy [54]. A further study, utilizing transgenic TGVII-7mice overexpressing a cardiac-specific Ang-(1–7) fusion pro-tein under control of the mouse α-MHC promoter also reportedreduced cardiac remodelling in response to AngII infusion viaosmotic mini-pump, with reductions in both fibrosis and hyper-trophy [55]. Protective cardiac effects have also been observedfollowing gene transfer of Ang-(1–7). In a study performed in a ratmodel of MI, employing lentiviral delivery of Ang-(1–7) via dir-ect injection to the left ventricle followed by ligation of the LAD(left anterior descending coronary artery), it was observed thatAng-(1–7) was able to attenuate cardiac hypertrophy and reduceLV (left ventricular) wall thinning [56]. Mechanistically, it wassuggested that the peptide may exert beneficial effects throughmodulation of ACE2 and the BK2R (bradykinin type 2 receptor),which were both found to be up-regulated in the myocardium ofAng-(1–7)-infused animals [56]. Furthermore, these findings in-dicated that Ang-(1–7) may also have anti-inflammatory proper-ties, as reduced levels of the pro-inflammatory cytokines TNF-α(tumour necrosis factor-α) and IL-6 (interleukin-6), but increasedexpression of the anti-inflammatory cytokine IL-10 (interleukin-10), were observed [56].

In vitro studies in both adult and neonatal rat cardiac fibro-blasts have demonstrated that Ang-(1–7) inhibits proliferationand collagen production [57,58]. In neonatal rat cardiac fibro-blasts, Ang-(1–7) achieved this via reduced AngII-mediatedphospho-ERK1 (extracellular-signal-regulated kinase 1) and-ERK2 levels and increasing DUSP1 (dual-specificity phos-phatase 1) activity, which in turn reduced MAPK (mitogen-activated protein kinase) activity and prevented production of pro-proliferative prostaglandin-2 [58]. Further work studying cardiacremodelling in vivo has identified a role for the ERK and proteinkinase pathways in the actions of Ang-(1–7). In the SHR, it hasbeen demonstrated that restoration of abnormal ACE2 in the heartresults in an increase in cardiac Ang-(1–7) production, which inturn leads to reduced ventricular collagen content [59]. This wasshown to be associated with a decrease in the phosphorylationof ERKs, which are known to participate in cardiac fibroblastcollagen synthesis [59]. In an AngII infusion model of cardiac re-modelling in ACE2-knockout mice infused with rhACE2 (recom-binant human ACE2), a significant reduction in multiple cardiacremodelling processes in an Ang-(1–7)-dependent manner wasreported [60]. Again, Ang-(1–7) reduced collagen and fibronectinproduction and attenuated cardiac hypertrophy. Furthermore, us-ing rhACE2 infusion, and thereby raising Ang-(1–7) levels ina wild-type mouse model of pressure-overload induced cardiacremodelling, partial attenuation of the development of dilatedcardiomyopathy was reported [60]. This was demonstrated to bevia inhibition of mitogen-activated JAK2 (Janus kinase 2)/STAT3(signal transducer and activator of transcription 3) and PKC (pro-tein kinase C) activity, as well as diminished AngII-mediatedERK1/2 activation [60].

As well as cardioprotective effects with regards to fibrosis andhypertrophy, Ang-(1–7) has recently been shown to promote an-giogenesis in infarcted rat hearts in a Mas-dependent manner viathe up-regulation of VEGF-D (vascular endothelial growth factorD) and MMP-9 (matrix metalloproteinase-9) [61]. Ang-(1–7) hasalso been demonstrated to reduce the production of cardiac ROS

(reactive oxygen species) in response to cardiac injury, as wellas increased NO production, thus further providing a cardiopro-tective effect [60]. Ang-(1–7) infusion in the SHR was shown toincrease expression levels of ventricular eNOS [endothelial NOS(NO synthase)] and nNOS (neuronal NOS), as well as causing anincrease in NOS phosphorylation and activity [62]. These effectswere via AT2R- and BK2R-dependent pathways, as demonstratedusing the receptor antagonists PD123,319 and icantibant respect-ively, as opposed to via the Mas axis, suggesting that some effectsof Ang-(1–7) may be mediated via a cross-talk between the AT2Rand BK2R [62]. The effect of Ang-(1–7) on oxidative stress wasdemonstrated further using chimaeric mice that mimic humanRAS activation [63]. These mice were generated by crossing hu-man renin (hRN) and human angiotensinogen (hANG) transgenicmice, which were then utilized as a mouse model of human hy-pertension [63]. Studies demonstrated that depletion of Ang-(1–7) via reduced ACE2 activity exacerbated hypertension-inducedcardiac remodelling. In chimaeric mice, L-NAME (NG-nitro-L-arginine methyl ester) treatment in order to persistently inhibitNO production resulted in an increase in blood pressure com-pared with wild-type mice, and was accompanied by acceleratedoxidative-stress induced cardiovascular remodelling, as well asreduced cardiac ACE2 expression [63]. Following AT1R ant-agonist (olmesartan) treatment, L-NAME-induced remodellingand cardiac ACE2 reduction was prevented, and this effect wasreversed by co-administration of the Mas antagonist A779. Acharacteristic of these animals is increased NADPH oxidase pro-duction, which results in an increase in ROS in the myocardiumas a result of remodelling. The use of the AT1R antagonist losar-tan reduced ROS levels through inhibition of the actions of AngIIand in a partially Ang-(1–7)-dependent manner.

The vast array of studies looking at the effects of Ang-(1–7)on structural remodelling has shown that it is an important reg-ulator in remodelling processes and key in counteracting AngIIsignalling, identifying it as a potential therapy for human cardiacremodelling.

Effects of Ang-(1–9)The first evidence to suggest that Ang-(1–9) elicits independentbiological effects in the heart was reported by Ocaranza et al.[34], using a rat coronary artery ligation model of MI, wherecardiac hypertrophy and dysfunction are present at 8 weeks, andin which they assessed peptide and enzyme levels. It was foundthat, 1 week post-MI, circulating levels of Ang-(1–9), along withlevels of AngII, ACE, and ACE2, were increased compared withcontrol animals; however, at 8 weeks, only AngII and ACE re-mained high, with circulating levels of ACE2 and Ang-(1–9)diminishing to levels lower than the control group [34]. Treat-ment of the animals with the ACE inhibitor enalapril preventedthe changes observed at 8 weeks, suggesting the generation ofAng-(1–9) via ACE2 is able to counter-regulate AngII-mediatedactions. Circulating Ang-(1–7) levels were reported to be un-changed throughout the study [37]. A later study demonstratedan antihypertrophic role for Ang-(1–9) in vitro using neonatalcardiac myocytes and in vivo in the rat model of MI. Importantly,it was demonstrated that these actions were independent of the



Figure 2 Ang-(1–7) and Ang-(1–9) signalling in the heartAng-(1–7) acting at Mas regulates cardioprotective effects in the heart via NO release through cGMP, reducing NFAT (nuclearfactor of activated T-cells) translocation to the heart, thereby preventing cardiac hypertrophy, preventing reductions incardiac FS (fractional shortening), LVSP (LV systolic pressure) and Ca2 + transient amplitude in isolated cardiomyocytes.NO generation also occurs indirectly via Ang-(1–7) cross-talk with the AT2R and BK2R, which increases phosphorylation ofeNOS and nNOS, also exerting cardioprotective effects. Ang-(1–7) signalling via Mas inhibits mitogen-activated JAK2 (Januskinase 2)/STAT3 (signal transducer and activator of transcription 3) and activates PKC (protein kinase C), decreasing ERKactivity, which has been found to attenuate cardiomyopathy. Decreases in ERK activity have also been associated withreduced susceptibility to AF in animal models, as well as increasing DUSP1 (dual-specificity phosphatase 1) activity,resulting in a decrease in MAPK activity reducing the production of prostaglandin-2, which acts on fibroblasts, preventingproliferation and collagen synthesis. Via Mas, Ang-(1–7) has also been shown to increase VEGF-D (vascular endothelialgrowth factor D) and MMP-9 (matrix metalloproteinase-9) levels, promoting cardiac angiogenesis. Ang-(1–9) signalling viathe AT2R reduces collagen synthesis thereby reducing cardiac fibrosis, and decreases Rho kinase activity which attenuatescardiac hypertrophy; however, the majority of Ang-(1–9) signalling pathways in the heart have yet to be characterized.

Ang-(1–7) receptor Mas using simultaneous delivery of the Masantagonist A779 [64].

Further in vitro studies by Flores-Munoz et al. [36] establishedan antihypertrophic role for Ang-(1–9) in primary adult rabbitcardiomyocytes and rat neonatal H9c2 cardiomyocytes stimulatedwith AngII. This study was the first to provide a direct compar-ison between Ang-(1–9) and Ang-(1–7). Importantly the effectof Ang-(1–9) was selectively inhibited by the AT2R antagonistPD123,319, whereas those of Ang-(1–7) were only inhibited inthe presence of the Mas antagonist A779, demonstrating for thefirst time a direct receptor-mediated biological action for Ang-(1–9) [36]. Intriguingly, the use of PD123,319 had no effect onAngII, suggesting that in this experimental setting selective Ang-(1–9) functional activity at the AT2R is important. The effects ofAng-(1–7) were only blocked by the Mas antagonist A779 andnot by the AT2R antagonist PD123,319, despite previous studiessuggesting a role for Ang-(1–7) mediating effects via the AT2R[28]. Clearly this requires further study and it may be that the ef-fects of individual angiotensin peptides at the angiotensin recept-ors are tissue- and/or pathology-specific. Ang-(1–9) was studiedfurther via peptide infusion in vivo and similar benefits to Ang-

(1–7) in cardiac structural remodelling was shown, but throughwhat appears to be an independent mechanism (Figure 2) [65].In the SHRSP, osmotic mini-pump-mediated infusion of Ang-(1–9) for 4 weeks resulted in a 50 % reduction in cardiac fibrosiscompared with control animals [65]. This was demonstrated to beAT2R-dependent via infusion of the antagonist PD123,319, whichattenuated the anti-fibrotic effects of the peptide. Unlike Ang-(1–7), very little is known about the potential mechanisms throughwhich Ang-(1–9) may act, as signalling via the AT2R is currentlypoorly characterized. Activation of various pathways that involvetyrosine or serine/threonine phosphatases have been suggested aspotential signalling mechanisms [66]. Phosphatases suggested tobe involved in AT2R signalling are MKP1 (MAPK phosphatase1), SHP1 (Src homology 2 domain-containing protein tyrosinephosphatase 1) and PP2A (protein phosphatase 2A) [67]. Inter-estingly, these phosphatases have been shown to interact withthe ERK1/2 pathway [68], which is known to regulate collagensynthesis, fibroblast proliferation and cardiac hypertrophy. There-fore further research is required to fully delineate the mechanismof action of Ang-(1–9) in the heart. Additionally, alternative ap-proaches to assess the role of the AT2R for Ang-(1–9) are required,

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since the pharmacological antagonist PD123,319 has also beenreported to be a competitive antagonist at the recently identifiedcounter-regulatory RAS receptor MrgD [39].

FUNCTIONAL AND ELECTRICALREMODELLING IN THE HEART

Structural remodelling changes are accompanied by detrimentalchanges in cardiac function and contractility. Fibrosis alters themechanical properties of the heart, leading to decreased com-pliance of the ventricle and impedance and desynchronizationof electrical conductance through the heart. Eventually this res-ults in diastolic dysfunction, followed by contractile dysfunction,leading to an increased risk of arrhythmia [44,69]. ECC, whereelectrical excitation of cardiomyocytes drives contraction of theheart, is also affected by AngII-remodelling processes, with al-terations in the calcium sensitivity of calcium-handling proteinsand changes in ion current expression in cardiomyocytes leadingto altered intracellular calcium concentrations, which in turn mayreduce cardiac contractility [70].

Studies on the effect of Ang-(1–7) in models of cardiacdysfunction have demonstrated the potential of the peptide toimprove or maintain cardiac contractility and that it exhibits anti-arrhythmogenic properties. Lentiviral-mediated delivery of Ang-(1–7) directly to the left ventricle of rat hearts which were thensubject to MI by ligation of the LAD not only prevented struc-tural changes, but also prevented a decline in cardiac function[56]. ECG and haemodynamic measurements demonstrated im-provements in fractional shortening and LV systolic pressure andan attenuation of the increase in LV diastolic pressure. The be-neficial functional effects of Ang-(1–7) were also shown usingpeptide infusion in a rat heart failure model, where restorationof LV systolic and diastolic pressure was observed in peptide-infused animals [71]. In isolated cardiomyocytes from transgenicTGR(A1-7)3292 rats, Ang-(1–7) preserved the calcium transi-ent compared with cardiomyocytes isolated from control AngII-infused rats, which presented a 26 % reduction in intracellularcalcium amplitude [72]. This is of note as changes in expressionlevels of cardiomyocyte calcium-handling proteins and especiallyalterations in intracellular and sarcoplasmic reticulum calciumconcentrations are prominent features of contractile dysfunctionand development of heart failure [41,72]. Moreover, this study[72] went on to identify possible mechanisms by which Ang-(1–7)exerts these effects, suggesting a role for NO/cGMP in regulat-ing the cardioprotective effects observed [72]. A similar studyin the TG(hA1-7)L7301 rat also demonstrated an enhancementof single cardiomyocyte calcium handling due to cardiac overex-pression of Ang-(1–7) [53]. Cardiomyocytes from these animalsexhibited an increased calcium transient amplitude, increased rateof transient decay and increased expression of SERCA2a (sar-coendoplasmic reticulum calcium transport ATPase 2a). Further-more, isolated hearts from these rats also showed a reduced rateof ischaemia/reperfusion injury arrhythmias, suggesting Ang-(1–7) can act directly on the heart to improve cardiac function[53]. Beneficial functional effects of Ang-(1–7) were also ob-

served in a study by Patel et al. [73] in ACE2-knockout mice inpressure-overload-induced heart failure. Ang-(1–7)-infused an-imals showed normalization of fractional shortening, LVEDPs(LV end-diastolic pressures) and +−dP/dtmax [73]. This was ac-companied by normalization of heart weight and hypertrophymarker expression levels, as well as attenuated NADPH oxidaseactivation. Moreover, the benefits observed were comparable withthose observed in animals treated with the AT1R blocker irbe-sartan [73].

Many studies on the electrical properties of Ang-(1–7) onthe heart have been performed using ex vivo whole-heart Lan-gendorff preparations, where anti-arrhythmogenic effects inischaemia/reperfusion injury have been clearly demonstrated[54,74–76]. Ang-(1–7) delivered via an osmotic mini-pump ina dog pacing model of AF (atrial fibrillation) reduced interstitialfibrosis and, as a result, showed reduced susceptibility to andduration of induced AF [77]. Again, this effect was believed tobe modulated by a reduction in ERK1/2 signalling [77]. In thismodel, Ang-(1–7) also attenuated the decrease in action potentialduration, characteristically observed in atrial myocytes during AF,as well as preventing the decrease in expression of ICaL (L-typecalcium channel) and ITO (outward potassium channel) observedin the model; however, the mechanism by which this occurs re-mains to be clarified [77]. This demonstrates that Ang-(1–7)not only has potential benefits for cardiac function through anti-structural remodelling, but also that it has the potential to alterion channel and calcium-handling protein expression, resultingin modulation of cardiac function. However, in vitro treatment ofisolated adult mouse cardiomyocytes with 10 nM Ang-(1–7) hasno direct effect on calcium-handling properties, whereas, con-versely, the absence of Mas in Mas− / − mouse cardiomyocytesresults in a reduced calcium transient peak and slower calciumtransient kinetics [78]. This suggests more work is needed to elu-cidate the role of Ang-(1–7) in modulating cardiac function. Therequirement for further work is highlighted by exciting findingsregarding Ang-(1–7). Ang-(1–7) infusion in a murine MI modelhas been demonstrated to improve cardiac function via stimula-tion of circulating and bone-marrow-derived EPCs (endothelialprogenitor cells), leading to enhanced EPC migration to the heart[79]. Therefore this provides evidence that the therapeutic ef-fects of Ang-(1–7) may also extend to enhancing regenerativecell therapy. These findings have been extended to show that len-tiviral overexpression of ACE2 in murine EPCs enhanced theirmigration and ability to form tubes in vitro [80]. Clinically, isol-ation of CD34+− cells from the blood of diabetic patients demon-strated that their NO bioavailability and migratory and proliferat-ive function could be improved via exposure to Ang-(1–7) [81].Furthermore, transduction of the EPCs with a lentiviral vectorexpressing Ang-(1–7) enhanced the reparative ability of the cellsin a murine model of diabetic retinopathy. As a point of note, incancer clinical trials Ang-(1–7) infusion has been demonstratedto mediate complete restoration of the haemopoietic cell profilefollowing chemotherapy in patients, highlighting the vast po-tential for this peptide in improving haemopoeitic and immunefunction [82].

There is currently very little evidence reported to suggest abeneficial functional effect of Ang-(1–9) in models of cardiac



dysfunction. Chronic infusion of Ang-(1–9) has been demon-strated by echocardiography to prevent LV wall thickening,LVESV (LV end-systolic volume) and LVEDV (LV end-diastolicvolume) in comparison with the MI animals [64]. However, therewas no change observed in cardiac function between MI andAng-(1–9)-infused MI animals as measured via LVEF (LV ejec-tion fraction) and LVFS (LV fractional shortening).

VASCULAR REMODELLING

Vascular remodelling, characterized by changes in the size andcomposition of adult blood vessels, is an adaptive process thatallows vessels to respond to changes in haemodynamic condi-tions, but also underlies the pathogenesis of various major CVDssuch as atherosclerosis. Damage or injury to the vessel wall res-ults in denudation of the endothelial cell layer and reduced NObioavailability [83]. It is this damage to the endothelium that pre-disposes to vascular remodelling by increasing the occurrence ofthrombosis, creating an inflammatory environment at the siteof injury and promoting growth and migration of VSMCs (vas-cular smooth muscle cells), and degradation and reorganizationof the extracellular matrix [84–89]. Dysregulation of the RAS isone of the key contributing factors to remodelling of the vascu-lature and the development of atherosclerosis, with the majorityof the pathological processes described above being mediated byAngII signalling at the AT1R [90]. In addition to its protectiveeffects in the heart, the components of the counter-regulatoryaxis of the RAS have been shown to play an important role in thevasculature by inhibiting the pathological actions of AngII (Fig-ure 3). In atherosclerotic human carotid arteries, ACE2 activitywas found to be increased in early-stage atherosclerosis or inunstable lesions in comparison with stable lesions, demonstrat-ing differential activity of ACE2 at different stages of the disease[91]. Although it is currently unclear how this differential activityis regulated, it may be that ACE2 is increased in the early stagesof atherosclerosis and in unstable lesions in an attempt to con-trol the disease pathology and lessen injury by reducing AngIIproduction and increasing production of Ang-(1–7). Addition-ally, overexpression of ACE2 using adenoviral vectors has beenshown to reduce atherosclerotic lesion size in ApoE (apolipo-protein E)− / − mice [22]. Moreover, ACE2 overexpression alsostabilizes existing atherosclerotic lesions, inhibiting progressionof lesion development at early stages of atherosclerosis, but notat advanced stages of the disease [92]. These effects were as-sociated with both reduced levels of AngII and increased levelsof Ang-(1–7), suggesting a key role for ACE2 in balancing theactivity of both axes of the RAS in atherosclerosis.

Effect of Ang-(1–7) in vascular remodellingTo date a large proportion of the protective effects of the counter-regulatory axis of the RAS in the vasculature have been attributedto the production of Ang-(1–7) and its resulting interaction withMas (Figure 3). Numerous in vitro studies have shown that Ang-(1–7) signalling via Mas inhibits MAPK signalling pathways,mainly via reduced activity and expression of ERK1/2, leading

to a reduction in VSMC proliferation [93] and migration [94]. TheAng-(1–7)–Mas interaction has also been shown to reduce VSMCproliferation and migration via release of prostaglandins includ-ing prostacyclin (PGI2) and PGE2 (prostaglandin E2), resultingin increased cAMP levels and inhibition of cyclo-oxygenases[95].

The anti-proliferative and anti-migratory role of Ang-(1–7)is also observed in rodent models of vascular disease. Ang-(1–7), via Mas, reduces neointimal formation following bal-loon injury [96], stent implantation in rats [21] and angioplastyin rabbits [97], and has been associated with reduced athero-sclerotic lesion size in ApoE− / − mice [22,98]. Additionally, anon-peptide agonist of Mas, AVE0991, which mimics the ac-tions of Ang-(1–7) [99], inhibits rat VSMC proliferation in vitro[100].

Ang-(1–7) increases NO release, thereby acting as a vas-odilator and improving vascular endothelial function [101,102].The increase in NO is achieved directly via Mas-mediated stim-ulation of eNOS and sustained Akt phosphorylation, or indir-ectly via production of bradykinin and receptor cross-talk withthe BK2R [31,103]. Additionally, Ang-(1–7) has been shown topromote vasodilation via the AT2R in the SHRSP during AT1Rblockade [28]. This was shown to involve interaction between theAT2R and BK2R, as vasodilation in response to Ang-(1–7) wasprevented by both PD123,319 and HOE 140 (a BK2R antagonist)[28].

An Ang-(1–7)-mediated increase in NO bioavailability hasalso been linked to reduced ROS production, thereby promotingimproved vascular function and reduced atherosclerosis [104]. InApoE− / − mice, chronic administration of Ang-(1–7) via osmoticmini-pump restored renal endothelial function, which was asso-ciated with increased NO bioavailability [104]. To investigate therelationship between ROS levels and NO bioavailability in thissetting, the ROS scavenger tempol was used. Although tempolimproved endothelial function in untreated ApoE− / − mice, it hadno effect on Ang-(1–7)-infused mice, indicating that these anim-als already have reduced ROS levels [104]. This was suggestedfurther by reduced levels of H2O2 and NADPH oxidase subunitexpression in Ang-(1–7)-infused animals. In addition to reducedROS activity, increased levels of eNOS were observed follow-ing treatment with Ang-(1–7), providing a further mechanism tosupport the findings of increased NO bioavailability [104].

In addition to enhancing vascular endothelial function, thisincrease in NO release has also been demonstrated to inhibitplatelet aggregation, demonstrating an anti-thrombotic role forAng-(1–7) [105]. Importantly, as well as mediating NO releasefrom endothelial cells [103,106] Ang-(1–7) has been shown topromote NO release from platelets, adding to its anti-thromboticeffects [105]. In vivo, the anti-thrombotic effect of Ang-(1–7)was blocked by pharmacological Mas receptor inhibition and isabsent in Mas− / − mice [105]. It was not established whetherthis was due to Ang-(1–7) actions in endothelial cells or plateletsleading to NO release, although it is likely to be a combinationof both [105]. Although further work is required to fully dis-sect the mechanisms involved in the anti-thrombotic properties,these findings have identified the Ang-(1–7)/Mas axis as a po-tential therapeutic target for the treatment of thrombotic events.

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Figure 3 Ang-(1–7) and Ang-(1–9) signalling in the vasculatureAng-(1–7) via Mas increases NO release, thereby acting as a vasodilator and reducing thrombosis either (i) directly viaactivation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, resulting in increased phosphorylation of eNOS,or (ii) indirectly via cross-talk with the BK2R, which increases PKA (protein kinase A)-mediated phosphorylation of eNOS.This pathway may also involve interaction with the AT2R. Ang-(1–7) via Mas also promotes vasodilation by stimulating PLA2(phospholipase A2), leading to an increased release of AA (arachidonic acid), which in turn produces the prostanoids PGI2(prostacyclin) and PGE2 (prostaglandin E2), both of which increase levels of cAMP. cAMP also reduces MAPK signalling andVSMC proliferation and migration. Ang-(1–7) via Mas also directly inhibits MAPK activity. Ang-(1–9) via the AT2R promotesvasodilation through increased NO either directly or via cross-talk with the BK2R. Ang-(1–9)-mediated vasodilation is alsoassociated with increased expression of NOX4.

The development of an orally available form of Ang-(1–7) [Ang-(1–7)-CyD], in which Ang-(1–7) has been incorporated into acyclodextrin (CyD), a cyclic oligosaccharide that enhances drugstability, absorption across biological barriers and provides gast-ric protection, has greatly increased the potential of utilizingAng-(1–7) in a therapeutic setting [107]. This compound hasbeen shown to be of particular use as an anti-thrombotic in-tervention, as it exerts anti-thrombotic effects in vivo, whichare associated with increased plasma levels of Ang-(1–7)[108].

Effects of Ang-(1–9) in vascular remodellingThe role of Ang-(1–9) in the vasculature is relatively unexplored;however, as Ang-(1–9) can also exert biological effects, some ofthe previously described vasculoprotective effects of the counter-regulatory axis of the RAS may be attributed to production of bothAng-(1–9) and Ang-(1–7) (Figure 3). As in the heart, signallingvia the AT2R in the vasculature is poorly defined and further workis required to delineate a role in this setting (Figure 3). Inhibi-tion of the ROCK (RhoA/Rho-associated kinase) signalling path-way results in increased activity and expression of ACE2 in theaorta and increased Ang-(1–9) plasma levels, reducing bloodpressure and vascular remodelling [109]. These effects arecoupled with reduced ACE activity and AngII levels and in-

creased expression of eNOS, identifying that the ROCK pathwaymay interact with the ACE2/Ang-(1–9) axis as a novel protect-ive interaction in the vasculature. Additionally, Ang-(1–9) hasalso been reported to indirectly contribute to improved vascularfunction by stimulating bradykinin release in endothelial cellsand enhancing the effects of bradykinin by augmenting NO andarachidonic acid release [31]. Although AngII is largely acceptedto be pro-thrombotic and Ang-(1–7) anti-thrombotic, evidencefor Ang-(1–9) is inconclusive. A study by Kramkowski et al.[110] has indicated that Ang-(1–9) enhances electrically stimu-lated thrombosis and increases platelet aggregation in rats via theAT1R. However, it is worthwhile to point out that electrical stimu-lation was used to injure the vessel and initiate thrombosis, whichwould not be the case clinically. In addition, the pro-thromboticeffect of Ang-(1–9) was much lower than that of AngII, as demon-strated in previous studies by the same group [111,112]. It wasconcluded that the actions of Ang-(1–9) were via metabolism intoAngII by an ACE-independent aminopeptidase, a rare process, asopposed to direct actions of Ang-(1–9) [110,113,114]. This mayhave implications for the therapeutic actions of Ang-(1–9) andrequires further study.

Florez-Munoz et al. [65] provided the first evidence tosuggest a direct beneficial effect of Ang-(1–9) in vascularfunction. Ang-(1–9) infusion in the SHRSP improved aortic



vasorelaxation and NO bioavailability via the AT2R [65]. Al-though the mechanisms involved are currently unknown, it ispossible that Ang-(1–9) may increase NO bioavailability by stim-ulating bradykinin release, as described previously in cardiac en-dothelial cells [31], or by enhancing the activity of eNOS, ashas been shown for Ang-(1–7) [115]. Additionally, Ang-(1–9)infusion and AT2R stimulation resulted in an increase in aor-tic expression of NOX4 (NADPH oxidase 4) [65], which hasbeen demonstrated previously to promote vasodilation via therelease of H2O2 [116]. However, this protective effect of NOX4is vascular-bed-specific [116] and therefore further investigationis required to fully assess the involvement of increased NOX4levels in the aorta in response to Ang-(1–9) infusion [68].

CONCLUSIONS

In summary, clear evidence identifies the peptides of the counter-regulatory axis of the RAS as modulators of the pathologicaleffects of AngII in the heart and vasculature. The effects of Ang-(1–7), signalling via Mas, are well established and there is clearevidence in support of its robust therapeutic potential in bothcardiac and vascular remodelling. The identification of receptorcross-talk pathways and potential interactions of Ang-(1–7) withother receptors may further highlight the broad therapeutic effectof Ang-(1–7), although further research is required to dissect themechanisms involved. The generation of transgenic animals hasgreatly expanded our knowledge of Ang-(1–7) and enabled indepth investigation into its biological properties. Additionally,the production of orally available forms of Ang-(1–7) has in-creased the potential of targeting this axis clinically. Althoughdeveloping therapeutic aspects of Ang-(1–7) is likely to be chal-lenging due to the general rapid pharmacokinetics of angiotensinpeptides in vivo, other approaches are being developed. AVE0991is the first non-peptide agonist of Mas which has been shown tohave therapeutic effects in a range of disease models, includingpulmonary, cardiac and renal injury and remodelling and in ather-osclerosis [117–119]. Further development of these approaches islikely to provide key data to develop the Ang-(1–7)/Mas axis as aclinical therapeutic target. More recently, Ang-(1–9) is emergingas another key peptide in this axis that exerts protective effectsin the heart and vasculature, potentially via the AT2R. Althoughfurther work is required to provide conclusive evidence in sup-port of this interaction, Ang-(1–9) may represent an alternativetherapeutic target within the counter-regulatory axis of the RASin the setting of cardiac and vascular remodelling, with the poten-tial of providing additive or synergistic effects to those mediatedvia Ang-(1–7).

FUNDING

The work in our laboratory is supported by the British Heart Found-ation [grant number PG/11/43/28901] and the Medical ResearchCouncil [grant number G0901161], and a Medical Research Coun-cil Doctoral Training Grant PhD Studentship.

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Received 29 July 2013/16 December 2013; accepted 17 January 2014

Published on the Internet 26 February 2014, doi: 10.1042/CS20130436

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Angiotensin-(1–7) and angiotensin-(1–9): function in cardiac and vascular remodelling