key: cord-0945873-uel0i3is
authors: Paz Ocaranza, Maria; Riquelme, Jaime A.; García, Lorena; Jalil, Jorge E.; Chiong, Mario; Santos, Robson A. S.; Lavandero, Sergio
title: Counter-regulatory renin–angiotensin system in cardiovascular disease
date: 2019-08-19
journal: Nat Rev Cardiol
DOI: 10.1038/s41569-019-0244-8
sha: e6d567a261b5a3967c0ea0e54680000f13fb5fb7
doc_id: 945873
cord_uid: uel0i3is

The renin–angiotensin system is an important component of the cardiovascular system. Mounting evidence suggests that the metabolic products of angiotensin I and II — initially thought to be biologically inactive — have key roles in cardiovascular physiology and pathophysiology. This non-canonical axis of the renin–angiotensin system consists of angiotensin 1–7, angiotensin 1–9, angiotensin-converting enzyme 2, the type 2 angiotensin II receptor (AT(2)R), the proto-oncogene Mas receptor and the Mas-related G protein-coupled receptor member D. Each of these components has been shown to counteract the effects of the classical renin–angiotensin system. This counter-regulatory renin–angiotensin system has a central role in the pathogenesis and development of various cardiovascular diseases and, therefore, represents a potential therapeutic target. In this Review, we provide the latest insights into the complexity and interplay of the components of the non-canonical renin–angiotensin system, and discuss the function and therapeutic potential of targeting this system to treat cardiovascular disease.

The renin-angiotensin system (RAS) has a critical role in cardiovascular physiology through its effects in regu lating blood pressure and electrolyte balance 1 . However, under pathophysiological conditions, the effects of the RAS can intensify to trigger inflammation and structural remodelling, thus promoting cardiac and vascular dam age 2, 3 . Researchers have studied the RAS for more than a century, not only to understand its role in normal physio logical function but also to develop effective therapies to treat its dysregulation 1,2 . These systematic research efforts have led to the discovery of a non canonical RAS, which has challenged the hypothesis that the RAS can only exert deleterious effects on the cardiovascular and renal systems. In the classical system, renin cleaves angiotensinogen to form angiotensin I, which is sub sequently converted to angiotensin II by angiotensin converting enzyme (ACE) (Fig. 1) . Conversely, ACE2 can cleave angiotensin II to produce angiotensin 1-7, and can cleave angiotensin I to generate angiotensin 1-9 1,3 . Increasing evidence supports the concept that these systems work to produce opposite effects, suggesting a counter balancing role for the two axes in cardiovascu lar physiology and disease. A timeline of key historical findings associated with the study and discovery of the counter regulatory RAS is shown in Box 1. In light of the emergence of multiple studies evaluating the effects and signalling pathways elicited by the counter regulatory RAS in the past decade, we sought to provide an update on the current understanding of the complex regulation of the non canonical RAS. In this Review, we discuss the cardioprotective effects of the non canonical RAS and provide a critical analysis of the current challenges that must be overcome to translate its therapeutic effects into the clinical context.

The counter regulatory RAS is made up of various pep tides, receptors and enzymes (Fig. 1 ). Whereas the effects of angiotensin 1-7 and angiotensin 1-9 on the cardio vascular system have been explored previously 3 , the potential roles of other counter regulatory RAS compo nents remain poorly understood. These non canonical RAS components include alamandine, angiotensin 1-12 and angiotensin 1-5, as well as angiotensin 2-8 and angio tensin 3-8, which are also known as angiotensin III and IV, respectively 3 . Figure 2 shows the molecular structures of these peptides.

In the past 10 years, new evidence has emerged about the signalling pathways triggered by the counter regulatory RAS, revealing their role as potential thera peutic targets for cardiovascular disease (CVD). Angiotensin 1-7 can act as a β arrestinbiased agonist of the type 1 angiotensin II receptor (AT 1 R) without activ ating the G q subunit. This mechanism might contribute to the anti hypertrophic properties of angiotensin 1-7, given that neither activation of the AT 1 R nor the proto oncogene Mas receptor antagonists prevented the bene ficial effects of this peptide 4 . Alamandine activates the AMPactivated protein kinase (AMPK)-nitric oxide (NO) pathway via the Mas related G protein coupled recep tor member D (MRGD), which prevents angiotensin II induced hypertrophy 5 . By contrast, angiotensin 1-9 stimulates the AT 2 R-AKT signalling pathway to pro tect the myocardium against reperfusion induced cell death 6 . Moreover, angiotensin 1-12 has been shown to regulate intracellular calcium transients and left ventri cular contractile function in both normal rats and rats with heart failure (HF) via a chymase dependent and cyclic AMP dependent mechanism 7 . Additionally, angio tensin 1-5 has been found to induce atrial natri uretic peptide (ANP) secretion from isolated perfused rat atria by binding to the Mas receptor and activating the phos phatidylinositol 3kinase-AKT-endothelial NO syn thase pathway 8 . Lastly, angiotensinogen is the precursor for the entire RAS family of peptides, but, to date, no studies have shown that angiotensinogen can elicit direct biological effects on the heart. Nonetheless, the aryl hydrocarbon receptor nuclear translocator like protein 1 has been shown to modulate blood pressure through a mechanism involving transcriptional regulation of angiotensinogen in a circadian manner in perivascu lar adipose tissue, which in turn increases local angio tensin II production 9 . These novel findings shed light on the complex regulation of the classical RAS and suggest a similar complexity for its counter regulatory system. In this context, circadian expression of local angiotensinogen might affect organ specific activity of peptides with known cardiovascular effects, such as angiotensin 1-7 or angiotensin 1-9, and requires further investigation.

In the non canonical RAS, angiotensin 1-7 and angio tensin 1-9 bind to the Mas receptor and AT 2 R, respec tively, whereas alamandine acts through the MRGD 3 (Fig. 3) . Angiotensin 1-7 can also bind to the MRGD, but the functional relevance of this association remains unclear 10 . Emerging evidence reveals a more complex interaction between components of the classical and the counter regulatory RAS than initially thought, given that angiotensin 1-7 has also been shown to bind to AT 2 R 11 . Moreover, AT 1 R can form heterodimers with the Mas receptor, which inhibits the activity of AT 1 R 12 . Using radiolabelling and dynamic mass redistribution experi ments in cells overexpressing the Mas receptor, Gaidarov and colleagues found that although angiotensin 1-7 can antagonize angiotensin II signalling, angiotensin 1-7 does not bind directly to the Mas receptor 13 . These data conflict with an earlier study that demonstrated binding of fluorescent or 125 I labelled angiotensin 1-7 to the Mas receptor 14 . Gaidarov and colleagues noted that in the absence of the Mas receptor, angiotensin 1-7 has no effect on angiotensin II signalling 13 . However, the investigators also reiterated that rigorously controlled experiments demonstrating interactions between angiotensin 1-7 and the Mas receptor are very scarce. Moreover, given that their findings suggest that angiotensin 1-7 does not bind to the Mas receptor, the researchers hypothe sized that any cardioprotective effects of angiotensin 1-7 might be attributable to antagonism of angio tensin II signalling 13 . Nevertheless, additional studies are required to confirm whether angiotensin 1-7 is an endogenous agonist of the Mas receptor.

Meems and colleagues designed and synthesized NPA7, a peptide that can simultaneously activate the Mas receptor and the particulate guanylyl cyclase A receptor 15 . NPA7, generated by the fusion of angio tensin 1-7 with a 22amino acid sequence of the B type natriuretic peptide (BNP), reduced blood pressure, car diac unloading and systemic vascular resistance, and exerted a more potent natriuretic and diuretic effect than separate administration of BNP and angiotensin 1-7. These observations raise the possibility that fusion of other counter regulatory RAS ligands that can target more than one receptor might also induce a synergistic effect to mediate potent cardioprotective benefits. AT 2 R can form functional heterodimers with Mas receptors, highlighting the possibility of developing drugs that can selectively target monomers or oligomers to upregulate or downregulate specific cell signalling cascades in the cardiovascular system 16 . In addition, the crystal struc tures of human AT 2 R bound to a selective ligand indicate that the ligand can induce an active conformation of the receptor, suggesting that AT 2 R does not bind to G pro teins or β arrestins 17 . Tetzner and colleagues showed that angiotensin 1-7 can bind to the MRGD and that the AT 2 R antagonist PD123319 can block both the Mas receptor and MRGD 10 . This latter finding is particularly important, given the large number of studies utiliz ing PD123319 to assess the effects of AT 2 R activation. Figure 3 provides an overview of the signalling pathways triggered by the counter regulatory RAS ligands upon binding to their receptors.

ACE inhibitors are a first line pharmacological therapy in the management of hypertension. Other proteases such as ACE2 and neprilysin (also known as neutral endopeptidase) have been identified as novel therapeutic targets, given that these enzymes can also reduce blood pressure. ACE2 might reduce blood pressure levels by generating angiotensin 1-7 from angiotensin II, whereas inhibition of neprilysin increases ANP levels 18 . In addi tion, the endogenous metabolic regulator fibroblast growth factor 21 (FGF21) can promote ACE2 genera tion in adipocytes and renal cells, thereby promoting the cleavage of angiotensin II to form angiotensin 1-7, sug gesting that FGF21 can reduce angiotensin II induced hypertension 19 .

• Chronic activation of the renin-angiotensin system (raS) promotes cardiovascular damage, an effect that is antagonized by components of the counter-regulatory raS. • Components of the counter-regulatory raS, including angiotensin 1-7, angiotensin 1-9, alamandine and their receptors have been found to be protective in multiple cardiovascular diseases, such as hypertension and heart failure.

• Numerous preclinical studies have demonstrated the beneficial effects of the counter-regulatory raS, but clinical trials confirming these observations are still scarce. • The challenges in quantitating angiotensin 1-7, angiotensin 1-9 and alamandine associated with their short plasma half-life and similarity in their molecular structures must be overcome before these peptides can be evaluated in the clinical setting.

NaTure revIewS | CARdiology Fig. 1 | Classical and counter-regulatory renin-angiotensin pathways. In the classical system, renin cleaves angiotensinogen to produce angiotensin I. This peptide can be processed by angiotensin-converting enzyme (ACE) to form angiotensin II, which in turn can bind to the type 1 angiotensin II receptor (AT 1 R) and AT 2 R 3 . AT 1 R activation increases aldosterone 165 and anti-diuretic hormone (ADH) 166 production, sympathetic nervous system (SNS) tone 167 , blood pressure 168 , vasoconstriction 169 , cardiac hypertrophy 170 , fibrosis 171 , inflammation 172 , vascular smooth muscle cell (VSMC) dedifferentiation 173 and reactive oxygen species (ROS) production 36 , while decreasing parasympathetic nervous system (PSNS) tone 174 , baroreflex sensitivity 175 , nitric oxide (NO) production 176 and natriuresis 177 . Angiotensin II can be further processed by aminopeptidase A (APA) to form angiotensin III, which also acts through AT 1 R . Angiotensin III can be cleaved by alanyl aminopeptidase N (APN) to generate angiotensin IV, which binds to AT 4 R , producing cardioprotective effects 178 , increasing natriuresis 179 and NO production 180 , as well as reducing vasoconstriction 181 , inflammation 178 and VSMC dedifferentiation 182 . Angiotensin I can also be cleaved by ACE2 and neprilysin (NEP) to produce angiotensin 1-9 and angiotensin 1-7 , respectively 3 . Angiotensin 1-9 can activate AT 2 R to trigger natriuresis 183 and NO production 73 , thus mediating vasodilatory effects 73 and reducing blood pressure 73 . In addition, this peptide is cardioprotective 6 and can attenuate inflammation 73 , cardiac hypertrophy 135 and fibrosis 73 . Angiotensin 1-7 binds to the proto-oncogene Mas receptor (MasR) and reduces both blood pressure 184 and noradrenaline release in hypertensive rodents 185 . Conversely , activation of MasR increases NO generation 186 , natriuresis 187 , vasodilatation 186 , PSNS tone and baroreflex sensitivity 188, 189 . Angiotensin 1-7 can also be formed from angiotensin II cleavage by ACE2 and be further metabolized to alamandine. Alternatively , angiotensin II can be processed by aspartate decarboxylase (AD) to produce angiotensin A , which can be converted to alamandine by ACE2. Upon binding to the Mas-related G protein-coupled receptor member D (MRGD), alamandine can promote the same effects reported for angiotensin 1-7 5, 67, 190 , with the exception of natriuresis. RAS, renin-angiotensin system.

The classical RAS can act at both local and systemic levels, but how these signals are coordinated is poorly understood. Exosomes, which are extracellular vesi cles of 50-100 nm in size, can transport and transmit molecules such as proteins and microRNAs from one cell to another, and can also transport components of the classical RAS 20 . Previously assumed to be scattered cellular waste, exosomes are attracting much research interest since the discovery of their role in intercellular communication 21 . Considering that these extracellular vesicles can communicate signals from afar and that the counter regulatory RAS can exert its effects on multiple cell types, these vesicles might have a role in orchestrat ing the effects of the counter regulatory RAS. In this context, Pironti and colleagues observed that exosomes induced by cardiac pressure overload in mice contain functional AT 1 R, which might influence AT 1 R mediated regulation of vascular tone 22 . Moreover, exosomes seem to have a role in the local RAS. Angiotensin II triggers exosome production in rat cardiac fibroblasts in vitro, and these exosomes in turn promote angiotensin II production and AT 1 R expression in rat cardiomyocytes in vitro, suggesting a positive feedback mechanism that might contribute to the exacerbation of cardiac hyper trophy elicited by angiotensin II 23 . However, this evi dence only supports a role for exosomes in orchestrating the effects of the canonical RAS. Whether extracellular vesicles contribute to the cardioprotective properties of the counter regulatory RAS remains to be determined.

The ACE2-angiotensin 1-7-Mas receptor axis. ACE2, first described as a receptor for severe acute respiratory syndrome coronavirus, is characterized by its marked homology with ACE 24 . The therapeutic potential of ACE2 agonists for pulmonary arterial hypertension (PAH) has been explored in a number of studies. In rats with monocrotaline induced PAH, Ace2 gene therapy prevented PAH mediated hypertrophy and functional impairment of the right ventricle 25 . Moreover, synthetic activators of ACE2 (XNT 26 and resorcinolnaphtha lein 27 ) improve pulmonary artery endothelial function by inducing phosphorylation of endothelial NO syn thase at Ser1177 and dephosphorylation at Thr495 27 , which consequently increases the bioavailability of NO. A meta analysis to assess the efficacy of 522 interven tions for PAH revealed that these ACE2 synthetic activ ators were among the most potent agents 28 . Although these findings strongly support the therapeutic poten tial of ACE2 activators, translation of these agents into a clinical setting remains challenging because ACE2 is a membrane bound enzyme. ACE2 can be cleaved and its soluble and catalytically active form can be secreted 29, 30 . Given that increasing the circulating levels

The timeline in the figure shows a historical perspective of the most important findings associated with the counter-regulatory reninangiotensin system (raS). The proto-oncogene mas receptor (masr) was initially described as an oncogene and detected through its tumorigenicity in nude mice 147 . angiotensin 1-9 and angiotensin 1-7 were first identified from hydrolytic cleavage of angiotensin I, and angiotensin I or angiotensin II, respectively 148, 149 . In 1989, angiotensin 1-7 was found to have antihypertensive effects in rats upon unilateral injection into the medial "nucleus of the solitary tract" and into the dorsal motor nucleus of the vagus 150 . The earliest patents related to the components of the counterregulatory raS described the use of the masr in an assay system for detecting angiotensin-blocking activity, a cDNa encoding the type 2 angiotensin II receptor (aT 2 r) in mice and rats, a nucleic acid encoding angiotensin 1-7, a cDNa encoding angiotensin-converting enzyme 2 (aCe2) and the use of angiotensin 1-9 to prevent, reverse, inhibit or reduce cardiovascular, pulmonary, cerebral or renal remodelling. aCe2 was simultaneously discovered by two independent research groups in 2000 151, 152 . angiotensin 1-7 was subsequently described as a cardioprotective peptide 153 with anti-inflammatory actions 110 and found to be activated through the masr 14 . The first clinical trial of angiotensin 1-7 assessed the effect of this peptide on the reduction of blood flow in solid tumours 154 , whereas the first trial of aCe2 evaluated the safety and tolerability of a recombinant form of aCe2 121 . The anti-hypertrophic 135 , anti-hypertensive 73 , anti-inflammatory 120 and cardioprotective 6 properties of angiotensin 1-9 have been described. alamandine was discovered in 2013 as an anti-hypertensive agent 67 , and the cardioprotective properties of this compound have since been described 155, 156 . NaTure revIewS | CARdiology of ACE2 might have a therapeutic effect, a recombinant human ACE2 (rhACE2) has been developed and tested in animal models. Administration of rhACE2 improved right ventricular function in mice subjected to pressure overload 31 and attenuated vascular remodelling in mice with bleomycin induced pulmonary hypertension 32 . A pilot study evaluated the effects of increasing the enzy matic activity of ACE2 through intravenous infusion of 0.2 mg/kg or 0.4 mg/kg of rhACE2 in patients with PAH 33 . The drug was well tolerated and had beneficial effects on pulmonary vascular resistance and cardiac output, in addition to reducing inflammatory markers and increasing superoxide dismutase 2 levels in plasma. Nonetheless, this proof ofconcept study included only five patients. In a separate study, rhACE2 administration was also shown to be well tolerated in 44 patients with acute respiratory distress syndrome 34 . The safety profile of rhACE2 needs to be further assessed in clinical studies. Angiotensin 1-7 and other Mas receptor activators might also have a protective role against the develop ment of PAH 35 . Notably, however, angiotensin 1-7 is not considered a good therapeutic candidate owing to its pharmacokinetic limitations. Angiotensin 1-7 is rapidly cleaved by peptidases and thus has a very short half life of ~10 s (reF. 36 ). However, cell signalling mechanisms and effects mediated by biological peptides are thought to persist despite their short half life 37 . Furthermore, stud ies in animal models have shown that administration of angiotensin 1-7 included in cyclodextrin complexes has neuroprotective effects and improves muscle dam age induced by eccentric cardiac overload [38] [39] [40] [41] [42] [43] [44] . A stable, cyclic analogue of angiotensin 1-7 moderately reduced right ventricular systolic pressure in a rat model of monocrotaline induced PAH, but no significant changes were observed in the medial wall thickness of pulmo nary arterioles 45 . To optimize the protective potential of this angiotensin 1-7 analogue for the treatment of PAH, the compound can potentially be combined with a neprilysin inhibitor or an ACE2 activator 46 ; whether this approach is effective in maintaining high levels of angiotensin 1-7 requires further investigation.

AT 2 R stimulation. AT 2 R activation can attenuate right ventricular and pulmonary remodelling 47 . AT 2 R stim ulation protected mice from severe lung injury induced by sepsis or acid aspiration 48 , whereas AT 2 R deficiency exacerbated HF in mice subjected to acute myocardial infarction 49 . Furthermore, activation of AT 2 R (using the agonist dKc angiotensin 1-7) in a rat model of chronic lung disease protected the heart and lungs from 48 . Adult rats with PAH treated with angioten sin 1-9 showed reduced right ventricular weight and systolic pressure, as well as diminished lung fibrosis, pulmonary arteriole thickness and endothelial damage compared with untreated controls. These effects were dependent on activation of the AT 2 R but not the Mas receptor. Treatment with angiotensin 1-9 also reduced plasma levels of the pro inflammatory markers tumour necrosis factor (TNF), CC chemokine ligand 2 (CCL2; also known as MCP1), IL1β and IL6 48 . 192 and mitogen-activated protein kinase-phosphatase 1 (MKP1) 193 , which can result in attenuation of cardiac hypertrophy. AT 2 R can also activate the transcription factor promyelocytic zinc finger protein (PLZF), thereby inducing the expression of ribosomal protein S6 kinase β1 (p70S6K) and p85α expression and, in turn, eliciting protein synthesis 194 . In addition, AT 2 R might trigger vasodilatation by activating the phosphatidylinositol-3-kinase (PI3K)-AKT-endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cGMP pathway either via angiotensin 1-9-mediated activation [194] [195] [196] or by heterodimerization with bradykinin B 2 receptor (B 2 R) 197 . Phosphorylation of AKT by activation of AT 2 R through angiotensin 1-9 binding has also been found to confer cardioprotection 6 . Angiotensin 1-7 might induce the NO-soluble guanylyl cyclase pathway , thereby triggering vasodilatation via proto-oncogene Mas receptor (MasR) activation. Activation of this receptor can also reduce cardiac fibrosis by stimulating SHP1 198 and dual-specificity phosphatase (DUSP) 199 , consequently inhibiting p38 mitogen-activated protein kinase (MAPK) and ERK1 and ERK2 200 . The K Ca 3.1 channel 201 and mothers against decantaplegic homologue 2 (SMAD2) and SMAD3 202 are downstream targets of ERK1 and ERK2, and are downregulated upon MasR activation. Additionally , angiotensin 1-7 exerts an anti-hypertrophic effect by inhibiting nuclear factor of activated T cells (NFAT) through a MasR-PI3K-AKT-NO-cGMP-dependent pathway 203 . This anti-hypertrophic effect also depends on atrial natriuretic peptide (ANP) secretion during atrial pacing and is associated with activation of the Na + /H + exchanger (NHE1) and calcium/calmodulin-dependent protein kinase II (CaMKII) via the PI3K-AKT pathway 200 . Cardiac hypertrophy can also be reduced by activation of the Mas-related G protein-coupled receptor member D (MRGD) by alamandine via adenylate cyclase (AC)-cAMP-protein kinase A (PKA) signalling 10 .

ACE2 activity. The synthetic ACE2activator XNT reduced blood pressure in an angiotensin II induced model of hypertension, but plasma concentrations of angiotensin II and angiotensin 1-7 remained unal tered 56 . Moreover, the antihypertensive effect of this drug was observed in ACE2deficient mice, and neither XNT nor DIZE induced the enzymatic activity of ACE2 in rat or mouse kidneys 56 . These findings raise the ques tion as to whether researchers should continue to focus on these drugs with unknown mechanisms of action. However, ACE2 remains an appealing therapeutic target for treating hypertension, especially in tissues in which expression of this enzyme is higher than in plasma 56 . The therapeutic potential of DIZE as an alternative treatment for hypertension and PAH has been shown in previous experimental studies 52, 57 . Moreover, deoxycorticosterone acetate (DOCA)-salt hypertensive rats treated with the Mas receptor agonist AVE0991 had lower blood pressure levels than untreated controls 58 . The anti hypertrophic effects of AVE0991 are, in part, mediated by inhibition of NADPH oxidase 2 and NADPH oxidase 4, as observed in hypertensive mice subjected to aortic banding 59 . At present, the effects of these ACE2 activators have only been evaluated in preclinical studies. A rigorous evalu ation of how these agents exert their beneficial effects is needed before they can be tested in the clinical setting, in order to identify off target and potentially toxic effects. ACE2 activity has also been assessed in patients with high blood pressure. The level of ACE2mediated angio tensin II degrading activity in monocyte derived macro phages in vitro has been found to be similar in cells from both healthy individuals and patients with hyper tension 60 . Of note, ACE2 activity is significantly higher in monocyte derived macrophages from patients with prehypertension than in those from patients with hyper tension, suggesting a potential role for ACE2 as an early marker of hypertension. This finding might also indicate a physiological protective mechanism against hypertension, most probably through the rapid degradation of angio tensin II 60 . By contrast, no correlation has been found between hypertension and ACE2 activity in patients with ST segment elevation myocardial infarction 61 .

Plasma ACE2 levels have been suggested to vary depending on sex 62, 63 , although most of the research exploring the role of ACE2 in CVD has not considered sex related differences in activity levels. During preg nancy, plasma levels of angiotensin II are significantly elevated, whereas angiotensin 1-7 levels are significantly diminished, which together might predispose preg nant women to hypertension related complications 64 . Furthermore, levels of urinary angiotensin 1-7 in patients with hypertension have been reported to be inversely proportional to blood pressure levels, implying a cru cial role for this peptide in the development of hyper tension 65 . Finally, angiotensin 1-7 has also been shown to alleviate obesity induced haemodynamic alterations 66 .

Alamandine. Alamandine is a heptapeptide formed by the catalytic action of ACE2 on angiotensin A or directly from angiotensin 1-7 in the heart. Oral admin istration of an inclusion compound of alamandine and β hydroxypropyl cyclodextrin reduced blood pressure in spontaneously hypertensive rats and diminished myocardial fibrosis in isoprenaline treated rats 67 . This anti hypertensive effect was shown to have two phases. Initially, mean arterial pressure and left ventricular sys tolic pressure increased briefly in an AT 1 R dependent manner, followed by a reduction in these parameters, which persisted throughout the rest of the infusion period. This anti hypertensive effect was reversed by PD123319, an AT 2 R antagonist 68 . Additionally, alaman dine treatment mitigated vascular remodelling in mice subjected to transverse aortic constriction 69 . Additional studies are required to further our understanding of the complex regulation of alamandine, the cell signal ling cascades it triggers, and its therapeutic implica tions for hypertension and other CVDs. The normal range of alamandine levels in both healthy individuals and patients with hypertension should be established to provide a better understanding of the effect of RAS inhibition on alamandine plasma concentrations in this clinical context. 70, 71 or over expressing this receptor 72 . Mice lacking the AT 2 R showed an increased response to angiotensin II and significantly elevated blood pressure levels 70, 71 , whereas transgenic overexpression of the AT 2 R in vascular smooth muscle cells of mice reduced angiotensin II induced vasocon striction 72 . The anti hypertensive effects of the AT 2 R selective agonists CGP42112A and angiotensin 1-9 have also been evaluated 73, 74 . CGP42112A treated obese rats had reduced blood pressure levels compared with untreated rats, which was associated with an increase in urinary sodium excretion 74 . This agonist also decreased blood pressure levels in spontaneously hypertensive rats 75 and prevented endothelial cell migration mediated by vascular endothelial growth factor signalling 76 .

The specific Rho kinase inhibitor fasudil significantly increased plasma levels of angiotensin 1-9 in both nor motensive and hypertensive rats 77 . In addition, fasudil reduced blood pressure levels and aortic Rho kinase and ACE activity, whereas mRNA and protein levels of ACE2 were increased in plasma and the aortic wall 77 . Interestingly, another study showed an increase in ACE and angiotensin II levels in patients at high risk of acute pulmonary embolism compared with healthy volunteers 78 . Moreover, in a rat model of acute pulmonary embolism, RhoA-ROCK signalling mediated an imbalance in RAS vasoconstrictors, which was reversed with ROCK inhibitors or an ACE2 activator 78 . These findings further highlight the protective effects that ROCK inhibition can exert in the setting of hypertension, atherosclerosis and pathological cardiovascular remodelling.

In a study by Ocaranza and colleagues, administra tion of angiotensin 1-9 reduced blood pressure levels in hypertensive rats and attenuated myocardial damage by inhibiting the development of ventricular hypertro phy and fibrosis; importantly, these effects were medi ated through AT 2 R but not Mas receptor signalling 73 . However, in a separate study, gene delivery of angio tensin 1-9 with an adeno associated virus (AAV) in mice subjected to coronary artery ligation completely restored www.nature.com/nrcardio systolic blood pressure levels and cardiac output com pared with sham treated mice, but histological analy sis revealed only mild effects on cardiac hypertrophy and fibrosis 79 . Notably, Ocaranza and colleagues only evaluated angiotensin 1-9 administration for 2 weeks 73 , compared with the latter study that examined the effects of this peptide for 8 weeks 79 . The conflicting findings between these two studies suggest that the attenuation of myocardial damage might be transient and not sus tained in the long term. However, the latter study did not measure plasma levels of angiotensin 1-9. AAV mediated gene delivery of angiotensin 1-9 might not have produced a therapeutic concentration of the pep tide in the blood that would be sufficient to protect the heart from adverse structural remodelling. A study that tested the anti hypertensive actions of angiotensin 1-9 in stroke prone spontaneously hypertensive rats also found no evidence of a protective effect 80 , but this study used a dose of angiotensin 1-9 that was six times lower than that used by Ocaranza and colleagues 73 . Additional studies are warranted to explore the anti hypertensive and anti remodelling effects of angiotensin 1-9 admin istration and the implications of the plasma levels of this peptide on cardioprotection. Although the efficacy of angiotensin 1-9 administration has not been explored in the clinical setting, in patients with acute respiratory distress syndrome, higher angiotensin 1-9 levels in plasma were associated with reduced mortality, whereas increased plasma angiotensin I levels were associated with increased mortality 81 .

Heart failure ACE2 is critical for heart function 82 , vasodilatation 83 and fluid balance 84 . Ace2 −/y mutant mice have impaired con tractility, increased expression of hypoxia markers and increased circulating levels of angiotensin II compared with control mice 82 . Furthermore, Ace2 −/y mutant mice develop angiotensin II mediated dilated cardiomyopathy that is characterized by an increase in markers of oxida tive stress and inflammation, pathological hypertrophy and impaired left ventricular function 85 . Interestingly, plasma levels of the soluble form of ACE2 have been reported to be elevated in patients with HF and reduced ejection fraction, suggesting that sustained activation of the counter regulatory RAS in HF might be a compen satory mechanism to attenuate cardiovascular dysfunc tion 86 . The mechanisms underlying HF with preserved ejection fraction (HFpEF) remain poorly defined, but the progression of this disease has been proposed to be linked to hypertension induced cardiac remodelling 87 . Given the antihypertensive and anti remodelling effects of the counterregulatory RAS described thus far, this non canonical signalling pathway might be a potential thera peutic target for the treatment of HFpEF. Angiotensin II infusion in wild type mice resulted in increased blood pressure levels, myocardial hypertrophy, fibrosis and diastolic dysfunction; these effects were exacerbated in Ace2 −/y mice 88 . Conversely, treatment of angiotensin II infused wild type mice with rhACE2 reduced angio tensin II induced superoxide production and blunted the cardiac hypertrophic response, highlighting a possible protective role for this enzyme in HFpEF 88 .

Other components of the non canonical RAS path way are also involved in HF. Mice deficient in the alamandine receptor MRGD have left ventricular remodelling and severe dysfunction, and present with pronounced dilated cardiomyopathy 89 . Furthermore, infusion of the AT 2 R agonist C21 for 7 days in rats with HF induced by coronary artery ligation led to a reduction in noradrenaline excretion, as well as decreased renal sympathetic nerve activity 90 . Additionally, C21 admin istration increased baroreflex sensitivity, suggesting a protective role for this drug in the setting of HF.

Collectively, these findings support a role for vari ous components of the counter regulatory RAS in HF, both as potential biomarkers and therapeutic targets. Additional clinical studies are needed to determine the levels of ACE2, angiotensin 1-9 and angiotensin 1-7 in patients with HF.

The role of non canonical RAS signalling in the devel opment of myocardial infarction has been described. ACE2 mRNA levels are elevated in the setting of myo cardial infarction 91 , whereas loss of ACE2 can further exacerbate cardiac damage 92 . By the same token, Ace2 overexpression has been shown to alleviate myocardial damage induced by ischaemia-reperfusion in rats 93 . Furthermore, administration of angiotensin 1-7 (added to the oligosaccharide hydroxypropyl β cyclodextrin) in rats with myocardial infarction improved cardiac function and reduced infarct size by 50% 42, 43 . Likewise, transgenic rats overexpressing a fusion protein that leads to a selective increase in angiotensin 1-7 levels were less susceptible to reperfusion induced arrhyth mias and isoproterenol induced hypertrophy than wildtype rats 94 .

The cardioprotective role of AT 2 R in preventing post ischaemic cardiac remodelling has been documented 95,96 . Mice lacking AT 2 R have aggravated myocardial infarction induced HF and reduced survival compared with sham treated mice 97 . Correspondingly, transgenic mice overexpressing AT 2 R showed improved left ven tricular function after myocardial infarction 98 , and simi lar results were observed in rats with cardiac specific overexpression of AT 2 R 99 . Administration of the AT 2 R agonist C21 to rats subjected to coronary artery ligation significantly improved recovery of left ventricular func tion and reduced cardiac remodelling after myocardial infarction 100 . Delivery of angiotensin 1-9 with an AAV vector into mice after the induction of myocardial infarc tion resulted in a reduction in sudden cardiac death and improved left ventricular function compared with con trol mice 79 . Importantly, angiotensin 1-9 had a positive inotropic effect, achieved by increasing calcium transient amplitude and contractility through a protein kinase A dependent mechanism 79 . Using an ex vivo approach with isolated rat hearts subjected to global ischae mia and reperfusion, Mendoza Torres and colleagues showed that angiotensin 1-9 infusion can also reduce infarct size and apoptotic and necrotic cell death, and improve left ventricular function in an AT 2 R dependent and AKT dependent mechanism 6 . Together, these data suggest that angiotensin 1-7 and angiotensin 1-9 might be valuable pharmacological tools for the treat ment of myocardial infarction, given their acute and long term cardioprotective effects.

Inflammatory processes are central to the develop ment and progression of CVDs such as atherosclero sis, hypertension, myocardial infarction and HF 101-105 . A link between inflammation and RAS has previously been observed. T cells have an endogenous RAS that can regulate T cell function, NADPH oxidase activity and superoxide production 106, 107 . Natural killer cells have also been shown to express renin, angiotensinogen, ACE and AT 2 R 107 . In line with these observations, the pro inflammatory state is thought to upregulate RAS signal ling in the setting of hypertension 108 . Interestingly, human monocytes also express ACE and ACE2 and can produce angiotensin 1-7 and angiotensin 1-9 109 . Taken together, these data suggest that the immune system might also be involved in regulating the non canonical RAS.

Activation of the Mas receptor has been shown to promote anti inflammatory effects 110 . Mice lacking this receptor have an exacerbated inflammatory reaction after treatment with lipopolysaccharides compared with wild type mice 111 . Therefore, Mas receptor activation might be a valuable therapeutic target to counteract the pro inflammatory processes that promote the develop ment and progression of atherosclerosis 112, 113 . Indeed, the Mas receptor agonist AVE0991 inhibits atherogenesis in Apoe −/− mice 114 . Moreover, long term angiotensin 1-7 treatment confers both vasoprotection (by improving endothelial function) and atheroprotection (by reducing lesion progression) in Apoe −/− mice 115 . Consistent with these observations, angiotensin 1-7 can activate signal ling pathways critical for the resolution of inflammatory processes involved in asthma 116 .

In addition to the Mas receptor, AT 2 R signalling has also been associated with the regulation of inflamma tion. The AT 2 R agonist C21 dose dependently attenuates lipopolysaccharide induced TNF and IL6 production, but increased production of the anti inflammatory cytokine IL10 117 . Consistent with these observations, a separate study showed that administration of C21 in prehypertensive, obese Zucker rats reduced plasma levels of TNF and IL6, whereas coadministration with the AT 2 R antagonist PD123319 decreased IL10 levels in the kidneys 118 . Furthermore, in Wistar rats subjected to left coronary artery ligation, C21 treatment reduced the production of the pro inflammatory cytokines IL1β, IL6 and IL2 in an AT 2 R dependent manner, improved systolic and diastolic ventricular function, and reduced scar size 119 . Angiotensin 1-9 administration has also been shown to reduce cardiac and renal inflammation in a DOCA-salt model of hypertension in rats, but this effect was independent of AT 2 R 120 .

Research into the counter regulatory RAS has resulted in the generation of a substantial amount of intellectual property related to its study and use. Currently, 184 pat ent applications associated with this system have been filed, most related to angiotensin 1-7 and its analogues, AT 2 R, the Mas receptor, ACE2 and angiotensin 1-9. Only 76 patents are related to cardiovascular applica tions involving the control of arterial pressure, vascular remodelling, cardiac remodelling and HF. Furthermore, the robust evidence collated from large numbers of pre clinical studies on the counter regulatory RAS has also prompted the initiation of numerous clinical trials. At the time of this report, 15 clinical trials that involve interventions with counter regulatory RAS molecules in CVDs were ongoing, including two studies designed to evaluate the safety of recombinant ACE2 and angio tensin 1-7 in treating thrombocytopenia 121,122 . A further nine trials aim to assess the use of ACE2 in the treatment of pulmonary hypertension 123,124 and the safety and use of angiotensin 1-7 in hypoxia, hypertension, HF and coro nary artery bypass surgery [125] [126] [127] [128] [129] [130] [131] . Two trials investigating the use of angiotensin 1-7 to treat peripheral arterial disease and obesity associated hypertension 132,133 are currently in the pre recruitment phase.

Despite the substantial amount of evidence suggesting a counter regulatory role for the non canonical RAS in protecting against the deleterious actions of a dysregu lated classical RAS, the complexity of the relationship between the two systems remains to be fully elucidated. For example, ACE2 is elevated in patients with HF 86 or pre hypertension 60 , but depressed in patients with PAH 33 . These discrepancies suggest that the components of the counter regulatory RAS are upregulated or down regulated depending on the stage, severity or type of CVD. Moreover, these conflicting findings reinforce our lack of knowledge of the physiological and pathophysio logical mechanisms involved in non canonical RAS regulation. For instance, elevated levels of soluble ACE2 might represent a compensatory mechanism in response to HF but might also be the result of increased cleav age of membrane ACE2 by disintegrin and metallopro teinase domain containing protein 17, which is known to be upregulated in HF 86 . In addition, RAS peptides can also be modulated by pharmacological treatment. In this regard, patients with chronic HF treated with ACE inhibitors have elevated plasma levels of angio tensin 1-7 and reduced plasma levels of angio tensin II, whereas patients with acute HF treated with angio tensin II receptor antagonists have decreased plasma levels of angio tensin 1-7 and increased plasma levels of angiotensin II 134 . Furthermore, the addition of rhACE2 to plasma samples from patients with HF induced the conversion of angiotensin I and angiotensin II into angiotensin 1-9 and angiotensin 1-7, respectively 134 .

In addition to the aforementioned challenges in inter preting the data on the non canonical RAS, the measure ment of angiotensin 1-7, angiotensin 1-9 or alamandine in a clinical context poses many challenges. The separa tion of these peptides from a biological sample is diffi cult, given the similarity in their molecular structures. Angiotensin 1-7 is only two amino acids shorter than angiotensin 1-9 135 , whereas angiotensin 1-7 and alaman dine only differ in their N terminal amino acid 67 (Fig. 2) .

Therefore, the identification of these peptides requires the use of high precision approaches, such as high performance liquid chromatography and mass spec trometry (Box 2). Furthermore, one of the fundamental problems associated with the use of these peptides in the clinical context is their short plasma half life, owing to rapid enzymatic degradation. In each of the numerous ongoing clinical trials assessing the effects of angioten sin 1-7 in CVDs, angiotensin 1-7 is administered via subcutaneous or intravenous injection 126-131 . However, a cyclized angiotensin 1-7 analogue has been described that has increased half life, improved resistance to enzy matic degradation and superior functional activity com pared with natural angiotensin 1-7 136 . Similar chemical modifications to the angiotensin 1-9 peptide might also prolong the half life of the peptide. However, the non peptide agonist C21, which has a half life of 4-6 h, can also induce AT 2 R activation 137 . This agonist has high selectivity for its receptor and is well tolerated 137, 138 . However, although the results to date are promising, angiotensin 1-9 still requires extensive safety and effi cacy assessment as a potential endogenous AT 2 R agonist.

The oral bioavailability of C21 is only 30%, and this agonist has also been reported to modulate epi genetic mechanisms associated with the pathophysio logy of dia betic nephropathy 137 , raising the possibility of unwanted off target effects if used to treat CVD. Studies compar ing the effects of C21 and angiotensin 1-9 will be useful to establish the potential differences between the two agents.

Once the challenges hindering clinical translation of counter regulatory RAS components for the treatment of CVD have been overcome, these therapeutic agents might be used to complement traditional pharmacolog ical treatments. Such complementary drugs are neces sary, because even gold standard drugs for hypertension are associated with issues such as suboptimal drug effi cacy and adherence. Most patients with hypertension, especially those with comorbidities, require two or more drugs to manage their blood pressure levels 139, 140 . Furthermore, many of these patients require two or more doses each day 140 , suggesting that the separate use of ACE inhibitors or angiotensin II receptor antagonists is not always effective. The use of more than one drug and the need for multiple doses per day can increase the incidence of adverse events, which can result in loss of adherence 140, 141 . In addition, a longitudinal study that evaluated the dosing histories of 4,783 patients taking antihypertensive drugs found that nearly half of the patient cohort discontinued the treatment 142 , which results in poorly controlled hypertension 143 . Combining these counter regulatory RAS peptides with the current gold standard antihypertensive drugs in one pill might overcome the need for patients with hypertension and other comorbidities to receive more than one drug or multiple dosages of drugs per day. Counter regulatory RAS peptides, such as angiotensin 1-7, alamandine or angiotensin 1-9, have been found to be effective in reducing blood pressure and attenuating cardiovas cular remodelling in preclinical studies 67, 73, 144 . These effects might be achieved with fewer adverse reactions in patients with hypertension compared with current antihypertensive therapies, which in turn might improve treatment adherence. Combining angiotensin 1-7 with the angiotensin receptor blocker losartan might increase or extend its blood pressure lowering capac ity 145 . Importantly, the anti atherosclerotic effects of dual angiotensin 1-7 and losartan therapy are synergistic 146 . Pharmacological synergy between current gold standard treatment for CVDs and counter regulatory RAS pep tides might decrease the dosages required to achieve efficacy, thereby reducing adverse effects. However, although the endogenous origin of counter regulatory RAS components suggests a safe pharmacological pro file, the current lack of robust evidence in patients means that this hypothesis remains to be tested.

The evidence supporting the protective role of the counter regulatory RAS in CVD is robust but incom plete. In addition to the methodological pitfalls that must be overcome, future research should also be

Three critical issues should be considered to ensure that the method for quantifying angiotensin peptides is reliable. First, the sampling procedure must be efficient because blood or tissue samples need to undergo immediate peptidase inhibition to ensure stabilization of angiotensin metabolites [157] [158] [159] [160] [161] [162] (see the figure, part a) . The sampling duration should be kept as short as possible to avoid unexpected shifts in angiotensin metabolite patterns. Second, the structural similarity of all angiotensin metabolites (Fig. 2) necessitates an effective separation procedure, usually liquid chromatography [157] [158] [159] [160] [161] [162] . once all peptides have been separated, angiotensin metabolites are quantified in the liquid chromatography eluate. Finally, the assay must be sensitive, given that angiotensin metabolites have been described at levels in the femtomolar range [157] [158] [159] [160] [161] [162] . Immunological assays (radioimmunoassay (rIa) or enzyme-linked immunosorbent assays (elISa)) [157] [158] [159] [160] and mass spectrometry 161, 162 have been used in this setting. both methods have lower limits of quantification for angiotensin metabolites (~1-2 fmol/ml in plasma and 5-10 fmol/g in tissue samples) [157] [158] [159] [160] [161] [162] . although rIa and elISa are the traditional methods for quantifying angiotensin peptides given their high sensitivity and specificity, these methods rely heavily on the characteristics of the antibodies. Therefore, liquid chromatography-mass spectrometry is a promising approach for obtaining reliable read-outs, given its capacity to detect renin-angiotensin system (raS) peptides by assessing their unique massto-charge spectra, which might also allow measurement of potential post-translational modifications in these peptides 163 . However, this technique is not without drawbacks. a complex spectrum resulting from measurement of multiple components with similar mass-to-charge ratios has been reported 164 . moreover, this approach is expensive and requires very specific expertise 163 . These considerations are of paramount importance, given that inaccurate measurement of raS peptides can lead to erroneous conclusions that might cloud our understanding of the non-canonical raS. NaTure revIewS | CARdiology conducted in large animals with high translational value to further confirm the data from the studies carried out in vitro and in small animal models. The roles of other RAS peptides, such as angiotensin III and angiotensin IV, in the cardiovascular system warrant further inves tigation. Furthermore, the assessment of classical and counter regulatory RAS peptides during routine clin ical evaluation in patients with CVD should be con sidered, although development of practical, affordable and accurate methods to assess these levels are required to achieve reliable readouts. The balance -or imbal ance -of the levels of these peptides in plasma or urine might be useful as markers of CVD. Moreover, a thor ough evaluation of the counter regulatory RAS profile of each patient might bring current therapeutic approaches a step closer to the goal of precision medicine, allowing tailored treatment plans for each patient to optimize drug efficacy and adherence.

Published online 19 August 2019

Role of angiotensin II in cardiovascular disease therapeutic implications of more than a century of research

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin receptors: interpreters of pathophysiological angiotensinergic stimuli

Angiotensin II signal transduction: an update on mechanisms of physiology and pathophysiology

Ang-(1-7) is an endogenous beta-arrestin-biased agonist of the AT1 receptor with protective action in cardiac hypertrophy

Alamandine acts via MrgD to induce AMPK/NO activation against ANG II hypertrophy in cardiomyocytes

Protection of the myocardium against ischemia/reperfusion injury by angiotensin-(1-9) through an AT2R and Akt-dependent mechanism

Critical role of the chymase/ angiotensin-(1-12) axis in modulating cardiomyocyte contractility

Angiotensin-(1-5), an active mediator of reninangiotensin system, stimulates ANP secretion via Mas receptor

Bmal1 in perivascular adipose tissue regulates resting-phase blood pressure through transcriptional regulation of angiotensinogen

G-protein-coupled receptor MrgD is a receptor for angiotensin-(1-7) involving adenylyl cyclase, cAMP, and phosphokinase A

Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors

G-protein-coupled receptor Mas is a physiological antagonist of the angiotensin II type 1 receptor

Angiotensin (1-7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor

Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas

This paper describes the synthesis of a peptide that simultaneously activates the Mas receptor and the particulate guanylyl cyclase A receptor

Evidence for heterodimerization and functional interaction of the angiotensin type 2 receptor and the receptor MAS

Structural basis for selectivity and diversity in angiotensin II receptors

This paper reports the crystal structure of human AT 2 R and evidence showing that this receptor does not bind to G proteins or β-arrestins

Interventional procedures and future drug therapy for hypertension

FGF21 Prevents angiotensin II-induced hypertension and vascular dysfunction by activation of ACE2/angiotensin-(1-7) axis in mice

Microvesicles and exosomes: new players in metabolic and cardiovascular disease

Exosomes: nanoparticles involved in cardioprotection?

Circulating exosomes induced by cardiac pressure overload contain functional angiotensin II type 1 receptors

A critical role of cardiac fibroblast-derived exosomes in activating renin angiotensin system in cardiomyocytes

The emerging role of ACE2 in physiology and disease

Prevention of pulmonary hypertension by angiotensin-converting enzyme 2 gene transfer

Evidence for angiotensinconverting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension

Angiotensin-converting enzyme 2 activation ameliorates pulmonary endothelial dysfunction in rats with pulmonary arterial hypertension through mediating phosphorylation of endothelial nitric oxide synthase

Systematic review and meta-analysis of interventions tested in animal models of pulmonary hypertension

Soluble angiotensin-converting enzyme 2 in human heart failure: relation with myocardial function and clinical outcomes

Increasing serum soluble angiotensinconverting enzyme 2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure

ACE2 improves right ventricular function in a pressure overload model

rhACE2 therapy modifies bleomycin-induced pulmonary hypertension via rescue of vascular remodeling

A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension

A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome

Intrapulmonary activation of the angiotensin-converting enzyme type 2/angiotensin 1-7/G-protein-coupled Mas receptor axis attenuates pulmonary hypertension in Ren-2 transgenic rats exposed to chronic hypoxia

Converting enzyme determines plasma clearance of angiotensin-(1-7)

Concepts for biologically active peptides

Neuroprotection by post-stroke administration of an oral formulation of angiotensin-(1-7) in ischaemic stroke

Eccentric overload muscle damage is attenuated by a novel angiotensin-(1-7) treatment

Chronic oral administration of Ang-(1-7) improves skeletal muscle, autonomic and locomotor phenotypes in muscular dystrophy

An orally active angiotensin-(1-7) inclusion compound and exercise training produce similar cardiovascular effects in spontaneously hypertensive rats

Beneficial effects of long-term administration of an oral formulation of angiotensin-(1-7) in infarcted rats

An oral formulation of angiotensin-(1-7) produces cardioprotective effects in infarcted and isoproterenol-treated rats

Study of angiotensin-(1-7) vasoactive peptide and its beta-cyclodextrin inclusion complexes: complete sequence-specific NMR assignments and structural studies

Dose-dependent, therapeutic potential of angiotensin-(1-7) for the treatment of pulmonary arterial hypertension

Concurrent neprilysin inhibition and renin-angiotensin system modulations prevented diabetic nephropathy

Protective role of the ACE2/Ang-(1-9) axis in cardiovascular remodeling

Angiotensin-(1-9) ameliorates pulmonary arterial hypertension via angiotensin type II receptor

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis

Agonists of MAS oncogene and angiotensin II type 2 receptors attenuate cardiopulmonary disease in rats with neonatal hyperoxia-induced lung injury

Structure-based identification of small-molecule angiotensin-converting enzyme 2 activators as novel antihypertensive agents

Anti-hypertensive effects of diminazene aceturate: an angiotensin-converting enzyme 2 activator in rats

AVE 0991, a nonpeptide mimic of the effects of angiotensin-(1-7) on the endothelium

Vascular relaxation, antihypertensive effect, and cardioprotection of a novel peptide agonist of the MAS receptor

Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation

Angiotensin-converting enzyme 2-independent action of presumed angiotensinconverting enzyme 2 activators: studies in vivo, ex vivo, and in vitro

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models

Effect of combination of renin inhibitor and Mas-receptor agonist in DOCA-salt-induced hypertension in rats

AVE 0991 attenuates cardiac hypertrophy through reducing oxidative stress

ACE2 activity is increased in monocyte-derived macrophages from prehypertensive subjects

Role of circulating angiotensin converting enzyme 2 in left ventricular remodeling following myocardial infarction: a prospective controlled study

Circulating ACE2 activity is increased in patients with type 1 diabetes and vascular complications

Angiotensin-converting enzyme 2 activity in patients with chronic kidney disease

Dynamics of renin, angiotensin II, and angiotensin (1-7) during pregnancy and predisposition to hypertension-associated complications

Characterization of angiotensin-(1-7) in the urine of normal and essential hypertensive subjects

Favorable vascular actions of angiotensin-(1-7) in human obesity

Discovery and characterization of alamandine: a novel component of the reninangiotensin system

Differences in cardiovascular responses to alamandine in two-kidney, one clip hypertensive and normotensive rats

Alamandine attenuates arterial remodelling induced by transverse aortic constriction in mice

Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor

Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice

Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation

Angiotensin-(1-9) reverses experimental hypertension and cardiovascular damage by inhibition of the angiotensin converting enzyme/Ang II axis

Chronic AT2 receptor activation increases renal ACE2 activity, attenuates AT1 receptor function and blood pressure in obese Zucker rats

AT2 receptor-mediated vasodilatation is unmasked by AT1 receptor blockade in conscious SHR

Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells

Rho kinase inhibition activates the homologous angiotensin-converting enzymeangiotensin-(1-9) axis in experimental hypertension

RhoA-Rho associated kinase signaling leads to renin-angiotensin system imbalance and angiotensin converting enzyme 2 has a protective role in acute pulmonary embolism

Gene therapy with angiotensin-(1-9) preserves left ventricular systolic function after myocardial infarction

a route to targeted therapies

Role of inflammation in heart failure

Inflammation and cardiovascular disease: from pathogenesis to therapeutic target

Oxidative stress, inflammation, and vascular aging in hypertension

Inflammation, immunity, and hypertensive end-organ damage

Regulation of T-cell function by endogenously produced angiotensin II

Human T and natural killer cells possess a functional renin-angiotensin system: further mechanisms of angiotensin II-induced inflammation

Roles of inflammation, oxidative stress, and vascular dysfunction in hypertension

Human monocyte subsets exhibit divergent angiotensin I-converting activity

Anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, MAS, in experimental models of arthritis

Mas receptor deficiency exacerbates lipopolysaccharide-induced cerebral and systemic inflammation in mice

Angiotensin-(1-7): beyond the cardio-renal actions

Angiotensin-(1-7) attenuates airway remodelling and hyperresponsiveness in a model of chronic allergic lung inflammation

Angiotensin-(1-7) receptor Mas agonist ameliorates progress of atherosclerosis in apoE-knockout mice

Vasoprotective and atheroprotective effects of angiotensin (1-7) in apolipoprotein E-deficient mice

Angiotensin-(1-7) promotes resolution of eosinophilic inflammation in an experimental model of asthma

Angiotensin AT2 receptor stimulation is anti-inflammatory in lipopolysaccharide-activated THP-1 macrophages via increased interleukin-10 production

Proximal tubule angiotensin AT2 receptors mediate an antiinflammatory response via interleukin-10: role in renoprotection in obese rats

Angiotensin II type 2 receptor stimulation: a novel option of therapeutic interference with the renin-angiotensin system in myocardial infarction?

Angiotensin-(1-9) reduces cardiovascular and renal inflammation in experimental renin-independent hypertension

Roles of angiotensin peptides and recombinant human ACE2 in heart failure

Angiotensin-(1-9) regulates cardiac hypertrophy in vivo and in vitro

Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog

AT2 receptor agonist compound 21: a silver lining for diabetic nephropathy

Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist

Combination therapy in hypertension: an update

American Society of Hypertension Writing Group. Combination therapy in hypertension

Adverse effects and non-adherence to antihypertensive medications in University of Gondar Comprehensive Specialized Hospital

Adherence to prescribed antihypertensive drug treatments: longitudinal study of electronically compiled dosing histories

Adherence in hypertension

Prevention of angiotensin II-induced cardiac remodeling by angiotensin-(1-7)

Therapeutic uses for angiotensin

Comparison of angiotensin-(1-7), losartan and their combination on atherosclerotic plaque formation in apolipoprotein E knockout mice

Isolation and characterization of a new cellular oncogene encoding a protein with multiple potential transmembrane domains

Hydrolysis of angiotensin I by peptidases in homogenates of rat lung and aorta

Converting enzyme activity and angiotensin metabolism in the dog brainstem

Cardiovascular effects of angiotensin-(1-7) injected into the dorsal medulla of rats

A human homolog of angiotensinconverting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase

A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9

Angiotensin-(1-7): cardioprotective effect in myocardial ischemia/reperfusion

Alamandine attenuates hypertension and cardiac hypertrophy in hypertensive rats

Alamandine protects the heart against reperfusion injury via the MrgD receptor

Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis

An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal-directed antisera: measurement of eight angiotensin peptides in human plasma

Angiotensinogen and angiotensinconverting enzyme gene copy number and angiotensin and bradykinin peptide levels in mice

Enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat

Pharmacodynamic effects of C-domainspecific ACE inhibitors on the renin-angiotensin system in myocardial infarcted rats

Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects

Biochemical evaluation of the reninangiotensin system: the good, bad, and absolute?

Detecting low-abundance vasoactive peptides in plasma: progress toward absolute quantitation using nano liquid chromatography-mass spectrometry

Role of angiotensin II receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat

Angiotensin II-induced vasopressin release is mediated through alpha-1 adrenoceptors and angiotensin II AT1 receptors in the supraoptic nucleus

Mineralocorticoid and AT1 receptors in the paraventricular nucleus contribute to sympathetic hyperactivity and cardiac dysfunction in rats post myocardial infarct

Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense

Comparative vasoconstrictor effects of angiotensin II, III, and IV in human isolated saphenous vein

Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype

Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat

Angiotensin II activates nuclear transcription factor-kappaB through AT1 and AT2 receptors

Balloon angioplasty enhances the expression of angiotensin II AT1 receptors in neointima of rat aorta

Tonin overexpression in mice diminishes sympathetic autonomic modulation and alters angiotensin type 1 receptor response

Angiotensin-II-derived reactive oxygen species on baroreflex sensitivity during hypertension: new perspectives

Angiotensin II inhibits interleukin-1 beta-induced nitric oxide production in cultured rat mesangial cells

Altered pressure natriuresis in chronic angiotensin II hypertension in rats

Angiotensin IV protects cardiac reperfusion injury by inhibiting apoptosis and inflammation via AT4R in rats

Angiotensin IV AT4-receptor system in the rat kidney

Role of nitric oxide in angiotensin IVinduced increases in cerebral blood flow

Autoradiographic identification of kidney angiotensin IV binding sites and angiotensin IV-induced renal cortical blood flow changes in rats

Effect of berberine on PPARalpha-NO signalling pathway in vascular smooth muscle cell proliferation induced by angiotensin IV

Conversion of renal angiotensin II to angiotensin III is critical for AT2 receptor-mediated natriuresis in rats

Evidence that angiotensin-(1-7) plays a role in the central control of blood pressure at the ventro-lateral medulla acting through specific receptors

Angiotensin-converting enzyme 2: central regulator for cardiovascular function

Angiotensin-(1-7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide

Natriuretic action of angiotensin(1-7)

-7) lowers blood pressure and improves baroreflex function in transgenic (mRen2)27 rats

Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen

Hypotensive effect induced by microinjection of alamandine, a derivative of angiotensin-(1-7), into caudal ventrolateral medulla of 2K1C hypertensive rats

The angiotensin II AT2 receptor is an AT1 receptor antagonist

Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase

Molecular and cellular mechanism of angiotensin II-mediated apoptosis

A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy

Angiotensin AT2 receptor agonist stimulates high stretch induced-ANP secretion via PI3K/NO/sGC/PKG/pathway

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor

Angiotensin II type 2 receptor-bradykinin B2 receptor functional heterodimerization

Angiotensin-(1-7) attenuates angiotensin II-induced signalling associated with activation of a tyrosine phosphatase in Sprague-Dawley rats cardiac fibroblasts

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Angiotensin-(1-7) stimulates high atrial pacing-induced ANP secretion via Mas/PI3-kinase/Akt axis and Na + /H + exchanger

Protective role of ACE2-Ang-(1-7)-Mas in myocardial fibrosis by downregulating KCa3.1 channel via ERK1/2 pathway

Angiotensin-(1-7) prevents angiotensin II-induced fibrosis in cremaster microvessels

Angiotensin-(1-7) prevents cardiomyocyte pathological remodeling through a nitric oxide/guanosine 3′,5′-cyclic monophosphatedependent pathway

and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The authors received funding from Comisión Nacional de Ciencia y Tecnología ( 

This study showed that the use of gene therapy to overexpress angiotensin 1-9 prevents cardiac dysfunction and improves survival after myocardial infarction in mice. 80 Inflammatory processes in cardiovascular disease:

All authors researched data for the article, contributed to discussion of the content and wrote, reviewed and edited the manuscript before submission.

M.P.O., M.C., J.E.J. and S.L. have patents related to the pharmacological effects of angiotensin 1-9. R.A.S.S. has patents related to the pharmacological effects of angiotensin 1-7 and alamandine. J.A.R. and L.G. declare no competing interests.

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