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Current Biology, Vol. 12, R745–R752, October 29, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)01255-1

Just The Beginning: Novel Functions Review for Angiotensin-Converting Enzymes

1 1 Urs Eriksson , Ursula Danilczyk and Josef M. results in the generation of the main effector molecule 2 Penninger angiotensin II, which acts both as a systemic and a locally generated paracrine or autocrine effector peptide [1–3] (Figure 1). The protease renin, which is Cardiovascular disease is predicted to be the secreted by juxtaglomerular cells at the renal afferent commonest cause of death worldwide by the year arterioles, cleaves the liver-derived precursor peptide 2020. Diabetes, smoking and hypertension are the angiotensinogen into the decameric peptide angio- main risk factors. The renin-angiotensin system plays tensin I. Angiotensin I is further hydrolyzed into the a key role in regulating blood pressure and fluid and vasoconstrictor octapeptide angiotensin II by the pro- electrolyte homeostasis in mammals. The discovery teolytic dipeptidase ACE, located on the luminal side of specific drugs that block either the key enzyme of of the vascular endothelium, and other non-ACE the renin-angiotensin system, angiotensin-convert- systems, such as tissue chymase at the tissue level [8]. ing enzyme (ACE), or the receptor for its main effec- ACE also inactivates the vasodilator peptides tor angiotensin II, was a major step forward in the bradykinin and kallidin. Thus, activation of the renin- treatment of hypertension and heart failure. In recent angiotensin system mainly results in a systemic vaso- years, however, the renin-angiotensin system has pressor response by blocking the hypotensive been shown to be a far more complex system than kinin-bradykinin pathway and by generation of the initially thought. It has become clear that additional vasoconstrictor angiotensin II [4]. peptide mediators are involved. Furthermore, a new Angiotensin II is not just a potent vasopressor which ACE, angiotensin-converting enzyme 2 (ACE2), has raises systemic blood pressure; it also mediates a been discovered which appears to negatively regu- broad array of physiological and pathophysiological late the renin-angiotensin system. In the heart, ACE2 effects by binding to specific cell membrane receptors deficiency results in severe impairment of cardiac [9,10]. So far, two distinct types of G-protein-coupled contractility and upregulation of hypoxia-induced receptors for angiotensin II have been cloned and char- genes. We shall discuss the interplay of the various acterised in humans and rodents. The angiotensin II effector peptides generated by angiotensin-convert- type 1 (AT1) and type 2 (AT2) receptors are heteroge- ing enzymes ACE and ACE2, highlighting the role of neously distributed in peripheral tissues and the brain ACE2 as a negative regulator of the renin-angiotensin [9,10]. The AT1 receptor is predominantly expressed in system. the kidneys, adrenal glands, vascular smooth muscle cells and the heart, and it is to this receptor that the regulatory actions of angiotensin II on blood pressure and salt/water balance have been attributed [9,11,12]. Introduction The AT2 receptor is present at high density during fetal The renin-angiotensin system is a complex regulatory development; in the adult, significant AT2 receptor system that plays a key role in maintaining blood expression occurs only in the adrenal medulla, uterus, pressure homeostasis, as well as fluid and salt ovary, vascular endothelium, adrenal glands and certain balance in mammals [1–4]. The angiotensin convert- areas in the brain [4,9]. The AT2 receptor is thought to ing-enzyme (ACE) is an important regulator of the counterbalance effects mediated by the AT1 receptor: it renin-angiotensin system. Recently, a homologue of appears to induce vasodilation and may be involved in human ACE, angiotensin-converting enzyme 2 (ACE2), the control of cell proliferation, differentiation and has been discovered [5,6]. Characterisation of its angiogenesis [4,10,11]. function and substrate specificity [5,6], together with data from ace2 mutant mice [7], suggests that ACE2 A Broad Array of Biologically Active Peptides negatively regulates the activated renin-angiotensin In vivo, angiotensin II is degraded into several meta- system. In this review we shall discuss the functions bolites by different enzymes (Figure 2). So far, it has of ACE and ACE2 and their substrates and products in been shown that at least five of these products are the regulation of the renin-angiotensin system. biologically active [4]. Interestingly, these metabolites have different biological effects. Angiotensin III, which Classical Model of the Renin-Angiotensin System is cleaved from angiotensin II by an aminopeptidase, In the classical pathway of the renin-angiotensin binds to both AT1 and AT2 receptors and acts similar system, activation of a cascade of enzymatic reactions to angiotensin II [13]. In contrast, angiotensin(1–7), a product of angiotensin II as well as of the angiotensin 1 IMBA, Institute for Molecular Biotechnology of the Austrian I metabolite angiotensin(1–9), appears to have vasodi- Academy of Sciences, C/o Dr. Bohr Gasse 7, A-1030 Vienna, latatory [14–16] and antiproliferative effects mediated 2 Austria. University Health Network, Departments of Medical by binding a novel, not yet defined, angiotensin(1–7) Biophysics and Immunology, University of Toronto, Toronto, receptor [17]. Ontario, Canada. 620 University Avenue, Toronto, Ontario, Angiotensin IV is generated from angiotensin III by an Canada M5G 2C1. E-mail: [email protected] as yet unknown pathway. In the kidneys, angiotensin IV Review R746

Figure 1. The renin-angiotensin system: Kininogen Angiotensinogen the classical picture.

Renin

Vasodilation B1 Kinin Angiotensin I

ACE Non-ACE enzymes

Inactive product Angiotensin II

AT 1 receptor AT 2 recpetor

Vasoconstriction Vasodilation Proliferation Apoptosis Hypertrophy Growth arrest

Current Biology enhances natriuresis and increases renal blood flow. of ACE remains unclear, but the fact that fertility is There is evidence that, in the central nervous system, impaired in ace-deficient mice [22,23], but not in AT1A angiotensin IV facilitates memory retention and retrieval receptor knock-out mice [24], suggests that testicular [4,12]. The effects of angiotensin IV involve a novel AT4 ACE does not mediate its effects via angiotensin receptor [18,19]. The AT4 receptor has also been iden- II–AT1A interactions. Both ACE isoforms are mem- tified as an insulin-regulated aminopeptidase [19], sug- brane-bound protein and, at the cell surface, they gesting an indirect mechanism whereby angiotensin IV function as ectoenzymes which hydrolyze circulating might extend the half-life of biologically active neu- peptides. ACE may be cleaved from the cell surface ropeptides by competitive inhibition of AT4’s enzyme and act as a soluble enzyme, but the biological signif- activity [4,19]. icance of soluble ACE remains unclear. The diversity of the renin-angiotensin system is not ACE is currently thought to have two major activities: only demonstrated by the different effects of the it acts as a peptidyl dipeptidase which removes the various degradation products of angiotensin I and carboxy-terminal dipeptide from its substrate, and as angiotensin II. Characterisation of receptor specificities endopeptidase on substrates that are amidated at the and enzymes involved in the degradation of angiotensin carboxyl terminus (Figure 3). The emerging picture of metabolites has revealed far more complex mediator ACE function is that it is more than just a key enzyme systems than previously recognised. It appears that the that catalyses cleavage of angiotensin I to the potent complexity of the renin-angiotensin system guarantees vasoconstrictor peptide angiotensin II [3,4,20]. ACE also well-tuned adaptation of the blood supply of the body hydrolyzes the inactive angiotensin(1-9) peptide into the compartments and organs to all possible physiologic vasodilator metabolite angiotensin(1–7) [25], and it is conditions. In this complex system, a key role has been additionally thought to inactivate the vasodilator pep- attributed to the rate-limiting enzymes, ACE and ACE2, tides bradykinin and kallidin [25,26]. Furthermore, it acts via their action in generating or degrading the various as endopeptidase on the multi-functional neuropep- active mediators. tides substance P and cholecystokinin, and may degrade the luteinizing hormone releasing hormone Angiotensin Converting-Enzyme (ACE) [4,27]. In this context, however, its biological signifi- ACE was termed a ‘hypertensin-converting enzyme’ cance is not known, nor has the possibility of functional when it was initially isolated in 1956 [20]. The human crosstalk between the systems been excluded. ace gene, located on chromosome 17, encodes a The finding that ACE catalyzes cleavage of angio- 180 kDa protein with two homologous domains. Each tensin I into angiotensin II, together with the eluci- domain has an active zinc-binding motif, His-Glu-X-X- dation of ACE’s three-dimensional structure, led to the His, which is found in many peptidases [3,21] (Figure design of specific ACE inhibitors. Inhibition of ACE 3). ACE is anchored to the plasma membrane through results in lowering of blood pressure by impaired for- a single carboxy-terminal transmembrane domain. In mation of angiotensin II and reduced degradation of humans, two distinct ACE isoenzymes have been the hypotensive peptides bradykinin and kallidin. described, an abundant somatic form found on the Numerous potent ACE inhibitors have been synthe- endothelial surface of the lungs and on brush-border sized subsequently and evaluated in clinical trials [28]. membranes of kidneys, intestine, placenta and Aside from lowering blood pressure, ACE inhibitors choroid plexus, and the germinal form of ACE found have been found to improve the prognosis of patients only in the testis [4]. The function of the germinal form with congestive heart failure and after myocardial Current Biology R747

infarction [29]. These clinical findings widened our understanding of the renin-angiotensin system and Amino acid sequences and ACE/ACE2 cleavage sites of RAS mediator peptides suggested that it must have additional functions. Pharmacological inhibition of ACE by ACE inhibitors Angiotensin I Asp Arg Val Tyr Ile His Pro Phe His Leu has become a mainstay in treatment of hypertension, cardiac failure and diabetic nephropathy. Angiotensin II Asp Arg Val Tyr Ile His Pro Phe

Angiotensin Converting-Enzyme 2 Angiotensin III Arg Val Tyr Ile His Pro Phe ACE2 has been recently identified as a homolog of ACE in human, mouse and rat [5–7]; the human ace2 Angiotensin IV Val Tyr Ile His Pro Phe gene has been cloned and mapped to the X chromo- some [7]. ACE2 is predicted to be a 805 amino acid, Angiotensin(1Ð7) Asp Arg Val Tyr Ile His Pro type I membrane-bound glycoprotein with a molecu- lar weight of about 120 kDa. Like ACE, ACE2 has two Angiotensin(1-Ð9) Asp Arg Val Tyr Ile His Pro Phe His domains: the amino-terminal catalytic domain and the carboxy-terminal domain (Figure 3). The catalytic Apelin-13 Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe domain has one active site — the zinc metallo-pepti- dase domain — and shows 41.8% sequence identity Bradykinin Arg Pro Pro Gly Phe Ser Pro Phe Arg with the amino domain of ACE [4–6]. ACE2’s carboxy- 9 terminal domain shows 48% sequence identity with [Des-Arg] Bradykinin Arg Pro Pro Gly Phe Ser Pro Phe collectrin [30] (Figure 3), a novel renal-specific type 1 transmembrane glycoprotein that lacks the metallo- Dynorphin A Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu Lys protease catalytic domain of ACE and ACE2. In con- β trast to ACE, ACE2 expression appears to be limited -Casomorphin Tyr Pro Phe Val Glu Ile Current Biology to heart, kidney and testis [6,7]. At the cellular level, ACE2 is mainly confined to the endothelium and renal Figure 2. Amino-acid sequences and ACE (red) and ACE2 tubular epithelium; occasionally it is identified on vas- (blue) cleavage sites of renin-angiotensin mediator peptides. cular smooth muscle cells [6,7]. As an enzyme, ACE2 functions as a carboxy- synthesized as 77 amino-acid preprohormone, which monopeptidase with a preference for carboxy-terminal is processed into the 36 amino acid peptide apelin-36; hydrophobic or basic residues [31]. So whereas ACE further proteolytic cleavage generates apelin-13 cleaves two amino acids, ACE2 cleaves only the [37,38]. The physiological role of the apelins is not yet carboxy-terminal amino acid (Figure 3). Although ACE fully understood [39–41], though apelin-13 has been and ACE2 share many biochemical properties, ACE2 shown to promote vasoconstriction in endothelium- activity is unaffected by classical ACE inhibitors, such as denuded coronary arteries [41] and systemic adminis- captopril or enalaprilat, and its specificity is distinct from tration of apelin-13 prompts hypotension in rats [42]. ACE [5,25]. ACE2 hydrolyses angiotensin I, the inactive Two opioid peptides, dynorphin A (1–13) and β-caso- precursor of angiotensin II, to generate angiotensin(1–9) morphin, are also substrates of ACE2 [31]. These κ δ peptides [25,31]. Angiotensin(1-9), in turn, can be con- peptides activate G-protein-coupled and opioid verted to the vasodilator peptide angiotensin(1–7) by receptors, which regulate pain perception and may ACE. ACE2 also directly converts angiotensin II to have negative affects on cardiomyocyte contractility angiotensin(1–7). Angiotensin(1–7) has been proposed to [43–45]. Other substrates for ACE2 include neurotensin be an important regulator of cardiovascular function, and the related peptide kinetensin, but several other promoting vasodilatation, apoptosis and growth arrest regulatory peptides, including the enkephalins, are not [4,32,33], but the significance of this metabolite in ACE2 substrates [31]. Together, the recent findings humans is controversial. Nevertheless, it will be inter- suggest a multi-functional role for ACE2, not only in the esting to test whether ACE2 modulates angiotensin II- renin-angiotensin system, but also in neurotransmis- mediated effects in the heart and kidneys in vivo by sion including pain perception. degradation of angiotensin II and/or the generation of additional vasodilator peptides. Blood Pressure Regulation The specificity of ACE2, like that of ACE, is not Hypertension is recognised to be a major risk factor limited to angiotensin I and II. ACE2 also acts on for arteriosclerosis [46]. The renin-angiotensin system several other classes of peptides [31] (Figure 2). The has been recognised as the main regulator linking the kinin metabolites, des-Arg10-kallidin (des-Arg10-Lys1- extracellular fluid volume to blood pressure homeo- bradykinin) and des-Arg9-bradykinin, but not brady- stasis. Reduction of the perfusion pressure in the kinin, itself are both hydrolyzed by ACE2 [30]. Both of afferent renal arterioles, as a result of a decrease in these metabolites activate the G-protein-coupled B1 either systemic blood pressure or extracellular receptor [34,35], which is upregulated in response volume, or deterioration of renal arteries, results in to tissue injury and may be important in mediating reduced glomerular filtration and a decreased amount inflammatory responses [32], including the local of sodium entering the proximal tubuli. In turn, this release of nitric oxide [36]. stimulates renin secretion in the kidneys. This mecha- ACE2 also acts on the peptides apelin-13 and nism, termed tubuloglomerular feedback (reviewed in apelin-36 with high catalytic efficiency [31]. Apelin is [47]), ultimately results in increased angiotensin II Review R748

Figure 3. Domain structures of ACE, 1 1306 ACE2 and collectrin. Somatic ACE N HEMGH HEMGH C Each protein is a type I integral protein with a signal peptide, depicted in green, and transmembrane domain in black. The zinc- 1 732 binding motif (HEMGH) repeats two times ACE2 N HEMGH C in ACE and once in ACE2, and is located within the homologous region denoted by the yellow box. Regions of homology 1 222 between ACE2 and collectrin are denoted Collectrin N C by dark orange. The numbers refer to the Current Biology amino acids in each protein. production. Angiotensin II raises systemic blood Mice deficient for the ACE2 gene have recently pressure by peripheral vasoconstriction and promotes been described [7]. These mice develop normally, and sodium reabsorption in the kidney, both indirectly by despite the genetic association of ACE2 with stimulating aldosterone secretion from the adrenal hypertension in rat strains they have normal blood cortex and directly by acting on the AT1 receptor [48]. pressure and renal histology [7]. But levels of the In over 90% of the cases hypertension is considered vasopressor angiotensin II are moderately increased to be ‘essential’, in the clinical sense of not having one in the ACE2-deficient mice: so is ACE2 a negative reg- distinct cause. Although in a high proportion of these ulator of blood pressure control in vivo? As ACE2 has patients screening of serum aldosterone and plasma a broad substrate specificity, it cannot be excluded renin activity reveals primary hyperaldosteronism [49], that in ACE2-deficient mice the hypertensive effects of the etiology of essential hypertension is not yet clear elevated angiotensin II levels are blunted by compen- [46]. Rather, it appears that essential hypertension is satory changes in other regulatory peptides, such as multi-factorial, depending on diet (for example a high angiotensin(1-7) [32,54], the kinin metabolites [35] or salt diet), environment and genetic factors. the newly discovered apelin system [42]. Given that Several quantitative trait loci (QTL) that affect the susceptibility to essential hypertension is multi- susceptibility to hypertension have been identified factorial, it appears that a lack of ACE2 activity pre- [50,51]. The possibility that ACE2 plays a role in disposes to, rather than causes, hypertension [50,55]. essential hypertension was suggested when ace2 was mapped to the QTL on the X chromosome in hyper- ACE2 is a Negative Regulator of ACE in the Heart tensive rats. This chromosomal region overlaps with As mentioned, ACE2-deficient mice show increased the SS-X QTL in salt-sensitive Sabra rats, the BP3 QTL tissue angiotensin II levels; they also show upregula- in stroke-prone spontaneously hypertensive rats and tion of hypoxia genes in the heart and severe cardiac the BB.Xs QTL in spontaneous hypertensive rats [7]. dysfunction [7]. Indeed, the most striking phenotype of In all these rat strains, ACE2 mRNA and protein levels the ACE2 mutant mice is of a profound decrease in were found to be greatly reduced and inversely corre- cardiac contractility [7]. Despite the elevated plasma lated with increased blood pressure [7]. and heart angiotensin II levels, there is no evidence in These data provide evidence that ACE2 has a role in these mutant mice for structural alterations such as the pathogenesis of hypertension. The fact that ACE2 myocardial fibrosis, hypertrophy or dilation. Rather is produced primarily in the heart and the kidneys intriguingly, ACE2 mutant hearts resemble certain reflects the critical role of these organs in controlling kinds of defective heart found in human cases of coro- blood pressure homeostasis. But the mechanisms by nary artery disease and after bypass surgery — in par- which ACE2 regulates blood pressure remain unclear. ticular, those cases referred to as ‘cardiac stunning’ Furthermore, collectrin, the ACE2 homolog made [56] and ‘hibernation’ [57]. In these human diseases exclusively in kidneys, may play an important role in and related animal models, chronic hypoxic conditions controlling ACE /ACE2 functions. Homology searches lead to compensatory changes in myocyte metabolism of the human genome indicate that the collectrin gene [56,58], upregulation of hypoxia-induced genes [59] is located on chromosome Xp22, about 26 kilobases and reduced heart function [56,57,60]. from the ace2 gene. As collectrin lacks the car- Importantly, the cardiac dysfunction observed in boxypeptidase domain it may regulate blood pressure ACE2 mutant mice is completely reversed by a second through a process not dependent on angiotensin II. mutation that causes ACE deficiency [7]. Normal In vivo models using mice deficient in the key cardiac function in ACE, ACE2 double mutant mice enzymes renin and ACE, angiotensinogen or the AT1a suggests that a catalytic product of ACE triggers con- receptor have greatly advanced our knowledge of the tractile impairment in the absence of ACE2, supporting renin-angiotensin system and confirmed its role in the hypothesis that ACE2 is indeed a critical negative blood pressure regulation. Indeed, under physiologi- regulator of the cardiac effects of the renin-angiotensin cal conditions these mutant mice all show reduced system. So far, the mechanism by which the absence blood pressure relative to wild-type. Mice deficient in of ACE2 induces impairment of cardiac contractility renin, angiotensinogen or ACE all show altered kidney has not been elucidated. morphology and function [22,52], and those deficient Given the moderately enhanced angiotensin II levels in ACE, angiotensinogen or AT1/AT2 receptors all in ACE2 mutant mice and the upregulation of hypoxia- show impaired somatic growth [22,52,53]. induced genes [7], it is tempting to speculate whether Current Biology R749

ACE2

Inactive product Angiotensin I Angiotensin(1-9) Inactive product

ACE2 ACE ACE ACE

ACE2 Apelin-13 Angiotensin II Angiotensin(1-7) Bradykinin

Vasoconstriction Vasodilation

ACE2 Des-arg9kinin Inactive product

Ð +

Ð Coronary blood flow Redistribution of blood flow towards inflamed areas ?

Cardiac function Current Biology

Figure 4. Simplified schematic representation of how ACE and ACE2 may regulate blood supply to coronary arteries. Dotted arrows represent pathways, which may be important under pathophysiological conditions only. the observed heart phenotype of ACE2 mutants is the depends on the maintenance of a constant blood result of angiotensin II-mediated hypoperfusion, which supply, even in the presence of a systemic vasopres- in turn is caused by vasoconstriction and endothelial sor response. dysfunction [61,62]. Indeed, in vitro studies have shown that angiotensin II directly induces hypoxia- Perspectives inducible factor-1α and VEGF in smooth muscle cells Studies of ACE2 single mutant and ACE, ACE2 double and cardiomyocytes [63]. Furthermore, it has been mutant mice indicate that ACE2 is a negative regula- shown that angiotensin II causes endothelial dysfunc- tor of the renin-angiotensin system. It is not clear, tion via the AT1A receptor and induction of oxidative however, whether this function is mediated by ACE2’s stress [61,64]. This view is supported by the fact that metallopeptidase or collectrin-like domain activities. ACE2 is expressed only in the vascular endothelium, ACE2’s carboxypeptidase activity may limit the avail- and not on cardiac myocytes [7]. Given the broad ability of angiotensin II, or may generate counterregu- specificity of ACE2 for various peptide systems, it is latory vasodilator peptide products. If the ACE2 not clear whether the elevated vascular tone of ACE2 mutant phenotype of decreased cardiac contractility mutant hearts is due simply to an unopposed effect of is indeed due to excess angiotensin II at the endothe- angiotensin II, or whether alterations in the local lium–smooth muscle interface, one would expect that availability of other mediators also play a part (Figure ACE2, angiotensin II receptor double mutant mice will 4). For example, angiotensin(1-7), which is cleaved from turn out to have normal cardiac function. Given the angiotensin I by ACE2, acts as vasodilatatory peptide complexity of the involved systems, in vivo studies on coronary arteries and preserves heart function and with mutant mice or specific blocking agents appear coronary artery perfusion in rats after myocardial to be the most straightforward approach to further infarction [65]. elucidating the roles of ACE and ACE2 in regulating It thus appears that ACE2 does negatively regulate the renin-angiotensin system. the activated renin-angiotensin system, probably by If the physiological role of ACE does not involve its diverting the generation of the vasoactive peptide metallopeptidase activity, it is conceivable that the angiotensin II towards inactive and vasodilatatory collectrin like-domain is involved. ACE2 and collectrin peptides. Given that ACE2 is primarily expressed in are both upregulated upon tissue injury: ACE2 in heart the kidneys and the heart only [6,7], it is intriguing to failure and collectrin in renal injury [60]. The speculate that ACE2 has been conserved during evo- development of novel inhibitors specific to the collec- lution as a negative regulator of the renin-angiotensin trin-domain of ACE2, and the generation of collectrin- system in those organs whose vital function critically deficient mice, should provide important information Review R750

on the physiological roles of this protein domain and M. Crackower, R. Sarao, G. Oudit, C. Yagil, Y. Yagil, M. address the question whether it is involved in the Chappel, P. Backx and many others for their excellent renin-angiotensin system. contributions and discussion. So far the ACE2 mutant mice have not added much References to our understanding of ACE2’s role in blood pressure 1. Inagami, T. (1994). The renin-angiotensin system. Essays Biochem. regulation. Key experiments remain to be done, such 28, 147–164. as testing whether increasing ACE2 expression in 2. Weinberg, M.S., Weinberg, A.J. and Zappe, D.H. (2000). Effectively targeting the renin-angiotensin-aldosterone system in cardiovascu- defined hypertensive rat strains ameliorates the lar and renal disease: rationale for using angiotensin II receptor increased blood pressure. Polymorphisms of ACE or blockers in combination with angiotensin-converting enzyme angiotensin have not yet been clearly shown yet to be inhibitors. 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