ACE 2: More of Ang 1-7 or less Ang II?

Abstract

Purpose of review

Previous concepts regarding the pathways involved in the generation of angiotensin II (Ang II) are been challenged by studies showing the existence of a peptide acting as an endogenous antagonist of Ang II. The discovery that angiotensin-(1-7) [Ang-(1-7)] opposes the pressor, proliferative, profibrotic, and prothrombotic actions mediated by Ang II has contributed to the realization that the renin angiotensin system is comprised by two opposing arms: the pressor arm constituted by the enzyme angiotensin converting enzyme (ACE), Ang II as the product, and the Ang II type 1 receptor (AT₁) as the main protein mediating the biological actions of Ang II; the second arm is composed by the mono carboxypeptidase –angiotensin converting enzyme 2 (ACE2)–, Ang-(1-7) produced through hydrolysis of Ang II, and the Mas receptor as the protein conveying the vasodilator, antiproliferative, anti-fibrotic, and anti-thrombotic effects of Ang-(1-7).

Recent findings

Experimental and clinical studies demonstrate a role for the Ang-(1-7)/ACE2/Mas-axis in the evolution of hypertension, the regulation of renal function, and the progression of renal disease including diabetic nephropathy. Additional evidence suggests that reduction in the expression and activity of this vasodepressor component may be a critical factor in mediating the progression of cardiovascular disease.

Summary

Further research on the contribution of the Ang-(1-7)/ACE2/Mas-axis to cardiovascular pathology will lead to the development of new pharmacological approaches resulting in the design of molecular or genetic means to increase the expression of ACE2, allow for increased tissue levels of Ang-(1-7), or both.

Introduction

Remarkable progress continues to be made in unraveling the contribution of the renin angiotensin system to cardiovascular pathology and, specifically the role of the vasodepressor pathway composed by the triad of the enzyme angiotensin converting enzyme 2 (ACE2), the heptapeptide product angiotensin-(1-7) [Ang-(1-7)], and the mas receptor. Work originating from our research program established the basis and initial mechanisms for the inclusion of the Ang-(1-7)/ACE2/Mas-axis as a critical counter balancing component of the pressor pathway composed by the angiotensin converting enzyme (ACE), the octapeptide angiotensin II (Ang II) and its signaling action following binding to the subtype I Ang II receptor. Over the last decade data continues to support the hypothesis that the pathological actions of Ang II on cardiovascular regulatory activity may in part result from a diminished expression or activity of the components of the Ang-(1-7)/ACE2/mas-R axis. This review provides a birds-eye-view on the progress made over the last two years. The fact that 2010 publications to date tally 48 in PubMed for the term Ang-(1-7) documents how significant progress is been made on this topic.

Angiotensin-(1-7) Formation and Signaling Mechanisms

There are two biochemical pathways that account for the formation of Ang-(1-7) (Figure 1). The first entails the hydrolysis of angiotensin I (Ang I) by the tissue endopeptidases: prolyl-endopeptidase (EC 3.4.21.26), neutral endopeptidase 24.11 (neprilysin; EC 3.4.24.11), and oligopeptidase 24.15 (thimet oligopeptidase EC 3.4.24.15) [1;2]. The second pathway entails the cleavage of the Pro⁷-Phe⁸ bond of Ang II by angiotensin converting enzyme 2 (E.C. 3.4.15.1; ACE2) [3;4], an exopeptidase which has also been shown to cleave Ang I into angiotensin-(1-9) [Ang-(1-9)] [3]. Among the Ang-(1-7) forming enzymes, the kcat/km of ACE2 is 500-fold higher for Ang II compared to the kcat/km for Ang I. A third pathway leading to the ultimate generation of Ang-(1-7) may result from the discovery of an extended form of Ang I, the dodecapeptide angiotensin-(1-12) [Ang-(1-12)] [5]. The studies reported by Nagata et al. [5] showed that this novel angiotensinogen derived peptide was capable of generating Ang II and possessed vasoconstrictor activity that were blocked by prior administration of the ACE inhibitor, captopril or the type 1 Ang II receptor (AT₁) candesartan. In confirming their findings, we showed in the isolated heart preparation, a late formation of Ang-(1-7) after administration of Ang-(1-12) [**6]. More detailed information regarding the biochemical pathways leading to Ang-(1-7) formation and metabolism are published [7–9].

Figure 1.

Schematic representation of the components of the renin angiotensin system.

The Mas receptor has been identified as the protein transducing the vasodilator and antiproliferative actions of Ang-(1-7) [10–12]. The Mas protein is a G-protein couple receptor originally linked to modulation of growth regulating pathways involved in oncogenic effects and high expression in testis, hypothalamus and amygdala [13;14]. In a detailed study of the function of the Mas receptor Kostenis et al. [15] showed that in transfected Mas cells this G-protein coupled receptor antagonized Ang II actions at the Ang II type 1 receptor (AT₁) owing to formation of a hetero-oligomeric complex. This important study explains previous findings of a weak direct vasodilator effect of Ang-(1-7) [16] that contrasts with effects of the heptapeptide in reducing pressor actions of Ang II both in vitro and in vivo [*17–29]. See also annotated references 20, 24, 26, 27 and 28. Although a search of the current literature did not reveal the existence of a detailed analysis of renal mas distribution, a picture of the mas receptor localization in rat kidney suggest that it may reside in proximal and distal tubules (Alomone Labs; http://www.alomone.com/p_postcards/database/510.htm). On the other hand, Metzger et al. [14] reported the presence of mas mRNA in renal tissues from mice and rats.

The signaling mechanisms by which Ang-(1-7) antagonizes the pleiotropic actions of Ang II remain under investigation. Earlier studies implicated a stimulatory action of the heptapeptide on release of endothelial derived nitric oxide (NO) and vasodilator prostaglandins [30–40] as well as potentiation of the vasodilator and metabolic actions of bradykinin [41–46]. Intracellular mediators accounting for the antagonistic actions of Ang-(1-7) on Ang II responses may include phosphorylation of both Akt (adipose tissue, skeletal muscle, and liver) and GSK-3β in liver and skeletal muscle of the rat [*47]. In keeping with this finding, Sampaio et al. [38] reported that Ang-(1-7) stimulates AKt kinase from human endothelial cells. In vascular smooth muscle cells Ang-(1-7) inhibited Ang II stimulation of mitogen-activated protein kinase activities (ERK1/2) [48]. A preliminary observation by Tallant and Gallagher, reported as abstracts [49;50] only, suggests that Ang-(1-7) may upregulate the mitogen-activated DUSP-1 in vascular smooth muscle cells.

As progress continues to be made in the understanding of the molecular mechanisms of Ang-(1-7) signaling, other new information is accumulating as to the actions of Ang II and Ang-(1-7) in the regulation of ACE2 gene and activity. Ishiyama et al. [51] first reported that blockade of AT₁ receptors was accompanied by increase cardiac ACE2 transcripts, a finding that was interpreted to indicate that Ang II may exert a negative influence in the regulation of ACE2. These findings were corroborated in further studies in astrocytes in culture [52], and later in neonatal cardiac myocytes and fibroblasts [53]. In these experiments, the observation that Ang-(1-7) by itself had no effect on ACE2 mRNA while it blocked the reduction in ACE2 transcripts produced by Ang II or endothelin-1 through activation of the extracellular signal-regulated kinase ERK1/ERK2 is reminiscent of the conclusions made by Kostenis et al [15] showing that Ang-(1-7) antagonizes AT₁ receptors.

A conflicting report in primary human cardiac fibroblast suggests that Ang II stimulates rather than suppress ACE2 gene expression [*54]. As recognized by the authors [54], their findings may be restricted to the type of cell employed in their experiments and the state of cell differentiation, as the work showing increased cardiac ACE2 mRNA following chronic blockade of Ang II production or activity by lisinopril or losartan [55] is in support of the view of a negative Ang II effect on ACE2 transcripts. Our studies [56;57] showing upregulation of ACE2 mRNA in the aorta of SHR given the AT₁ receptor blocker olmesartan or in the neointima of olmesartan-treated SHR are also at variance with the idea that Ang II exerts a positive regulatory action on ACE2.

The Ang-(1-7)/ACE2/Mas-axis in Hypertension. Focus on the Kidney

Since we first proposed that a deficit in the counter regulatory effects of Ang-(1-7) may directly contribute to the pathogenesis of human essential hypertension [58] evidence for this possibility continues to gain support mostly from data obtained in experimental models of the disease. As commented by us elsewhere [59], the paucity of clinical studies directed to address this possibility are rooted in the belief that the antihypertensive actions resulting from inhibition of Ang II synthesis or activity suffice to explain their cardio-renal protective effects. Advances in the understanding of the molecular and physiological mechanisms of ACE2 has began to reverse this tide but the task is a difficult one as it entails the design of agents that will increase the activity of ACE2, augment tissue levels of Ang-(1-7), or both in combination [60–62].

Clinical studies supporting the view of hemodynamic and end-organ effects of Ang-(1-7) are summarized elsewhere [59]. A first glimpse to the possible favorable actions of Ang-(1-7) in contributing to the antihypertensive actions of ACE inhibition was gained from the observation of an increased excretion of Ang-(1-7) in the urine of essential hypertensives whose blood pressure was controlled by a 6-month treatment with captopril [63]. Two years later, a more detailed study was performed in 31 normal healthy volunteers and 18 untreated essential hypertensive subjects [64]. This study showed that urinary excretion rates of Ang-(1-7) in the essential hypertensive subjects averaged almost one-half of those measured in the normotensive controls [64]. Additional studies showed that administration of the Ang II receptor blocker (ARB) irbesartan was associated with increases in plasma Ang-(1-7) concentrations [64;65]. Similar findings were reported in salt-sensitive hypertensive subjects medicated with omapatrilat, a potent dual inhibitor of ACE and neprilysin [66]. Changes in the sodium status may regulate the influence of Ang-(1-7) on the action of ACE inhibitors as in healthy normal volunteers, the increases in Ang-(1-7) produced by the combination of enalapril and a low salt diet were much greater than those observed in the same subjects when exposed to the low salt diet alone [67].

Along the animal and human nephron, multiple studies have documented the presence of renin angiotensin system components underscoring the concept that the kidney is a site at which an intrarenal system participates in the regulation of glomerular-tubular balance in health and disease [9;68;69]. Increase expression of cortical and medullary Ang II and cortical ACE2 activity was found in a model of hypertension with increased tissue renin [70]. And the blood pressure lowering action of administering lisinopril or losartan to normotensive rats were associated with increased urinary excretion of Ang-(1-7), augmented expression of cortical renin and angiotensinogen gene transcripts, and higher cortical ACE2 activity in renal membranes from the same treated animals [71]. In an experimental model of renal hypertension, the administration of a selective Ang-(1-7) receptor blocker or an ACE2 inhibitor was associated with worsening of hypertension and renal function [17].

The experimental evidence demonstrating a palette of actions of Ang-(1-7) and ACE2 in the regulation of nephron function [72;73] correlates with newer studies showing that altered ACE2 expression or activity contributes to the progression of renal disease and diabetic nephropathy [74]. Lely et al. [74;75] showed neo-expression of ACE2 in the glomerular and peritubular capillary endothelium of biopsied kidneys of patients with primary and secondary forms of renal disease as well as renal transplants. In patients with diabetes and overt nephropathy, an increase in the ACE/ACE2 ratio resulted from a decreased expression of tubulointerstitium and glomeruli ACE2 [76]. Similar findings were also reported in patients with diabetic nephropathy [*77]. These findings are in keeping with the observation of increased deposition of collagen I, collagen III and fibronectin in the glomeruli and increased urinary albumin excretion compared to age-matched control mice in mutant male ACE (−/−) mice [78]. Of additional interest is the recent report demonstrating the presence of serum ACE2 autoantibodies in patients with connective tissue diseases to constrictive vasculopathy, pulmonary arterial hypertension (PAH), or persistent digital ischemia [77;79]. These findings are in keeping with the observation of reduced plasma Ang I, Ang II, and Ang-(1-7) levels in patients with systemic sclerosis compare to controls [80]. Since the ratio of Ang II/Ang-(1-7) indicated a prevalence of Ang II over the countervailing Ang-(1-7), these data provide additional support for an important interaction among ACE2 and Ang-(1-7) in multiple disease states in which the actions of Ang II favor proliferative, profibrotic, and thrombotic effects.

Conclusion

The question posed on the title as to whether more of Ang-(1-7) or less of Ang II may participate in the pathogenesis of hypertensive vascular disease cannot be answered with certainty but the research done to-date continues to support the hypothesis that a decrease in the expression or activity of Ang-(1-7) renders the cardiovascular system more susceptible to the pathological actions of Ang II. In the kidney, the opposing effects of low and high doses of Ang-(1-7) on tubular sodium reabsorption needs further study, as binding of Ang-(1-7) to mas may alter expression or activity of AT₁ or AT₂ receptors [9,15]. In addition, accumulating evidence suggest that ACE2 may play a critical role in modulating the relative contributions of Ang II and Ang-(1-7) to blood pressure regulation and that changes in the relative tissue expression of ACE and ACE2 activities may be deterministic in diseases of the heart, the blood vessels, and the kidneys. While the experimental evidence is rather appealing, there is a real need to extend these findings to human’s conditions such as essential hypertension, chronic renal disease, and diabetes.