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New findings What is the central question of this study? What are the effects of the antifibrotic peptide acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on the angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas axis during the occurrence and progression of silicosis? What is the main finding and its importance? Ac-SDKP inhibited lung fibrosis in rats exposed to silica by activation of the ACE2-angiotensin-(1-7)-Mas axis. Angiotensin-(1-7) potentially promotes Ac-SDKP by increasing the level of meprin α, the major synthetase of Ac-SDKP. Thus, the interaction Ac-SDKP and angiotesin-(1-7) in silicosis could provide a new therapeutic strategy. Abstract The central role of angiotensin-converting enzyme (ACE) in the occurrence and progression of silicosis has been established. The antifibrotic peptide acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) can be degraded by ACE. The ACE2-angiotensin-(1-7)-Mas axis is protective and acts to counterbalance the detrimental effects of ACE-angiotensin II (Ang II)-Ang II type 1 receptor and exerts antifibrotic effects. Here, we demonstrate an interaction between Ac-SDKP and Ang-(1-7) in the inhibition of collagen deposition and myofibroblast differentiation in rats exposed to silica. Treatment with Ac-SDKP increased the level of ACE2-Ang-(1-7)-Mas in rats or in cultured fibroblasts and decreased the levels of collagen type I and α-smooth muscle actin. Furthermore, exogenous Ang-(1-7) had similar antifibrotic effects and increased the level of meprin α, a major Ac-SDKP synthetase, both in vivo and in vitro. Compared with non-silicotic patients exposed to silica, the level of serum ACE was increased in patients with silicosis phase III; the levels of Ang II and Ang-(1-7) were high in patients with silicosis phase II; and the level of Ac-SDKP was high in the silicosis phase III group. These data imply that Ac-SDKP and Ang-(1-7) have an interactive effect as regulatory peptides of the renin-angiotensin system and exert antifibrotic effects.

What is the central question of this study? The central goal of this study was to understand the effects of central angiotensin-(1-7) on basal and osmotically stimulated water intake in rats. What is the main finding and its importance? This study demonstrated that central administration of angiotensin-(1-7) did not induce thirst in basal conditions but increased water intake after osmotic stimulation, such as water deprivation and salt loading. These results indicate a new function for this peptide, which, in turn, allows for future research on the mechanisms through which angiotensin-(1-7) influences osmotic thirst. Angiotensin-(1-7) [Ang-(1-7)] is generated by type 2 angiotensin-converting enzyme (ACE2) and binds to the MAS receptor. Although it is well known that Ang-(1-7) functionally antagonizes the effects of the classical renin-angiotensin system in several situations, the role of Ang-(1-7) in hydromineral homeostasis is not clear. The aim of this study was to assess the role of Ang-(1-7) on neuroendocrine responses to hyperosmolality in rats. Male Wistar rats were divided into the following three groups: control; 24 h of water deprivation (WD); and 24 h of salt loading (SL; 1.8% NaCl). Intracerebroventricular (i.c.v.) injections of Ang-(1-7) or vehicle were given to assess water intake and plasma concentration of vasopressin. Additionally, the brains from control and WD groups were collected to evaluate gene expression in the subfornical organ (SFO), paraventricular nucleus (PVN) and supraoptic nucleus (SON). It was found that i.c.v. Ang-(1-7) did not change water and salt intake in control rats; however, Ang-(1-7) increased water intake after WD and SL, with no change in salt intake. Plasma vasopressin was not changed by i.c.v. Ang-(1-7) in control or WD rats. Moreover, WD increased Mas gene expression in the SON and PVN, with no changes in Ace2 mRNA levels. In conclusion, Ang-(1-7) increases thirst after osmotic stimuli, indicating that a previous sensitization to its action is necessary. This finding is consistent with the increased Mas gene expression in the PVN and SON after water deprivation.

Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin II (Ang II) have been implicated in the pathogenesis of pancreatitis. Angiotensin-converting enzyme 2 (ACE2) degrades Ang II to angiotensin-(1-7) [Ang-(1-7)] and has recently been described to have an antagonistic effect on ACE signalling. However, the specific underlying role of ACE2 in the pathogenesis of severe acute pancreatitis (SAP) is unclear. In the present study, the local imbalance of ACE and ACE2, as well as Ang II and Ang-(1-7) expression, was compared in wild-type (WT) and ACE2 knock-out (KO) or ACE2 transgenic (TG) mice subjected to cerulein-induced SAP. Serum amylase, tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-10 levels and histological morphometry were used to determine the severity of pancreatitis. In WT mice, pancreatic ACE and Ang II and serum Ang II expression increased (P < 0.05), while pancreatic ACE2 and Ang-(1-7) and serum Ang-(1-7) levels were also significantly elevated (P < 0.05) from 2 to 72 h after the onset of SAP. However, the ratio of pancreatic ACE2 to ACE expression was significantly reduced (from 1.46 ± 0.09 to 0.27 ± 0.05, P < 0.001) and paralleled the severity of pancreatitis. The Ace2 KO mice exhibited increased levels of tumour necrosis factor-α, IL-1β, IL-6, multifocal coagulative necrosis and inflammatory infiltrate, and lower levels of serum IL-10 and pancreatic Ang-(1-7) (4.70 ± 2.13 versus 10.87 ± 2.51, P < 0.001) compared with cerulein-treated WT mice at the same time point. Conversely, Ace2 TG mice with normal ACE expression were more resistant to SAP challenge as evidenced by a decreased inflammatory response, attenuated pathological changes and increased survival rates. These data suggest that the ACE2-ACE imbalance plays an important role in the pathogenesis of SAP and that pancreatic ACE2 is an important factor in determining the severity of SAP.

What is the central question of this study? What are the effects of exercise training on the hepatic renin-angiotensin system and their contribution to damage resulting from fructose overload in rats? What is the main finding and its importance? Exercise training attenuated the deleterious actions of the angiotensin-converting enzyme/angiotensin II/angiotensin II type 1 receptor axis and increased expression of the counter-regulatory (angiotensin-converting enzyme 2/angiotensin (1-7)/Mas receptor) axis in the liver. Therefore, our study provides evidence that exercise training modulates the hepatic renin-angiotensin system, which contributes to reducing the progression of metabolic dysfunction and non-alcoholic fatty liver disease in fructose-fed rats. The renin-angiotensin system (RAS) has been implicated in the development of metabolic syndrome. We investigated whether the hepatic RAS is modulated by exercise training and whether this modulation improves the deleterious effects of fructose overload in rats. Male Wistar rats were divided into (n = 8 each) control (CT), exercise control (CT-Ex), high-fructose (HFr) and exercise high-fructose (HFr-Ex) groups. Fructose-drinking rats received d-fructose (100 g l

Angiotensin-converting enzyme 2 (ACE2), a monocarboxypeptidase capable of metabolizing angiotensin II (Ang II) into angiotensin-(1-7) [Ang-(1-7)], has emerged as a potential therapeutic target. We hypothesized that ACE2 is a negative regulator of Ang II-mediated pathological effects in vivo. In Wistar-Kyoto (WKY) rats, Ang II infusion (0.1 μg min(-1) kg(-1)) induced a pressor response, activation of NADPH oxidase and generation of superoxide in the heart, kidney and blood vessels; these effects were significantly blunted by recombinant human ACE2 (rhACE2; 2 mg kg(-1)), in association with a lowering of plasma Ang II and elevation of Ang-(1-7) levels. In the spontaneously hypertensive rat (SHR) model, rhACE2 (2 mg kg(-1) day(-1)) delivered over a 14 day period partly corrected the hypertension, the NADPH oxidase activation and the increased superoxide generation in the heart, kidney and blood vessels. Treatment with rhACE2 inhibited Ang II-mediated phosphorylation of the myocardial extracellular signal-regulated kinase 1/2 pathway in WKY rats, with congruent results seen in SHR hearts. Hence, rhACE2 is an important negative regulator of the Ang II-induced pressor response and NADPH oxidase activation and suppresses pathological myocardial signalling, thereby providing a novel therapeutic agent with which to antagonize an activated renin-angiotesin system.

Several studies have shown the presence of components of the renin-angiotensin system in mammalian ovaries and their involvement in ovarian physiology. We have previously shown the presence of angiotensin-(1-7) [Ang-(1-7)], an important biologically active component of the renin-angiotensin system, and its receptor, Mas, in rat, rabbit and human ovaries. We have also shown the involvement of Ang-(1-7) in the rabbit ovulatory process in vitro. In the present study, we observed that Ang-(1-7) stimulated the resumption of meiosis in oocytes of rat preovulatory follicles, reaching more than 30% of oocytes with germinal vesicle breakdown. The specific antagonist of the Mas receptor, A-779, inhibited the germinal vesicle breakdown induced by Ang-(1-7) and reduced the oocyte maturation stimulated by luteinizing hormone (LH). Immunohistochemistry showed that LH increased both Ang-(1-7) and angiotensin-converting enzyme 2 (ACE2) staining in preovulatory follicles. The effect of gonadotrophins on mRNA expression of Mas and ACE2 in ovaries of immature equine chorionic gonadotrophin-primed rats was analysed by real-time PCR after 6 h of human chorionic gonadotrophin (hCG) injection, which exhibits LH-like effects. After hCG treatment, ACE2 mRNA expression was higher in the ovaries of treated rats than in the ovaries of control rats, whereas Mas mRNA levels were unchanged. A-779 changed the steroidogenesis stimulated by LH. An increased testosterone concentration and decreased progesterone levels were measured in the follicle medium. In conclusion, our results suggest that LH upregulates the ACE2-Ang-(1-7)-Mas axis and that Ang-(1-7) promotes meiotic resumption, possibly as a gonadotrophin intermediate.

Activation of angiotensin-converting enzyme 2 (ACE2), production of angiotensin-(1–7) [Ang-(1–7)] and stimulation of the Ang-(1–7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang-(1–7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang-(1–7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang-(1–7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET-1-induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang-(1–7) and DIZE were reversed by co-intracerebroventricular administration of the Mas receptor inhibitor, A-779. Neither the Ang-(1–7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET-1. Lastly, intracerebroventricular administration of Ang-(1–7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET-1-induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2–Ang-(1–7)–Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang-(1–7) protective action includes blunting of inducible nitric oxide synthase expression.

Our previous studies have indicated that chronic treatment with XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), an angiotensin-converting enzyme2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHR). Furthermore, XNT prevented pulmonary vascular remodeling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200ng/kg/min for 28 days) SHR. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and western blot analysis, respectively. Extracellular signal-regulated kinases (p44 and p42; ERKs) and AT1 receptor levels were quantitated by western blotting. Cardiac ACE2 protein levels were ~15% lower in SHR compared with WKY controls (1.00±0.02 vs. 0.87±0.01 ACE2/GAPDH ratio in SHR). However, treatment of SHR with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHR. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the anti-fibrotic effect of XNT correlated with increased cardiac Ang-(1–7) immunostaining, though no change in cardiac AT1 protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (WKY: 1.00±0.04; Control-SHR: 1.46±0.25; SHR-treated: 0.86±0.02 phospho ERK/total ERK ratio). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in Ang-(1–7) and inhibition of cardiac ERK signaling.

Overactivity of the renin-angiotensin system (RAS) is involved in the pathogenesis of hypertension, and an overactive brain RAS has been highlighted in several genetic and experimental models. Until now, angiotensin II (Ang II) was thought to be the main effector of this system, and the angiotensin-converting enzyme (ACE)-Ang II-Ang II type 1 receptor axis was the main target for antihypertensive therapies. A new member of the RAS, ACE2 (angiotensin-converting enzyme type 2), has been identified in organs and tissues related to cardiovascular function (e.g. heart, kidney and blood vessels) and appears to be part of a counter-regulatory pathway to buffer the excess of Ang II. We recently identified the ACE2 protein in brain regions involved in the central regulation of blood pressure and showed that it regulates, and is regulated by, other components of the RAS. Here, we present evidence for the involvement of brain ACE2 in the central regulation of blood pressure, autonomic and cardiac function. We show that lack of ACE2 is deleterious for the central regulation of blood pressure and that brain ACE2 gene therapy can restore baroreflex and autonomic functions and prevent the development of hypertension. Additionally, and independently of a reduction in Ang II levels, we will highlight some of the mechanisms responsible for the beneficial effects of central ACE2 in cardiovascular function.

As a major regulator of blood pressure homeostasis, the renin-angiotensin system (RAS) has been the subject of extensive scientific investigation. While the RAS was first discovered more than 100 years ago, several novel components of the system have been identified only in the last decade. One of these newer members of the RAS family is angiotensin-converting enzyme 2 (ACE2). Among the approaches used to establish a physiological role for ACE2 has been the generation of ACE2-null mouse lines using homologous recombination in embryonic stem cells. In the literature, there have been at least three lines of ACE2 knockout mice generated by gene targeting by different investigative groups. Interestingly, there are significant differences in some of the reported phenotypes of these distinct lines, especially with regard to their cardiovascular physiology. In this paper, we will review the results of published experiments using these ACE2-null mouse lines, highlighting similarities and differences in these studies and summarizing their contributions to our understanding of the physiological functions of this novel member of the RAS.

During several months of 2002, severe acute respiratory syndrome (SARS) caused by SARS-coronavirus (SARS-CoV) spread rapidly from China throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ARDS), which is the severe form of acute lung injury (ALI). Interestingly, a novel homologue of angiotensin-converting enzyme, termed angiotensin-converting enzyme 2 (ACE2), has been identified as a receptor for SARS-CoV. Angiotensin-converting enzyme and ACE2 share homology in their catalytic domain and provide different key functions in the renin-angiotensin system (RAS). Angiotensin-converting enzyme cleaves angiotensin I to generate angiotensin II, which is a key effector peptide of the system and exerts multiple biological functions, whereas ACE2 reduces angiotensin II levels. Importantly, our recent studies using ACE2 knockout mice have demonstrated that ACE2 protects murine lungs from ARDS. Furthermore, SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin-angiotensin pathway, suggesting that the activation of the pulmonary RAS influences the pathogenesis of ALI/ARDS and SARS.

Angiotensin-converting enzyme 2 (ACE2), a homologue of angiotensin-converting enzyme (ACE), converts angiotensin (Ang) I to Ang(1−9) and Ang II to Ang(1−7), but does not directly process Ang I to Ang II. Cardiac function is compromised in ACE2 null mice; however, the importance of ACE2 in the processing of angiotensin peptides within the murine heart is not known. We determined the metabolism of angiotensins in wild-type (WT), ACE (ACE−/−) and ACE2 null mice (ACE2−/−). Angiotensin II was converted almost exclusively to Ang(1−7) in the cardiac membranes of WT and ACE−/− strains, although generation of Ang(1−7) was greater in the ACE−/− mice (27.4 ± 4.1 versus 17.5 ± 3.2 nmol−1 mg h−1 for WT). The ACE2 inhibitor MLN4760 significantly attenuated Ang II metabolism and the subsequent formation of Ang(1−7) in both strains. In the ACE2−/− hearts, Ang II metabolism and the generation of Ang(1−7) were significantly attenuated; however, the ACE2 inhibitor reduced the residual Ang(1−7)-forming activity in this strain. Angiotensin I was primarily converted to Ang(1−9) (WT, 28.9 ± 3.1 nmol−1 mg h−1; ACE−/−, 49.8 ± 5.3 nmol−1 mg h−1; and ACE2−/−, 35.9 ± 5.4 nmol−1 mg h−1) and to smaller quantities of Ang(1−7) and Ang II. Although the ACE2 inhibitor had no effect on Ang(1−9) formation, the carboxypeptidase A inhibitor benzylsuccinate essentially abolished the formation of Ang(1−9) and increased the levels of Ang I in cardiac membranes. In conclusion, our studies in the murine heart suggest that ACE2 is the primary pathway for the metabolism of Ang II and the subsequent formation of Ang(1−7), a peptide that, in contrast to Ang II, exhibits both antifibrotic and antiproliferative actions.

Injections of the angiotensin(1-7) [Ang(1-7)] antagonist [d-Ala7]-Ang(1-7) into the nucleus of the solitary tract (NTS) of Sprague-Dawley rats reduce baroreceptor reflex sensitivity (BRS) for control of heart rate by approximately 40%, whereas injections of the angiotensin II (Ang II) type 1 receptor antagonist candesartan increase BRS by 40% when reflex bradycardia is assessed. The enzyme angiotensin-converting enzyme 2 (ACE2) is known to convert Ang II to Ang(1-7). We report that ACE2 activity, as well as ACE and neprilysin activities, are present in plasma membrane fractions of the dorsomedial medulla of Sprague-Dawley rats. Moreover, we show that BRS for reflex bradycardia is attenuated (1.16 +/- 0.29 ms mmHg-1 before versus 0.33 +/- 0.11 ms mmHg-1 after; P < 0.05; n = 8) 30-60 min following injection of the selective ACE2 inhibitor MLN4760 (12 pmol in 120 nl) into the NTS. These findings support the concept that within the NTS, local synthesis of Ang(1-7) from Ang II is required for normal sensitivity for the baroreflex control of heart rate in response to increases in arterial pressure.

In the past few years, the classical concept of the renin-angiotensin system (RAS) has experienced substantial conceptual changes. The identification of: the renin/prorenin receptor; the angiotensin-converting enzyme homologue, ACE2, as an angiotensin peptide-processing enzyme and a virus receptor for severe acute respiratory syndrome, the Mas as a receptor for angiotensin (1-7) [Ang(1-7)], and the possibility of signaling through ACE have contributed to switch our understanding of the RAS from the classical limited-proteolysis linear cascade to a cascade with multiple mediators, multiple receptors and multifunctional enzymes. With regard to Ang(1-7), the identification of ACE2 and of Mas as a receptor implicated in its actions contributed to decisively establish this heptapeptide as a biologically active member of the RAS cascade. In this review, we will focus on the recent findings related to the ACE2-Ang(1-7)-Mas axis and, in particular, on its putative role as an ACE-Ang II-AT(1) receptor counter-regulatory axis within the RAS.

Angiotensin (Ang) AT1 receptors and Ang-converting enzymes (ACE and ACE2) are expressed in the dorsal vagal complex (DVC) of the brainstem. The aim of this study was to examine in vivo interactions between brainstem Ang AT1 receptors, ACE and ACE2 using small, hairpin RNA (shRNA) gene-silencing methods. The study takes advantage of the bilateral brainstem expression of renin–angiotensin system (RAS) markers. Adenovirus vectors (Ad, 2.0 × 109 c.f.u. ml−1, 200 nl) carrying interference small hairpin RNA (shRNA) for either AngAT1a (Ad-AT1a-shRNA) or AngAT1b (Ad-AT1b-shRNA) were microinjected into the right side of the brainstem DVC. The Ad-LacZ control was injected into the left side. Brainstems were processed with in situ hybridization and immunochemistry. Results showed that: (1) Ad-AT1a-shRNA downregulated Ang AT1a mRNA by 61.2 ± 6.8% (P < 0.01) and Ad-AT1b-shRNA downregulated Ang AT1b mRNA by 51.6 ± 5.2% (P < 0.01); (2) downregulation of Ang AT1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 ± 14.5%, P < 0.01), while reduction in Ang Ad-AT1b mRNA had no effect; (3) ACE mRNA expression was not altered by either RNA interference (RNAi) treatment; and (4) immunochemical staining for Ang AT1 receptors, ACE and ACE2 were in agreement with the mRNA changes observed. These results demonstrate the utility of in vivo gene silencing to examine functional specificity. Both Ad-AT1a-shRNA and Ad-AT1b-shRNA induced site- and subtype-specific downregulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT1a receptors and the RAS regulatory enzyme, ACE2.

Angiotensin (Ang)-converting enzyme 2 (ACE2) metabolizes Ang II to the vasodilatory peptide Ang(1-7), while neprilysin (NEP) generates Ang(1-7) from Ang I. Experiments used novel Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) mass spectroscopic (MS) assays to study Ang processing. Mass spectroscopy was used to measure proteolytic conversion of Ang peptide substrates to their specific peptide products. We compared ACE/ACE2 activity in plasma, brain and kidney from C57BL/6 and NEP(-/-) mice. Plasma or tissue extracts were incubated with Ang I or Ang II (1296 or 1045, m/z, respectively), and generated peptides were monitored with MS. Angiotensin-converting enzyme 2 activity was detected in kidney and brain, but not in plasma. Brain ACE2 activity was highest in hypothalamus. Angiotensin-converting enzyme 2 activity was inhibited by the specific ACE2 inhibitor, DX600 (10 microm, 99% inhibition), but not by the ACE inhibitor, captopril (10 microm). Both MS and colorimetric assays showed high ACE activity in plasma and kidney with low levels in brain. To extend these findings, ACE measurements were made in ACE overexpressing mice. Angiotensin-converting enzyme four-copy mice showed higher ACE activity in kidney and plasma with low levels in hypothalamus. In hypothalamus from NEP-/- mice, generation of Ang(1-7) from Ang I was decreased, suggesting a role for NEP in Ang metabolism. With Ang II as substrate, there was no difference between NEP-/- and wild-type control mice, indicating that other enzymes may contribute to generation of Ang(1-7). The data suggest a predominant role of hypothalamic ACE2 in the processing of Ang II, in contrast to ACE, which is most active in plasma.

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. Angiotensin-converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague-Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n = 10), and STNx rats received the ACE inhibitor (ACEi) ramipril, 1 mg kg(-1) day(-1) (n = 15) or vehicle (n = 13) orally for 10 days after surgery. Rats with AKI had polyuria (P < 0.001), proteinuria (P < 0.001) and hypertension (P < 0.001). Cardiac structural changes were present and characterized by LVH (P < 0.001), fibrosis (P < 0.001) and increased cardiac brain natriuretic peptide (BNP) mRNA (P < 0.01). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P < 0.01) and ACE2 activity (P < 0.05). Ramipril decreased blood pressure (P < 0.001), LVH (P < 0.001), fibrosis (P < 0.01) and BNP mRNA (P < 0.01). These changes occurred in association with inhibition of cardiac ACE (P < 0.05) and a reduction in cardiac ACE2 activity (P < 0.01). These data suggest that AKI, even at 10 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. Angiotensin-converting enzyme 2, by promoting the production of the antifibrotic peptide angiotensin(1-7), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.

Angiotensin-converting enzyme 2 (ACE2) is thought to act in an opposing manner to its homologue, angiotensin-converting enzyme (ACE), by inactivating the vasoconstrictor peptide angiotensin II and generating the vasodilatory fragment, angiotensin(1-7). Both ACE and ACE2 are membrane-bound ectoenzymes and may circulate in plasma as a consequence of a proteolytic shedding event. In this study, we show that ACE2 circulates in human plasma, but its activity is suppressed by the presence of an endogenous inhibitor. Partial purification of this inhibitor indicated that the inhibitor is small, hydrophilic and cationic, but not a divalent metal cation. These observations led us to develop a method for removal of the inhibitor, thus allowing detection of plasma ACE2 levels using a sensitive quenched fluorescent substrate-based assay. Using this technique, ACE2 activity measured in plasma from healthy volunteers (n = 18) ranged from 1.31 to 8.69 pmol substrate cleaved min-1 ml-1 (mean +/- s.e.m., 4.44 +/- 0.56 pmol min-1 ml-1). Future studies of patients with cardiovascular, renal and liver disease will determine whether plasma ACE2 is elevated in parallel with increased tissue levels observed in these conditions.

Angiotensin-converting enzyme (ACE) 2 is a homologue of ACE with enzymatic activity that seems to counterbalance the angiotensin II-promoting effect of ACE. While ACE promotes angiotensin (Ang) II formation from Ang I, ACE2 degrades Ang II and Ang I. In this review, we discuss recent studies that have delineated the localization of ACE2 within the kidney, an organ that highly expresses this enzyme. In models of diabetic kidney, pharmacological ACE2 inhibition is associated with albuminuria and worsening of glomerular injury. Similarly, genetic ablation of ACE2 causes glomerular lesions in male mice and worsens the renal lesions seen in diabetic Akita mice. Taken together, these findings suggest that a decrease in ACE2 may be involved in diabetic kidney disease, possibly by disrupting the metabolism of angiotensin peptides in such a way that angiotensin II degradation within the glomerulus may be diminished.

The renin-angiotensin system (RAS), in particular angiotensin II, plays an important role in cardiac remodelling. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key players in the RAS and act antagonistically to regulate the levels of angiotensin II. In this study, we reveal the functional expression of ACE2 in human cardiac myofibroblasts, cells that are essential to the maintenance of normal cardiac architecture and also play a key role in myocardial remodelling. The observed reciprocal expression of ACE and ACE2 in these cells may reflect the possible opposing activity of these two enzymes. In this study, we demonstrate the presence of ACE2 as an ectoenzyme and reveal that ACE2 undergoes phorbol-12-myristate-13-acetate-inducible ectodomain shedding from the membrane. When cells were exposed to a number of pathophysiological stimuli, modulation of ACE2 levels was not detected. Importantly, whilst we found ACE2 to be expressed constitutively in cardiac myofibroblasts there were no detectable levels in either vascular smooth muscle cells or vascular endothelium, indicating that ACE2 expression is not ubiquitous. In paraffin sections of atrial appendage tissue, we observed a distinct staining pattern for ACE2 which appeared different from that of ACE. In conclusion, this study is the first to report co-expression of ACE and ACE2 in human cardiac myofibroblasts and may therefore present a model primary system for study of the comparative cell biology of ACE2 and ACE and their potentially opposing roles in myocardial remodelling.

Angiotensin (Ang)-converting enzyme 2 (ACE2) metabolizes Ang II to the vasodilatory peptide Ang(1-7), while neprilysin (NEP) generates Ang(1-7) from Ang I. Experiments used novel Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF) mass spectroscopic (MS) assays to study Ang processing. Mass spectroscopy was used to measure proteolytic conversion of Ang peptide substrates to their specific peptide products. We compared ACE/ACE2 activity in plasma, brain and kidney from C57BL/6 and NEP(-/-) mice. Plasma or tissue extracts were incubated with Ang I or Ang II (1296 or 1045, m/z, respectively), and generated peptides were monitored with MS. Angiotensin-converting enzyme 2 activity was detected in kidney and brain, but not in plasma. Brain ACE2 activity was highest in hypothalamus. Angiotensin-converting enzyme 2 activity was inhibited by the specific ACE2 inhibitor, DX600 (10 microm, 99% inhibition), but not by the ACE inhibitor, captopril (10 microm). Both MS and colorimetric assays showed high ACE activity in plasma and kidney with low levels in brain. To extend these findings, ACE measurements were made in ACE overexpressing mice. Angiotensin-converting enzyme four-copy mice showed higher ACE activity in kidney and plasma with low levels in hypothalamus. In hypothalamus from NEP-/- mice, generation of Ang(1-7) from Ang I was decreased, suggesting a role for NEP in Ang metabolism. With Ang II as substrate, there was no difference between NEP-/- and wild-type control mice, indicating that other enzymes may contribute to generation of Ang(1-7). The data suggest a predominant role of hypothalamic ACE2 in the processing of Ang II, in contrast to ACE, which is most active in plasma.

Angiotensin-converting enzyme 2 (ACE2) is a novel enzyme with possible implications in the treatment of blood pressure disorders. Recent evidence suggests that an upregulation of ACE2 can be stimulated by all-trans retinoic acid (at-RA); however, at-RA also affects regulation of the stem-cell marker octomer-4 (Oct-4) and thus cellular differentiation. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for ACE2, Oct-4 and the haematopoietic stem-cell marker CD34. Thus, to provide evidence that possible at-RA treatment could affect both plaque cellular biology (via effects on cellular differentiation) and blood pressure (via ACE2), it is vital to show that cells with atherosclerotic plaques co-express all three markers. Thus, we sought to provide evidence that a subset of cells within atherosclerotic plaques is positive for ACE2, Oct-4 and CD34. We used New Zealand White rabbits that were fed a control diet supplemented with 0.5% cholesterol plus 1% methionine for 4 weeks and then allowed to consume a normal diet for 10 weeks. Immunohistochemistry was performed by standard techniques. We report that ACE2, Oct-4 and CD34 were all present within atherosclerotic plaques. Although macrophages were positive for all three markers, spindle-shaped cells in the media did not show all three markers. The endothelium overlying normal arterial wall showed positive Oct-4 and ACE2 immunoreactivity, but CD34 immunoreactivity was patchy, indicating that such cells might not have fully differentiated. It is concluded that cells in atherosclerotic plaques express co-express ACE2, Oct-4 and CD34. Further studies aimed at establishing the effects of all-trans retinoic acid on blood pressure and atherosclerotic cell differentiation are warranted.

Angiotensin converting enzyme 2 (ACE2), a newly discovered member of the renin-angiotensin system (RAS), is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides from angiotensin II. The aim of the present study was to evaluate whether overexpression of ACE2 could protect the heart from angiotensin II-induced hypertrophy and fibrosis. Lentiviral vector encoding mouse ACE2 (lenti-mACE2) or GFP was injected intracardially in 5-day-old Sprague-Dawley rats. This resulted in expression of mACE2 in cardiac tissue for the duration of the study. Infusion of 200 ng kg-1 min-1 angiotensin II for 4 weeks resulted in an 80 mmHg increase in systolic blood pressure, a significant increase in the heart weight to body weight ratio (HW:BW), and marked myocardial fibrosis in control rats. Transduction with lenti-mACE2 resulted in significant attenuation of the increased HW:BW and myocardial fibrosis induced by angiotensin II infusion. These observations demonstrate that ACE2 overexpression results in protective effects on angiotensin II-induced cardiac hypertrophy and fibrosis.

Less than one-third of patients with hypertension have their blood pressures (BP) controlled with current traditional therapeutic approaches for the treatment and control of hypertension. Pharmacological approaches may have reached a plateau in their effectiveness and thus newer innovative strategies need to be studied not only to increase the number of patients that can achieve BP control, but also to find a way to cure, not just manage, the disease. Continuous advances in gene delivery systems coupled with the completion of the Human Genome Project, now make it possible to investigate genetic means for the treatment and possible cure for hypertension. The renin-angiotensin system (RAS) has long been known to regulate BP, and salt and water metabolism. This system is unique in having both a peripheral circulating system and a tissue-based system. Each of these components have been ascribed a variety of physiological effects that have been associated with not only an increase in BP, but also in a variety of the pathophysiological manifestations associated with hypertension, such as cardiac hypertrophy and kidney dysfunction. We and others have used an antisense gene therapy approach, targeting the classical components of the RAS, to effectively attenuate the development of hypertension and related cardiovascular pathophysiologies in numerous experimental models of hypertension. Recently other components of the RAS have been elucidated and some of these components may be potential targets in a gene therapy approach. This article will focus on angiotensin-converting enzyme 2 (ACE2) as a new, potential target of gene therapy for hypertensive disorders.

Angiotensin 1-7 is the product of the catalytic activity of angiotensin converting enzyme type 2 (ACE2) upon angiotensin II. Angiotensin 1-7 appears to signal through the G protein coupled receptor Mas. Mas activation causes vasodilation, decreases formation of reactive oxygen species and increases NO production in vascular tissues (Passos-Silva et al., 2013). Thus, angiotensin 1-7 generally is a functional antagonist of angiotensin II. Little is known about functions of angiotensin 1-7 in brain arteries or cerebrovascular disease. Recent studies published in Experimental Physiology have shed some light on protective actions of angiotensin 1-7 in experimental models of stroke (Mecca et al., 2011; Regenhardt et al., 2013b). The angiotensin 1-7 / ACE2 / Mas axis may be a therapeutic target in cerebrovascular disease. Levels of angiotensin 1-7 in serum and brain cortex increase during the first 12 hours after middle cerebral artery occlusion (MCAO) in rats (Lu et al., 2013). Increases in angiotensin 1-7 levels after MCAO are accompanied by increased expression of ACE2 and Mas in brain tissue (Lu et al., 2013). Intracerebroventricular (ICV) administration of angiotensin 1-7 decreases formation of autophagosomes (Jiang et al., 2013b), oxidative stress, and apoptosis in the brain of hypertensive rats (Jiang et al., 2013a). Angiotensin 1-7 also decreases angiotensin II levels, and reduces expression of angiotensin II type 1 (AT1) and 2 (AT2) receptors in the brain of hypertensive rats (Jiang et al., 2013a). Thus angiotensin 1-7 appears to protect from effects of hypertension on the brain. Mecca et al., (2009) developed a model of ischemic stroke produced by endothelin-1 – induced occlusion of the middle cerebral artery (MCAO) in rats. Using this model, they demonstrated that continuous ICV administration of angiotensin 1-7, from one week before MCAO until sacrifice, decreases infarct size and neurological deficit 72 hours after occlusion (Mecca et al., 2011). Importantly these results have been reproduced as angiotensin 1-7 decreased infarct size in two separate studies (Jiang et al., 2012; Regenhardt et al., 2013a). Mechanisms by which angiotensin 1-7 exerts its neuroprotective actions are not clear, but appear to involve activation of Mas receptors and modulation of inflammation and oxidative stress because 1) an antagonist of Mas receptors prevented the reduction in infarct size induced by angiotensin 1-7 after MCAO (Mecca et al., 2011; Jiang et al., 2012); 2) angiotensin 1-7 attenuated activation of the transcriptional regulator of inflammation NFkB, and decreased expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) (Jiang et al., 2012; Regenhardt et al., 2013a); and 3) angiotensin 1-7 attenuated the increase in lipid peroxidation and decrease in superoxide dismutase activity after brain ischemia (Jiang et al., 2012). In the current issue of Experimental Physiology, Regenhardt et al., (2013b) show that the protective effects of angiotensin 1-7 are also seen in stroke prone spontaneously hypertensive rats (SHRSP). SHRSP develop multiple vascular lesions in the brain including alterations in blood brain barrier permeability and cerebral blood flow, microthrombosis, and intracerebral hemorrhage (Schreiber et al., 2012). Regenhardt et al., (2013b) treated SHRSP early in life (49 days of age) with ICV angiotensin 1-7 during 6 weeks, and monitored neurological function and survival. Angiotensin 1-7 treatment did not attenuate hypertension in SHRSP. Angiotensin 1-7 extended the median survival of SHRSP from 108 to 154 days. Angiotensin 1-7 also decreased the number of subcortical, but not cortical, hemorrhages. Because the variance in the sunflower test was high, it is difficult to definitely state that angiotensin 1-7 preserves neurological function, although angiotensin 1-7 appeared to be associated with increased spontaneous movement late in the course of the study. An antiinflammatory effect of angiotensin 1-7 could account for neuroprotective effects. But this study appears to be underpowered to detect differences in expression of cytokines after brain ischemia. The authors however noted a decrease in microglial activation in the striatum of SHRSP. Thus, angiotensin 1-7 appears to have several neuroprotective actions in the context of hypertension and stroke. The angiotensin 1-7 axis is activated after ischemic stroke. Angiotensin 1-7 appears to reduce apoptosis, oxidative stress and autophagosome formation in brains of hypertensive rats. It also appears to reduce brain damage after ischemic stroke and the number of hemorrhages in SHRSP. These results were obtained in rats in which angiotensin 1-7 was delivered directly to the cerebral ventricles. These are important findings, but several questions remain unanswered. 1) Are beneficial actions of angiotensin 1-7also seen in other species or are they specific for rats? 2) Why is activation of endogenous Ang1-7 /ACE2 /Mas axis after stroke (Lu et al., 2013) insufficient to protect the brain? Mecca et al., (2011) demonstrated that ICV administration of an ACE2 activator reduces brain damage after MCAO. Thus, it is possible that an additional “boost” is needed to induce neuroprotection by the endogenous brain angiotensin 1-7. It would also be interesting to study effects of angiotensin 1-7 on stroke in animals deficient in Mas receptor or ACE2. ACE2 deficiency, for example, is associated with vascular dysfunction in brain arteries from mice (Pena Silva et al., 2012), but it is not known if Mas or ACE2 deficiency is associated with worse outcomes after stroke. 3) What are baseline concentrations of angiotensin 1-7 in the CSF, and what concentrations are needed to induce a neuroprotective effect in the brain? 4) Would systemic angiotensin 1-7 have neuroprotective effects? Hypertension and stroke are associated with increases in blood brain barrier permeability, and it would be interesting to see if increased permeability would facilitate diffusion of systemic angiotensin 1-7 to the brain, and thereby allow a protective effect. Research in neuroprotective actions of angiotensin 1-7 is still a nascent field, but the current results are certainly promising. We can foresee development of therapeutics that could target the brain and systemic angiotensin 1-7 /ACE2 /Mas axis for treatment and prevention of cerebrovascular disease and neuroinflammation.

Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin II (Ang II) have been implicated in the pathogenesis of pancreatitis. Angiotensin-converting enzyme 2 (ACE2) degrades Ang II to angiotensin-(1-7) [Ang-(1-7)] and has recently been described to have an antagonistic effect on ACE signalling. However, the specific underlying role of ACE2 in the pathogenesis of severe acute pancreatitis (SAP) is unclear. In the present study, the local imbalance of ACE and ACE2, as well as Ang II and Ang-(1-7) expression, was compared in wild-type (WT) and ACE2 knock-out (KO) or ACE2 transgenic (TG) mice subjected to cerulein-induced SAP. Serum amylase, tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-10 levels and histological morphometry were used to determine the severity of pancreatitis. In WT mice, pancreatic ACE and Ang II and serum Ang II expression increased (P0.05), while pancreatic ACE2 and Ang-(1-7) and serum Ang-(1-7) levels were also significantly elevated (P0.05) from 2 to 72 h after the onset of SAP. However, the ratio of pancreatic ACE2 to ACE expression was significantly reduced (from 1.46 ± 0.09 to 0.27 ± 0.05, P0.001) and paralleled the severity of pancreatitis. The Ace2 KO mice exhibited increased levels of tumour necrosis factor-α, IL-1β, IL-6, multifocal coagulative necrosis and inflammatory infiltrate, and lower levels of serum IL-10 and pancreatic Ang-(1-7) (4.70 ± 2.13 versus 10.87 ± 2.51, P0.001) compared with cerulein-treated WT mice at the same time point. Conversely, Ace2 TG mice with normal ACE expression were more resistant to SAP challenge as evidenced by a decreased inflammatory response, attenuated pathological changes and increased survival rates. These data suggest that the ACE2-ACE imbalance plays an important role in the pathogenesis of SAP and that pancreatic ACE2 is an important factor in determining the severity of SAP.

Several studies have shown the presence of components of the renin-angiotensin system in mammalian ovaries and their involvement in ovarian physiology. We have previously shown the presence of angiotensin-(1-7) [Ang-(1-7)], an important biologically active component of the renin-angiotensin system, and its receptor, Mas, in rat, rabbit and human ovaries. We have also shown the involvement of Ang-(1-7) in the rabbit ovulatory process in vitro. In the present study, we observed that Ang-(1-7) stimulated the resumption of meiosis in oocytes of rat preovulatory follicles, reaching more than 30% of oocytes with germinal vesicle breakdown. The specific antagonist of the Mas receptor, A-779, inhibited the germinal vesicle breakdown induced by Ang-(1-7) and reduced the oocyte maturation stimulated by luteinizing hormone (LH). Immunohistochemistry showed that LH increased both Ang-(1-7) and angiotensin-converting enzyme 2 (ACE2) staining in preovulatory follicles. The effect of gonadotrophins on mRNA expression of Mas and ACE2 in ovaries of immature equine chorionic gonadotrophin-primed rats was analysed by real-time PCR after 6 h of human chorionic gonadotrophin (hCG) injection, which exhibits LH-like effects. After hCG treatment, ACE2 mRNA expression was higher in the ovaries of treated rats than in the ovaries of control rats, whereas Mas mRNA levels were unchanged. A-779 changed the steroidogenesis stimulated by LH. An increased testosterone concentration and decreased progesterone levels were measured in the follicle medium. In conclusion, our results suggest that LH upregulates the ACE2-Ang-(1-7)-Mas axis and that Ang-(1-7) promotes meiotic resumption, possibly as a gonadotrophin intermediate.

Renin-angiotensin system blockade slows but does not prevent the cardiovascular complications of chronic kidney disease (CKD). Angiotensin-converting enzyme (ACE) 2 is differentially regulated in acute kidney injury, with increased cardiac ACE2 but decreased kidney ACE2 levels. This study investigated the effect of long-term ACE inhibition on cardiac and renal ACE2 in rats with CKD induced by subtotal nephrectomy (STNx). Sprague-Dawley rats had sham (control) or STNx surgery. Control rats received vehicle (n = 9) and STNx rats ramipril (1 mg kg(-1) day(-1); n = 10) or vehicle (n = 10) for 28 days. Subtotal nephrectomy resulted in impaired creatinine clearance (P0.05), proteinuria (P0.05), renal fibrosis (P0.05) and reduced renal cortical ACE2 mRNA (P0.05) and activity (P0.05). In rats with CKD, ramipril improved creatinine clearance (P0.05) and was associated with an increase in cortical but not medullary ACE2 activity (P0.05). Compared with control rats, STNx rats were hypertensive (P0.01), with increased left ventricular end-diastolic pressure (LVEDP; P0.01), left ventricular hypertrophy (LVH; P0.05) and interstitial (P0.05) and perivascular fibrosis (P0.01). In rats with CKD, ramipril decreased blood pressure (P0.001) and reduced LVEDP (P0.01), LVH (P0.01) and perivascular fibrosis (P0.05) but did not significantly reduce interstitial fibrosis. There was no change in cardiac ACE2 in rats with CKD compared with control rats. In rats with CKD, ACE inhibition had major benefits to reduce blood pressure and cardiac hypertrophy and to improve creatinine clearance, but did not significantly impact on cardiac ACE2, cardiac interstitial fibrosis, renal fibrosis or proteinuria. Thus, in rats with CKD, renal ACE2 deficiency and lack of activation of cardiac ACE2 may contribute to the progression of cardiac and renal tissue injury. As long-term ACE inhibition only partly ameliorated the adverse cardio-renal effects of CKD, adjunctive therapies that lead to further increases in ACE2 activity may be needed to combat the cardio-renal complications of CKD.

Activation of angiotensin-converting enzyme 2 (ACE2), production of angiotensin-(1-7) [Ang-(1-7)] and stimulation of the Ang-(1-7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang-(1-7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang-(1-7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang-(1-7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET-1-induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang-(1-7) and DIZE were reversed by co-intracerebroventricular administration of the Mas receptor inhibitor, A-779. Neither the Ang-(1-7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET-1. Lastly, intracerebroventricular administration of Ang-(1-7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET-1-induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2-Ang-(1-7)-Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang-(1-7) protective action includes blunting of inducible nitric oxide synthase expression.

During several months of 2002, severe acute respiratory syndrome (SARS) caused by SARS‐coronavirus (SARS‐CoV) spread rapidly from China throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ARDS), which is the severe form of acute lung injury (ALI). Interestingly, a novel homologue of angiotensin‐converting enzyme, termed angiotensin‐converting enzyme 2 (ACE2), has been identified as a receptor for SARS‐CoV. Angiotensin‐converting enzyme and ACE2 share homology in their catalytic domain and provide different key functions in the renin–angiotensin system (RAS). Angiotensin‐converting enzyme cleaves angiotensin I to generate angiotensin II, which is a key effector peptide of the system and exerts multiple biological functions, whereas ACE2 reduces angiotensin II levels. Importantly, our recent studies using ACE2 knockout mice have demonstrated that ACE2 protects murine lungs from ARDS. Furthermore, SARS‐CoV infections and the Spike protein of the SARS‐CoV reduce ACE2 expression. Notably, injection of SARS‐CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin–angiotensin pathway, suggesting that the activation of the pulmonary RAS influences the pathogenesis of ALI/ARDS and SARS.

Angiotensin (Ang) AT1 receptors and Ang-converting enzymes (ACE and ACE2) are expressed in the dorsal vagal complex (DVC) of the brainstem. The aim of this study was to examine in vivo interactions between brainstem Ang AT1 receptors, ACE and ACE2 using small, hairpin RNA (shRNA) gene-silencing methods. The study takes advantage of the bilateral brainstem expression of renin-angiotensin system (RAS) markers. Adenovirus vectors (Ad, 2.0 x 10(9) c.f.u. ml(-1), 200 nl) carrying interference small hairpin RNA (shRNA) for either AngAT1a (Ad-AT1a-shRNA) or AngAT1b (Ad-AT1b-shRNA) were microinjected into the right side of the brainstem DVC. The Ad-LacZ control was injected into the left side. Brainstems were processed with in situ hybridization and immunochemistry. Results showed that: (1) Ad-AT1a-shRNA downregulated Ang AT1a mRNA by 61.2 +/- 6.8% (P0.01) and Ad-AT1b-shRNA downregulated Ang AT1b mRNA by 51.6 +/- 5.2% (P0.01); (2) downregulation of Ang AT1a mRNA was associated with decreased ACE2 mRNA expression (decrease of 29.0 +/- 14.5%, P0.01), while reduction in Ang Ad-AT1b mRNA had no effect; (3) ACE mRNA expression was not altered by either RNA interference (RNAi) treatment; and (4) immunochemical staining for Ang AT1 receptors, ACE and ACE2 were in agreement with the mRNA changes observed. These results demonstrate the utility of in vivo gene silencing to examine functional specificity. Both Ad-AT1a-shRNA and Ad-AT1b-shRNA induced site- and subtype-specific downregulation of receptor expression. Gene silencing showed that there were interactions between brainstem Ang AT1a receptors and the RAS regulatory enzyme, ACE2.

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. Angiotensin-converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague-Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n = 10), and STNx rats received the ACE inhibitor (ACEi) ramipril, 1 mg kg(-1) day(-1) (n = 15) or vehicle (n = 13) orally for 10 days after surgery. Rats with AKI had polyuria (P0.001), proteinuria (P0.001) and hypertension (P0.001). Cardiac structural changes were present and characterized by LVH (P0.001), fibrosis (P0.001) and increased cardiac brain natriuretic peptide (BNP) mRNA (P0.01). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P0.01) and ACE2 activity (P0.05). Ramipril decreased blood pressure (P0.001), LVH (P0.001), fibrosis (P0.01) and BNP mRNA (P0.01). These changes occurred in association with inhibition of cardiac ACE (P0.05) and a reduction in cardiac ACE2 activity (P0.01). These data suggest that AKI, even at 10 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. Angiotensin-converting enzyme 2, by promoting the production of the antifibrotic peptide angiotensin(1-7), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.

The renin-angiotensin system (RAS), in particular angiotensin II, plays an important role in cardiac remodelling. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key players in the RAS and act antagonistically to regulate the levels of angiotensin II. In this study, we reveal the functional expression of ACE2 in human cardiac myofibroblasts, cells that are essential to the maintenance of normal cardiac architecture and also play a key role in myocardial remodelling. The observed reciprocal expression of ACE and ACE2 in these cells may reflect the possible opposing activity of these two enzymes. In this study, we demonstrate the presence of ACE2 as an ectoenzyme and reveal that ACE2 undergoes phorbol-12-myristate-13-acetate-inducible ectodomain shedding from the membrane. When cells were exposed to a number of pathophysiological stimuli, modulation of ACE2 levels was not detected. Importantly, whilst we found ACE2 to be expressed constitutively in cardiac myofibroblasts there were no detectable levels in either vascular smooth muscle cells or vascular endothelium, indicating that ACE2 expression is not ubiquitous. In paraffin sections of atrial appendage tissue, we observed a distinct staining pattern for ACE2 which appeared different from that of ACE. In conclusion, this study is the first to report co-expression of ACE and ACE2 in human cardiac myofibroblasts and may therefore present a model primary system for study of the comparative cell biology of ACE2 and ACE and their potentially opposing roles in myocardial remodelling.

Angiotensin-converting enzyme (ACE) and ACE2 and the AT1 and AT2 receptors are pivotal points of regulation in the renin-angiotensin system. ACE and ACE2 are key enzymes in the formation and degradation of angiotensin II (Ang II) and angiotensin-(1-7)(Ang-(1-7)). Ang II acts at either the AT1 or the AT2 receptor to mediate opposing actions of vasoconstriction or vasodilatation respectively. While it is known that oestrogen acts to downregulate ACE and the AT(1) receptor, its regulation of ACE2 and the AT2 receptor and the involvement of a specific oestrogen receptor subtype are unknown. To investigate the role of oestrogen receptor-alpha (ERalpha) in the regulation by oestrogen of ACE/ACE2 and AT1/AT2 mRNAs in lung and kidney, ovariectomized female mice lacking apolipoprotein E (ee) with the ERalpha (AAee) or without the ERalpha (alphaalphaee) were treated with 17beta-oestradiol (6 microg day(-1)) or placebo for 3 months. ACE, ACE2, AT1 receptor and AT2 receptor mRNAs were measured using reverse transcriptase, real-time polymerase chain reaction. In the kidney, 17beta-oestradiol showed 1.7-fold downregulation of ACE mRNA in AAee mice, with 2.1-fold upregulation of ACE mRNA in alphaalphaee mice. 17beta-Oestradiol showed 1.5- and 1.8-fold downregulation of ACE2 and AT1 receptor mRNA in AAee mice; this regulation was lost in alphaalphaee mice. 17beta-Oestradiol showed marked (81-fold) upregulation of the AT(2) receptor mRNA in AAee mice. In the lung, 17beta-oestradiol treatment had no effect on AT1 receptor mRNA in AAee mice, but resulted in a 1.5-fold decreased regulation of AT1 mRNA in alphaalphaee mice. There was no significant interaction of oestrogen with ERalpha in the lung for ACE, ACE2 and AT2 receptor genes. These studies reveal tissue-specific regulation by 17beta-oestradiol of ACE/ACE2 and AT1/AT2 receptor genes, with the ERalpha receptor being primarily responsible for the regulation of kidney ACE2, AT1 receptor and AT2 receptor genes.

Angiotensin-converting enzyme (ACE) 2 is thought to counterbalance ACE by breakdown of angiotensin (Ang) II and formation of Ang(1-7). Both enzymes are highly expressed in the kidney, but reports on their regulation differ. To enhance our understanding of the regulation of renal ACE and ACE2, we investigated renal ACE and ACE2 expression during conditions of physiological (low-sodium diet) and pharmacological changes (ACE inhibition) in activity of the renin-angiotensin-aldosterone system (RAAS). Healthy rats were treated with vehicle or lisinopril with either a control or a low-sodium diet, and renal ACE2, ACE and plasma angiotensins were studied. During vehicle treatment, low sodium reduced renal ACE mRNA and activity without affecting ACE2 mRNA or activity and plasma Ang(1-7) and Ang II balance. Lisinopril significantly reduced renal ACE activity without affecting renal ACE2 activity. During ACE inhibition, low sodium reduced both ACE and ACE2 mRNA without affecting ACE2 activity or further reducing ACE activity. Measurements of renal neprilysin activity revealed no significant differences between any of the treatment groups. Plasma Ang(1-7) and Ang II balance is positively shifted towards the beneficial vasopeptide Ang(1-7) by the ACE inhibitor lisinopril, especially during a low sodium intake. In conclusion, modulation of the RAAS, by low sodium intake or ACE inhibition, does not affect renal ACE2 despite major variations in renal ACE. Thus, ACE and ACE2 are differentially regulated by low sodium and ACE inhibition. Therefore, we propose that the beneficial effects of ACE inhibitors are predominantly mediated by modulation of ACE and not ACE2. Whether this also applies to renal disease conditions should be investigated in future studies.

Angiotensin‐converting enzyme 2 (ACE2) is a novel enzyme with possible implications in the treatment of blood pressure disorders. Recent evidence suggests that an upregulation of ACE2 can be stimulated by all‐trans retinoic acid (at‐RA); however, at‐RA also affects regulation of the stem‐cell marker octomer‐4 (Oct‐4) and thus cellular differentiation. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for ACE2, Oct‐4 and the haematopoietic stem‐cell marker CD34. Thus, to provide evidence that possible at‐RA treatment could affect both plaque cellular biology (via effects on cellular differentiation) and blood pressure (via ACE2), it is vital to show that cells with atherosclerotic plaques co‐express all three markers. Thus, we sought to provide evidence that a subset of cells within atherosclerotic plaques is positive for ACE2, Oct‐4 and CD34. We used New Zealand White rabbits that were fed a control diet supplemented with 0.5% cholesterol plus 1% methionine for 4 weeks and then allowed to consume a normal diet for 10 weeks. Immunohistochemistry was performed by standard techniques. We report that ACE2, Oct‐4 and CD34 were all present within atherosclerotic plaques. Although macrophages were positive for all three markers, spindle‐shaped cells in the media did not show all three markers. The endothelium overlying normal arterial wall showed positive Oct‐4 and ACE2 immunoreactivity, but CD34 immunoreactivity was patchy, indicating that such cells might not have fully differentiated. It is concluded that cells in atherosclerotic plaques express co‐express ACE2, Oct‐4 and CD34. Further studies aimed at establishing the effects of all‐trans retinoic acid on blood pressure and atherosclerotic cell differentiation are warranted.

Angiotensin converting enzyme 2 (ACE2), a newly discovered member of the renin-angiotensin system (RAS), is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides from angiotensin II. The aim of the present study was to evaluate whether overexpression of ACE2 could protect the heart from angiotensin II-induced hypertrophy and fibrosis. Lentiviral vector encoding mouse ACE2 (lenti-mACE2) or GFP was injected intracardially in 5-day-old Sprague-Dawley rats. This resulted in expression of mACE2 in cardiac tissue for the duration of the study. Infusion of 200 ng kg-1 min-1 angiotensin II for 4 weeks resulted in an 80 mmHg increase in systolic blood pressure, a significant increase in the heart weight to body weight ratio (HW:BW), and marked myocardial fibrosis in control rats. Transduction with lenti-mACE2 resulted in significant attenuation of the increased HW:BW and myocardial fibrosis induced by angiotensin II infusion. These observations demonstrate that ACE2 overexpression results in protective effects on angiotensin II-induced cardiac hypertrophy and fibrosis.