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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Hypertension. 2013 Apr 22;61(6):1233–1238. doi: 10.1161/HYPERTENSIONAHA.111.00627

ANGIOTENSIN-CONVERTING ENZYME 2 ACTIVATION IMPROVES ENDOTHELIAL FUNCTION

Rodrigo A Fraga-Silva 1,6, Fabiana P Costa-Fraga 2, Tatiane M Murça 3, Patrícia L Moraes 3, Augusto Martins Lima 1,2, Roberto Q Lautner 1,2, Carlos H Castro 1,4, Célia Maria A Soares 4, Clayton L Borges 4, Ana Paula Nadu 2, Marilene L Oliveira 2, Vinayak Shenoy 5, Michael J Katovich 5, Robson AS Santos 1,2, Mohan K Raizada 6, Anderson J Ferreira 1,3
PMCID: PMC3733257  NIHMSID: NIHMS475952  PMID: 23608648

Abstract

Diminished release and function of endothelium-derived nitric oxide (NO) coupled with increases in reactive oxygen species (ROS) production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme2 (ACE2), Angiotensin-(1-7) [Ang-(1-7)] and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic ACE2 would improve endothelial function by decreasing the ROS production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule ACE2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1mg/kg/day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7) and it was reduced in Mas knockout mice. These effects were associated with reduction in ROS production. In addition, Ang II-induced ROS production in human aortic endothelial cells was attenuated by pre-incubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that ACE2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Keywords: Oxidative stress, Angiotensin-(1-7), renin-angiotensin system, endothelium dysfunction, diabetes

INTRODUCTION

Endothelial cells play a central role in maintaining vascular homeostasis and their dysfunction is associated with many forms of cardiovascular and metabolic diseases.1 Diminished production and function of endothelium-derived nitric oxide (NO) and other vasoprotective factors and the exaggerated production of reactive oxygen species (ROS) and vasoconstrictors eventually lead to endothelial dysfunction, resulting in elevated vascular tone, which contributes to the development and progression of cardiovascular and metabolic diseases.1,2

The renin-angiotensin system (RAS) is a pivotal modulator of the vascular function and its hyperactivity is involved in the endothelial dysfunction.3,4 The RAS is regulated by two opposite axes.5,6 The effector of the first one is Angiotensin II (Ang II) which is generated by coordinated enzymatic reactions involving, mainly, the angiotensin converting-enzyme (ACE). This peptide acts primarily through the AT1 receptor, promoting vasoconstriction, proliferation and oxidative stress.4,7 Thus, this axis is composed by ACE, Ang II and AT1 receptor. The second axis, formed by ACE2, Angiotensin-(1-7) [Ang-(1-7)] and Mas,5 is activated by Ang-(1-7) binding to its own receptor Mas.8 Generally, this axis promotes opposite effects to those elicited by the ACE/Ang II/AT1 receptor branch.5,6 Ang-(1-7) may be generated by different enzymatic pathways;9 however it has been proposed that ACE2 is the main enzyme involved in the Ang-(1-7) formation.10,11 ACE2 is a monocarboxypeptidase which primarily catalyzes the conversion of Ang II into Ang-(1-7),10 thereby contributing to the balance between the two peptides and, consequently, it is a key modulator of the two axes of the RAS.6

The role of the ACE/Ang II/AT1 axis in the development and progression of the endothelial dysfunction is well-recognized, especially in terms of ROS production by endothelial and vascular smooth muscle cells.3,4,7 Treatment with free radical scavengers, such as superoxide dismutase (SOD), catalase and tempol, reduces blood pressure and vascular damage in response to Ang II.12,13 On the other hand, it was reported that activation of the ACE2/Ang-(1-7)/Mas axis ameliorates the endothelial function in many animal models.14 Indeed, short-term infusion of Ang-(1-7) improved endothelial response to acetylcholine15 and Mas deficiency caused endothelial dysfunction and increases in blood pressure associated with elevation of the ROS production.16

Altogether, these findings led us to postulate that activation of intrinsic ACE2 would improve endothelial dysfunction by decreasing the production of ROS. To test this hypothesis, we evaluated the effects of 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule that has been reported to activate intrinsic ACE2,17 on endothelial dysfunction. XNT was discovered based on the crystal structure of ACE2 using a virtual screening strategy.17 Administration of this compound in spontaneously hypertensive rats (SHR) decreased blood pressure, improved cardiac function and reversed myocardial and perivascular fibrosis through a mechanism involving reductions in extracellular signal-regulated kinases expression.17,18 Moreover, XNT reduced pulmonary hypertension induced by monocrotaline,19 attenuated thrombus formation and platelet attachment to vessels of hypertensive rats,20 and ameliorated cardiac and autonomic function of diabetic rats.21,22

METHODS

The procedures used for the measurement of ACE2 activity, isolated aortic rings preparation, radioimmunoassay, western blotting and immunohistochemistry are described in the online Data Supplement (please see at http://hyper.ahajournals.org).

Animals

Male Sprague-Dawley rats and SHR (12 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA, USA). Male Wistar rats (12 weeks of age) were obtained from the CEBIO-Federal University of Minas Gerais (Belo Horizonte, MG, Brazil). Male Wild type (Mas+/+) and Mas knockout (Mas−/−) mice (FVB/N background, 12 weeks of age) were bred at the transgenic animal facility of the Laboratory of Hypertension, Federal University of Minas Gerais (Belo Horizonte, MG, Brazil). All animals were kept in temperature-controlled rooms with 12/12 hour light/dark cycle and had free access to water and food. All experimental protocols were performed in accordance with the University of Florida (USA) and the Federal University of Minas Gerais (Brazil) Institutional Animal Care and Use Committees, which are in compliance with the National Institutes of Health (NIH) guidelines.

Statistical Analysis

The results are presented as mean ± SEM. Two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test was used to compare the curves obtained in the ACE2 activity and aortic rings preparation protocols. In addition, one-way analysis of variance (ANOVA) followed by the Bonferroni’s post-test was utilized to analyze the western blotting, immunohistochemistry and ROS production data, and Student t-test was used to analyze the radioimmunoassay results. All statistical analyses were considered significant when p<0.05.

RESULTS

XNT Improves Endothelial Function of SHR and Diabetic Rats

In accordance with previous studies by Hernández Prada and co-authors,17 we observed that incubation of rhACE2 with XNT in vitro increased the activity of ACE2, thereby confirming the ability of XNT to activate this enzyme (please see Figure S1 of the online Data Supplement at http://hyper.ahajournals.org). Similar data were observed when aortic samples from normal and diabetic animals were incubated with the fluorogenic substrate (please see Figure S2 of the online Data Supplement at http://hyper.ahajournals.org). As a consequence, ACE2 activation significantly increased the concentration of Ang-(1-7) in plasma of diabetic animals, but not in aorta (please see Figure S3 of the online Data Supplement at http://hyper.ahajournals.org). Although approximately 30% of decrease in plasma Ang II levels was observed in diabetic-treated rats, it did not reach statistical significance (please see Figure S4 of the online Data Supplement at http://hyper.ahajournals.org). Importantly, the ACE2 activity in diabetic rats was significantly lower when compared with normal animals (please see Figure S2 of the online Data Supplement at http://hyper.ahajournals.org). Treatment with XNT was unable to change the ACE2 protein expression in diabetic rats (please see Figure S5 and S6 of the online Data Supplement at http://hyper.ahajournals.org).

To examine the effects of chronic XNT treatment on the endothelial function, the vasorelaxant responses to ACh and SNP were evaluated in aortic rings from hypertensive and diabetic rats. The vasodilatory responses to ACh were markedly enhanced in both SHR (Figure 1A) and diabetic Wistar rats (Figure 1B) treated with XNT. In contrast, the endothelial-independent responses to SNP were not affected by XNT when compared with vessels from untreated SHR (Figure 1C) and diabetic rats (Figure 1D). These results showed that the endothelium-dependent vascular responses were improved by ACE2 activation in SHR and diabetic rats.

Figure 1.

Figure 1

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XNT improves endothelial function of hypertensive and diabetic rats. Vasodilation produced by increasing cumulative concentrations of acetylcholine (A) and sodium nitroprusside (B) in aortic rings of XNT-treated or untreated hypertensive rats (SHR). Vasodilatory effects induced by increasing cumulative concentrations of acetylcholine (C) or sodium nitroprusside (D) in aortic rings of XNT-treated or untreated diabetic rats. *p<0.05 (Two-way ANOVA followed by the Bonferroni’s multiple comparison test). Each point represents the mean ± SEM (n=7 to 10). n.s.: non-significant.

XNT Produces Vasorelaxant Responses Associated with Mas Activation

XNT caused concentration dependent vasorelaxation in aortic rings of Sprague-Dawley rats pre-constricted with phenylephrine (Figure 2A). Using the submaximal concentration of 10μmol/L, XNT promoted a time-dependent vasorelaxation with maximal effect reached after 7 minutes (Figure 2B). To evaluate the participation of the endothelium in the vasorelaxant effects of XNT, aortic rings of rats with or without intact endothelium were incubated with this compound. It was found that the XNT effects were dependent on the endothelial cells (Figure 2C).

Figure 2.

Figure 2

Figure 2

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XNT produces Mas-mediated vasodilation. Vasodilation produced by increasing cumulative concentrations of XNT (A) and time curve of the submaximal concentration (10μmol/L) of XNT (B). The vasodilatory effects of XNT were dependent on the intact endothelium (C). The effects of XNT (10μmol/L) were partially blocked by D-pro7-Ang-(1-7) (D) but not by A-779 (E). Also, the vasorelaxant effect of XNT (10μmol/L) was absent in aortic rings of Mas−/− mice after 5 minutes of incubation (F). The AT1 receptor antagonist Losartan did not affect the response of XNT in Mas−/− (G) and Mas+/+ (H) mice. **p<0.01 and ***p<0.001 (Two-way ANOVA followed by the Bonferroni’s multiple comparison test). Each point represents the mean ± SEM (n=8 to 10). n.s.: non-significant.

To address the mechanism by which XNT produces vasorelaxation, the vessels were pre-incubated with two different Mas antagonists. It was observed that the vasorelaxant effect of XNT was attenuated by D-pro7-Ang-(1-7) (10μmol/L) (Figure 2D). Interestingly, pre-incubation with A-779 (10μmol/L), a classical Mas antagonist, did not change its vasorelaxant activity (Figure 2E). Based on the possible participation of Mas in the vasorelaxant effects of XNT, we further evaluated the actions of this compound in vessels of Mas−/− mice. We found that XNT at 10μmol/L caused similar vasorelaxation in Mas−/− and Mas+/+ mice during the first 5 minutes of incubation. However, after this initial period the XNT effect was absence in Mas−/− mice when compared with Mas+/+ mice (Figure 2F). The initial vasodilatory effect observed in Mas−/− mice was not associated to Ang II degradation by ACE2 since incubation with losartan did not affect the vasodilator response of XNT (Figures 2G and 2H).

XNT Attenuates Oxidative Stress

Consistent evidences indicate that ROS play an important role in the development of endothelial dysfunction. Thus, we investigated the participation of the oxidative stress in the effects of XNT on the endothelial function of diabetic Wistar rats. Specifically, we evaluated the ROS production in aortic vessels of diabetic animals. Diabetes caused an increase in the generation of ROS, which was significantly reduced by XNT treatment (Figure 3). No significant changes were observed in the expression of catalase, SOD and NOX2 in aorta of diabetic rats treated or not with XNT (please see Figure S7 of the online Data Supplement at http://hyper.ahajournals.org).

Figure 3.

Figure 3

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XNT treatment reduces reactive oxygen species (ROS) content in aortic rings of diabetic rats. Representative photomicrographs of descending thoracic aorta sections showing the ROS production using DHE (2μmol/L) in non-diabetic rats (A), vehicle-treated diabetic rats (B) and XNT-treated diabetic rats (C). Quantification of ROS content (D). *p<0.05 (One-way ANOVA followed by the Bonferroni’s multiple comparison test). Each column represents the mean ± SEM (n=6 to 8) of relative fluorescence in arbitrary unit (A.U.).

Additionally, we tested if XNT is able to reduce the Ang II inducing ROS production in HAEC. It was observed that XNT treatment attenuated the ROS production stimulated by Ang II. XNT alone did not change the basal level of ROS (please see Figure S8 of the online Data Supplement at http://hyper.ahajournals.org).

DISCUSSION

The beneficial role of ACE2 in the pathophysiology of cardiovascular and metabolic diseases is currently under intensive investigation. In fact, the involvement of this enzyme in cardiac function, hypertension, atherosclerosis and other cardiovascular diseases has recently been demonstrated.6,17,18-20,23-27 The most significant finding of the present study is that pharmacological ACE2 activation using XNT exerts protective effects on endothelial function. Furthermore, we showed that this action involves the Mas receptor and reduction of ROS production.

As the main ACE2 enzymatic function is degrading Ang II with consequent production of Ang-(1-7), the likely mechanism underlying the XNT effects is balancing the bioavailability of these two peptides. Indeed, we observed that the Mas antagonist D-pro7-Ang-(1-7) attenuated the vasorelaxant response elicited by XNT, thereby indicating the involvement of Ang-(1-7)/Mas in this effect. Unexpectedly, A-779, a classical Mas antagonist, did not affect this action. In spite of these apparent contradictory results, these findings are in keeping with previous studies showing that the vasorelaxant effect of Ang-(1-7) in aortic rings of Sprague-Dawley rats was blocked by D-pro7-Ang-(1-7) but not by A-779.28 Nowadays, there is only one Ang-(1-7) receptor identified, i.e. Mas receptor.8 Usually, this receptor is blocked by A-779 and D-Pro7-Ang-(1-7).29 However, as mentioned above, in certain situations one of these antagonists is not efficient or is only partially effective in blocking the Ang-(1-7) effects.28 This strongly suggests the existence of other unidentified Ang-(1-7) receptors. To further evaluate the role of Ang-(1-7)/Mas in the effects of XNT, we measured the plasma and aortic Ang-(1-7) levels and tested the XNT effects in isolated aortic rings of Mas deficient mice. It was observed that ACE2 activation significantly increased the concentration of this peptide in the plasma. The results obtained in Mas−/− mice showed that, during the first five minutes of incubation, XNT induced vasorelaxation in Mas−/− and Mas+/+ mice. However, after ten minutes the effects of XNT were absence in Mas−/−, indicating that Mas is involved in the vascular response of XNT. One may suggest that the initial vasodilatory effect observed in Mas−/− mice is due to degradation of Ang II by ACE2. Nevertheless, this hypothesis is not plausible since incubation of XNT associated with losartan did not block this vasodilatory response. Therefore, further experiments are required to explain this observation. Altogether, these findings indicated that the vasorelaxant effects of XNT are dependent on Ang-(1-7)/Mas axis.

The partial blockade (~50%) of the XNT actions by D-Pro7-Ang-(1-7) was an expected finding since ACE2 is an enzyme with a dual role within the RAS, i.e. it degrades Ang II with consequent production of Ang-(1-7). Thus, the residual effect of XNT in the presence of D-Pro7-Ang-(1-7) might be due to the reduction of the Ang II content. However, we did not observe any significant decrease in plasma Ang II levels in diabetic rats treated with XNT. Furthermore, increases in ACE2 protein and mRNA expression are a frequent finding when using ACE2 activators.18 This suggests that these compounds not only induce their beneficial effects by forming Ang-(1-7) and/or degrading Ang II, but a non-identified mechanism is also present. Nevertheless, in our study XNT was unable to increase the ACE2 protein expression maybe due to the duration of the experimental protocol. Thus, further investigations are necessary to clearly identify the mechanisms of action of XNT.

The ROS are intracellular and intercellular second messengers that modulate the endothelial function.30 Under pathological conditions, elevation of the ROS content, the so-called oxidative stress, results in vascular dysfunction.31 In fact, consistent evidences indicate that ROS play an important role in the development of organ damage in diabetes and hypertension.30,32 Ang II elicits many of its pathophysiological effects by stimulating ROS generation through the reduction of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Moreover, treatment with free radical scavengers, such as SOD, catalase and tempol, reduces the vascular damage in response to Ang II.12,13 In contrast, it has been reported that Ang-(1-7) via Mas ameliorates endothelial dysfunction in animal models by decreasing the oxidative stress.14-16 In accordance with these findings, we showed that chronic treatment with XNT reduced the ROS content in aorta of diabetic rats to a similar level observed in non-diabetic animals. Additionally, XNT treatment was also able to reduce the Ang II-induced ROS production in HAEC. No significant changes were observed in the aortic expression of catalase, SOD and NOX2 among any of the groups. Taking into account that these enzymes are critical ROS scavengers, our results suggest that the modulation of the expression of these enzymes is not a mechanism by which XNT reduces the ROS production in aorta. Moreover, it is pertinent to note that the effects of XNT on ROS generation must be evaluated under in vivo condition to further confirm the association between XNT treatment and oxidative stress reduction.

In attempting to explain the mechanisms by which XNT improves the endothelial function of diabetic rats, acute experiments evaluating the role of the endothelium and Mas receptor were run in rings extracted from normal animals. Thus, the translation of these data obtained in normal rats to diabetic and hypertensive animals must be done carefully. However, they are an evidence of the mechanisms involved in the beneficial endothelial actions of XNT in these pathological conditions. Also, the hypertensive model was less explored in our study and it was used with the intention of adding more generality to our data.

PERSPECTIVES

Our present study demonstrated that XNT improves the endothelial function of hypertensive and diabetic rats. These actions involved the Mas receptor and decreases in ROS production. Thus, these results indicate that pharmacological ACE2 activation by XNT promotes beneficial effects on the endothelial function and that this compound is a lead molecule to develop potential therapeutic strategies and drugs to treat cardiovascular and metabolic diseases by improving the endothelial function.

Supplementary Material

Supplementary Material

NOVELTY AND SIGNIFICANCE.

1. What Is New

Our study indicates that activation of intrinsic angiotensin-converting enzyme2 (ACE2), a recently described member of the renin-angiotensin system, using a small molecule ACE2 activator named XNT improves endothelial function by decreasing the generation of reactive oxygen species (ROS).

2. What Is Relevant?

The balance between release and function of endothelium-derived nitric oxide and ROS production is critical in endothelial dysfunction. Therapies targeting the reestablishment of this balance might have significant implications in hypertension and cardiovascular diseases management. Thus, our study proposes ACE2 activation as a potential target for treating hypertension and related cardiovascular diseases.

3. Summary

Our results show that XNT administration ameliorates the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which is mediated by Mas.

Acknowledgments

SOURCES OF FUNDING

This work was partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-Brazil) and National Institutes of Health (NIH-USA).

Footnotes

CONFLICT OF INTEREST/DISCLOSURE

None.

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Supplementary Materials

Supplementary Material
NIHMS475952-supplement-01.pdf (1.2MB, pdf)

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