Possible Role of Angiotensin-Converting Enzyme 2 and Activation of Angiotensin II Type 2 Receptor by Angiotensin-(1−7) in Improvement of Vascular Remodeling by Angiotensin II Type 1 Receptor Blockade Kousei Ohshima, Masaki Mogi, Hirotomo Nakaoka, Jun Iwanami, Li-Juan Min, Harumi Kanno, Kana Tsukuda, Toshiyuki Chisaka, Hui-Yu Bai, Xiao-Li Wang, Akiyoshi Ogimoto, Jitsuo Higaki and Masatsugu Horiuchi Hypertension. 2014;63:e53-e59; originally published online December 30, 2013; doi: 10.1161/HYPERTENSIONAHA.113.02426 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2013 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/63/3/e53
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2013/12/30/HYPERTENSIONAHA.113.02426.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Downloaded from http://hyper.ahajournals.org/ at UNIVERSITY OF LEEDSPERIODICA on August 20, 2014
Original Articles Part 2 Possible Role of Angiotensin-Converting Enzyme 2 and Activation of Angiotensin II Type 2 Receptor by Angiotensin-(1–7) in Improvement of Vascular Remodeling by Angiotensin II Type 1 Receptor Blockade Kousei Ohshima, Masaki Mogi, Hirotomo Nakaoka, Jun Iwanami, Li-Juan Min, Harumi Kanno, Kana Tsukuda, Toshiyuki Chisaka, Hui-Yu Bai, Xiao-Li Wang, Akiyoshi Ogimoto, Jitsuo Higaki, Masatsugu Horiuchi Abstract—Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis and the ACE2/Ang-(1–7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Furthermore, possible stimulation of the Ang II type 2 (AT2) receptor by Ang-(1–7) has been highlighted as a new pathway. Therefore, we examined the possibility of whether the ACE2/Ang-(1–7)/Mas axis and Ang-(1–7)/AT2 receptor axis are involved in the inhibitory effects of AT1 receptor blockers on vascular remodeling. Wild-type, M as-knockout, and AT2 receptor knockout mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. Some mice were treated with azilsartan, an AT1 receptor blocker, or Ang-(1–7). Neointimal formation 2 weeks after cuff placement was more marked in Mas-knockout mice compared with wild-type mice. Treatment with azilsartan or Ang-(1–7) attenuated neointimal area, vascular smooth muscle cell proliferation, increases in the mRNA levels of monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1β, and superoxide anion production in the injured artery; however, these inhibitory effects of azilsartan and Ang-(1–7) were less marked in Mas-knockout mice. Administration of azilsartan or Ang-(1–7) attenuated the decrease in ACE2 mRNA and increased AT2 receptor mRNA but did not affect AT1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1–7) on neointimal formation was less marked in AT2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1–7)/Mas axis and ACE2/Ang-(1–7)/AT2 receptor axis, thereby inhibiting neointimal formation. (Hypertension. 2014;63:e53-e59.) Online Data Supplement
•
Key Words: angiotensin ◼ angiotensin-converting enzyme 2 ◼ inflammation ◼ oxidative stress ◼ receptors ◼ remodeling
I
t has been suggested that the angiotensin-converting enzyme (ACE) 2/angiotensin-(1–7) (Ang-(1–7))/Mas pathway exerts an antagonistic action in many physiological and pathophysiological processes in several systems and organs, including cardiovascular remodeling, via opposing the classical ACE/Ang II/Ang II type 1 (AT1) receptor axis–mediated action.1,2 Recent studies have suggested that Ang-(1–7), generated by ACE2, has vasoprotective and atheroprotective effects in several animal models.3–5 Ang-(1–7) is synthesized from Ang I and Ang II mainly via ACE2 activity. The effects of Ang-(1– 7) could appear as the balance between the ACE2/Ang-(1–7)/ Mas axis and the ACE/Ang II/AT1 receptor axis, which seems to be switched on by ACE2. Accordingly, it is possible that the increase in Ang-(1–7) level during ACE inhibition and AT1
receptor blockade could result in Mas receptor activation and the induction of cardioprotective and renoprotective effects.6 In addition, it has been suggested that AT1 receptor blockade directly activates the ACE2/Ang-(1–7)/Mas pathway. A previous report suggested that AT1 receptor stimulation regulated ACE2 and Ang-(1–7) expression in the aorta of spontaneously hypertensive rats.7 AT1 receptor stimulation downregulated ACE2 via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway mediated by AT1 receptor stimulation.8 These results suggest that AT1 receptor stimulation inhibited ACE2 expression, whereas AT1 receptor blockade increased it. Consistent with these observations, we demonstrated that in AT1a receptor knockout mice, mRNA expression and immunostaining of ACE2 and
Received September 25, 2013; first decision October 9, 2013; revision accepted November 19, 2013. From the Departments of Molecular Cardiovascular Biology and Pharmacology (K.O., M.M., H.N., J.I., L.-J.M., H.K., K.T., H.-Y.B., X.-L.W., M.H.), Cardiology, Pulmonology, Hypertension and Nephrology (K.O., A.O., J.H.), and Pediatrics (T.C.), Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 113.02426/-/DC1. Correspondence to Masatsugu Horiuchi, Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. E-mail [email protected] © 2013 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.113.02426
Downloaded from http://hyper.ahajournals.org/ at UNIVERSITY OF LEEDSPERIODICA on August 20, 2014 e53
e54 Hypertension March 2014 Mas in the injured artery were greater, with less neointimal formation than in wild-type (WT) mice, and that increases in ACE2 expression and neointimal formation in the injured artery were also observed with treatment with an AT1 receptor blocker (ARB), olmesartan, suggesting that AT1 receptor blockade by ARBs seemed to alter the balance between the ACE2/Ang-(1–7)/Mas axis and the ACE/Ang II/AT1 receptor axis to improve vascular remodeling.9 Ang II type 2 (AT2) receptor stimulation is known to be involved in the beneficial effects of ARBs on cardiovascular remodeling. In addition to cross talk between the AT1 receptor and Mas, the role of the AT2 receptor in terms of the effects mediated by the ACE2/Ang-(1–7)/Mas pathway, distinguishing the Mas and AT2 receptor signaling pathways, has been highlighted.10 Previously, it was reported that Ang-(1–7) exerts a role as a vasodepressor via AT2 receptor activation in the presence of partial AT1 receptor blockade11 and that the vasoprotective and atheroprotective effects of Ang-(1–7) are mediated by restoration of nitric oxide bioavailability via both the Mas and AT2 receptors.4 It is reported that AT2 receptor expression in the femoral artery was increased after cuff placement.12 These results led us to examine the possibility that the Ang-(1–7)/AT2 receptor pathway is involved in the inhibitory effects of ARB on vascular remodeling using Mas-knockout (MasKO) mice and AT2 receptor knockout (AT2KO) mice.
Methods This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The experimental protocol was approved by the Animal Studies Committee of Ehime University.
Animals and Treatment AT2KO (C57BL/6J background)13 mice and MasKO mice (C57BL/6J background) aged 10 to 11 weeks, weighing 25 to 30 g, were used. WT (C57BL/6J) mice were used as control. Animal treatment and targeted disruption of the Mas gene are presented in Methods in the online-only Data Supplement.
Morphometric Analysis and Immunohistochemical Staining The femoral arteries were taken 14 days after cuff placement and fixed with 10% neutral-buffered formalin. Paraffin-embedded cross-sections were prepared as described previously.12 Samples were examined with a Zeiss Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system.14
Dihydroethidium Staining Superoxide generation in cryostat frozen section was evaluated using fluorogenic dihydroethidium (5 μmol/L), as described previously.15 The intensity of fluorescence was analyzed and quantified using computer imaging software (Densitograph, ATTO Corp).
Quantitative Real-Time Polymerase Chain Reaction Please see the online-only Data Supplement.
Statistical Analysis All values are expressed as mean±SD in the text and figures. Data were evaluated by ANOVA. If a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. Values of P
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/63/3/e53
Data Supplement (unedited) at: http://hyper.ahajournals.org/content/suppl/2013/12/30/HYPERTENSIONAHA.113.02426.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Hypertension is online at: http://hyper.ahajournals.org//subscriptions/
Downloaded from http://hyper.ahajournals.org/ at UNIVERSITY OF LEEDSPERIODICA on August 20, 2014
Original Articles Part 2 Possible Role of Angiotensin-Converting Enzyme 2 and Activation of Angiotensin II Type 2 Receptor by Angiotensin-(1–7) in Improvement of Vascular Remodeling by Angiotensin II Type 1 Receptor Blockade Kousei Ohshima, Masaki Mogi, Hirotomo Nakaoka, Jun Iwanami, Li-Juan Min, Harumi Kanno, Kana Tsukuda, Toshiyuki Chisaka, Hui-Yu Bai, Xiao-Li Wang, Akiyoshi Ogimoto, Jitsuo Higaki, Masatsugu Horiuchi Abstract—Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis and the ACE2/Ang-(1–7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Furthermore, possible stimulation of the Ang II type 2 (AT2) receptor by Ang-(1–7) has been highlighted as a new pathway. Therefore, we examined the possibility of whether the ACE2/Ang-(1–7)/Mas axis and Ang-(1–7)/AT2 receptor axis are involved in the inhibitory effects of AT1 receptor blockers on vascular remodeling. Wild-type, M as-knockout, and AT2 receptor knockout mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. Some mice were treated with azilsartan, an AT1 receptor blocker, or Ang-(1–7). Neointimal formation 2 weeks after cuff placement was more marked in Mas-knockout mice compared with wild-type mice. Treatment with azilsartan or Ang-(1–7) attenuated neointimal area, vascular smooth muscle cell proliferation, increases in the mRNA levels of monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1β, and superoxide anion production in the injured artery; however, these inhibitory effects of azilsartan and Ang-(1–7) were less marked in Mas-knockout mice. Administration of azilsartan or Ang-(1–7) attenuated the decrease in ACE2 mRNA and increased AT2 receptor mRNA but did not affect AT1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1–7) on neointimal formation was less marked in AT2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1–7)/Mas axis and ACE2/Ang-(1–7)/AT2 receptor axis, thereby inhibiting neointimal formation. (Hypertension. 2014;63:e53-e59.) Online Data Supplement
•
Key Words: angiotensin ◼ angiotensin-converting enzyme 2 ◼ inflammation ◼ oxidative stress ◼ receptors ◼ remodeling
I
t has been suggested that the angiotensin-converting enzyme (ACE) 2/angiotensin-(1–7) (Ang-(1–7))/Mas pathway exerts an antagonistic action in many physiological and pathophysiological processes in several systems and organs, including cardiovascular remodeling, via opposing the classical ACE/Ang II/Ang II type 1 (AT1) receptor axis–mediated action.1,2 Recent studies have suggested that Ang-(1–7), generated by ACE2, has vasoprotective and atheroprotective effects in several animal models.3–5 Ang-(1–7) is synthesized from Ang I and Ang II mainly via ACE2 activity. The effects of Ang-(1– 7) could appear as the balance between the ACE2/Ang-(1–7)/ Mas axis and the ACE/Ang II/AT1 receptor axis, which seems to be switched on by ACE2. Accordingly, it is possible that the increase in Ang-(1–7) level during ACE inhibition and AT1
receptor blockade could result in Mas receptor activation and the induction of cardioprotective and renoprotective effects.6 In addition, it has been suggested that AT1 receptor blockade directly activates the ACE2/Ang-(1–7)/Mas pathway. A previous report suggested that AT1 receptor stimulation regulated ACE2 and Ang-(1–7) expression in the aorta of spontaneously hypertensive rats.7 AT1 receptor stimulation downregulated ACE2 via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway mediated by AT1 receptor stimulation.8 These results suggest that AT1 receptor stimulation inhibited ACE2 expression, whereas AT1 receptor blockade increased it. Consistent with these observations, we demonstrated that in AT1a receptor knockout mice, mRNA expression and immunostaining of ACE2 and
Received September 25, 2013; first decision October 9, 2013; revision accepted November 19, 2013. From the Departments of Molecular Cardiovascular Biology and Pharmacology (K.O., M.M., H.N., J.I., L.-J.M., H.K., K.T., H.-Y.B., X.-L.W., M.H.), Cardiology, Pulmonology, Hypertension and Nephrology (K.O., A.O., J.H.), and Pediatrics (T.C.), Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 113.02426/-/DC1. Correspondence to Masatsugu Horiuchi, Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. E-mail [email protected] © 2013 American Heart Association, Inc. Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.113.02426
Downloaded from http://hyper.ahajournals.org/ at UNIVERSITY OF LEEDSPERIODICA on August 20, 2014 e53
e54 Hypertension March 2014 Mas in the injured artery were greater, with less neointimal formation than in wild-type (WT) mice, and that increases in ACE2 expression and neointimal formation in the injured artery were also observed with treatment with an AT1 receptor blocker (ARB), olmesartan, suggesting that AT1 receptor blockade by ARBs seemed to alter the balance between the ACE2/Ang-(1–7)/Mas axis and the ACE/Ang II/AT1 receptor axis to improve vascular remodeling.9 Ang II type 2 (AT2) receptor stimulation is known to be involved in the beneficial effects of ARBs on cardiovascular remodeling. In addition to cross talk between the AT1 receptor and Mas, the role of the AT2 receptor in terms of the effects mediated by the ACE2/Ang-(1–7)/Mas pathway, distinguishing the Mas and AT2 receptor signaling pathways, has been highlighted.10 Previously, it was reported that Ang-(1–7) exerts a role as a vasodepressor via AT2 receptor activation in the presence of partial AT1 receptor blockade11 and that the vasoprotective and atheroprotective effects of Ang-(1–7) are mediated by restoration of nitric oxide bioavailability via both the Mas and AT2 receptors.4 It is reported that AT2 receptor expression in the femoral artery was increased after cuff placement.12 These results led us to examine the possibility that the Ang-(1–7)/AT2 receptor pathway is involved in the inhibitory effects of ARB on vascular remodeling using Mas-knockout (MasKO) mice and AT2 receptor knockout (AT2KO) mice.
Methods This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The experimental protocol was approved by the Animal Studies Committee of Ehime University.
Animals and Treatment AT2KO (C57BL/6J background)13 mice and MasKO mice (C57BL/6J background) aged 10 to 11 weeks, weighing 25 to 30 g, were used. WT (C57BL/6J) mice were used as control. Animal treatment and targeted disruption of the Mas gene are presented in Methods in the online-only Data Supplement.
Morphometric Analysis and Immunohistochemical Staining The femoral arteries were taken 14 days after cuff placement and fixed with 10% neutral-buffered formalin. Paraffin-embedded cross-sections were prepared as described previously.12 Samples were examined with a Zeiss Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system.14
Dihydroethidium Staining Superoxide generation in cryostat frozen section was evaluated using fluorogenic dihydroethidium (5 μmol/L), as described previously.15 The intensity of fluorescence was analyzed and quantified using computer imaging software (Densitograph, ATTO Corp).
Quantitative Real-Time Polymerase Chain Reaction Please see the online-only Data Supplement.
Statistical Analysis All values are expressed as mean±SD in the text and figures. Data were evaluated by ANOVA. If a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. Values of P
