loss of angiotensin-converting enzyme 2 exacerbates myocardial injury via activation of the...

Circulation Journal Vol.77, December 2013

Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

he renin-angiotensin system (RAS) has been implicat-ed in pathological hypertrophy and myocardial re-modeling,1–3 changes that may cause cardiac dysfunc-

tion, myocardial stiffness, and increased clinical risk of heart failure and sudden death.4,5 Angiotensin (Ang) II has recently been shown to augment release of monocyte chemoattractant protein-1 (MCP-1) via its type I (AT1) receptor,6,7 leading to myocardial remodeling and injury.8,9 In addition, AngII pro-motes activation of mitogen-activated protein kinase (MAPK)/

extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2)1,5,10,11 and connective tissue growth factor (CTGF) signaling,10,12 which may further increase the release of inflammatory chemo-kines such as fractalkine (FKN) and MCP-1.10 Activation of the MAPK/ERK signaling pathways by various stimuli has been correlated with several cellular processes such as cell prolifera-tion and remodeling of extracellular matrix (ECM).11,13,14 Fur-thermore, AngII increases the synthesis and deposition of ECM proteins by modulation of the expression and activities

T

Received June 26, 2013; revised manuscript received August 2, 2013; accepted August 23, 2013; released online October 26, 2013 Time for primary review: 13 days

State Key Laboratory of Medical Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (B.S., Z.-Z.Z., J.-C.Z., H.-Y.J., Y.-L.X., P.-J.G., D.-L.Z.); Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Shanghai (B.S., Z.-Z.Z., J.-C.Z., Y.-L.X., P.-J.G., D.-L.Z.); Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (L.L., W.-F.S.); Research Center of Medical Science, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou (X.-Y.Y.), China; and Division of Cardiology, Department of Medicine, University of Alberta, Ma-zankowski Alberta Heart Institute, Edmonton (G.Y.O., Z.K.), Canada

The first two authors contributed equally to this work (B.S., Z.-Z.Z.).*Mailing address: Jiu-Chang Zhong, MD, 197 Ruijin 2nd Road, State Key Laboratory of Medical Genomics and Shanghai Institute of

Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. E-mail: [email protected]

ISSN-1346-9843 doi: 10.1253/circj.CJ-13-0805All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Myocardial Injury via Activation of the CTGF-Fractalkine Signaling Pathway

Bei Song, BSc; Zhen-Zhou Zhang, BSc; Jiu-Chang Zhong, MD*; Xi-Yong Yu, MD, PhD; Gavin Y. Oudit, MD, PhD; Hai-Yan Jin, MD; Lin Lu, MD; Ying-Le Xu, BSc; Zamaneh Kassiri, PhD;

Wei-Feng Shen, MD; Ping-Jin Gao, MD, PhD; Ding-Liang Zhu, MD, PhD

Background: Angiotensin-converting enzyme 2 (ACE2) has been implicated in human heart failure, but the mech-anism remains elusive. We hypothesized that ACE2 deficiency would exacerbate angiotensin (Ang) II-mediated myocardial injury.

Methods and Results: 10-week-old ACE2 knockout (ACE2KO) and wild-type mice received by mini-osmotic pump either AngII (1.5 mg · kg–1 · day–1) or saline for 2 weeks. ACE2 deficiency triggered greater increases in the expression of connective tissue growth factor (CTGF), fractalkine (FKN) and phosphorylated ERK1/2 in AngII-treated ACE2KO hearts. These changes were associated with greater activation of matrix metalloproteinase (MMP) 2, MMP9 and MT1-MMP and exacerbation of myocardial injury and dysfunction. In cultured cardiofibroblasts, exposure to AngII (100 nmol/L) for 30 min resulted in marked increases in superoxide production and expression of CTGF, FKN and phosphorylated ERK1/2, which were strikingly prevented by recombinant human ACE2 (rhACE2; 1 mg/ml) and the CTGF-neutralizing antibody (5 μg/ml), but were aggravated by ACE2 inhibitor DX600 (0.5 μmol/L). These protective effects of rhACE2 were eradicated by the Ang-(1–7) antagonist A779 (1 μmol/L). More intriguingly, rhACE2 treatment significantly abolished AngII-mediated increases in MMP2, MMP9 and MT1-MMP in cardiofibroblasts.

Conclusions: Loss of ACE2 exacerbates AngII-mediated inflammation, myocardial injury and dysfunction in ACE2-deficient hearts via activation of the CTGF-FKN-ERK and MMP signaling. ACE2 gene may represent a potential candidate to prevent and treat myocardial injury and heart diseases. (Circ J 2013; 77: 2997 – 3006)

Key Words: Angiotensin-converting enzyme 2; Connective tissue growth factor; Fractalkine; Matrix metalloprotein-ase; Myocardial injury

ORIGINAL ARTICLEMyocardial Disease


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School of Medicine and the Canadian Council on Animal Care.

Echocardiography and Myocardial Ultrastructure ObservationTransthoracic echocardiography was performed and analyzed in a blinded manner, as described previously, with a Vevo 770 high-resolution imaging system equipped with a 30-MHz trans-ducer (RMV-707B; Visualsonics, Toronto, ON, Canada).3,8 For transmission electron microscopic analysis, samples of mice heart tissues (left ventricles) were immediately cut into small pieces and immersed in 2.5% glutaraldehyde as described previ-ously.19 The myocardial ultrastructure of the WT and ACE2KO mice was observed on a HITACHI-600 electron microscope (Hitachi, Japan).

Culture of CardiofibroblastsAdult murine LV cardiofibroblasts were isolated and cultured as described previously.3 The cardiofibroblasts were put into serum-free DMEM for 18 h prior to treatment. Recombinant human ACE2 (rhACE2; 1 mg/ml), the Ang-(1–7) antagonist, A779 (1 μmol/L; Sigma-Aldrich, St. Louis, MO, USA), ACE2 inhibitor DX600 (0.5 μmol/L), ERK1/2 inhibitor PD98059 (10 μmol/L), Ang-(1–7) (100 nmol/L), CTGF-neutralizing an-tibody (monoclonal anti-CTGF, 5 μg/ml; Sigma-Aldrich), and IgG (5 μg/ml) were added to the cardiofibroblasts for 30 min prior to 30-min exposure to AngII (100 nmol/L; Sigma-Al-drich). The treated cells further underwent dihydroethidium (DHE) staining or were collected for TaqMan real-time poly-merase chain reaction (PCR) and Western blotting analyses.

Gelatin Zymography and TaqMan Real-Time PCR AnalysisPro- and cleaved forms of MMP2 and MMP9 were detected by gelatin zymography analysis as described previously.11 The mRNA expression levels were evaluated by TaqMan real-time reverse transcription PCR as described before.8,9,17 Total RNA was extracted from heart tissues and cardiofibroblasts using TRIzol reagent. The primers and probes for α-smooth muscle actin (α-SMA), MCP-1, tumor necrosis factor-α (TNFα), MMP2, MMP9 and MT1-MMP are listed in Table S1.

Western Blot AnalysisThe proteins from heart tissues or cardiofibroblasts were mea-

of matrix metalloproteinase (MMP), an effect that may also drive pathological remodeling and cardiac injury.5 In mice with cardiac remodeling and injury, MMP-2, MMP-9 and mem-brane type 1 (MT1)-MMP, a cell surface activator of MMP-2, were overexpressed in the heart associated with activation of CTGF signaling.1,5,11 However, the roles of the CTGF-FKN and MMP signaling pathways involved in myocardial injury remain to be fully elucidated.

A new counterbalancing arm of the RAS is now known to exist, in which angiotensin-converting enzyme 2 (ACE2) de-grades AngII to the beneficial heptapeptide Ang-(1–7), thereby functioning essentially as a negative regulator of the RAS.2,3,15–19 In the heart, ACE2 is expressed in cardiomyocytes, fibro-blasts, and endothelial cells.3,8,11,18 Our previous studies have demonstrated that ACE2 overexpression prevents AngII-me-diated cardiovascular inflammation and injury associated with reduced MMP2 level,3,8,11 suggesting a critical role of ACE2 in the regulation of cardiovascular injury and dysfunction. However, the exact roles and mechanisms of ACE2 in the cardiovascular system remain largely unknown. In this work, we assessed the hypothesis that loss of ACE2 would acceler-ate myocardial injury. We randomized ACE2 knockout mice (ACE2KO, Ace2–/y) and wild-type littermates (WT, Ace2+/y) to either AngII or saline infusion, as in a previous study.3,20

MethodsExperimental Animals and Protocols10-week-old male WT and ACE2KO mice were randomized to either AngII (1.5 mg · kg–1 · day–1) or saline (control) infusion with an osmotic minipump (model 1002, Alzet Corp, Palo Alto, CA, USA) for 2 weeks, as in previous studies.3,20 Systolic blood pressure (SBP) was measured noninvasively by the tail-cuff method with an IITC Blood Pressure Monitoring System (Life Science Inc, St. Petersburg, Fl, USA). Plasma AngII and Ang-(1–7) levels were measured by radioimmunoassay as described previously.3,11 All experiments were approved and performed in accordance with the Guide for the Care and Use of Labora-tory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), the Animal Re-search Ethics Committee at Shanghai Jiao Tong University

Table. General Data for the Study Mice

Ace2+/y control Ace2+/y+AngII Ace2–/y control Ace2–/y+AngII

n 6 8 6 8

SBP (mmHg) 105.2±2.8　　　　156.4±4.0**　　 123.5±2.5Ø 178.8±5.3**,#　　　　HR (beats/min) 496±11 501±12 491±15 503±11　　　　　　　BW (g) 26.53±0.72 25.83±0.61 26.27±0.69 25.81±0.50　　　　　　　LVW (mg) 9.95±0.38 　12.20±0.27* 11.95±0.29Ø 14.18±0.38*,#　　　

LVW/BW (mg/g) 0.37±0.01 　0.48±0.02* 0.46±0.01Ø 0.55±0.02*,#　　　

LVEDD (mm) 3.83±0.03 3.85±0.03 3.91±0.05 4.02±0.04#　　　　　

LVESD (mm) 1.75±0.04 1.82±0.03 1.83±0.04 2.07±0.04**,#　　

LVFS (%) 54.324±0.928 52.860±0.704 53.291±0.664 48.394±0.682*,#　　　

LVEF (%) 62.033±3.002 57.501±1.813 58.450±2.869 45.275±2.174**,##

LVPWT (mm) 0.61±0.03 　　0.84±0.04** 0.76±0.02Ø 1.05±0.03**,#　　

Plasma AngII (pg/ml) 43.9±2.8 　　481.9±19.5** 58.3±3.0Ø 640.4±22.3**,##

Plasma Ang-(1–7) (pg/ml) 56.2±2.8 　　104.0±5.4**　　 38.85±2.2Ø 46.8±2.5**,#　　

Results are presented as mean ± SEM. *P<0.05, **P<0.01 compared with corresponding control group; #P<0.05, ##P<0.01 compared with Ace2+/y+AngII group; ØP<0.05 compared with Ace2+/y control group.BW, body weight; HR, heart rate; LV, left ventricular; LVEDD, LV end-diastolic diameter; LVEF, LV ejection fraction; LVESD, LV endsystolic diameter; LVFS, LV fractional shortening; LVPWT, LV posterior wall thickness; LVW, LV weight; SBP, systolic blood pressure.


2999Loss of ACE2 Exacerbates Myocardial Injury

described previously.20 Data were calculated as the changes in the rate of luminescence per minute.

Statistical AnalysisValues are shown as mean ± SEM. All statistical analyses were performed with SPSS software (Version 11.5) either by Stu-dent’s t test for comparison between groups or by ANOVA followed by the Student-Newman-Keuls test for multiple-comparison testing as appropriate. A value of P<0.05 was considered statistically significant.

ResultsLoss of ACE2 Facilitated AngII-Mediated Hypertension and Myocardial Inflammation With Greater Activation of the CTGF-FKN and ERK SignalingWe firstly evaluated the effects of ACE2 deficiency on the SBP and cardiac CTGF-FKN signaling. As shown in Table, compared with WT mice, SBP was obviously elevated in

sured by Western blot analysis as described previously.3 Total protein was extracted using the BCA Protein Array Kit (Pierce, Rockford, IL, USA). The primary antibodies against ERK1/2 (44/42 kD), phospho-ERK1/2 (44/42 kD), CTGF (38 kDa), FKN (76 kDa), MMP2 (72 kDa; 63 kDa), MMP9 (90 kDa) and α-tubulin (55 kDa) were obtained from Abgent Biotech Co (San Diego, CA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively.

Measurement of Superoxide Production and NADPH Oxidase ActivityOxidative stress is generally identified by indirect markers of the oxidant injury, such as superoxide. To evaluate superoxide production in cardiofibroblasts, the oxidative fluorescent dye, DHE, was used as described previously.8,17 The NADPH oxi-dase activity in cardiofibroblasts was quantified by lucigenin-enhanced chemiluminescence assay using a Berthold FB12 luminometer (Berthold Technologies, Germany) at 37°C, as

Figure 1. Cardiac expression of CTGF, FKN, phosphorylated ERK1/2 and inflammatory cytokines in mice. (A–C) Representative Western blot images of the cardiac expression of CTGF (A), FKN (B) and phosphorylated ERK1/2 (C) in wild-type (WT) and an-giotensin-converting enzyme 2 (ACE2) knockout (KO) mice in response to angiotensin (Ang) II (n=5 for each group). (D–F) TaqMan real-time PCR analysis shows mRNA expression of inflammatory cytokines MCP-1 (D), TNFα (E) and α-SMA (F) in the heart of mice (n=6 for the Ace2+/y control group and Ace2–/y control group; n=8 for the Ace2+/y AngII group and Ace2–/y AngII group). α-tubulin or 18S rRNA was used as an endogenous control. **P<0.01 compared with corresponding control group; #P<0.05 compared with Ace2+/y AngII group; ØP<0.05 compared with Ace2+/y control group. R.E., relative expression; CTGF, connective tissue growth factor; FKN, fractalkine; ERK1/2, extracellular signal-regulated kinase 1/2; MCP-1, monocyte chemoattractant protein-1; TNF, tumor necrosis factor; SMA, smooth muscle actin.


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tion, another key determinant of adverse myocardial injury.11 We next investigated AngII-induced cardiac inflammation and the associated signaling cascades in ACE2-deficiency status. Compared with WT mice, there were marked increases in the expression of CTGF (Figure 1A), FKN (Figure 1B), phos-phorylated ERK1/2 (Figure 1C) and MCP-1 (Figure 1D) in ACE2-deficient hearts (n=5–6; P<0.05, respectively). Western blotting and TaqMan real-time PCR analyses revealed that lack of ACE2 resulted in greater increases in AngII-induced expression of CTGF (Figure 1A), FKN (Figure 1B), phos-

ACE2 mutant mice associated with a marked increase in the plasma AngII level and a reduction in the plasma Ang-(1–7) level (n=6; P<0.05). Chronic AngII infusion resulted in a predicted pressor response in both WT and ACE2-deficient mice (n=6–8; P<0.01, respectively). Moreover, loss of ACE2 resulted in greater increases in SBP at 2 weeks after AngII infusion in ACE2KO mice than in WT mice (n=6–8; P<0.05, respectively), together with a greater elevation in the AngII level and lower Ang-(1–7) level (Table).

Activation of the RAS has also been linked to inflamma-

Figure 2. Cardiac levels of MMP2, MMP9, and MT1-MMP in mice. (A–C) TaqMan real-time PCR analysis shows the mRNA expres-sion of the key MMPs, including MMP2 (A), MMP9 (B) and membrane type 1 (MT1)-MMP (C) in mice hearts. 18S rRNA was used as an endogenous control. (n=6 for the Ace2+/y control group and Ace2–/y control group; n=8 for the Ace2+/y AngII group and Ace2–/y AngII group.) (D) Gelatin zymography showing greater cleavage and activation of MMP2, which is quantified and shown with greater MMP9 levels in the ACE2KO heart (n=5 for control group; n=6 for all other groups). (E) Representative Western blot analysis shows the cardiac protein expression of MMP2 and MMP9 in wild-type (WT) and ACE2 knockout (KO) mice in response to AngII. α-tubulin was used as an endogenous control. n=5 for each group. **P<0.01 compared with corresponding control group; #P<0.05 compared with Ace2+/y AngII group; ØP<0.05 compared with Ace2+/y control group. R.E., relative expression; AU, arbitrary units; ACE2, angiotensin-converting enzyme 2; Ang, angiotensin; MMP, matrix metalloproteinase.



increased levels of MMP2 (cleaved- and pro-MMP2) and MMP9 (Figure 2D) in ACE2-deficient hearts in response to AngII (n=5–6; P<0.05, respectively). Increased levels of MMP2 and MMP9 were confirmed independently by Western blotting analysis (Figure 2E), which showed substantially greater increases in the ACE2-deficient hearts (n=5; P<0.05, respectively). Although we have not provided direct evidence for collagen degradation, these increases in MMP expression and activities provide some evidence for increased degrada-tion and dissolution of myocardial myofilaments in the ACE2-deficient hearts as assessed by ultrastructure observation with transmission electron microscopic analysis (Figures 3D,H). Compared with WT mice (Figures 3A,E), more severe myo-cardial ultrastructural injury was observed in ACE2KO hearts, including disarranged myofilaments and mild swollen mito-chondria (Figures 3C,G). Notably, in response to chronic stim-ulation by AngII, loss of ACE2 resulted in aggravated myocar-dial ultrastructural injury in ACE2-null mice (Figures 3D,H), characterized by disruption or dissolution of myocardial myo-filaments and mitochondrial vacuolar degeneration with mem-brane lysis.

Inhibition of ACE2 Facilitated AngII-Mediated Oxidative Stress and Inflammation in Cardiofibroblasts With Greater Activation of the CTGF-FKN-ERK SignalingEnhanced oxidative stress has been linked to activation of MMPs and degradation of key components of the ECM, there-by contributing to myocardial injury.21 In cultured cardiofibro-blasts, exposure to AngII (100 nmol/L) resulted in significant increases in the expression of CTGF (Figures 4A,D), FKN (Figures 4B,E), phosphorylated ERK1/2 (Figures 4C,F) and MCP-1 (Figure 6A) as well as superoxide production (Figure 5) and NADPH oxidase activity (Figure 5), which were largely aggravated by pharmacological inhibition of ACE2 with DX600 (0.5 μmol/L) (n=5–6; P<0.01 or P<0.05, respectively). More intriguingly, pretreatment with Ang-(1–7) (100 nmol/L), ERK1/2 inhibitor PD98059 (10 μmol/L) and CTGF-neutraliz-

phorylated ERK1/2 (Figure 1C) and MCP-1 (Figure 1D) in the AngII-infused ACE2KO mice (n=5–8; P<0.05, respec-tively), without having a differential effect on the expression of TNFα (Figure 1E). These observations confirmed the det-rimental effects of ACE2 deficiency on AngII-mediated hyper-tension and cardiac inflammation associated with enhanced activation of the CTGF-FKN and ERK phosphorylation sig-naling in ACE2-null mice.

Loss of ACE2 Exacerbated AngII-Mediated Cardiac Hypertrophy and DysfunctionThe echocardiographic data revealed normal systolic function with no change in LV fractional shortening (FS) or ejection fraction (EF) between WT and ACE2KO mice (Table). There were obvious increases in LV posterior wall thickness (LVPWT) and the ratio of LV weight (LVW) and body weight (BW) in ACE2KO mice (Table; n=6; P<0.05, respectively). Notably, chronic stimulation by AngII resulted in greater concentric remodeling in the ACE2KO mice with greater increases in LVPWT, LVW and the LVW/BW ratio (Table), as well as mRNA expression of α-SMA (Figure 1E; n=6–8; P<0.05, re-spectively). Consistent with the exacerbation of AngII patho-logical hypertrophy in ACE2KO mice, AngII-induced cardiac dysfunction was greater in ACE2KO mice, based on the de-creased LVFS and LVEF (Table; n=8; P<0.05 or P<0.01, re-spectively), indicating that loss of ACE2 facilitated AngII-mediated pathological hypertrophy and cardiac dysfunction in ACE2-null mice.

Loss of ACE2 Exacerbated AngII-Induced Myocardial Ultrastructure Injury With Greater Activation of MMP SignalingTaqMan real-time PCR analysis revealed that loss of ACE2 resulted in greater increases in AngII-induced mRNA expres-sion of MMP2 (Figure 2A), MMP9 (Figure 2B) and MT1-MMP (Figure 2C) in ACE2-deficient hearts (n=6–8; P<0.05, respectively). Gelatin zymography demonstrated markedly

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Figure 3. Myocardial ultrastructural changes in wild-type (WT: Ace2+/y) and ACE2 knockout (KO; Ace2–/y) mice by transmission electron microscopy (A–D, ×7,400; E–H, ×17,500). Compared with the WT mice, myocardial ultrastructural injury was aggravated in ACE2KO mice in response to angiotensin (Ang) II, characterized by disruption or dissolution of myocardial myofilaments, myo-filaments arranged irregularly and loosely (pink arrow), and vacuolar degeneration and swollen mitochondria (yellow star).


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treatment with rhACE2 (1 mg/ml) but were largely aggravated by ACE2 inhibitor DX600 (0.5 μmol/L) (n=5–6; P<0.05, re-spectively). Decreased levels of MMP2 and MMP9 after treat-ment with rhACE2 (1 mg/ml) were confirmed by Western blotting analysis, which showed reduction in the AngII-medi-ated increased protein expressions of MMP2 and MMP9 in cardiofibroblasts in response to rhACE2 (Figure 6G) (n=5–6; P<0.05, respectively). Intriguingly, pretreatment with rhACE2 (1 mg/ml) dramatically prevented AngII-induced increases in superoxide production, NADPH oxidase activity (Figure 5) and expression of CTGF (Figure 4D), FKN (Figure 4E), and phosphorylated ERK1/2 (Figure 4F) in cardiofibroblasts (n=5–6; P<0.05, respectively). These changes were completely suppressed by the administration of the Ang-(1–7) antagonist A779 (1 μmol/L) (n=5–6; P<0.05, respectively), suggesting the beneficial effects of rhACE2 on AngII-mediated pathological actions via the Ang-(1–7) signaling. Collectively, these find-

ing antibody (anti-CTGF; 5 μg/ml), but not IgG (5 μg/ml), ab-rogated AngII-induced upregulation of superoxide production and NADPH oxidase activity in cardiofibroblasts, along with marked reductions in protein expression of CTGF (Figure 4D), FKN (Figure 4E) and phosphorylated ERK1/2 (Figure 4F) (n=5–6; P<0.05, respectively), indicating the detrimental ef-fect of ACE2 inhibition on AngII-mediated oxidative stress via the CTGF-FKN-ERK signaling.

Treatment With rhACE2 Suppressed AngII-Mediated Oxidative Stress, Inflammation and Activation of the CTGF-FKN-ERK and MMP Signaling in CardiofibroblastsAs shown in Figure 6, TaqMan real-time PCR revealed that stimulation with AngII (100 nmol/L) resulted in significant increases in the expression of MCP-1, TNFα, α-SMA, MMP2, MMP9, and MT1-MMP in cultured cardiofibroblasts (n=5–6; P<0.01, respectively), which were strikingly prevented by

Figure 4. Effects of rhACE2 and DX600 on activation of CTGF-FKN-ERK signaling in cardiofibroblasts. Representative Western blot analysis shows expression of connective tissue growth factor (CTGF: A,D), fractalkine (FKN: B,E) and phosphorylated extra-cellular signal-regulated kinase (ERK) 1/2 (C,F) in cultured cardiofibroblasts treated with AngII (100 nmol/L) in the absence and presence of rhACE2 (1 mg/ml), the Ang-(1–7) antagonist A779 (1 μmol/L), ACE2 inhibitor DX600 (0.5 μmol/L), ERK1/2 inhibitor PD98059 (10 μmol/L), Ang-(1–7) (100 nmol/L), anti-CTGF (5 μg/ml) and IgG (5 μg/ml). n=5 for each group. **P<0.01 compared with control group; #P<0.05 compared with AngII group; ØP<0.05 compared with AngII+rhACE2 group. R.E., relative expression; AngII, angiotensin II; rhACE2, recombinant human angiotensin-converting enzyme 2 (ACE2); anti-CTGF, CTGF-neutralizing antibody.



structural injury.22,23 CTGF has been shown to enhance the level of phosphorylated ERK1/2 and promote the expression of the inflammatory chemokine, FKN (also called CX3CL1), which is a newly identified membrane-bound chemokine that plays a vital role in the transition from compensated ventricu-lar remodeling hypertrophy to heart failure.10 Moreover, FKN augments myocardial injury and accelerates the progress of heart failure with activation of MAPK/ERK signaling through the G protein-coupled chemokine receptor CX3CR1.24 The activation of MAPK pathways plays a key role in the progres-sion to cardiac hypertrophy and heart failure.3 Intriguingly,

ings implied regulatory roles of ACE2 in oxidative stress, and the CTGF-FKN-ERK and MMP signaling pathways.

DiscussionThe major findings in our study are the regulatory roles of ACE2 in the CTGF-FKN-ERK signaling pathway in mice with myocardial hypertrophy and cardiac injury. It is well established that CTGF is a matricellular protein that regulates diverse cel-lular processes such as ECM deposition, cell migration, sur-vival and proliferation, leading to cardiac remodeling and

Figure 5. Effects of rhACE2 and DX600 on oxidative stress levels in cardiofibroblasts. Representative dihydroethidium (DHE) fluorescence images (A–J), relative fluorescence values (K) and lucigenin-enhanced chemiluminescence assay (L) show super-oxide generation and NADPH oxidase activity in cardiofibroblasts treated with angiotensin II (AngII: 100 nmol/L) in the absence and presence of rhACE2 (1 mg · ml), the Ang-(1–7) antagonist A779 (1 μmol/L), ACE2 inhibitor DX600 (0.5 μmol/L), ERK1/2 inhibitor PD98059 (10 μmol/L), Ang-(1–7) (100 nmol/L), anti-CTGF (5 μg/ml) and IgG (5 μg/ml) (n=5 for control group; n=6 for all other groups). **P<0.01 compared with control group; #P<0.05 compared with AngII group; ØP<0.05 compared with AngII+rhACE2 group. anti-CTGF, connective tissue growth factor-neutralizing antibody; ERK, extracellular signal-regulated kinase; Ig, immuno-globulin; rhACE2, recombinant human angiotensin-converting enzyme 2 (ACE2).


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portant role in the development of cardiac hypertrophy and myocardial injury with enhanced expression of FKN and phosphorylated ERK1/2.10,23 More importantly, absence of ACE2 triggers greater increases in cardiac CTGF-FKN and ERK phosphorylation signaling in ACE2-null mice in re-sponse to chronic AngII infusion. These changes were associ-ated with exacerbation of AngII-induced myocardial hypertro-phy and ultrastructural injury, characterized by disruption or dissolution of myocardial myofilaments and mitochondrial vacuolar degeneration. Consistent with exacerbation of AngII pathological hypertrophy and ultrastructural injury, AngII-in-duced cardiac dysfunction was greater in ACE2KO mice than

activation of endogenous ACE2 by treatment with the ACE2 agonist, XNT, or rhACE2 has been shown to improve cardiac hypertrophy and dysfunction with downregulation of MAPK/ERK signaling and upregulation of the ACE2/ACE ratio.3,18 In this work, we demonstrated that in AngII-induced hyperten-sive mice with myocardial hypertrophy and ultrastructure de-terioration, the levels of CTGF and FKN were upregulated in the heart, with marked increases in phosphorylated ERK1/2 and expression of MCP-1. Our data confirm the activation of the myocardial CTGF-FKN-ERK signaling pathway in Ace2–/y mutant mice. This is in agreement with previous reports dem-onstrating that activation of the CTGF signaling plays an im-

Figure 6. Effects of rhACE2 and DX600 on inflammation and matrix metalloproteinase (MMP) activation in cardiofibroblasts. Taq-Man real-time PCR (A–F) and representative Western blot (G) show the expression of MCP-1 (A), TNFα (B), α-SMA (C), membrane type 1 (MT1)-MMP (D), MMP2 (E,G) and MMP9 (F,G) in cardiofibroblasts treated with angiotensin II (AngII: 100 nmol/L) in the absence and presence of rhACE2 (1 mg/ml) and the ACE2 inhibitor DX600 (0.5 μmol/L) (n=5 for control group; n=6 for all other groups). **P<0.01 compared with control group; #P<0.05 compared with AngII group. R.E., relative expression; rhACE2, recom-binant human angiotensin-converting enzyme 2; SMA, smooth muscle actin; TNF, tumor necrosis factor.



thesis and deposition of ECM proteins. Particularly, MMP-2 and MMP-9 contribute to the degradation and reorganization of ECM components and are involved in the pathophysiology of cardiovascular remodeling and injury.1,5,31,33,34 In our previ-ous work,11 we showed that in ACE2 deficient-myocardial infarction mice, loss of ACE2 leads to increased MMP2 and MMP9 levels with MMP2 activation, as well as increased neutrophilic infiltration in the infarct and peri-infarct regions, ultimately resulting in a disrupted ECM and ventricular dys-function. In this study, we demonstrated that loss of ACE2 augmented AngII-induced CTGF expression and myocardial ultrastructural injury in ACE2-deficient hearts, with greater activation of MMP2, MMP9 and MT1-MMP. This is in agree-ment with other reports demonstrating that enhanced CTGF is associated with increased MMP activation in mice with car-diac hypertrophy and myocardial injury.1,5 AngII-mediated oxidative stress is known to activate MMPs, leading to degra-dation of ECM proteins and cardiac injury.5,21,33 In our in vitro study, the downregulation of AngII-induced oxidative stress by rhACE2 was responsible for inhibition of MMP activation in cardiofibroblasts, including MMP2, MMP9 and MT1-MMP. Conversely, pharmacological inhibition of ACE2 by DX600 largely aggravated AngII-induced enhancement of superoxide production and NADPH oxidase activity in cardiofibroblasts associated with MMP activation. Taken together, our data demonstrate that loss of ACE2 leads to greater increases in AngII-mediated inflammation and activation of the CTGF-FKN-ERK and MMP signaling, ultimately contributing to ad-verse myocardial injury.

In conclusion, we provide the first evidence that loss of ACE2 exacerbates AngII-mediated inflammation, myocardial injury and cardiac dysfunction associated with greater activa-tion of the CTGF-FKN-ERK and MMP signaling. ACE2 exerts protective effects on AngII-induced cellular oxidative stress and inflammation with the suppression of the CTGF-FKN-ERK and MMP signaling pathways in cardiofibroblasts. These results indicate a critical role of ACE2 in the prevention of myocardial inflammation and ultrastructural deterioration and support the notion that ACE2/Ang-(1–7) signaling is a nega-tive regulator of the RAS. Targeting ACE2 has potential ther-apeutic importance for modulating myocardial injury and heart failure. Future investigations are required to more precisely clarify the role of ACE2 as a biomarker of myocardial injury and cardiovascular disease and assess whether measurement of ACE2 will improve heart disease risk prediction.

AcknowledgmentsThis work was supported by the National Natural Science Foundation of China (No.s 81170246, 81370362, 30973522 & 81070103), the Shanghai Pujiang Talents Program of Shanghai Science and Technology Committee (11PJ1408300), the Canadian Institute for Health Research (86602 & 84279) and Scientific Research Project of Health Bureau of Shanghai (2011347). Dr Zhong is one of the SMC Morningstar Distinguished Young Scholars of Shanghai Jiao Tong University in China and a Scholar of Alberta Innovates-Health Solution in Canada. We gratefully acknowl-edge technical assistance from the University of Alberta.

DisclosuresThe authors have no disclosures.

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Supplementary FilesSupplementary File 1

Methods.

Table S1. Primer and probe sequences for TaqMan real-time PCR analysis*

Please find supplementary file(s);http://dx.doi.org/10.1253/circj.CJ-13-0805

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