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. 2020 Mar 2;177(8):1719–1734. doi: 10.1111/bph.14906

Angiotensin‐(1‐7) mitigated angiotensin II‐induced abdominal aortic aneurysms in apolipoprotein E‐knockout mice

Fei Xue 1, Jianmin Yang 1,✉, Jing Cheng 1, Wenhai Sui 1, Cheng Cheng 1, Hongxuan Li 1, Meng Zhang 1, Jie Zhang 1, Xingli Xu 1, Jing Ma 1, Lin Lu 1, Jinfeng Xu 2, Meng Zhang 1, Yun Zhang 1,✉, Cheng Zhang 1,✉
PMCID: PMC7070176  PMID: 31658493

Abstract

Background and Purpose

To test the hypothesis that angiotensin‐(1‐7) [Ang‐(1‐7)] may attenuate abdominal aortic aneurysm (AAA) via inhibiting vascular inflammation, extracellular matrix degradation, and smooth muscle cell (SMC) apoptosis, an animal model of AAA was established by angiotensin II (Ang II) infusion to apolipoprotein E‐knockout (ApoE‐/‐) mice.

Experimental Approach

All mice and cultured SMCs or macrophages were divided into control, Ang II‐treated, Ang II + Ang‐(1‐7)‐treated, Ang II + Ang‐(1‐7) + A779‐treated and Ang II + Ang‐(1‐7) + PD123319‐treated groups respectively. In vivo, aortic mechanics and serum lipids were assessed. Ex vivo, AAA were examined by histology, immunohistochemistry and zymography. Cultured cells were analysed by RT‐PCR, western blots and TUNEL assays.

Key Results

In vivo, Ang‐(1‐7) reduced the incidence and severity of AAA induced by Ang II infusion, by inhibiting macrophage infiltration, attenuating expression of IL‐6, TNF‐α, CCL2 and MMP2, and decreasing SMC apoptosis in abdominal aortic tissues. Addition of A779 or PD123319 reversed Ang‐(1‐7)‐mediated beneficial effects on AAA. In vitro, Ang‐(1‐7) decreased expression of mRNA for IL‐6, TNF‐α, and CCL2 induced by Ang II in macrophages. In addition, Ang‐(1‐7) suppressed apoptosis and MMP2 expression and activity in Ang II‐treated SMCs. These effects were accompanied by inhibition of the ERK1/2 signalling pathways via Ang‐(1‐7) stimulation of Mas and AT2 receptors.

Conclusion and Implications

Ang‐(1‐7) treatment attenuated Ang II‐induced AAA via inhibiting vascular inflammation, extracellular matrix degradation, and SMC apoptosis. Ang‐(1‐7) may provide a novel and promising approach to the prevention and treatment of AAA.

Abbreviations

AAA

abdominal aortic aneurysm

Ang II

angiotensin II

Ang‐(1‐7)

angiotensin‐(1‐7)

ApoE−/−

apolipoprotein E‐knockout

Bax

Bcl‐2‐associated X

Bcl‐2

B‐cell lymphoma‐2

HSMCs

human primary aortic smooth muscle cells

p‐ERK1/2

phospho‐ERK1/2

RAS

renin‐angiotensin system

SMCs

smooth muscle cells

TG

triglyceride

What is already known

  • ACE2 deficiency aggravated, whereas ACE2 activation attenuated the severity of AAA in hyperlipidaemic mice.

What this study adds

  • Ang‐(1‐7) treatment attenuated Ang II‐induced AAA in ApoE−/− mice.

  • Underlying mechanisms involve inhibited inflammatory responses, extracellular matrix degradation, and SMCs apoptosis via Ang‐(1‐7).

What is the clinical significance

  • Ang‐(1‐7) may provide a novel and promising approach to the prevention and treatment of AAA.

1. INTRODUCTION

Abdominal aortic aneurysm (AAA) is defined as a segmental fusiform or cystiform dilatation of the abdominal aorta exceeding the normal vessel diameter by 50%, although in clinical practice, an aneurysm diameter of 3.0 cm is commonly used as a diagnostic criterion (Kent, 2014). Ultrasound screening studies have reported that the prevalence of AAA is 4–7% in males over the age of 65 and 1–2% in females (Lederle, 2003). As most patients with AAA are asymptomatic until rupture develops, leading to a high mortality rate of 85–90%, an effective approach to preventing AAA formation and progression is highly warranted. Although the pathological features of AAA have been elucidated as degraded matrix of the vascular media, decrease of vascular smooth muscle cells (SMCs), and infiltration of inflammatory cells such as macrophages and lymphocytes (Satoh et al., 2009; Zhang, Vincelette, et al., 2009), the pathogenesis driving these pathological changes remains poorly understood, resulting in the status quo of “still no pills” for AAA (Lederle, 2013).

In the past few years, with the rapid progress in molecular biology and pharmacology, the essential role of the renin‐angiotensin system (RAS) in the pathogenesis of AAA has received renewed attention. As https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2504 (Ang II) is a key member of the RAS in promoting cardiovascular diseases including AAA, medications aiming at antagonizing Ang II have been developed such as ACE inhibitors and angiotensin receptor antagonists. Unfortunately, however, due to the inherent mechanisms offsetting the therapeutic effects of ACE inhibitors and angiotensin receptor antagonists, these drugs did not meet the original clinical expectations (Davis, Rateri, & Daugherty, 2014; Messerli, Bangalore, Bavishi, & Rimoldi, 2018).

Recently, the newly found members of the RAS, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1614 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=582 [Ang‐(1‐7)], have attracted increased interest in the field of cardiovascular research (Jiang et al., 2014; Patel, Zhong, Grant, & Oudit, 2016). The major physiological role of ACE2 is to degrade Ang II, a vasoconstrictor octapeptide, to Ang‐(1‐7), a vasodilator heptapeptide. Overexpression of ACE2 attenuated plaque formation and progression in a rabbit model of atherosclerosis, by activating several signalling pathways in endothelial cells and in SMCs (Dong et al., 2008; Zhang et al., 2010). Whole‐body ACE2 deficiency aggravated, whereas administration of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=10307 to activate ACE2 markedly attenuated, the formation and severity of Ang II‐induced AAAs in LDLr−/− mice (Thatcher et al., 2014). More recently, our group further demonstrated that ACE2 gene transfer significantly prevented AAA formation in apolipoprotein E‐knockout (ApoE−/−) mice by inhibiting the inflammatory response and MMP activation (Hao et al., 2017). However, significant side effects and toxicity of diminazene aceturate and the immune response triggered by injection of recombinant ACE2 protein may preclude clinical applications of these treatments in patients with AAA. Recent studies in our and other laboratories showed that Ang‐(1‐7) inhibited atherosclerotic lesion formation and enhanced plaque stability in ApoE−/− mice, thus acting as a physiological antagonist of Ang II (Tesanovic, Vinh, Gaspari, Casley, & Widdop, 2010; Yang et al., 2013). However, the direct effect of Ang‐(1‐7) treatment on the incidence and severity of AAA remains to be demonstrated.

In the present study, we hypothesized that Ang‐(1‐7) treatment may attenuate AAA in ApoE−/− mice induced by Ang II infusion, via the activation of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=150 and of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=35, thus inhibiting vascular inflammation, extracellular matrix degradation, and SMCs apoptosis. A series of in vivo and in vitro experiments were designed and performed to test this hypothesis.

2. METHODS

2.1. Animal model

All animal care and experimental protocols were approved by the Ethics Committee on Animal Experiment of Shandong University Qilu Hospital and complied with the guidelines of the Animal Management Rules of the Chinese Ministry of Health (Document No. 55, 2001). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. One hundred male ApoE−/− mice (RRID:IMSR_JAX:002052) aged 8 weeks were purchased from Beijing Weishanglide Animal Experimental Center. All mice were housed in standard cages in a specific pathogen‐free environment and kept on a 12‐hr light/12‐hr dark cycle with food and water freely available. All the animals were fed with a high‐fat diet (0.25% cholesterol and 15% cocoa butter) during the entire experimental period.

In the in vivo study, the mice were randomly divided into five groups (n = 20 per group) after 4 weeks high‐fat feeding using the random number table method, and all mice were treated for another 4 weeks with continuous subcutaneous infusion of different drugs by an osmotic pump (Alzet model 2004, Alza Corp, Palo Alto, CA, USA), implanted under aseptic conditions, after anaesthesia with sodium pentobarbital. The control group received an infusion of saline, the Ang II‐treated group received an infusion of Ang II (1,000 ng·kg−1·min−1), the Ang II + Ang‐(1‐7)‐treated group received an infusion of Ang II (1,000 ng·kg−1·min−1) plus Ang‐(1‐7) (400 ng·kg−1·min−1), the Ang II + Ang‐(1‐7) + https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6081 (a known Mas receptor antagonist)‐treated group received an infusion of Ang II (1,000 ng·kg−1·min−1) plus Ang‐(1‐7) (400 ng·kg−1·min−1) plus A779 (400 ng·kg−1·min−1), and the Ang II + Ang‐(1‐7) + https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=597 (an AT2 receptor antagonist)‐treated group received an infusion of Ang II (1,000 ng·kg−1·min−1) plus Ang‐(1‐7) (400 ng·kg−1·min−1) plus PD123319 (3 mg·kg−1·day−1). After drug treatment for 4 weeks, all mice were humanely killed (overdose of pentobarbital) and samples taken for further histological studies.

2.2. Measurement of body weight and blood pressure

Body weight and blood pressure were measured in all mice at the start and the end of the experiment. A non‐invasive tail‐cuff system (Softron BP‐98A, Tokyo, Japan) was used to measure blood pressure, and all mice were trained first to adapt to the device to ensure reproducible measurements. Blood pressure was measured between 9:00 a.m. and noon by the same researcher and the values of blood pressure in each mouse were recorded as the mean of three consecutive measurements.

2.3. Serum lipid assay

At the end of the experiment, all mice were fasted 12 hr and then blood samples were taken from the cardiac apex of each mouse after anaesthesia with sodium pentobarbital. Finally, all mice were humanely killed with an overdose of sodium pentobarbital. The serum levels of total cholesterol, triglyceride (TG), LDL cholesterol, and HDL cholesterol were measured by enzymic assays, using a automatic biochemistry analyser (Chemray 240, Rayto, Shenzhen, China).

2.4. AAA quantification

The aortas were dissected and fixed in 4% paraformaldehyde overnight, after exsanguination and saline perfusion in all mice. AAA was defined as ≥50% dilation of the external diameter of the abdominal aorta compared with the normal abdominal aorta in saline‐infused mice of the control group (Kent, 2014). The adventitia of the suprarenal aortas was removed and the maximal outer diameter of the abdominal aorta measured by the use of Image‐Pro Plus 6.0 (Media Cybernetics, USA). All aneurysms were classified as saccular or fusiform types according to their pathological morphology (Biasetti, Gasser, Auer, Hedin, & Labruto, 2010).

2.5. Cell culture

In the first part of the in vitro study, RAW264.7 mouse macrophage cell line (RRID:CVCL_0493), purchased from the American Type Culture Collection (Manassas, VA) and cultured in RPMI‐1640 medium (ScienCell, California, USA), was chosen for the experiment. The macrophages were cultured followed by trypsin digestion with trypsin/EDTA, and only cells grown to 80% confluence were used and seeded in 75‐cm2 flasks. These cells were subcultured three times a week. Then, the macrophages were divided into control group, Ang II‐treated group (10−7 M), Ang II (10−7 M) + Ang‐(1‐7) (10−6 M)‐treated group, Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + A779 (10−6 M)‐treated group, and Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + PD123319 (10−6 M)‐treated group and treated for 24 hr. Prior to treatment, all cells were placed in serum‐depleted medium for 24 hr to maintain quiescence. The doses of Ang II, Ang‐(1‐7), A779, and PD123319 were based on previous experiments (Chen et al., 2016; Daugherty, Manning, & Cassis, 2000; Esteban et al., 2005; Kong, Zhang, Meng, Zhang, & Zhang, 2015; Liles et al., 2015; Patel, Ali, & Hussain, 2016; Yang et al., 2013).

In the second part of the in vitro study, human primary aortic smooth muscle cells (HSMCs), purchased from ScienCell, were cultured in SMC medium (ScienCell), as described previously (Yang et al., 2013). The HSMC medium consisted of 500 ml of basal medium, 10 ml of FBS (Cat. No. 0010), 5 ml of smooth muscle cell growth supplement (Cat. No. 1152), and 5 ml of penicillin/streptomycin solution (Cat. No. 0503). These cells were subcultured twice a week, harvested by trypsin digestion with trypsin/EDTA, and seeded in 75‐cm2 flasks. Cells from passages 4–6 at 80% confluence in culture wells were used after serum depletion for 24 hr and then divided into control group, Ang II‐treated group (10−7 M), Ang II (10−7 M) + Ang‐(1‐7) (10−6 M)‐treated group, Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + A779 (10−6 M)‐treated group, and Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + PD123319 (10−6 M)‐treated group that were treated for 24 hr. Prior to treatment, all cells were placed in serum‐depleted medium for 24 hr to maintain quiescence.

2.6. Quantitative real‐time PCR

Total RNA was extracted from macrophages by using the TRIzol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. The extracted RNA was dissolved in RNase free water, and concentration of the total RNA was determined with a spectrophotometer. RNA was then reverse‐transcribed using the Applied Biosystems cDNA Reverse Transcription kit (Applied Biosystems, Life Technologies), and the amplification of cDNA was measured by use of SYBR Green PCR Master Mix (RR420A, Takara Bio Inc., Otsu, Shiga, Japan). Quantitative values were obtained from the threshold cycle value (Ct) and the relative mRNA expression levels were analysed by the 2−△△Ct method. The primer sequences were listed in Table S1.

2.7. Histology and immunohistochemical staining

At the end of the study, the mice were killed by an overdose of pentobarbital (i.p.). The abdominal aortas were removed and fixed in 4% paraformaldehyde overnight. Then, the aortic tissues were embedded with paraffin and cut into 5‐μm‐thick cross sections, which were stained with HE and Verhoeff‐Van Gieson (Abcam, Cambridge, UK) in accordance with standard protocol. To quantitate elastin degradation, a previously described grading method was used (Satoh et al., 2009; Wang et al., 2012). Grade 1: no elastin degradation; Grade 2: mild elastin degradation; Grade 3: severe elastin degradation; and Grade 4: aortic rupture. The aortic wall thickness (T) was measured by the following formula: T = (D − d)/2, where D was the vessel diameter, d was the lumen diameter, and both D and d were measured along the largest diameter of the normal abdominal aorta in the control group or of the AAA in the four treatment groups respectively. The incidence of luminal thrombosis was calculated to assess the severity of luminal thrombosis, and the incidence of AAA was used to indicate the severity of breakdown of the aortic media and adventitia.

For immunohistochemical staining, 10 tissue sections, 5 μm thick, were derived from the AAA portion with the largest diameter in each mouse, which were used for staining of vascular cells and cytokines. The paraffin sections required antigen retrieval with citrate buffer after deparaffinization and were then incubated with 3% H2O2 for 10 min at room temperature. Thereafter, the sections of AAA were blocked with 5% BSA for 30 min at 37°C and incubated with the corresponding primary antibodies for α SM‐actin antibody (1:300, Abcam, ab5694, RRID:AB_2223021), CD68 antibody (1:300, Boster, BA3638, Wuhan, China, RRID:AB_2813855), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4998 antibody (1:200, Abcam, ab7737, RRID:AB_306031), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4406 antibody(1:200, Abcam, ab25124, RRID:AB_448636), and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5074 antibody (1:200, Abcam, ab6671, RRID:AB_305641), at 4°C overnight, and then the tissue sections were incubated with appropriate HRP‐conjugated secondary antibodies for 30 min at 37°C after washing with PBS. The positive reactions of tissue sections were developed using the peroxidase substrate solution including 0.02% H2O2 and 0.1% 3,3′‐diaminobenzidine tetrahydrochloride (ZLI‐9018, ZSGB‐BIO, China) with the reaction products displayed as a brown colour. Finally, the sections were counterstained with haematoxylin, and the integration optical density values of positive staining were calculated using Image‐Pro Plus software (Media Cybernetics, RRID:SCR_007369). The relative content of SMCs, macrophages, and inflammatory cytokines was quantitated as the ratio of the positive staining area to the cross‐sectional area of the aortic wall (Chen et al., 2016). Sections reacted with non‐immune IgG, secondary antibody only, and no primary and secondary antibodies were used as negative controls. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

2.8. Zymography

Zymography was performed with an MMP gelatin zymography kit (GenMed Scientific Inc., USA). The protein extracts from the mouse aorta or culture medium were separated by electrophoresis in SDS‐PAGE gels containing 0.1% gelatin to measure the activities of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1629. Gels were washed with renaturing buffer for 2 hr and further incubated with developing buffer at 37°C overnight. After incubation, gels were stained with Coomassie Brilliant Blue and then destained with the buffer until clear white bands appeared on the blue background.

2.9. TUNEL assay

Apoptosis of HSMCs was detected by TUNEL using the In Situ Cell Death Detection Kit, TMR red (Roche, Germany) following the manufacturer's instructions. HSMCs were cultured in 12‐well plates and divided into five treatment groups: control group, Ang II‐treated group (10−7 M), Ang II (10−7 M) + Ang‐(1‐7) (10−6 M)‐treated group, Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + A779 (10−6 M)‐treated group, and Ang II (10−7 M) + Ang‐(1‐7) (10−6 M) + PD123319 (10−6 M)‐treated group. The doses of Ang II, Ang‐(1‐7), A779, and PD123319 were based on earlier reports (Liles et al., 2015; Patel, Ali, & Hussain, 2016; Yang et al., 2013). After incubation for 24 hr, cells were collected and fixed with 4% paraformaldehyde for 10 min in room temperature. Then, cells were washed twice with PBS for 10 min and permeabilized with 0.1% Triton X‐100 for 5 min. Cellular nuclei were stained with DAPI (Sigma‐Aldrich) for 10 min at 37°C. Samples were analysed under a fluorescence microscope with an excitation wavelength in the range of 570–620 nm.

2.10. Western blot analysis

Proteins were extracted from cells or fresh tissue lysates of the abdominal aortas of ApoE−/− mice (RRID:IMSR_JAX:002052). The extracts of proteins were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA), blocked with 5% BSA for 2 hr at room temperature, and incubated with corresponding primary antibodies for β‐actin antibody (1:1,000, Abcam, ab8227, RRID:AB_2305186), B‐cell lymphoma‐2 (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2844) antibody (1:1,000, Abcam, ab182858, RRID:AB_2715467), Bcl‐2‐associated X (https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=910) antibody (1:1,000, Abcam, ab32503, RRID:AB_725631), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4471 antibody (1:500, Abcam, ab37150, RRID:AB_881512), https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=514 antibody (1:2,000, Cell Signaling Technology, 4696, USA, RRID:AB_390780), and phospho‐ERK1/2 (p‐ERK1/2) antibody (1:2,000, Cell Signaling Technology, 4370, RRID:AB_2315112) overnight at 4°C. Then, the membranes were incubated with appropriate HRP‐conjugated secondary antibodies for 2 hr at room temperature after being washed by tris‐buffered saline‐tween three times for 10 min. Finally, the membranes were visualized with the use of chemiluminescent HRP substrate (Millipore). The levels of protein expression of Bcl‐2, Bax, and MMP2 were normalized to that of β‐actin. Five independent experiments were performed to derive the mean values calculated for statistical analysis.

2.11. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. The five experimental groups of mice were labelled as A ‐ E and thus data collection and analysis were carried out blindly, without knowledge of the treatment in each group, particularly with the histological procedures. All data are expressed as means ± SEM from five independent assays and the statistical analyses involved were from SPSS 19.0 (SPSS, Inc., Chicago, IL, RRID:SCR_002865). A normal distribution of data was ensured for parametric analysis. An unpaired Student t‐test (parametric analysis) was used to analyse between‐group difference. One‐way ANOVA test with Tukey post hoc analysis was used to compare data among multiple groups. After analysis of variance, post hoc tests were run only if F achieved the necessary level of statistical significance (P<0.05) and there was no significant variance inhomogeneity. Differences in the incidence and grading of AAA were analysed with chi‐squared (Χ2) tests. P<0.05 was considered statistically significant.

3. MATERIALS

Ang II, Ang‐(1‐7) and A779 were supplied by GL Biochem Ltd (Shanghai, China) and PD123319 was supplied by Lintai Biological Technology Company (Xi'an, China).

3.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos, et al., 2019; Alexander, Fabbro, et al., 2019).

4. RESULTS

4.1. Body weight, blood pressure, and serum lipid profile

At the end of the study, there were no significant differences in body weight and serum lipid levels among different groups. Although the serum lipid levels in the Ang II + Ang‐(1‐7) + PD123319‐treated group seemed higher than those in other groups, the difference did not reach statistical significance (Table 1).

Table 1.

Body weight, blood pressure, and serum lipid levels at the end of the experiment, in the five experimental groups of mice

Group BW (g) SBP (mmHg) DBP (mmHg) TC (mmol·L−1) TG (mmol·L−1) HDL‐C (mmol·L−1) LDL‐C (mmol·L−1)
Control 28.1 ± 1.2 99.9 ± 2.5 70.7 ± 2.4 15.6 ± 1.7 2.0 ± 0.2 6.0 ± 0.4 4.3 ± 0.3
Ang II 28.0 ± 0.4 150.1 ± 5.9* 108.3 ± 4.6* 13.9 ± 2.3 1.9 ± 0.2 4.9 ± 0.6 4.2 ± 0.4
Ang II + Ang‐(1‐7) 28.5 ± 0.3 156.0 ± 3.3* 104.3 ± 3.5* 15.5 ± 3.2 2.0 ± 0.3 5.7 ± 0.7 4.3 ± 0.6
Ang II + Ang‐(1‐7) + A779 29.2 ± 0.6 157.3 ± 4.8* 102.4 ± 2.0* 15.2 ± 2.8 2.0 ± 0.2 4.9 ± 0.6 4.2 ± 0.5
Ang II + Ang‐(1‐7) + PD123319 27.6 ± 0.9 157.0 ± 3.8* 110.0 ± 4.5* 23.0 ± 2.0 2.6 ± 0.3 6.7 ± 0.4 5.9 ± 0.4
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Abbreviations: BW, body weight; DBP, diastolic blood pressure; HDL‐C, HDL cholesterol; LDL‐C, LDL cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride.

*

P < .05, significantly different from control group.

Ang II infusion significantly increased systolic and diastolic blood pressure levels in the four treatment groups as compared with those in the control group. However, the addition of Ang‐(1‐7), A779, or PD123319 treatment to Ang II infusion did not lower blood pressure, compared with the Ang II treatment group (Table 1).

4.2. Effect of Ang‐(1‐7) on the incidence and severity of AAA

At the end of this experiment, the aortic rupture rate was 0% in the control group, 30% in the Ang II‐treated group, 5% in the Ang II + Ang‐(1‐7)‐treated group, 30% in the Ang II + Ang‐(1‐7) + A779‐treated group, and 40% in the Ang II + Ang‐(1‐7) + PD123319‐treated group respectively. As aortic rupture inevitably resulted in sudden death, the survival rate was correspondingly 100% in the control group, 70% in the Ang II‐treated group, 95% in the Ang II + Ang‐(1‐7)‐treated group, 70% in the Ang II + Ang‐(1‐7) + A779‐treated group, and 60% in the Ang II + Ang‐(1‐7) + PD123319‐treated group respectively (Figure S1D).

The incidence of AAA was 0% in the control group, 75% in the Ang II‐treated group, 25% in the Ang II + Ang‐(1‐7)‐treated group, 65% in the Ang II + Ang‐(1‐7) + A779‐treated group, and 80% in the Ang II + Ang‐(1‐7) + PD123319‐treated group respectively. The incidence of AAA was markedly reduced in the Ang II + Ang‐(1‐7)‐treated group relative to the Ang II‐treated group. In contrast, A779 or PD123319 administration essentially blocked the beneficial effects of Ang‐(1‐7) on AAA (Figure 1). In addition, compared with the control group, Ang II infusion significantly increased the diameter of abdominal aortas, whereas the diameter of AAA was significantly decreased in Ang II + Ang‐(1‐7)‐treated group. In contrast, A779 and PD123319 treatment virtually reversed the salutary effects of Ang‐(1‐7) on AAA diameter (Figure 1). Taken together, these results indicated that Ang‐(1‐7) treatment reduced the incidence and severity of Ang II‐induced AAA in ApoE−/− mice.

Figure 1.

Figure 1

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Effect of Ang‐(1‐7) on the incidence of AAA and components of the abdominal aortic wall in ApoE−/− mice. (a) Representative abdominal aortic specimens from the five experimental groups of mice. (b) Incidence of AAA in five groups of mice (n = 20 in each group). (c) Maximal diameters of the abdominal aorta of five groups of mice (n = 5 in each group). (d) Representative HE and Verhoeff staining of the abdominal aorta in the five groups of mice. Dotted boxes in the middle panel indicated a local area in the low power field (scale = 50 μm), which was enlarged in the high power field (scale = 20 μm) in the lower panel. (e) Grading of elastin degradation in the aorta wall. *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

4.3. Effect of Ang‐(1‐7) on pathological remodelling of the aortic wall

At the end of the experiment, the number of saccular aneurysms in surviving mice with AAA was 6 (66.7%) in Ang II‐treated group, 1 (25%) in Ang II + Ang‐(1‐7)‐treated group, 5 (71%) in Ang II + Ang‐(1‐7) + A779‐treated group, and 6 (75%) in Ang II + Ang‐(1‐7) + PD123319‐treated group respectively. The number of fusiform aneurysms in surviving mice with AAA was 3 (33.3%) in Ang II‐treated group, 3 (75%) in Ang II + Ang‐(1‐7)‐treated group, 2 (28.6%) in Ang II + Ang‐(1‐7) + A779‐treated group, and 2 (25%) in Ang II + Ang‐(1‐7) + PD123319‐treated group. The Ang II‐induced pathological remodelling in AAA consisted of discontinuity of elastin fibres, disruption of intima with thrombus formation, a thickened aortic wall, and breakdown of adventitia (Zhang, Naggar, et al., 2009; Zhang, Vincelette, et al., 2009). Staining with HE and Verhoeff stains in the present study revealed disrupted medial elastin, aortic wall thickening, luminal thrombosis, and breakdown of the aortic media and adventitia. Compared with the control group, these pathological changes occurred more frequently in Ang II‐treated, Ang II + Ang‐(1‐7) + A779‐treated, and Ang II + Ang‐(1‐7) + PD123319‐treated groups, but to a much lesser extent in Ang II + Ang‐(1‐7)‐treated group (Figures 1e and S1). These results suggested that Ang‐(1‐7) effectively alleviated pathological remodelling of the aortic wall induced by Ang II infusion in ApoE−/− mice.

4.4. Effect of Ang‐(1‐7) on macrophage infiltration and inflammatory cytokine expression

Macrophage infiltration and chronic inflammation of the aortic wall is a pathological feature of AAA (Shimizu, Mitchell, & Libby, 2006). In the present study, macrophage infiltration and the expression of IL‐6, TNF‐α, and CCL2 were examined to compare the inflammatory state in different mouse groups. Relative to the control group, the relative content of macrophages and mRNA and protein expression of IL‐6, TNF‐α, and CCL2 in the abdominal aortic wall were significantly increased in the Ang II‐treated group. In contrast, Ang‐(1‐7) treatment markedly decreased macrophage infiltration and mRNA and protein expression of IL‐6, TNF‐α, and CCL2 induced by Ang II infusion, whereas A779 and PD123319 treatment reversed these therapeutic effects of Ang‐(1‐7) (Figure 2).

Figure 2.

Figure 2

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Effect of Ang‐(1‐7) on macrophage infiltration and inflammatory cytokine expression in ApoE−/− mice and macrophages. (a) Representative immunohistochemical staining for CD68 in the abdominal aorta from the five experimental groups of mice. High magnification pictures are shown in the lower panel. (b) Quantitative analysis of the positive CD68 staining (n = 5 in each group). (c) Representative staining for IL‐6, TNF‐α, and CCL2 in the abdominal aorta of the five groups of mice. Dotted boxes indicated a local area in the low power field (scale = 50 μm), which was enlarged in the high power field (scale = 20 μm). (d), (e), and (f) Quantitative analysis of the positive staining for IL‐6, TNF‐α, and CCL2, in five groups of mice (n = 5 in each group). (g), (h), and (i) Quantitative analysis of the mRNA expression of IL‐6, TNF‐α, and CCL2 in the abdominal aortic tissues of five groups of mice (n = 5 in each group). (j), (k), and (l) Quantitative analysis of the mRNA expression levels of IL‐6, TNF‐α, and CCL2 in five groups of cultured macrophages in vitro. Five independent experiments were performed to derive the mean values. *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

In cultured RAW macrophages, relative to the control group, the mRNA expression levels of IL‐6, TNF‐α, and CCL2 were substantially enhanced by Ang II but reduced by Ang‐(1‐7) treatment. However, these therapeutic effects were virtually reversed by A779 and PD123319 treatment (Figure 2).

4.5. Effect of Ang‐(1‐7) on apoptosis of the aortic tissues and HSMCs

One striking pathological feature of AAA is the reduced number of vascular SMCs (Kuivaniemi, Ryer, Elmore, & Tromp, 2015). Thus, we examined the content of SMCs in the abdominal aortic wall by immunohistochemical staining for the marker protein, α‐smooth muscle actin (SMA; Figure 3a). The relative content of SMCs in the AAA from the Ang II‐treated group was lower than that in the control group (Figure 3b). The addition of Ang‐(1‐7) to Ang II treatment exerted a beneficial effect on the content of SMCs, while A779 or PD123319 treatment abolished these beneficial effects of Ang‐(1‐7) (Figure 3b).

Figure 3.

Figure 3

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Effect of Ang‐(1‐7) on Ang II‐induced apoptosis of abdominal aortic tissues of ApoE−/− mice. (a) Representative immunohistochemical staining for smooth muscle α‐actin (SMA) in the abdominal aorta from the five experimental groups of mice. High magnification pictures are shown in the lower panel. (b) Quantitative analysis of relative content of SMA in five groups of mice (n = 5 in each group). (c) Representative western blot analysis of protein expression of Bax and Bcl‐2 in the abdominal aorta of five groups of mice. (d) and (e) Quantitative analysis of Bax protein expression and Bax/Bcl‐2 in five groups of mice (n = 5 in each group). (f) and (g) Quantitative analysis of Bax and Bcl‐2 mRNA expression in the abdominal aortic tissues of five groups of mice (n = 5 in each group). *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

To assess the effects of Ang‐(1‐7) on Ang II‐induced apoptosis, the expression levels of Bax and Bcl‐2 in the abdominal aortic tissues and cultured SMCs were measured. In the in vivo experiment, compared with the control group, the mRNA and protein expression levels of Bax were significantly up‐regulated in the Ang II‐treated group, while addition of Ang‐(1‐7) to the Ang II treatment markedly down‐regulated the expression of Bax compared with the Ang II‐treated group. However, the expression of Bax was again raised in Ang‐(1‐7) + A779‐treated and Ang‐(1‐7) + PD123319‐treated groups when compared with the values in the Ang II + Ang‐(1‐7)‐treated group (Figure 3). As the ratio of Bax/Bcl‐2 is commonly used to quantitate the degree of apoptosis, we measured this parameter in this experiment. Compared with the control group, the Bax/Bcl‐2 ratio was significantly increased in Ang II‐treated group and decreased in the Ang‐(1‐7) treatment group, compared with the Ang II‐treated group. In contrast, A779 and PD123319 treatment reversed the beneficial effect of Ang‐(1‐7) on the Bax/Bcl‐2 ratios (Figure 3). In the in vitro experiment, the expression levels of Bax and Bcl‐2 were examined in cultured HSMCs and the results showed changes similar to those in the experiments in vivo (Figure 4).

Figure 4.

Figure 4

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Effect of Ang‐(1‐7) on Ang II‐induced apoptosis in cultured SMCs. (a) Representative western blot analysis of protein expression of Bax and Bcl‐2 in the five experimental groups of HSMCs. (b) and (c) Quantitative analysis of protein expression of Bax and Bax/Bcl‐2 in five groups of HSMCs. Five independent experiments were performed to derive the mean values. (d) Representative TUNEL images in five groups of HSMCs. Dotted boxes in the third panel indicated a local area in the low power field (scale = 100 μm), which was enlarged in the high power field in the bottom panel. (e) Quantitative analysis of TUNEL images in five groups of HSMCs. Five independent experiments were performed to derive the mean values. *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

To further assess the effect of Ang‐(1‐7) on apoptosis of SMCs in AAA samples, TUNEL assays were performed. The results showed that Ang II treatment significantly increased apoptosis, whereas Ang‐(1‐7) markedly attenuated apoptosis induced by Ang II. In contrast, A779 and PD123319 treatment abolished the effect of Ang‐(1‐7) on SMCs (Figure 4). Altogether, these results indicated that Ang‐(1‐7) alleviated apoptosis of SMCs induced by Ang II.

4.6. Effect of Ang‐(1‐7) on expression and activity of MMP

Previous studies found that degradation of the extracellular matrix in AAA was attributed to up‐regulated MMPs, especially MMP2 (Xiong et al., 2009). In order to assess the role of Ang‐(1‐7) in the homeostasis of extracellular MMPs, we examined expression of MMP2 in vivo and in vitro. In vivo, compared with the control group, the protein expression levels of MMP2 in AAA tissues were significantly increased in Ang II‐treated group but returned to control values in Ang II + Ang‐(1‐7)‐treated group. In contrast, A779 and PD123319 treatment reversed the effect of Ang‐(1‐7) on MMP2 expression (Figure 5). The activity of MMP2 by zymography in five groups of mice showed changes similar to those of the protein expression of MMP2 (Figure 5). Our assessments of MMP2 expression and activity in cultured HSMCs showed changes similar to those from the experiments in vivo (Figure 5).

Figure 5.

Figure 5

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Effect of Ang‐(1‐7) on the expression of MMP2 in vivo and in vitro. (a) and (b) Representative western blot analysis of protein expression and quantitative analysis of MMP2 in the abdominal aortic tissues from the five experimental groups of mice (n = 5 in each group). (c) and (d) Representative western blot analysis of protein expression and quantitative analysis of MMP2 in five groups of HSMCs. Five independent experiments were performed to derive the mean values. (e) Gelatin zymography of MMP2 activity in five groups of mice (n = 5 in each group). (f) Gelatin zymography of MMP2 activity in five groups of HSMCs. Five independent experiments were performed to derive the mean values. (g) Quantitative analysis of MMP2 activity in five groups of mice. (h) Quantitative analysis of MMP2 activity in five groups of HSMCs. *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

4.7. Effect of Ang‐(1‐7) on MAPK signalling

Previous studies reported that the MAPK signalling pathway was involved in the pathogenesis of AAA and in particular, ERK1/2 was associated with MMPs activity and inflammatory signalling (Zhang, Naggar, et al., 2009). In vivo, compared with the control group, expression of p‐ERK1/2 was increased in the Ang II‐treated group, whereas the addition of Ang‐(1‐7) to Ang II treatment down‐regulated the expression of p‐ERK1/2. On the other hand, compared with the Ang‐(1‐7)‐treated group, A779 and PD123319 treatment reversed the beneficial effect of Ang‐(1‐7) and enhanced the expression of p‐ERK1/2 again (Figure 6). Furthermore, we examined the p‐ERK1/2 expression levels in cultured HSMCs and RAW macrophages. Similarly, Ang‐(1‐7) treatment attenuated p‐ERK1/2 elevation induced by Ang II infusion, while A779 and PD123319 treatment blocked the beneficial effects of Ang‐(1‐7), in HSMCs and macrophages (Figure 6).

Figure 6.

Figure 6

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Effect of Ang‐(1‐7) on p‐ERK1/2 expression in vivo and in vitro. (a) and (b) Representative western blot analysis of phosphorylated (p) and total (T) ERK1/2 and quantitative analysis of p‐ERK1/2 normalized to T‐ERK1/2 protein expression in the abdominal aorta from the five experimental groups of mice (n = 5 in each group). (c) and (d) Representative western blot analysis of p‐ERK1/2 and T‐ERK1/2 and quantitative analysis of p‐ERK1/2 normalized to T‐ERK1/2 protein expression in five groups of HSMCs. Five independent experiments were performed to derive the mean values. (e) and (f) Representative western blot analysis of p‐ERK1/2 and T‐ERK1/2 and quantitative analysis of p‐ERK1/2 normalized to T‐ERK1/2 protein expression in five groups of macrophages. Five independent experiments were performed to derive the mean values. *P < .05, significantly different from control group; # P < .05, significantly different from Ang II group; ▲ P < .05, significantly different from Ang II + Ang‐(1‐7) group [Colour figure can be viewed at http://wileyonlinelibrary.com]

4.8. Effect of Ang‐(1‐7) on expression of AT1 receptors, AT2 receptors and Mas receptors in AAA tissues

The expression of mRNA for Mas receptors in the abdominal aortic tissues showed no significant difference among the five experimental groups of mice. Compared with the control group, the expression of mRNA for https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=34 was increased in the four groups receiving Ang II treatment and, similarly, mRNA for AT2 receptors in the four groups receiving Ang II treatment was higher than that in the control group. However, expression of mRNA for AT2 receptors in the Ang II + Ang‐(1‐7)‐treated group was higher those in in the groups receiving Ang II alone, A779 or PD123319. (Figure S2).

5. DISCUSSION

Although the beneficial effects of Ang‐(1‐7) on atherosclerosis, heart failure, diabetic nephropathy, hypertension and intracranial aneurysm, and haemorrhage have been reported in experimental studies (Duprez, 2006; Pena Silva et al., 2014; Shimada et al., 2015; Yang et al., 2013; Zhang et al., 2015), the exact role of Ang‐(1‐7) in AAA remains unclear. The major finding of the present study was that Ang‐(1‐7) treatment attenuated the severity of AAA in ApoE−/− mice induced by Ang II infusion, by inhibiting vascular inflammation, extracellular matrix degradation, and SMC apoptosis. To the best of our knowledge, this is the first study to report the beneficial effects and the relevant mechanisms of Ang‐(1‐7) in a mouse model of AAA.

Ang II infusion in ApoE−/− mice has become a widely used animal model of AAA that shares some of the same characteristic features as human AAA, including macrophage infiltration, medial elastolysis, luminal expansion, and thrombus formation (Daugherty et al., 2000; Daugherty, Cassis, & Lu, 2011; Rateri et al., 2011; Saraff, Babamusta, Cassis, & Daugherty, 2003). Nonetheless, this model does exhibit some important differences from human AAA. First, a meta‐analysis of Ang II infused ApoE−/− mice showed that the overall incidence of AAA was only 60% but the mortality rate was as high as 20% (Trachet, Fraga‐Silva, Jacquet, Stergiopulos, & Segers, 2015). Second, unlike human AAA, the AAAs produced in this model were almost always suprarenal (Daugherty et al., 2000; Manning, Cassi, Huang, Szilvassy, & Daugherty, 2002). Finally, the progression of luminal dilatation in this animal model was often rapid, rather than gradual (Daugherty et al., 2011; Saraff et al., 2003). These differences may explain why the therapeutic targets derived from animal models of AAA have ended in failure in clinical trials.

Clinically, AAA is characterized by an asymptomatic but progressive dilation of the abdominal aorta, leading to aortic rupture and sudden death in the majority of patients (Khosla et al., 2014). Unfortunately, there is, so far, no effective preventive or therapeutic approach to the treatment of AAA. Recent experimental studies reported that Ang‐(1‐7) counteracted the detrimental effects induced by Ang II in a range of cardiovascular diseases such as atherosclerosis, heart failure, diabetic nephropathy, and hypertension (Jiang et al., 2014). However, whether Ang‐(1‐7) alleviated Ang II‐induced AAA and, if it did, what mechanisms were responsible, remained to be determined. In the current study, Ang‐(1‐7) treatment reduced both formation and severity of Ang II‐induced AAA. As Ang‐(1‐7) treatment did not affect the serum lipid or blood pressure levels in ApoE−/− mice, such therapeutic effects cannot contribute to biochemical or haemodynamic changes. In addition, Ang‐(1‐7) had little or no effect on blood pressure, which was consistent with our previous results (Yang et al., 2013). This lack of effect on blood pressure may be due to the following reasons: First, Ang‐(1‐7) decreased peripheral arterial resistance by ≈26%, while increasing cardiac output by ≈17% in rats (Botelho‐Santos et al., 2007; Wysocki et al., 2010); second, Ang II infusion in the four treatment groups of mice in this study may activate AT1 receptors, causing vasoconstriction and thus attenuating vasodilatation mediated by Mas and AT2 receptors (Yang et al., 2013). Ang‐(1‐7) lowered blood pressure only when used in combination with an AT1 receptor antagonist (candesartan) in SHR rats (Walters, Gaspari, & Widdop, 2005).

The Mas receptor is a GPCR with a high affinity for Ang‐(1‐7) and previous studies have suggested that most cardiovascular effects of Ang‐(1‐7) are likely to be mediated by the Mas receptors. Thus, we used A779, a known Mas receptor antagonist, to examine the role of these receptors in Ang‐(1‐7)‐mediated protective effects on AAA. Our results demonstrated that A779 substantially reversed the protective effects of Ang‐(1‐7) on AAA, demonstrating a crucial role of Mas receptors in the therapeutic effects of Ang‐(1‐7). In addition, recent studies have indicated that Ang‐(1‐7) had a weak affinity for AT2 receptors (Bosnyak et al., 2011), and AT2 receptor deficiency accelerated atherosclerosis (Iwai et al., 2005). Our previous study demonstrated that AT2 receptors were involved in Ang‐(1‐7)‐mediated anti‐atherosclerotic effects (Yang et al., 2013). In the present study, we used PD123319, an AT2 receptor antagonist, to test the role of these receptors in Ang‐(1‐7)‐mediated effects on AAA. To our surprise, administration of PD123319 not only completely reversed the therapeutic effects of Ang‐(1‐7) on AAA but also aggravated AAA formation and severity. Daugherty et al. have elegantly demonstrated that AT2 receptor deficiency did not affect the incidence of AAA, whereas administration of PD123319 aggravated AAA induced by Ang II, not only in wild‐type (with AT2 receptors) mice but also in mice lacking AT2 receptors (Daugherty, Manning, & Cassis, 2001; Daugherty, Rateri, Howatt, Charnigo, & Cassis, 2013). For this reason, selection of an appropriate dose of PD123319 is necessary to minimize the non‐specific effects of this compound. In this study, we chose 3 mg·kg−1·day−1 of PD123319 because such a dose had no effect on atherosclerosis and systolic blood pressure in Ang II‐infused mice lacking AT2 receptors (Daugherty et al., 2013). In addition, as the IC50 of PD123319 for the AT2 receptor was approximately 10 nM, 3 mg·kg−1·day−1 of PD123319 infusion resulted in an effective block of AT2 receptors without affecting the AT1 receptors (Kuizinga, Smits, Arends, & Daemen, 1998).

In the current study, we found that Ang II treatment increased the expression of mRNA for both AT1 and AT2 receptors, whereas Ang‐(1‐7) treatment further up‐regulated the mRNA for AT2 receptors and tended to increase the expression of mRNA for Mas receptors, but without statistical significance. These results suggested that AT2 receptors might play an important role in the effects of Ang‐(1‐7) on AAA. However, the binding affinity of Ang II to AT2 receptors is much higher than that of Ang‐(1‐7) and Ang‐(1‐7) has a high affinity for Mas receptors and a weak affinity for AT1 and AT2 receptors (Bosnyak et al., 2011; Yang et al., 2013). Thus, the role of MasR in the effect of Ang‐(1‐7) on AAA is indispensable.

Anti‐inflammatory and vasodilator effects are mediated by AT2 receptors and Mas receptors (Iwai & Horiuchi, 2009; Jones, Vinh, McCarthy, Gaspari, & Widdop, 2008; Villela et al., 2015). We and others have demonstrated that the protective effects of Ang‐(1‐7) on atherosclerosis were largely blocked by A779 (Yang et al., 2013) or PD123319 (Tesanovic et al., 2010). Recently, AT2 and Mas receptors were found to share 31% sequence identity (Villela et al., 2015), and these two receptors were colocalized and functionally interdependent in obese Zucker rat kidney (Patel, Ali, Samuel, Steckelings, & Hussain, 2017). More recently, Leonhardt et al. elegantly demonstrated the formation of heterodimers of Mas and AT2 receptors and thus inhibition of one of the heterodimer partners may cause inhibition of the other (Leonhardt et al., 2017). These observations may explain why, in our work, Ang‐(1‐7) activated both AT2 and Mas receptors, and A779 and PD123319 might inhibit their effects on both AT2 and Mas receptors. In further experiments, it would be interesting to clarify this relationship by using a Mas receptor deletion mouse model.

There was a noticeable difference between aneurysmal and non‐aneurysmal aortic wall in the number of vascular SMCs. Histological studies have shown that adventitial and medial inflammatory cell infiltration contributed to extracellular matrix degradation and loss of vascular SMCs, resulting in weakening and remodelling of the aortic wall (Raffort et al., 2017). Our results showed that Ang II treatment significantly reduced the content of SMCs in the wall of the abdominal aorta, while Ang‐(1‐7) treatment returned the SMC content to control levels in the vessel. Furthermore, the ratio of Bcl‐2/Bax, an index of cell apoptosis, was decreased after Ang II infusion but returned to baseline levels after Ang‐(1‐7) treatment in the aneurysmal aortic tissues and cultured HSMCs. These results were corroborated by TUNEL assay which showed that Ang II infusion enhanced while Ang‐(1‐7) treatment alleviated SMCs apoptosis. As MMPs play an essential role in the degradation of extracellular matrix and the destruction of the aortic wall integrity in AAA (Davis et al., 2014; Humphrey, Schwartz, Tellides, & Milewicz, 2015), we examined the expression level and activity of MMP2 in vitro and in vivo, and our results demonstrated that MMP2 expression and activity was significantly up‐regulated in the Ang II‐treated group but returned to control levels after Ang‐(1‐7) treatment. However, this beneficial effect on MMP2 by Ang‐(1‐7) was abolished by administration of A779 and PD123319. These results indicated that decreased SMC apoptosis and decreased expression and activity of MMP2 may be another fundamental mechanism involved in the therapeutic effect of Ang‐(1‐7) on AAA.

Previous studies found that ERK1/2, a member of the MAPKs family, was associated with MMP activity and inflammatory signalling and involved in the pathogenesis of AAA. In a mouse model of AAA, suppression of ERK1/2 signalling prevented the progression of AAA (Meng et al., 2017; Zhang, Naggar, et al., 2009). In our study, the expression level of p‐ERK1/2 was significantly increased by Ang II infusion which was attenuated by Ang‐(1‐7) in both the aneurysmal tissues and in cultures of HSMCs. In contrast, administration of A779 and PD123319 blocked these salutary effects of Ang‐(1‐7). Thus, down‐regulation of ERK1/2 via Mas and AT2 receptors may be involved in the molecular mechanism of the therapeutic effect of Ang‐(1‐7) on AAA.

In conclusion, our study has demonstrated that in a mouse model of AAA induced by Ang II infusion, Ang‐(1‐7) treatment markedly attenuated the formation and severity of AAA, as well as the remodelling of the aortic wall, through the inhibition of the inflammatory response, expression of MMPs, and the apoptosis of SMCs. The underlying molecular mechanisms may involve inhibited ERK1/2 signalling pathways via Ang‐(1‐7) stimulation of Mas and AT2 receptors. Thus, Ang‐(1‐7) may provide a novel and promising approach to the prevention and treatment of AAA.

AUTHOR CONTRIBUTIONS

C. Z., Y. Z. and J. Y. designed the project and corrected the manuscript, F. X., C. C., H. L. and M. Z. performed in vivo experiments, J. Z., X. X. and J. M. performed in vitro experiments, M. Z. and L. L. conducted data analysis, J. X. engaged in discussion of the experiments and F. X., J. C. and W. S. wrote the manuscript. The manuscript has been reviewed and approved by all authors.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1. Quantitative assessment of pathological remodeling of AAA in vivo. A, Measurement of the aortic wall thickness in 5 groups of mice (n = 5 in each group). B, The incidence of luminal thrombosis in 5 groups of mice (n = 20 in each group). C, The incidence of AAA rupture in 5 groups of mice (n = 20 in each group). D, The survival rate in 5 groups of mice (n = 20 in each group). *P < 0.05 vs. control group; #P < 0.05 vs. Ang II group; ▲P < 0.05 vs. Ang II + Ang‐(1‐7) group.

Click here for additional data file. (9.6MB, tif)

Figure S2. Effect of Ang‐(1‐7) on mRNA expression of AT1R, AT2R and MasR in the abdominal aortic tissues of ApoE−/− mice. A, B and C, Quantitative analysis of mRNA expression of AT1R, AT2R and MasR in the abdominal aortic tissues of 5 groups of mice (n = 5 in each group). *P < 0.05 vs. control group; #P < 0.05 vs. Ang II group; ▲P < 0.05 vs. Ang II + Ang‐(1‐7) group.

Click here for additional data file. (6MB, tif)

Table S1. Primers used for RT‐PCR analysis

Click here for additional data file. (15.3KB, docx)

ACKNOWLEDGEMENTS

This work was supported by the Program of Introducing Talents of Discipline to Universities (Grant B07035), the State Key Program of National Natural Science of China (Grant 81530014), the grants of the National Natural Science Foundation of China (Grants 81425004, 81770442, and 81570324), and the Taishan Scholars Program of Shandong Province, China.

Xue F, Yang J, Cheng J, et al. Angiotensin‐(1‐7) mitigated angiotensin II‐induced abdominal aortic aneurysms in apolipoprotein E‐knockout mice. Br J Pharmacol. 2020;177:1719–1734. 10.1111/bph.14906

Fei Xue and Jianmin Yang contributed equally to this study.

Contributor Information

Jianmin Yang, Email: yangjianminsdu@163.com.

Yun Zhang, Email: zhangyun@sdu.edu.cn.

Cheng Zhang, Email: zhangc@sdu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Quantitative assessment of pathological remodeling of AAA in vivo. A, Measurement of the aortic wall thickness in 5 groups of mice (n = 5 in each group). B, The incidence of luminal thrombosis in 5 groups of mice (n = 20 in each group). C, The incidence of AAA rupture in 5 groups of mice (n = 20 in each group). D, The survival rate in 5 groups of mice (n = 20 in each group). *P < 0.05 vs. control group; #P < 0.05 vs. Ang II group; ▲P < 0.05 vs. Ang II + Ang‐(1‐7) group.

Click here for additional data file. (9.6MB, tif)

Figure S2. Effect of Ang‐(1‐7) on mRNA expression of AT1R, AT2R and MasR in the abdominal aortic tissues of ApoE−/− mice. A, B and C, Quantitative analysis of mRNA expression of AT1R, AT2R and MasR in the abdominal aortic tissues of 5 groups of mice (n = 5 in each group). *P < 0.05 vs. control group; #P < 0.05 vs. Ang II group; ▲P < 0.05 vs. Ang II + Ang‐(1‐7) group.

Click here for additional data file. (6MB, tif)

Table S1. Primers used for RT‐PCR analysis

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