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Scientific RepoRts | 6:34621 | DOI: 10.1038/srep34621
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Angiotensin-(1–7) abrogates
angiotensin II-induced
proliferation, migration and
inammation in VSMCs through
inactivation of ROS-mediated
PI3K/Akt and MAPK/ERK signaling
pathways
Feng Zhang, Xingsheng Ren, Mingxia Zhao, Bing Zhou & Ying Han
The proliferation, migration and inammation of vascular smooth muscle cells (VSMCs) contribute
to the pathogenesis and progression of several cardiovascular diseases such as atherosclerosis
and hypertension. Angiotensin (Ang)-(1–7) and Ang II are identied to be involved in regulating
cardiovascular activity. The present study is designed to determine the interaction between Ang-(1–7)
and Ang II on VSMCs proliferation, migration and inammation as well as their underlying mechanisms.
We found that Ang-(1–7) signicantly suppressed the positive eects of Ang II on VSMCs proliferation,
migration and inammation, as well as on induction of the phosphorylation of Akt and ERK1/2 and
increase of superoxide anion level and NAD(P)H oxidase activity in VSMCs, whereas Ang-(1–7) alone had
no signicant eects. This inhibitory eects of Ang-(1–7) were abolished by Mas receptor antagonist
A-779. In addition, Ang II type 1 (AT
1
) receptor antagonist losartan, but not A-779, abolished Ang II
induced VSMCs proliferation, migration and inammation responses. Furthermore, superoxide anion
scavenger N-acetyl-L-cysteine (NAC) or NAD(P)H oxidase inhibitor apocynin inhibited Ang II-induced
activation of Akt and ERK1/2 signaling. These results indicate that Ang-(1–7) antagonizes the Ang
II-induced VSMC proliferation, migration and inammation through activation of Mas receptor and then
suppression of ROS-dependent PI3K/Akt and MAPK/ERK signaling pathways.
Vascular smooth muscle cells (VSMCs) are an essential component of vascular walls and their highly dierenti-
ated contractile phenotype is critically responsible for maintaining vascular tone
1
. e proliferation and migra-
tion of VSMCs play crucial roles in atherosclerotic lesion formation and restenosis aer angioplasty
2
. Accelerated
proliferation of VSMCs is closely linked with hypertension
3
and atherosclerosis
4
. e VSMC migration from
media into intima is a critical event in the neointima formation in restenosis and is also an important feature in
the formation of atherosclerotic plaques
5
. Vascular inammation is considered to be involved in vascular remod-
eling in various cardiovascular diseases including hypertension and atherosclerosis
6
. Atherosclerotic plaques are
complex lesions associated with chronic vascular inammation, which contributes to VSMC proliferation and
migration
7
.
Ang II is one of the biologically active peptides of the renin-angiotensin system (RAS). Ang II is pivotally
involved in endothelial dysfunction, vascular inammation and brosis in many cardiovascular diseases
8–11
. Most
of the pathophysiologic actions of Ang II are largely mediated by AT
1
receptors
12
. e Ang II/AT
1
R activity plays
Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical
University, Nanjing, Jiangsu 211166, China. Correspondence and requests for materials should be addressed to
Y.H. (email: yhancn@njmu.edu.cn)
Received: 18 May 2016
Accepted: 24 August 2016
Published: 30 September 2016
OPEN
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Scientific RepoRts | 6:34621 | DOI: 10.1038/srep34621
an important role in the proliferation and migration of VSMCs in the development of atherosclerosis
13
. Ang II/AT
1
receptor is also demonstrated to be pro-inammatory in the progression of atherosclerosis
14
. Ang II binding to
the AT
1
receptor results in stimulation of a variety of intracellular signaling pathways, including the phosphatidy-
linositol 3-kinase (PI3K)/Akt
15
and the mitogen-activated protein kinases (MAPKs), contributing to the prolifer-
ation, migration and inammation of VSMCs
16,17
. Prior studies have indicated that the diverse actions of Ang II in
the vasculature are mediated by NAD(P)H oxidase derived reactive oxygen species (ROS), via activating multiple
signaling molecules, including PI3K/Akt or MAPK
18–20
.
Angiotensin converting enzyme 2 (ACE2) was recently identied as a multifunctional monocarboxypeptidase
responsible for the conversion of Ang II to Ang-(1–7)
21
. Ang-(1–7) is recognized to be an important therapy for
vascular disorders associated with vascular remodeling that are likely mediated by specic Mas receptors and are
selectively blocked by its specic antagonist D-Alanine-Ang-(1–7) (A-779)
22,23
. Ang-(1–7) is recently found to
have a protective role in systemic hypertension, oxidative stress and tubulointerstitial brosis in diabetic mice
24
.
e Ang-(1–7) levels were signicantly decreased 2 weeks aer vascular balloon injury, which was rescued by
rosuvastatin treatment
25
. Ang-(1–7) abolishes advanced glycated end product (AGE) -induced cellular hypertro-
phy and myobroblast transformation via inhibition of ERK1/2
26
. Oral formulation of Ang-(1–7) improves lipid
metabolism and prevents high-fat diet-induced hepatic steatosis and inammation in mice
27
. Ang-(1–7) prevents
Ang II-induced brosis in cremaster microvessels
28
. However, little is known in regard to the interaction of Ang-
(1–7) and Ang II in VSMC proliferation, migration and inammation, which are critical events in the process of
atherosclerosis. e present study was designed to explore whether Ang-(1–7) ameliorated the Ang II-induced
proliferation, migration and inammation in VSMCs and the exact cellular mechanisms.
Results
Ang-(1–7) inhibited Ang II-induced proliferation in VSMCs. e conuent VSMCs were seeded into
96 well-plates and incubated with dierent concentrations of Ang II and Ang-(1–7) separately for 24 h. All the
pretreatments were administered 5 min before Ang II or Ang-(1–7) treatment. Cell Counting Kit-8 (CCK-8)
assay showed that Ang-(1–7) treatment had no signicant eect on the VSMC proliferation (Fig.1A), but Ang II
exerted an approximately 3-fold increase of VSMC proliferation at the dose of 100 nmol/L (Fig.1B). Pretreatment
of VSMCs with Ang-(1–7) signicantly attenuated Ang II-induced VSMC proliferation, which was abolished
by Mas receptor antagonist A-779 application before Ang-(1–7) (Fig.1C). In addition, Ang II up-regulated pro-
liferating cell nuclear antigen (PCNA) protein expression in VSMCs which were also obviously suppressed by
pretreatment with Ang-(1–7), and this eect of Ang-(1–7) was inhibited by A-779 (Fig.1E ). Pre-incubation of
AT
1
receptor antagonist losartan, but not A–779 blocked the stimulatory eect of Ang II on VSMC proliferation.
Neither losartan nor A-779 alone has eect to induce proliferation of VSMCs (Fig.1D).
Ang-(1–7) suppressed Ang II-induced migration in VSMCs. e transwell Boyden chamber exper-
iment exhibited that treatment VSMCs with Ang II for 24 h markedly increased the number of VSMCs that
migrated through transwell chamber. More important, aer the VSMCs were pre-incubated with Ang-(1–7) for
5 min, Ang II-induced VSMC migration was signicantly depressed. is inhibitory eect of Ang-(1–7) on Ang
II was abolished by A-779 (Fig.2A). AT
1
receptor antagonist losartan eectively inhibited Ang II-induced VSMCs
migration, but A-779 pretreatment was unable to prevent the VSMC migration induced by Ang II. Neither losar-
tan nor A-779 alone has eect to induce migration of VSMCs (Fig.2B).
Ang-(1–7) retarded Ang II-induced inammation in VSMCs. To examine whether Ang-(1–7) inhib-
ited the inammatory responses of VSMCs induced by Ang II, the expressions of inammatory mediators of
VSMCs including monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1)
and interleukin (IL)-1β were determined by Western Blot aer Ang II application for 24 h. Pretreatment with
Ang-(1–7) signicantly retarded Ang II-induced inammatory responses of VSMCs associated with up-regulated
MCP-1, VCAM-1 and IL-1β expressions, and this effect of Ang-(1–7) was blocked by A-779 (Fig.3A). As
expected, pretreatment with losartan, rather than A-779 blocked Ang II-induced VSMC inammation. Neither
losartan nor A-779 alone has eect to induce inammatory response of VSMCs (Fig.3B).
Ang-(1–7) prevented Ang II-induced phosphorylation of Akt and ERK1/2. e phosphorylation of
Akt and ERK1/2 were markedly increased aer treatment VSMC with Ang II for 30 min, lasting at least 60 min.
However, total ERK1/2 (t-ERK1/2) and total Akt (t-Akt) protein levels were not inuenced by Ang II (Fig.4A).
Pretreatment VSMCs with Ang-(1–7) for 5 min signicantly inhibited Akt and ERK1/2 phosphorylation induced
by Ang II, and this eect was also blocked by A-779 (Fig.4B). e increased phosphorylation of Akt and ERK1/2
induced by Ang II were eectively blocked by pre-incubation of losartan, rather than A-779, while either losartan
or A-779 alone has no eect to induce phosphorylation of Akt and ERK1/2 in VSMCs (Fig.4C).
Ang-(1–7) diminished Ang II-induced ROS production. e ROS production was detected aer the
conuent VSMCs were stimulated by Ang II (100 nmol/l) for dierent time (0, 5, 10, 30 min). Both dihydro-
ethidium (DHE) uorescence intensity and lucigenin-derived chemiluminescence method results showed that
the superoxide anions production in VSMCs was remarkably enhanced upon Ang II stimulation for 30 min
(Fig.5A,C). NAD(P)H oxidase activity in VSMCs was also augmented in Ang II-treated VSMCs (Fig.5C). e
dramatic increases in superoxide anion level and NAD(P)H oxidase activity of VSMCs induced by Ang II were
diminished by pre-treated with Ang-(1–7), and this eect of Ang-(1–7) was blocked by A-779 (Fig.5B,C). e
increased superoxide anion level and NAD(P)H oxidase activity in VSMCs induced by Ang II were blocked by
pre-incubation of losartan, but not A-779. Both losartan and A-779 alone have no eect to inuence superoxide
anion level and NAD(P)H oxidase activity of VSMCs (Fig.5D).
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Scientific RepoRts | 6:34621 | DOI: 10.1038/srep34621
NAD(P)H oxidase-derived superoxide anion activated the PI3K/Akt and MAPK/ERK signaling
pathways in Ang II simulated VSMCs. Pretreatment with either N-acetyl-L-cysteine (NAC) (a superoxide
anions scavenger) or apocynin (a NAD(P)H oxidase inhibitor) abolished the eects of Ang II on the activation
of Akt and ERK1/2 (Fig.6).
Discussion
Growing evidences suggest that dysfunction of VSMCs including proliferation, migration and inammation is
involved in the development of several diseases such as atherosclerosis and hypertension
13
. e Ang II/AT
1
recep-
tor activity plays an important role in the proliferation, migration and inammation of VSMCs in the develop-
ment of the above diseases
29
. e present study demonstrates new ndings that Ang-(1–7)/Mas receptor activity
Figure 1. Eects on VSMCs proliferation. (A) Eects of 4 doses of Ang-(1–7) (0.1, 1, 10, 100 nmol/L)
treatment on VSMCs proliferation; (B) eects of 4 doses of Ang II (0.1, 1, 10, 100 nmol/L) on VSMCs
proliferation; (C) eects of control, Ang-(1–7) (100 nmol/L), Ang II (100 nmol/L), Ang-(1–7)+ Ang II, A-779
(100 nmol/L), A-779+ Ang-(1–7)+ Ang II on VSMCs proliferation; (D) eects of A-779 (100 nmol/L) and
losartan (100 nmol/L) on VSMCs proliferation or on Ang II (100 nmol/L) induced VSMCs proliferation
response; (E) eects of control, Ang-(1–7) (100 nmol/L), Ang II (100 nmol/L), Ang-(1–7)+ Ang II, A-779
(100 nmol/L), A-779+ Ang-(1–7)+ Ang II on PCNA expression in VSMCs. Values are mean ± SE. *P < 0.05 vs.
control;
†
P < 0.05 vs. Ang II. (A–D), n = 6 for each group; (E) n = 3 for each group.
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Scientific RepoRts | 6:34621 | DOI: 10.1038/srep34621
signicantly inhibited Ang II-induced VSMC proliferation, migration and inammation through inactivation of
superoxide anions-mediated PI3K/Akt and MAPK/ERK signaling pathways in VSMCs.
VSMCs are quiescent in healthy mature vascular tissue, however, they are activated and initiated abnormal
proliferation and migration in response to vascular injury
30
. Vascular injury also causes vascular inammation
which combining with proliferation and migration are recently accepted to be as the key contributors in the
pathophysiology of hypertension and atherosclerosis
10
. Inhibition of VSMC proliferation, migration and inam-
mation is an important strategy for therapy of atherosclerosis-related diseases
31
. Among various circulatory fac-
tors, Ang II is a well-characterized pathophysiological culprit peptide which has various actions for not only
maintaining the blood volume, modulating blood pressure, but also promoting the VSMC proliferation, migra-
tion, inammation and apoptosis via AT
1
receptors in hypertension and atherosclerosis
32,33
. Incubation of rat
tubular epithelial cells with Ang II signicantly increased key pro-inammatory cytokines expressions, which
was blocked by losartan
34
. It is known that employment of Ang II receptor blockers may be helpful to abate
inammation processes and disease progression
35
. Inhibition of Ang II/AT
1
activity has protective eects on
VSMCs
36,37
. Ang-(1–7) is another major active component in the RAS, which can be converted from Ang II by
ACE2
21
. Studies reported that Ang-(1–7) and Ang II have complicated interactions in dierent parts of the body
in dierent animal models. Some studies have shown that Ang-(1–7) plays an opposite role to Ang II and inhib-
its the eects of Ang II in some peripheral tissues
38,39
. Ang-(1–7) prevents Ang II-induced brosis in cremaster
microvessels
28
. e Ang-(1–7)/Mas receptor axis is counteractive to Ang II-induced proliferation, migration and
inammation in human brain VSMC and cerebral microvessels
40,41
. ACE2 deciency accelerated Ang II-induced
mRNA expressions of inammatory cytokines, including MCP-1 and IL-1β
42
. In the present study, we found that
Ang II drastically stimulated the VSMC proliferation and migration, and markedly up-regulated the expressions
of inammatory mediators including MCP-1, VCAM-1 and IL-1β , while Ang-(1–7) had no signicant eects
to induce VSMC proliferation, migration and inammatory responses. However pretreatment with Ang-(1–7)
Figure 2. Eects on VSMCs migration. (A) Eects of control, Ang-(1–7) (100 nmol/L), Ang II (100 nmol/L),
Ang-(1–7)+ Ang II, A-779 (100 nmol/L), A-779+ Ang-(1–7)+ Ang II on VSMCs migration; (B) eects of A-779
(100 nmol/L) or losartan (100 nmol/L) on VSMCs migration and Ang II-induced VSMCs migration response.
Values are mean ± SE. *P < 0.05 vs. control;
†
P < 0.05 vs. Ang II. n = 3 for each group.
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Scientific RepoRts | 6:34621 | DOI: 10.1038/srep34621
on VSMCs signicantly inhibited Ang II-induced proliferation, migration and inammatory responses. ese
protective eects of Ang-(1–7) on VSMCs were blocked by Mas receptors inhibitor A-779. ese results indicate
that Ang-(1–7) is a potential antagonist in the disruption of Ang II induced excessive proliferation, migration and
inammation of VSMCs and these eects of Ang-(1–7) are done through activation of Mas receptors. We also
found that blockade of AT
1
receptors, but not Mas receptors, obviously diminished the proliferation, migration
and inammatory responses in VSMCs induced by Ang II, which indicates that the positive eects of Ang II on
the VSMCs to induce proliferation, migration and inammation is AT
1
receptor dependent.
Oxidative stress is a critical modulator in the progression of hypertension and atherosclerotic lesions
35
.
NAD(P)H oxidase is emerged as a multi-component enzyme complex and one of major origins of the superoxide
anions in vascular system
43
. NAD(P)H oxidase-derived ROS play a critical role in Ang II-induced proliferation
and migration of VSMCs
44,45
. Increased ROS is involved in the pathogenesis of Ang II-dependent hyperten-
sion
46
. Suppression of ROS by knockdown of NAD(P)H oxidase largely inhibited the Ang II-induced prolifera-
tion, inammation in human mesangial cells
47
. We also previously revealed that the NAD(P)H oxidase-derived
superoxide anions in the paraventricular nucleus (PVN) are responsible for Ang II-induced sympathetic outow
in hypertensive rats
48
. Ang-(1–7) is recently reported to counteract the Ang II-induced ROS over-production in
cerebral endothelial cells
8
. e nonpeptide AVE0991, an agonist of the Mas receptor, signicantly attenuated ROS
production in Ang II-treated VSMCs
49
. Our results in the present study also showed that both NAD(P)H oxidase
activity and superoxide anion level increased signicantly in Ang II-treated VSMCs. Pretreatment with Ang-
(1–7) inhibited Ang II induced increases in NAD(P)H oxidase activity and superoxide anion level in VSMCs. is
eect of Ang-(1–7) was also abolished by Mas receptors inhibitor A-779. ese results indicated that NAD(P)
H oxidase derived ROS may be responsible for mediating the eects of Ang II to induce VSMCs proliferation,
Figure 3. Eects on VSMCs inammation. (A) Eects of control, Ang-(1–7) (100 nmol/L), Ang II
(100 nmol/L), Ang-(1–7)+ Ang II, A-779 (100 nmol/L), A-779+ Ang-(1–7)+ Ang II on MCP-1, VCAM-1 and
IL-1β protein expressions in VSMCs; (B) eects of A-779 (100 nmol/L) or losartan (100 nmol/L) on the MCP,
VCAM-1 and IL-1β protein expressions or Ang II (100 nmol/L) induced inammation response in VSMCs.
Values are mean ± SE. *P < 0.05 vs. control;
†
P < 0.05 vs. Ang II. n = 3 for each group.