ORIGINAL RESEARCH

Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Axis

Protects against Lung Fibrosis by Inhibiting the MAPK/NF-kB Pathway
Ying Meng1, Chang-Hui Yu1, Wei Li1, Ting Li1, Wei Luo2, Shan Huang3, Ping-Sheng Wu4, Shao-Xi Cai1, and Xu Li3
1
Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China; 2Guangdong Provincial Key
Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou,
China; 3Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China; and 4Department of
Cardiovascular Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China

Abstract attenuating inflammation and a-collagen I production, which could

Accumulating evidence has demonstrated that up-regulation of the
angiotensin (Ang)-converting enzyme (ACE)/AngII/AngII type 1
receptor (AT1R) axis aggravates pulmonary fibrosis. The recently
discovered ACE2/Ang-(1-7)/Mas axis, which counteracts the activity
of the ACE/AngII/AT1R axis, has been shown to protect against
pulmonary fibrosis. However, the mechanisms by which ACE2 and
Ang-(1-7) attenuate pulmonary fibrosis remain unclear. We
hypothesized that up-regulation of the ACE2/Ang-(1-7)/Mas axis
protects against bleomycin (BLM)-induced pulmonary fibrosis by
inhibiting the mitogen-activated protein kinase (MAPK)/NF-kB
pathway. In vivo, Ang-(1-7) was continuously infused into Wistar
rats that had received BLM or AngII. In vitro, human fetal lung-1 cells
were pretreated with compounds that block the activities of AT1R,
Mas (A-779), and MAPKs before exposure to AngII or Ang-(1-7).
The human fetal lung-1 cells were infected with lentivirus-mediated
ACE2 before exposure to AngII. In vivo, Ang-(1-7) prevented
BLM-induced lung fibrosis and AngII-induced lung inflammation
by inhibiting the MAPK phosphorylation and NF-kB signaling
cascades. However, exogenous Ang-(1-7) alone clearly promoted
lung inflammation. In vitro, Ang-(1-7) and lentivirus-mediated
ACE2 inhibited the AngII-induced MAPK/NF-kB pathway, thereby
be reversed by the Mas inhibitor, A-779. Ang-(1-7) inhibited
AngII-induced lung fibroblast apoptotic resistance via inhibition
of the MAPK/NF-kB pathway and activation of the BCL-2-
associated X protein/caspase-dependent mitochondrial apoptotic
pathway. Ang-(1-7) alone markedly stimulated extracellular
signal–regulated protein kinase 1/2 phosphorylation and the NF-kB
cascade. Up-regulation of the ACE2/Ang-(1-7)/Mas axis protected
against pulmonary fibrosis by inhibiting the MAPK/NF-kB
pathway. However, close attention should be paid to the
proinflammatory effects of Ang-(1-7).
Keywords: renin–angiotensin system; angiotensin-converting
enzyme 2/angiotensin-(1-7)/Mas axis; pulmonary fibrosis;
mitogen-activated protein kinases; NF-kB
Clinical Relevance
The antifibrotic effects of angiotensin-(1-7) make the
heptapeptide a candidate for a therapeutic target in humans
with pulmonary fibrosis.

Pulmonary fibrosis is a progressive and fatal
lung disease characterized by chronic
inflammation, the migration and
proliferation of fibroblasts, the accumulation
of the extracellular matrix (ECM), and
remodeling of the lung parenchyma (1, 2).
Currently, there are no effective antifibrotic
therapies for pulmonary fibrosis (3).
A growing body of evidence indicates
that angiotensin (Ang) II, a bioactive
peptide of the renin–Ang system (RAS),
plays a key role in the initiation and the
maintenance of lung fibrosis (4). Ang-
converting enzyme (ACE) inhibitors (5)
and AngII type 1 receptor (AT1R) blockers

( Received in original form December 9, 2012; accepted in final form September 14, 2013 )
This work was supported by National Science Foundation of China research grant 30900659.
Author Contributions: Conception and design, X.L., S.X.-C.; analysis and interpretation, Y.M., C.-H.Y., W. Li, T.L., W. Luo, S.H., P.-S.W.; drafting the
manuscript for important intellectual content, Y.M., C.-H.Y.
Correspondence and requests for reprints should be addressed to Xu Li, Ph.D., Department of Emergency Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, China. E-mail: mylx99@gmail.com
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Am J Respir Cell Mol Biol Vol 50, Iss 4, pp 723–736, Apr 2014
Copyright © 2014 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2012-0451OC on October 29, 2013
Internet address: www.atsjournals.org

Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis 723
 ORIGINAL RESEARCH

Figure 1. Effects of constant infusion with angiotensin (Ang)-(1-7) or AngII on bleomycin (BLM)-induced pulmonary fibrosis. Representative lung images
stained with hematoxylin and eosin (A) or Masson’s trichrome (B) from rats treated with BLM, BLM plus Ang-(1-7), or BLM plus AngII (n = 6 rats per group).

724 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
ORIGINAL RESEARCH
(6) have been used for the treatment of
pulmonary fibrosis in animal models and
humans. Consequently, down-regulation of
the ACE/AngII/AT1R axis may be a viable
therapeutic strategy for pulmonary fibrosis.
The recent discovery of the ACE2/Ang-
(1-7)/Mas axis offers an alternative approach
for counter-regulating the ACE/AngII/
AT1R axis to produce beneficial effects (4, 7).
The heptapeptide Ang-(1-7), an enzymatic
product of ACE2 (8), has been shown to
counteract the detrimental effects of AngII
in the liver (9), heart (10), and kidneys (11).
Recently, Ang-(1-7) and ACE2 have been
shown to prevent bleomycin (BLM)-induced
pulmonary fibrosis (4). Hence, the ACE2/
Ang-(1-7)/Mas axis may potentially offer
a novel therapeutic strategy for pulmonary
fibrosis. However, the exact molecular
mechanism by which the ACE2/Ang-(1-7)/
Mas axis protects against pulmonary fibrosis
remains unclear.
NF-kB, which can be activated by
mitogen-activated protein kinases
(MAPKs) (12), is responsible for the
transcription of inflammatory factors and
profibrotic cytokines, which promote an
inflammatory response and fibrosis (13,
14). Moreover, as a cell survival pathway,
the activation of NF-kB signaling results in
the apoptotic resistance of lung fibroblasts,
which facilitates the accumulation of lung
fibroblasts (15), all of which contribute
equally to pulmonary fibrogenesis. AngII
has been reported to activate fibroblasts and
promote collagen accumulation by
activating MAPKs (16) and NF-kB (17).
However, Ang-(1-7) inhibited the MAPK
and NF-kB pathways in the liver (18) and
in a murine model of asthma (19). The loss
of ACE2 enhanced NF-kB–driven renal
inflammation in a mouse model of
obstructive nephropathy (20). Whether the
ACE2/Ang-(1-7)/Mas axis inhibits the
MAPK and NF-kB pathways stimulated by
AngII in lung fibroblasts has not yet been
investigated. We hypothesized that the
ACE2/Ang-(1-7)/Mas axis protects against
pulmonary fibrosis by inhibiting the
MAPK/NF-kB pathway.
Hence, our study aimed to investigate
the mechanism by which the ACE2/
Figure 1. (Continued). Original magnification, 320. Scale bar, 200 mm. (C) The morphological changes in fibrotic lungs were quantified using the Ashcroft
score. (D) Measurement of the collagen area in the lungs of animals in designated treatment groups stained with Masson’s trichrome. (E) The
hydroxyproline content of the lungs in animals in the various treatment groups. (F) Quantitative real-time RT-PCR (qRT-PCR) was used to determine the
plaminogen activator inhibitor-1 (PAI-1) mRNA levels. (G and H) Protein levels of connective tissue growth factor (CTGF), a-smooth muscle actin (a-SMA),
and a-collagen I in lung homogenates were determined using a Western blot assay. The data are presented as means 6 SEM. *P , 0.05 versus
control group; †P , 0.05 versus BLM group.
Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis
Ang-(1-7)/Mas axis protects against
pulmonary fibrosis in vivo and in vitro.
We found that Ang-(1-7) and lentivirus-
mediated ACE2 (lenti-ACE2) protected
against BLM- and AngII-induced
inflammation and ECM accumulation by
inhibiting the MAPK/NF-kB pathway.
Ang-(1-7) inhibited AngII-induced
apoptotic resistance of lung fibroblasts by
inhibiting the MAPK/NF-kB pathway and
activating the BCL-2-associated X protein
(bax)/caspase–dependent mitochondrial
apoptotic pathway.
Materials and Methods
Materials
AngII, Ang-(1-7), A-779 (a selective Mas
inhibitor), SB203580 (a p38 MAPK
inhibitor), PD98059 (a specific extracellular
signal–regulated protein kinase [ERK] 1/2
inhibitor), SP600125 (a Jun N-terminal
kinase [JNK]/MAPK inhibitor), and
BAY117082 (an IkK inhibitor) were
purchased from Sigma-Aldrich (St. Louis,
MO). Irbesartan (an AT1R blocker) was
kindly provided by Merck and Co.
(Darmstadt, Germany). BLM was
purchased from Nippon Kayaku (Tokyo,
Japan). Alzet osmotic pumps (models 2004
and 2ML4) were purchased from Durect
Corporation (Cupertino, CA). The other
reagents are described subsequently here.
Animals
Male Wistar rats weighting 200–300 g were
provided by the Central Animal Care
Facility of Southern Medical University
(Permission No. SCXK 2009-015). The
animals were housed in a controlled
environment (12 h light/12 h dark at
22–248 C), and received food and water ad
libitum. All of the animals received humane
care in compliance with the Chinese
Animal Protection Act, which is in
accordance with the National Research
Council criteria.
Animal Treatment Regimens
We established two animal models. In the
first model, 24 male Wistar rats were
randomly divided into four groups of six
rats each: a control group, a BLM treatment
group; a BLM plus Ang-(1-7) treatment
group; and a BLM plus AngII treatment
group. All of the rats received a single
intratracheal instillation of 200 ml of sterile
saline while under pentobarbital anesthesia.
The three BLM groups received sterile
saline that contained 5 mg/kg of BLM
sulfate. While the animals were under
anesthesia, micro-osmotic pumps were
implanted subcutaneously to enable 28 days
of continuous infusion with AngII or
Ang-(1-7) at a rate of 25 mg/kg/h. The
animals in the control group and the BLM
treatment groups received a constant
subcutaneous infusion of saline.
In the second study model, 24 male
Wistar rats were randomly divided into four
groups: a control group; an Ang-(1-7)
treatment group; an AngII treatment group;
and an AngII plus Ang-(1-7) treatment
group. Micro-osmotic pumps were
implanted subcutaneously in these animals
to enable 28 days of infusion with AngII
and/or Ang-(1-7) at a rate of 25 mg/kg/h.
The control animals each received a
constant subcutaneous infusion of saline.
Cell Culture
Human fetal lung (HFL)-1 cells (fibroblasts)
were obtained from the American Type
Culture Collection (Manassas, VA).
Pulmonary primary fibroblasts were isolated
from rat lungs as previously described (21).
The cells were cultured in a-Dulbecco’s
modified Eagle medium (GIBCO BRL,
Gaithersburg, MD) with antibiotics and
15% FBS. Next, the cells were preincubated
for 1 hour with irbesartan (1025 mol/L),
A-779 (1025 mol/L), PD98059 (1025 mol/L),
SB203580 (1025 mol/L), SP600125 (1025
mol/L), or BAY117082 (1025 mol/L) before
being exposed to AngII (1027 mol/L) or
Ang-(1-7) (1027 mol/L) for 1 hour.
Statistical Analysis
All of the data are expressed as means
(6 SEM). The data were analyzed using an
ANOVA with least significant difference for
multiple comparisons. The differences were
considered significant at P values less than
725
 ORIGINAL RESEARCH

0.05. All of the data were analyzed using
SPSS 13.0 (SPSS, Inc., Chicago, IL).
Complementary Assays
The use of hematoxylin and eosin,
Masson’s trichrome, and terminal
deoxynucleotidyl transferase dUTP nick
end labeling staining, the hydroxyproline
assay, immunohistochemistry,
immunofluorescence, quantitative real-time
RT-PCR, the production of ACE2 lentiviral
particles, and Western blot analysis are
described in the online supplement.
RESULTS
Effects of Ang-(1-7) or AngII on
BLM-Induced Pulmonary Fibrosis
The animals in the BLM group exhibited
higher Ashcroft scores (22) compared with
the control animals. The BLM-treated
animals presented characteristic
histological changes in lung tissue,
including areas of inflammatory
infiltration, thickening of the alveolar walls,
increased interstitial collagen deposition,
and a fibroblastic appearance. Chronic
infusion with Ang-(1-7) resulted in
a significantly lower Ashcroft score,
indicating a protective effect of Ang-(1-7)
against lung fibrosis. However, treatment
with AngII significantly aggravated BLM-

Figure 2. Ang-(1-7) counter-regulates the balance between the Ang-converting enzyme (ACE)/AngII/AngII type 1 receptor (AT1R) axis and the ACE2/Ang-(1-
7)/Mas axis in fibrotic lungs. Immunohistochemistry was performed to determine the localization of ACE2 (A) and ACE (B) proteins. Original magnification,
340. Scale bars, 60 mm. (C) Quantitative immunohistochemistry using ACE2 and ACE staining in rats depicted in A and B. (D) The protein changes in ACE2
and ACE were analyzed using Western blot assays. (E–H) The mRNA levels of ACE2, ACE, Mas, and AT1R of lung tissues with various treatments were
determined by qRT-PCR. The data are presented as means 6 SEM. *P , 0.05 versus the control group; †P , 0.05 versus the BLM group.

726 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
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Figure 3. The effects of Ang-(1-7) and AngII on the mitogen-activated protein kinase (MAPK)/NF-kB cascade in BLM-treated rats. (A) Lung phospho-
MAPK levels were significantly increased by BLM administration; this increase was prevented by constant infusion with Ang-(1-7) and augmented by
AngII treatment (A). (B and C) BLM treatment significantly increased the lung cytoplasmic levels of phospho-IkBa/b and NF-kB and the nuclear protein
levels of NF-kB, and decreased the cytoplasmic levels of IkBb; these changes were prevented by Ang-(1-7) treatment. AngII had a synergistic effect
with BLM. (D) BLM treatment significantly increased mRNA levels for the NF-kB target genes, intercellular cell adhesion molecule-1 (ICAM-1), TNF-a, an
IL-6 mRNA in the lungs; these increases were prevented with Ang-(1-7) treatment and augmented with AngII treatment. The data are presented as
means 6 SEM. *P , 0.05 versus the control group; †P , 0.05 versus the BLM group.

Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis 727
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Figure 4. (See figure legend on following page)

728 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
ORIGINAL RESEARCH

induced fibrosis, resulting in a higher
Ashcroft score for the BLM plus AngII
treatment group than for the BLM group
(Figures 1A and 1C). Along similar lines, an
increase in lung collagen accumulation
(assessed by measuring the collagen area
and lung hydroxyproline levels) was
observed in the BLM group; this increase
was significantly reduced by Ang-(1-7)
treatment and exacerbated by AngII
treatment (Figures 1D and 1E). Plaminogen
activator inhibitor-1 (PAI-1) massage RNA
(mRNA) levels and connective tissue growth
factor (CTGF), a-smooth muscle actin
(a-SMA), and a-collagen I protein levels
increased in the lungs of the BLM group; the
increases were significantly reduced by
Ang-(1-7) treatment and augmented by
AngII treatment (Figures 1F–1H; see also
Figure E2 in the online supplement).
Moreover, lung fibroblast apoptosis in vivo
was confirmed using immunofluorescence
with an FITC-conjugated terminal
deoxynucleotidyl transferase dUTP nick end
labeling reaction mixture in combination
with CY3 NHS ester–conjugated anti-
Vimentin antibody (Figure E1).
Exogenous Ang-(1-7) Regulated the
Balance between the ACE/AngII/
AT1R Axis and the ACE2/Ang-(1-7)-
Mas Axis
The locally based RAS is involved in the
development of lung fibrosis, and the ACE2/
Ang-(1-7)/Mas axis counteracts the
profibrotic effects of the ACE/AngII/AT1R
axis. Hence, the balance of these two axes
determines the development of pulmonary
fibrosis. To evaluate the influences of Ang-
(1-7) and AngII on the balance of these two
axes in BLM-induced pulmonary fibrosis,
the lung protein levels of ACE2 and ACE
were assessed using immunohistochemistry
(Figures 2A–2C) and Western blot
(Figure 2D); the lung mRNA levels of
components of the RAS, including ACE2,
Mas, ACE, and AT1R, were measured using
quantitative real-time RT-PCR (Figures
2E–2H). The results show that BLM
treatment resulted in an obvious up-
regulation of the ACE protein level and
increases of approximately 21-fold and 32-
fold in the ACE and AT1R mRNA levels,
respectively. However, BLM treatment
augmented the ACE2 protein level and
increased the ACE2 and Mas mRNA levels
roughly 1.7-fold and 7-fold, respectively,
compared with the control group. Thus,
BLM treatment shifted the balance toward
the ACE/AngII/AT1R axis. Strikingly, Ang-
(1-7) promoted the ACE2 and Mas levels
and decreased the ACE and AT1R levels
compared with the BLM group, regulating
the balance from the ACE/AngII/AT1R axis
toward the ACE2/Ang-(1-7)/Mas axis. In
contrast, AngII shifted the balance toward
the ACE/AngII/AT1R axis (Figure 2).
Constant Infusion with Exogenous
Ang-(1-7) Inhibited the
MAPK/NF-kB Pathway
It is well known that the MAPKs, including
ERK1/2, p38, JNK, and NF-kB, are crucial for
lung fibrogenesis. Increased phosphorylation
of MAPKs can stimulate NF-kB activation;
increased nuclear translocation of activated
NF-kB initiates a cascade of responses,
including abundant expression of
proinflammatory factors, adhesion molecules,
and cytokines that have been shown to be
involved in the pathology of lung fibrosis. We
determined the effects of administering
Ang-(1-7) or AngII on the MAPK/NF-kB
pathway induced by BLM. The results of
these experiments revealed that exogenous
Ang-(1-7) treatment inhibits the BLM-
induced phosphorylation of ERK1/2, p38
MAPK, and JNK (Figure 3A), and inhibits
the activation of the NF-kB cascade (Figures
3B–3D) in lung tissue. In contrast, the
coadministration of AngII and BLM resulted
in marked increases in the activation of the
MAPK/NF-kB pathway (Figures 3A–3D).
Dual Effects of Exogenous Ang-(1-7)
on the MAPK/NF-kB Pathway and
Collagen Deposition in Lung Tissue
To determine whether Ang-(1-7) has a dual
effect on the MAPK/NF-kB pathway in
lung tissue, we performed a second
experiment. The morphological results
show higher scores for both the AngII and
the Ang-(1-7) treatment groups compared
with the control group, suggesting that the
administration of Ang-(1-7) alone initiated
lung inflammation and ECM accumulation.
However, the coadministration of AngII
and Ang-(1-7) resulted in a significantly
lower score than administering AngII
alone, demonstrating that Ang-(1-7) has
a protective effect against lung injury,
induced by AngII (Figures 4A–4D). Both
Ang-(1-7) and AngII significantly increased
the levels of ACE, AT1R, and Mas
mRNA; thus, both up-regulated the two
axes of the local RAS. Interestingly, the
coadministration of Ang-(1-7) and AngII
led to obviously higher ACE2 and Mas
mRNA levels and markedly lower ACE
and AT1R mRNA levels than the
administration of AngII alone, suggesting
that Ang-(1-7) facilitates the regulation of
the balance of the two axes from the ACE/
AngII/AT1R axis toward the ACE2/Ang-
(1-7)/Mas axis (Figure 4E). Finally, both
Ang-(1-7) and AngII significantly
augmented the phosphorylation of ERK1/2,
p38 MAPK, and JNK (Figure 4G), in
addition to augmenting the activation of
the NF-kB cascade (Figures 4F and 4H) and
the protein levels of CTGF,a-SMA, and
collagen (Figure 4I) in lung tissue. In
contrast, the coadministration of Ang-(1-7)
and AngII markedly attenuated the
activation of the MAPK/NF-kB pathway
and the deposition of collagen compared
with the AngII treatment group (Figures
4F–4I).
Effects of Ang-(1-7) on the
Phosphorylation of MAPKs in
HFL-1 Cells
As demonstrated previously here, Ang-(1-7)
plays a dual role in the phosphorylation of
MAPKs in lung tissue. To determine
whether Ang-(1-7) has similar effects in
HFL-1 cells, we performed a third
experiment. As shown in Figures 5A–5B,
the Ang-(1-7) treatment alone stimulated
the phosphorylation of ERK1/2 in HFL-1

Figure 4. The dual effects of Ang-(1-7) on intrapulmonary renin–Ang system (RAS), the MAPK/NF-kB cascade, and extracellular matrix (ECM)
accumulation in rat lungs. These representative photographs of lung tissue were stained with hematoxylin and eosin (A) or Masson’s trichrome (B).
Original magnification, 320. Scale bar, 200 mm. (C) Morphological changes in fibrotic lungs were quantified using the Ashcroft score. (D) The collagen area
in animals from designated treatment groups was measured after staining with Masson’s trichrome. qRT-PCR was used to measure the mRNA levels of
the state of local RAS (including ACE, AT1R, ACE2, and Mas) (E) and inflammatory cytokines (including ICAM-1, TNF-a, and IL-6) (F). The
phosphorylation of MAPKs (G), the activation of the NF-kB pathway (H), and the protein levels of CTGF, a-SMA, and a-collagen I (I) were detected with
a Western blot assay. The data are presented as means 6 SEM. *P , 0.05 versus the control group; †P , 0.05 versus the Ang-(1-7) group; ‡P , 0.05
versus the AngII group.

Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis 729
 ORIGINAL RESEARCH

Figure 5. Ang-(1-7) inhibited the MAPKs/NF-kB pathway induced by AngII in human fetal lung (HFL)-1 cells. MAPK phosphoproteins were measured
using a Western blot assay. (A) HFL-1 cells in defined serum-free media were incubated with increasing concentrations of Ang-(1-7) (1025, 1027, and 1029
mol/L) for 15 minoute. Ang-(1-7) treatment alone stimulated the phosphorylation of extracellular signal–regulated protein kinase (ERK) 1/2 and
blunted the phosphorylation of Jun N-terminal kinase (JNK) at 1027 mol/L, but did not affect the phosphorylation of p38. (B) HFL-1 cells were incubated with
1027 mol/L Ang-(1-7) for increasing amounts of time. Ang-(1-7) treatment stimulated the phosphorylation of ERK1/2 in a time-dependent manner, peaking at
15 minutes. However, Ang-(1-7) markedly blunted the phosphorylation of JNK 15 minutes later, and did not affect the phosphorylation of p38. (C) HFL-1
cells were pretreated with irbesartan (1025 mol/L) or A-779 (1025 mol/L) for 1 hour before exposure to Ang-(1-7) (1027 mol/L) or AngII (1027 mol/L), as indicated,

730 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
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cells in a dose- and time-dependent suppressed by AngII (Figure 6C), resulting pathway and the synthesis of a-collagen I
manner. However, Ang-(1-7) markedly in a reversal of the decrease in apoptosis via Mas.
blunted the phosphorylation of JNK, and induced by AngII (Figure 6A). Interestingly,
did not affect the phosphorylation of p38. Ang-(1-7) alone either stimulated NF-kB
The Ang-(1-7) treatment also significantly signaling (Figure 5D) or activated the bax/ Discussion
decreased the phosphorylation of MAPKs caspase–dependent mitochondrial apoptotic
stimulated by AngII; this effect could be pathway in lung fibroblasts (Figure 6C), This study reached four novel conclusions
reversed by A-779, a Mas antagonist resulting in an increase in apoptosis. with respect to the antifibrotic effect of the
(Figure 5C). Our study also found that AngII ACE2/Ang-(1-7)/Mas axis on pulmonary
treatment resulted in significant increases in fibrosis. First, Ang-(1-7) regulates the
the mRNA levels of transforming growth balance of the RAS from the ACE/AngII/
Ang-(1-7) Promoted Apoptosis and
factor (TGF)-b and PAI-1 (Figure 6E) and AT1R axis toward the ACE2/Ang-(1-7)/Mas
Suppressed AngII-Induced Collagen
the protein levels of CTGF, a-SMA, and axis. Second, Ang-(1-7) and lenti-ACE2
Production via Mas in
a-collagen I (Figure 6D) compared with protect against BLM- or AngII-induced
Lung Fibroblasts
those in the control group. However, the inflammation and ECM accumulation by
It is well known that the apoptosis resistance
effects of AngII were suppressed by Ang- inhibiting the MAPK/NF-kB pathway.
of lung fibroblasts facilitates the
(1-7), MAPK blockers (Figures 6D and 6E), Third, Ang-(1-7) inhibits the AngII-
accumulation of fibroblasts and contributes
and the inhibitor of nuclear factor kappa-B induced apoptotic resistance of lung
to pulmonary fibrosis. To assess the effects of
kinase (IKK) inhibitor, BAY117082 fibroblasts by inhibiting the MAPK/NF-kB
Ang-(1-7) or AngII on fibroblastic apoptosis,
(Figures E4B and E4C). The effects of pathway and activating the mitochondrial
flow cytometry was performed (Figures 6A
Ang-(1-7) were negated by A-779 apoptotic pathway. Fourth, Ang-(1-7)
and 6B). The rate of early apoptosis of
(Figures 6D and 6E). These results alone initiated the proinflammatory
primary lung fibroblasts treated with AngII
demonstrate that AngII protects lung response in vivo and in vitro.
was lower (0.03%) than that in the control
fibroblasts against apoptosis and promotes Research has found that the ACE/
group (0.1%). In contrast, treatment with
the deposition of ECM, which can be AngII/AT1R axis is up-regulated in BLM-
Ang-(1-7) alone markedly enhanced the rate
inhibited by Ang-(1-7). induced lung fibrosis (4), resulting in
of early apoptosis (14.1%) compared with
increases in the ACE level, local AngII
the AngII-treated group and the control
production, and the AT1R level, all of
group. Moreover, the inhibitory effect of
Lenti-ACE2 Inhibited the MAPK/NF-kB which contribute to the initiation and
AngII on the early apoptosis rate could be
Pathway and the a-Collagen I progression of lung fibrosis (23). Consistent
reversed by Ang-(1-7), PD98059, SB203580,
Level Stimulated by AngII in with these reports, we observed that BLM
SP600125, and BAY117082. In addition,
HFL-1 Cells enhanced the ACE and AT1R levels in the
A-779 clearly abolished the effect of Ang-(1-7).
To determine whether ACE2 protects lungs. Constant infusion with exogenous
Next, we investigated the concrete
against lung fibrosis in vitro, HFL-1 cells AngII not only significantly aggravated
mechanism. On the one hand, AngII
were transfected with lenti-ACE2. The lung fibrosis in BLM-treated rats, but also
inhibited the bax/caspase–dependent
overexpression of ACE2 protein in the shifted the balance toward the ACE/AngII/
mitochondrial apoptotic pathway by up-
transfected cells was verified with AT1R axis. On the other hand, constant
regulating the B-cell lymphoma-2 (bcl-2)
a Western blot assay (Figure 7A). We found infusion with exogenous Ang-(1-7) down-
protein level and down-regulating the bax
that lenti-ACE2 attenuated the increase in regulated the ACE/AngII/AT1R axis by
and caspase 3 protein levels, resulting in
the phosphorylation of MAPKs (Figure 7B), decreasing the mRNA or protein levels of
a decrease in lung fibroblast apoptosis
the activation of the NF-kB cascade ACE and AT1R, which led to the
(Figure 6C). On the other hand, AngII
(Figures 7C and 7D), and the increase in amelioration of lung fibrosis in BLM-
stimulated NF-kB signaling (Figure 5D),
the CTGF, a-SMA, and a-collagen I treated rats. Interestingly, we found that the
a cell survival pathway, leading to apoptosis
protein levels (Figure 7E) induced by AngII; levels of both Mas and ACE2 were higher in
resistance in lung fibroblasts. Ang-(1-7)
these effects of lenti-ACE2 could be fibrotic lungs than in normal lungs, which
markedly inhibited the AngII-induced
reversed by A-779. The results indicate that was not consistent with previous reports
MAPK/NF-kB pathway (Figure 5D) and
ACE2, by cleaving AngII onto Ang-(1-7), on lung fibrosis (4, 24). The inconsistent
activated the bax/caspase–dependent
inhibits the AngII-induced MAPK/NF-kB results may be due to the differences in the
mitochondrial apoptotic pathway

Figure 5. (Continued). for 15 minutes. Ang-(1-7) treatment significantly decreased the MAPK phosphorylation stimulated by AngII, which could be
reversed by A-779. (D) The nuclear protein levels of NF-kB and the cytoplasmic protein levels of phospho-IkKa/b and IkBa were analyzed using
a Western blot assay. AngII treatment significantly increased the phospho-IkKa/b and NF-kB levels and decreased the IkBa levels; these effects could
be prevented by Ang-(1-7), irbesartan, PD98059, SB203580, and SP600125. The inhibitory effect of Ang-(1-7) could be reversed by A-779. Ang-(1-7)
alone stimulated NF-kB activity. qRT-PCR was used to measure the expression of NF-kB target genes (E). AngII treatment increased the mRNA
levels of the NF-kB target genes, ICAM-1, TNF-a, and IL-6; this effect could be inhibited by Ang-(1-7), irbesartan, PD98059, SB203580, and
SP600125. The inhibitory effect of Ang-(1-7) could be reversed by A-779. Exposure to Ang-(1-7) alone elevated the TNF-a mRNA levels. All of the
assays were performed in triplicate; the data are presented as means 6 SEM. *P , 0.05 versus the control group; †P , 0.05 versus the AngII group;
‡
P , 0.05 versus the Ang-(1-7) 1 AngII group.

Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis 731
 ORIGINAL RESEARCH

Figure 6. Ang-(1-7) promoted cell apoptosis and reduced the collagen levels after AngII administration in pulmonary primary fibroblasts. The cells were
preincubated for 1 hour with irbesartan (1025 mol/L), A-779 (1025 mol/L), PD98059 (1025 mol/L), SB203580 (1025 mol/L), SP600125 (1025 mol/L),
and BAY117082 before being exposed to AngII (1027 mol/L) or Ang-(1-7) (1027 mol/L) for 24 hours. A flow cytometry assay was used to detect cell
apoptosis by Annexin V-FITC 1 propidium iodide (PI) staining. (A and B) The early apoptotic cells located in the bottom right quadrant represent early

732 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
ORIGINAL RESEARCH
dosage and the duration of time used in our
animal model and other investigators’
studies. In our study, BLM (5 mg/kg)
-induced lung fibrosis lasted for 4 weeks. In
a study by Li and colleagues (24), however,
BLM (1 mg/kg) -induced lung fibrosis only
lasted for 2 weeks. The differences in the
dosage and the duration of animal model
studies may affect the ACE2 protein levels.
We suppose that, in the early stages of
a BLM-induced lung injury (2 wk), the
protective protein, ACE2, was inhibited;
after that period, ACE2 gradually increased
to protect against the injury. However, this
explanation could be refuted by the finding
that ACE2 is severely down-regulated in
humans with idiopathic pulmonary fibrosis
(24). Hence, the dynamics of ACE2 in
fibrotic tissue deserve further study.
Although the ACE2/Ang-(1-7)/Mas axis
ameliorated lung fibrosis in rats, the
molecular mechanism remains unclear.
Therefore, we performed additional
experiments to determine whether the ACE2/
Ang-(1-7)/Max axis attenuated lung fibrosis
by inhibiting the MAPK/NF-kB pathway
stimulated by AngII. The MAPK family
(ERK 1/2, p38 kinase, and JNK) and NF-kB
regulate cellular growth and inflammation,
leading to lung fibrosis (13, 25, 26).
Consistent with these reports, we found that
the MAPKs and the NF-kB cascade were
activated by BLM or AngII in vivo or
in vitro. The study by Bancroft and
colleagues (27) and our in vitro data show
that inhibiting MAPKs suppressed the
AngII-stimulated NF-kB cascade.
Furthermore, the increased levels of TGF-b,
CTGF, a-SMA, and a-collagen I induced by
AngII could be inhibited by MAPK
inhibitors and an IkK blocker. These data
highlight the important role of the MAPK/
NF-kB pathway in the pathogenesis of
AngII-mediated lung fibrosis. However, in
BLM- or AngII-treated rats and in lung
fibroblasts, Ang-(1-7) prevented the
phosphorylation of MAPKs and the NF-kB
cascade; these results align with the results
from other cell systems of lung (28–30).
Similarly, we also observed that the
overexpression of ACE2 alleviated the
proinflammatory and profibrotic effects of
AngII. A-779, a Mas antagonist, was able to
Figure 6. (Continued). apoptotic cells. (C and D) In addition, the protein levels of B-cell lymphoma-2 (bcl-2), the BCL-2–associated X protein (bax),
caspase3, CTGF, a-SMA, and a-collagen I were analyzed using a Western blot assay. (E) qRT-PCR was performed to determine the mRNA levels of PAI-1
and transforming growth factor (TGF)-b. All of the assays were performed in triplicate; the data are presented as means 6 SEM. *P , 0.05 versus the
control group; †P , 0.05 versus the AngII group; ‡P , 0.05 versus the Ang-(1-7) 1 AngII group.
Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis
reverse the suppressive effects of ACE2,
suggesting that ACE2 exerted its effect by
generating Ang-(1-7) (8). These results
indicate that the ACE2/Ang-(1-7)/Mas axis
inhibited the MAPK/NF-kB pathway
induced by AngII, leading to the attenuation
of inflammation and collagen secretion.
A large number of reports have
demonstrated that increased apoptosis of
alveolar epithelial cells and decreased
apoptosis of fibroblasts play important roles
in the pathogenesis of pulmonary fibrosis
(2, 31). Previous study has shown that
apoptosis of alveolar epithelial cells was
induced by AngII through the
mitochondrial pathway (29). In contrast,
we found that AngII up-regulated the bcl-2
protein level and down-regulated bax and
caspase 3 protein levels, resulting in
a decrease in lung fibroblast apoptosis. As
a cell survival pathway, activation of the
NF-kB pathway promotes the apoptotic
resistance of fibroblasts (15). Consistent
with this, we found that inhibition of the
MAPK/NF-kB pathway could reverse down-
regulation of bax and caspase 3 protein
levels and the decreased apoptosis of lung
fibroblasts induced by AngII. These results
indicate that AngII promoted pulmonary
fibrosis by inhibiting lung fibroblast
apoptosis through activation of the MAPK/
NF-kB pathway and inhibition of the bax/
caspase–dependent mitochondrial apoptotic
pathway. On the other hand, Studies by
Uhal and colleagues (29, 32) showed that
Ang-(1-7) could inhibit AngII- and
endoplasmic reticulum stress–induced
epithelial apoptosis. However, we found that
Ang-(1-7) markedly promoted fibroblast
apoptosis and activated the bax/
caspase–dependent mitochondrial apoptotic
pathway inhibited by AngII. The
proapoptotic effects of Ang-(1-7) were
reversed by pretreatment with A-779.
Hence, we supposed that, besides inhibiting
epithelial apoptosis, Ang-(1-7) also
ameliorated lung fibrosis by suppressing the
inflammation and the apoptotic resistance
of lung fibroblasts via inhibition of the
AngII-induced MAPK/NF-kB pathway.
Many studies on the biological
properties of Ang-(1-7) have led to
widespread consensus that Ang-(1-7) acts as
a counter-regulatory peptide in the RAS,
often opposing the inflammatory response
and the proliferative actions of AngII.
However, the effects of Ang-(1-7) are
variable: inflammation and growth-
stimulatory pathways are activated in some
cases (33–35). In agreement with these
studies, the present study shows that
constant infusion of Ang-(1-7) in rats
significantly stimulated the MAPK/NF-kB
pathway and increased collagen deposition
in the lungs. Furthermore, Ang-(1-7) alone
stimulated ERK1/2 phosphorylation in
a time- and dose-dependent manner, but it
blunted the phosphorylation of JNK in
HFL-1 cells. Interestingly, although Ang-(1-7)
alone stimulated NF-kB signaling (a cell
survival pathway), it also promoted the
apoptosis of primary lung fibroblasts by
activating the bax/caspase–dependent
mitochondrial apoptotic pathway. Therefore,
in comparison with the NF-kB cascade,
bax/caspase–dependent mitochondrial
apoptotic signaling may be a main
apoptosis-regulating pathway for Ang-(1-7).
How can Ang-(1-7) exert both
protective and deleterious effects? To some
extent, the variable responses to Ang-(1-7)
can be explained by the state of local
RAS activation. In the presence of high
glucose levels, Ang-(1-7) attenuated the
epithelial–mesenchymal transition and
TGF-b1 production in rat kidney epithelial
cells (36). In contrast, in the absence of RAS
activation or high glucose levels, Ang-(1-7)
induced epithelial–mesenchymal transition
and increased TGF-b1 and CTGF
production in rat kidney epithelial cells
(37). In accordance with these results,
we found that Ang-(1-7) attenuated
inflammation and the accumulation of
collagen in BLM- and AngII-treated
animals, whereas Ang-(1-7) alone
significantly induced an inflammatory
response and collagen deposition. A
plausible interpretation of these results
is that, when the ACE/AngII/AT1R axis
is activated by BLM or AngII, exogenous
Ang-(1-7) not only significantly down-
regulates the ACE/AngII/AT1R axis,
directly attenuating lung fibrosis, but
also up-regulates the ACE2/Ang-(1-7)/
Mas axis, thereby facilitating the
733
 ORIGINAL RESEARCH

Figure 7. The effects of lentiviral-mediated overexpression of ACE2 on AngII-treated HFL-1 cells. (A) The HFL-1 cells were transfected with lentiviral-
mediated ACE2 (lenti-ACE2) for 72 hours, and cell extracts were prepared to determine the ACE2 protein levels with a Western blot assay. (B) The
cells were transfected with lenti-ACE2 and then exposed in AngII (1027 mol/L) for 30 minutes after pretreating the cells with A779 for 1 hour. ERK1/2, p38,
and JNK phosphorylation were measured with a Western blot assay. (C) Experiments were performed as described in (B) for 1 hour. The nuclear protein
levels of NF-kB and the cytoplasmic protein levels of phospho-IkKa/b and IkBa were analyzed by performing a Western blot assay. (D and E)
Experiments were performed as described in (B) for 24 hours. The mRNA levels of the NF-kB target genes, ICAM-1, TNF-a, and IL-6 were detected by
performing a qRT-PCR (D). A Western blot assay was performed to measure the CTGF, a-SMA, and a-collagen I protein levels (E). All of the assays
were performed in triplicate; the data are presented as means 6 SEM. *P , 0.05 versus the control group; †P , 0.05 versus the AngII group; ‡P , 0.05
versus the Ang-(1-7) 1 AngII group.

734 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
ORIGINAL RESEARCH

hetero-oligomerization of Mas (a
physiological antagonist of AT1R) with the
AT1R and interfering with AngII action
(38). In contrast, in the absence of ACE/
AngII/AT1R axis activation, Ang-(1-7)
exerts proliferative and proinflammatory
effects by interacting with the AT1R (39,
40). These results raise the possibility that
the dual effects of Ang-(1-7) observed in
the present study were determined by the
state of activation of the ACE/AngII/AT1R
axis. Nevertheless, in human aortic
endothelial cells (41), Ang-(1-7) alone
inhibited MAPK phosphorylation,
reflecting the functional diversity of the
heptapeptide in different cell types. The
exact molecular mechanism of Ang-(1-7)/
Mas signaling in different cells deserves
further examination.
In summary, our study demonstrate
that exogenous Ang-(1-7) and ACE2
overexpression protect against BLM- or
AngII-induced pulmonary fibrosis by
down-regulating the MAPK/NF-kB
pathway. However, constant infusion of
Ang-(1-7) paradoxically initiates an
inflammatory response in the lungs. The
antifibrotic effects of Ang-(1-7) noted
here make the heptapeptide a strong
candidate for a therapeutic target in
humans with pulmonary fibrosis. Further
studies to identify the precise mechanism of
its action are needed to fully understand its
role and to determine the therapeutic
possibilities. n
Author disclosures are available with the text
of this article at www.atsjournals.org.
Acknowledgments: The authors thank Prof.
Ji-Man He for his critical review of this manuscript.

References
1. Wilson MS, Wynn TA. Pulmonary fibrosis: pathogenesis, etiology and
regulation. Mucosal Immunol 2009;2:103–121.
2. Ajayi IO, Sisson TH, Higgins PD, Booth AJ, Sagana RL, Huang SK, White
ES, King JE, Moore BB, Horowitz JC. X-linked inhibitor of apoptosis
regulates lung fibroblast resistance to Fas-mediated apoptosis. Am J
Respir Cell Mol Biol 2013;49:86–95.
3. Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, Colby
TV, Cordier JF, Flaherty KR, Lasky JA, et al.; ATS/ERS/JRS/ALAT
Committee on Idiopathic Pulmonary Fibrosis. An official ATS/ERS/
JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based
guidelines for diagnosis and management. Am J Respir Crit Care Med
2011;183:788–824.
4. Shenoy V, Ferreira AJ, Qi Y, Fraga-Silva RA, Dı́ez-Freire C, Dooies A,
Jun JY, Sriramula S, Mariappan N, Pourang D, et al. The angiotensin-
converting enzyme 2/angiogenesis-(1-7)/Mas axis confers
cardiopulmonary protection against lung fibrosis and pulmonary
hypertension. Am J Respir Crit Care Med 2010;182:1065–1072.
5. Molteni A, Wolfe LF, Ward WF, Ts’ao CH, Molteni LB, Veno P, Fish BL,
Taylor JM, Quintanilla N, Herndon B, et al. Effect of an angiotensin
II receptor blocker and two angiotensin converting enzyme
inhibitors on transforming growth factor-beta (TGF-beta) and alpha-
actomyosin (alpha SMA), important mediators of radiation-induced
pneumopathy and lung fibrosis. Curr Pharm Des 2007;13:1307–1316.
6. Couluris M, Kinder BW, Xu P, Gross-King M, Krischer J, Panos RJ.
Treatment of idiopathic pulmonary fibrosis with losartan: a pilot
project. Lung 2012;190:523–527.
7. Ferreira AJ, Shenoy V, Yamazato Y, Sriramula S, Francis J, Yuan L,
Castellano RK, Ostrov DA, Oh SP, Katovich MJ, et al. Evidence for
angiotensin-converting enzyme 2 as a therapeutic target for the
prevention of pulmonary hypertension. Am J Respir Crit Care Med
2009;179:1048–1054.
8. Raizada MK, Ferreira AJ. ACE2: a new target for cardiovascular disease
therapeutics. J Cardiovasc Pharmacol 2007;50:112–119.
9. Herath CB, Warner FJ, Lubel JS, Dean RG, Jia Z, Lew RA, Smith AI,
Burrell LM, Angus PW. Upregulation of hepatic angiotensin-
converting enzyme 2 (ACE2) and angiotensin-(1-7) levels in
experimental biliary fibrosis. J Hepatol 2007;47:387–395.
10. Katovich MJ, Grobe JL, Raizada MK. Angiotensin-(1-7) as an
antihypertensive, antifibrotic target. Curr Hypertens Rep 2008;10:
227–232.
11. Lo CS, Liu F, Shi Y, Maachi H, Chenier I, Godin N, Filep JG, Ingelfinger
JR, Zhang SL, Chan JS. Dual RAS blockade normalizes angiotensin-
converting enzyme-2 expression and prevents hypertension and
tubular apoptosis in Akita angiotensinogen–transgenic mice. Am J
Physiol Renal Physiol 2012;302:F840–F852.
12. Luo SF, Fang RY, Hsieh HL, Chi PL, Lin CC, Hsiao LD, Wu CC, Wang
JS, Yang CM. Involvement of MAPKs and NF-kappaB in tumor
necrosis factor alpha–induced vascular cell adhesion molecule 1
expression in human rheumatoid arthritis synovial fibroblasts.
Arthritis Rheum 2010;62:105–116.
13. Christman JW, Sadikot RT, Blackwell TS. The role of nuclear
factor-kappa B in pulmonary diseases. Chest 2000;117:
1482–1487.
14. Wong CK, Ip WK, Lam CW. Interleukin-3, -5, and granulocyte
macrophage colony-stimulating factor–induced adhesion
molecule expression on eosinophils by p38 mitogen-activated
protein kinase and nuclear factor-kB. Am J Respir Cell Mol Biol
2003;29:133–147.
15. Golan-Gerstl R, Wallach-Dayan SB, Zisman P, Cardoso WV, Goldstein
RH, Breuer R. Cellular flice-like inhibitory protein deviates
myofibroblast fas-induced apoptosis toward proliferation during lung
fibrosis. Am J Respir Cell Mol Biol 2012;47:271–279.
16. Gu J, Liu X, Wang QX, Tan HW, Guo M, Jiang WF, Zhou L. Angiotensin
II increases CTGF expression via MAPKs/TGF-b1/TRAF6 pathway in
atrial fibroblasts. Exp Cell Res 2012;318:2105–2115.
17. Cai Y, Yu SS, Chen TT, Gao S, Geng B, Yu Y, Ye JT, Liu PQ. EGCG
inhibits CTGF expression via blocking NF-kB activation in cardiac
fibroblast. Phytomedicine 2013;20:106–113.
18. Santos SH, Andrade JM, Fernandes LR, Sinisterra RD, Sousa FB,
Feltenberger JD, Alvarez-Leite JI, Santos RA. Oral angiotensin-(1-7)
prevented obesity and hepatic inflammation by inhibition of resistin/
TLR4/MAPK/NF-kB in rats fed with high-fat diet. Peptides 2013;46:
47–52.
19. El-Hashim AZ, Renno WM, Raghupathy R, Abduo HT, Akhtar S, Benter
IF. Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1
receptor, through suppression of ERK1/2– and NF-kB–dependent
pathways. Br J Pharmacol 2012;166:1964–1976.
20. Liu Z, Huang XR, Chen HY, Penninger JM, Lan HY. Loss of
angiotensin-converting enzyme 2 enhances TGF-b/smad-mediated
renal fibrosis and NF-kB–driven renal inflammation in a mouse model
of obstructive nephropathy. Lab Invest 2012;92:650–661.
21. White ES, Thannickal VJ, Carskadon SL, Dickie EG, Livant DL,
Markwart S, Toews GB, Arenberg DA. Integrin a4b1 regulates
migration across basement membranes by lung fibroblasts: a role for
phosphatase and tensin homologue deleted on chromosome 10.
Am J Respir Crit Care Med 2003;168:436–442.
22. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating
severity of pulmonary fibrosis on a numerical scale. J Clin Pathol
1988;41:467–470.
23. Marshall RP, McAnulty RJ, Laurent GJ. Angiotensin II is mitogenic for
human lung fibroblasts via activation of the type 1 receptor. Am J
Respir Crit Care Med 2000;161:1999–2004.
24. Li X, Molina-Molina M, Abdul-Hafez A, Uhal V, Xaubet A, Uhal BD.
Angiotensin converting enzyme-2 is protective but downregulated in
human and experimental lung fibrosis. Am J Physiol Lung Cell Mol
Physiol 2008;295:L178–L185.
25. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways
mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:
1911–1912.
26. Krug LT, Torres-González E, Qin Q, Sorescu D, Rojas M, Stecenko A,
Speck SH, Mora AL. Inhibition of NF-kappaB signaling reduces virus
load and gammaherpesvirus-induced pulmonary fibrosis. Am J
Pathol 2010;177:608–621.

Meng, Yu, Li, et al.: ACE2/Ang-(1-7)/Mas Axis and Pulmonary Fibrosis 735

27. Bancroft CC, Chen Z, Dong G, Sunwoo JB, Yeh N, Park C, Van Waes
C. Coexpression of proangiogenic factors IL-8 and VEGF by human
head and neck squamous cell carcinoma involves coactivation by
MEK-MAPK and IKK-NF-kappaB signal pathways. Clin Cancer Res
2001;7:435–442.
28. Gallagher PE, Tallant EA. Inhibition of human lung cancer cell growth by
angiotensin-(1-7). Carcinogenesis 2004;25:2045–2052.
29. Uhal BD, Li X, Xue A, Gao X, Abdul-Hafez A. Regulation of alveolar
epithelial cell survival by the ACE-2/angiotensin 1-7/Mas axis. Am J
Physiol Lung Cell Mol Physiol 2011;301:L269–L274.
30. Ni L, Feng Y, Wan H, Ma Q, Fan L, Qian Y, Li Q, Xiang Y, Gao B.
Angiotensin-(1-7) inhibits the migration and invasion of A549 human
lung adenocarcinoma cells through inactivation of the PI3K/Akt and
MAPK signaling pathways. Oncol Rep 2012;27:783–790.
31. Drakopanagiotakis F, Xifteri A, Polychronopoulos V, Bouros D. Apoptosis
in lung injury and fibrosis. Eur Respir J 2008;32:1631–1638.
32. Uhal BD, Nguyen H, Dang M, Gopallawa I, Jiang J, Dang V, Ono S,
Morimoto K. Abrogation of ER stress–induced apoptosis of alveolar
epithelial cells by angiotensin 1-7. Am J Physiol Lung Cell Mol
Physiol 2013;305:L33–L41.
33. Zimpelmann J, Burns KD. Angiotensin-(1-7) activates growth-
stimulatory pathways in human mesangial cells. Am J Physiol Renal
Physiol 2009;296:F337–F346.
34. Nie W, Yan H, Li S, Zhang Y, Yu F, Zhu W, Fan F, Zhu J. Angiotensin-(1-7)
enhances angiotensin II induced phosphorylation of ERK1/2 in
mouse bone marrow–derived dendritic cells. Mol Immunol 2009;46:
355–361.
35. Heringer-Walther S, Eckert K, Schumacher SM, Uharek L, Wulf-
Goldenberg A, Gembardt F, Fichtner I, Schultheiss HP, Rodgers K,
736 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 4 | April 2014
ORIGINAL RESEARCH
Walther T. Angiotensin-(1-7) stimulates hematopoietic progenitor
cells in vitro and in vivo. Haematologica 2009;94:857–860.
36. Zhou L, Xue H, Wang Z, Ni J, Yao T, Huang Y, Yu C, Lu L. Angiotensin-(1-7)
attenuates high glucose–induced proximal tubular epithelial-to-
mesenchymal transition via inhibiting ERK1/2 and p38 phosphorylation.
Life Sci 2012;90:454–462.
37. Burns WC, Velkoska E, Dean R, Burrell LM, Thomas MC. Angiotensin II
mediates epithelial-to-mesenchymal transformation in tubular cells
by ANG 1-7/MAS-1–dependent pathways. Am J Physiol Renal
Physiol 2010;299:F585–F593.
38. Kostenis E, Milligan G, Christopoulos A, Sanchez-Ferrer CF, Heringer-
Walther S, Sexton PM, Gembardt F, Kellett E, Martini L,
Vanderheyden P, et al. G-protein–coupled receptor Mas is
a physiological antagonist of the angiotensin II type 1 receptor.
Circulation 2005;111:1806–1813.
39. Lara LdaS, Cavalcante F, Axelband F, De Souza AM, Lopes AG,
Caruso-Neves C. Involvement of the Gi/o/cGMP/PKG pathway in the
AT2-mediated inhibition of outer cortex proximal tubule Na1-ATPase
by Ang-(1-7). Biochem J 2006;395:183–190.
40. Lara LS, Correa JS, Lavelle AB, Lopes AG, Caruso-Neves C. The
angiotensin receptor type 1-Gq protein-phosphatidyl inositol
phospholipase Cbeta–protein kinase C pathway is involved in
activation of proximal tubule Na1-ATPase activity by angiotensin(1-7)
in pig kidneys. Exp Physiol 2008;93:639–647.
41. Verano-Braga T, Schwämmle V, Sylvester M, Passos-Silva DG, Peluso
AA, Etelvino GM, Santos RA, Roepstorff P. Time-resolved
quantitative phosphoproteomics: new insights into angiotensin-(1-7)
signaling networks in human endothelial cells. J Proteome Res 2012;
11:3370–3381.