Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice: ANP for pulmonary fibrosis
Okamoto et al. Respiratory Research
Atrial natriuretic peptide protects against bleomycin-induced pulmonary fibrosis via vascular endothelial cells in mice
Atsuko Okamoto 0 1
Takashi Nojiri 1
Kazuhisa Konishi 0
Takeshi Tokudome 1
Koichi Miura 1
Jun Hino 1
Mikiya Miyazato 1
Yohkoh Kyomoto 0 1
Kazuhisa Asai 0
Kazuto Hirata 0
Kenji Kangawa 1
0 Department of Respiratory Medicine, Osaka City University Graduate School of Medicine , 1-4-3, Asahi-machi, Abeno-ku, Osaka-City, Osaka 545-8585 , Japan
1 Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute , 5-7-1, Fujishirodai, Suita-City, Osaka 565-8565 , Japan
Background: Pulmonary fibrosis is a life-threatening disease characterized by progressive dyspnea and worsening pulmonary function. Atrial natriuretic peptide (ANP), a heart-derived secretory peptide used clinically in Japan for the treatment of acute heart failure, exerts a wide range of protective effects on various organs, including the heart, blood vessels, kidneys, and lungs. Its therapeutic properties are characterized by anti-inflammatory and anti-fibrotic activities mediated by the guanylyl cyclase-A (GC-A) receptor. We hypothesized that ANP would have anti-fibrotic and anti-inflammatory effects on bleomycin (BLM)-induced pulmonary fibrosis in mice. Methods: Mice were divided into three groups: normal control, BLM with vehicle, and BLM with ANP. ANP (0.5 μg/kg/min via osmotic-pump, subcutaneously) or vehicle administration was started before BLM administration (1 mg/kg) and continued until the mice were sacrificed. At 7 or 21 days after BLM administration, fibrotic changes and infiltration of inflammatory cells in the lungs were assessed based on histological findings and analysis of bronchoalveolar lavage fluid. In addition, fibrosis and inflammation induced by BLM were evaluated in vascular endothelium-specific GC-A overexpressed mice. Finally, attenuation of transforming growth factor-β (TGF-β) signaling by ANP was studied using immortalized mouse endothelial cells stably expressing GC-A receptor. Results: ANP significantly decreased lung fibrotic area and infiltration of inflammatory cells in lungs after BLM administration. Furthermore, similar effects of ANP were observed in vascular endothelium-specific GC-A overexpressed mice. In cultured mouse endothelial cells, ANP reduced phosphorylation of Smad2 after TGF-β stimulation. Conclusions: ANP exerts protective effects on BLM-induced pulmonary fibrosis via vascular endothelial cells.
Atrial natriuretic peptide; Pulmonary fibrosis; Bleomycin; Vascular endothelial cell; Transforming growth factor-β
Pulmonary fibrosis is a life-threatening disease
characterized by progressive dyspnea and worsening
pulmonary function . Although the pathologic processes that
cause disease progression are not fully understood, the
common pathological features of pulmonary fibrosis are
infiltration by inflammatory cells, including activated
macrophages . No effective therapeutic strategies for
this condition have been established; therefore,
development of effective treatment options is desirable.
Bleomycin (BLM)-induced pulmonary fibrosis is the
rodent model most commonly used to study idiopathic
pulmonary fibrosis . Administration of BLM causes
epithelial injury, followed by neutrophil-dominant and
lymphocyte-dominant inflammation that leads to fibrosis
. Vascular endothelial cells are a major target of
BLMinduced pulmonary fibrosis [4, 5].
Atrial natriuretic peptide (ANP) is a heart-derived
secretory peptide that mediates a wide range of
biological functions including diuresis, natriuresis,
vasorelaxation, and inhibition of the
renin–angiotensin–aldosterone system. These effects are mediated by
specific binding of ANP to the guanylyl cyclase-A
(GC-A) receptor [6, 7]. The GC-A receptor is
predominantly expressed in the heart and vascular
endothelium, indicating that the cardiovascular system
is the main target for treatments using ANP [6–8].
ANP exerts protective effects in a wide range of
organs, including the heart, blood vessels, kidneys, and
lungs, in which it exhibits both anti-inflammatory and
anti-fibrotic activities [6–10]. Previous studies showed
that ANP administration has beneficial effects on
acute lung injury in patients requiring mechanical
ventilation  or who experience postoperative
respiratory and cardiovascular complications following
lung cancer surgery [12, 13]. Recently, we reported
that ANP exerts a protective effect against
lipopolysaccharide-induced acute lung injury in mice
by suppressing vascular E-selectin expression . On
the basis of these studies, we hypothesized that ANP
may reduce BLM-induced pulmonary fibrosis, in
particular via vascular endothelial cells. In this study,
we investigated the protective effects of ANP on
BLM-induced pulmonary fibrosis using vascular
endothelium–specific GC-A overexpressed mice.
C57BL/6 N mice (male, 7 weeks old, weighing 21–23 g
each) were purchased from Japan SLC (Shizuoka, Japan).
We previously established the overexpressed mice for
Tie2-Cre-inducible overexpression of GC-A, which is
termed the endothelium-specific GC-A overexpressed
mice in this study. We previously confirmed that
Tie2Cre-GC-A overexpression mice showed GC-A protein of
vascular endothelial cells in the lung was upregulated
compared to wild type mice . Animals were
maintained at a controlled temperature of 24 °C ± 1 °C under
a 12:12 h light–dark cycle, and were fed a standard diet.
Water was freely available. All experimental protocols
described herein were approved by the Animal Care
Ethics Committee of the National Cerebral and
Cardiovascular Center Research Institute, Japan.
BLM administration and ANP treatment
The mice were anesthetized with 3% isoflurane delivered
in a box, and BLM (1 mg/kg, Nippon Kayaku Co, Tokyo,
Japan) in 80 μl of saline was administered via
oropharyngeal aspiration as previously described ; an identical
volume of sterile saline was administered to normal
control mice. ANP (0.5 μg/kg/min, Peptide Institute Inc,
Osaka, Japan) or vehicle was subcutaneously infused via
an osmotic mini-pump (Alzet Model 2004, Duret
Corporation, Cupertino, CA, USA), and the pumps were
implanted 72 h before BLM administration, as
previously described [10, 14, 15]; the infusion continued until
the mice were euthanized. Mice were divided into three
groups: normal control mice, BLM-treated mice
receiving ANP, and BLM-treated mice receiving vehicle
(n = 20 in each group).
On day 7 after BLM administration, mice were assessed
by measuring cell counts in bronchoalveolar lavage
(BAL) fluid, as described below, and immunostaining.
The remainder of the mice were euthanized for
histological and gene expression analysis of the lung on
day 21 after BLM administration. Vascular
endothelium–specific GC-A overexpressed mice and WT
littermates were also subjected to bleomycin inhalation, and
then sacrificed at 21 days after BLM administration. The
left lung was fixed by intratracheal instillation of 4%
paraformaldehyde for 7 days, and subsequently
embedded in paraffin. Paraffin sections were stained with
hematoxylin–eosin and Masson trichrome (MT).
Quantitative evaluation of lung fibrosis
Lung sections were stained with Masson trichrome, and
then each slide was scanned completely in a zigzag
fashion, and the percentage of fibrotic area in the whole
lung field was assessed. Brightfield images of Masson
trichrome-stained slides were acquired on an FSX100
system (Olympus, Tokyo, Japan) and the fibrotic area
(expressed as a percentage of the whole lung field) was
analyzed by using CellSens Dimension software version
Immunostaining of lung
For Mac-3 staining, tissue sections were deparaffinized,
and endogenous peroxidase was blocked with 3% H2O2
for 30 min. After each step, the tissue sections were rinsed
twice in phosphate-buffered saline (PBS) for 5 min. The
deparaffinized tissue sections were incubated with Protein
Block (DakoCytomation, Glostrup, Denmark) for 15 min.
The rat anti-mouse Mac-3 antibody was diluted in an
antibody diluent buffer (dilution 1:500; BioLegend, San
Diego, CA, USA) and applied overnight at 4 °C. After
incubation with primary antibodies, the slides were
incubated with biotinylated rabbit anti-rat IgG for 60 min,
followed by incubation with peroxidase-conjugated
avidin–biotin complex (Vectastain ABC kit; Vector
Laboratories, Burlingame, CA, USA) for 30 min. Antigen–
antibody complexes were visualized with 0.5%
diaminobenzidine (DakoCytomation) and 0.3% hydrogen peroxide,
and then counterstained with hematoxylin.
Gene expression analysis
Total RNA from lung was homogenized in
guanidiumphenol-chloroform and isolated using the RNeasy mini kit
(Qiagen, Hilden, Germany). The RNA was then
reversetranscribed into cDNA using a QuantiTect Reverse
Transcription kit (Qiagen). Quantitative PCR assays were
conducted in a 96-well plate using SYBR Premix Ex Taq
(Takara, Siga, Japan) on a Light Cycler 480 System II
(Roche Applied Science, Indianapolis, IN, USA). Primer
sequences are provided in Table 1. PCR settings were as
follows: initial denaturation for 30 s at 95 °C, followed by
38 cycles of 5 s at 95 °C and 20 s at 57 °C (interleukin
[IL]-6); 5 s at 95 °C, 10 s at 56 °C, and 15 s at 72 °C
(IL1β); 5 s at 95 °C and 20 s at 60 °C (basic fibroblast growth
factor [bFGF], transforming growth factor-β [TGF-β],
connective tissue growth factor [CTGF], and 36B4); or 5 s
Table 1 Sequence of primers used in the study
at 95 °C and 20 s at 58 °C (monocyte chemoattractant
protein-1 [MCP-1], collagen 1A, and tissue inhibitor of
metalloproteinases type1 [TIMP1]). Melting curve analysis
was conducted with temperature increasing from 72 to
98 °C. Gene expression levels were normalized against
corresponding levels of the housekeeping gene 36B4.
Cell culture analysis of mouse immortalized endothelial cells
SVEC, a mouse immortalized endothelial cell line, was
obtained from Y. Takuwa (Kanazawa University). SVEC
cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) supplemented with 10% fetal calf
serum (FCS). SVEC cells stably expressing GC-A-FLAG
(SVEC/GC-A) were established using ecotropic
retrovirus expressing the tagged protein. The details of the
methodology will be described elsewhere (K. Miura et
al., manuscript in revision). The SVEC/GC-A cells were
treated with or without TGF-β (1 ng/ml) and/or ANP
(0.1 μM) for 30 min for western blot analysis, and for
4 h for gene expression analysis.
Western blot analysis
Cultured cells were lysed in RIPA buffer (1% Nonidet
P-40, 50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM
EDTA, 0.1% SDS, 1% sodium deoxycholate)
supplemented with protease and phosphatase inhibitor cocktail
(Nacalai Tesque, Inc., Kyoto, Japan). The lysate was
centrifuged at 12,000 rpm at 4 °C for 20 min, and the
supernatant was collected. Equal amount of lysates were
separated by 4–15% SDS-PAGE (Bio-Rad, Hercules, CA,
USA) and transferred to a polyvinylidene fluoride
membrane (Millipore, Billerica, MA, USA). The
membrane was incubated in polyvinylidene blocking reagent
(Toyobo, Tokyo, Japan) at room temperature for 20 min,
and then incubated at 4 °C overnight with the
appropriate primary antibody diluted in Can Get Signal Solution
1 (Toyobo). The primary antibody was detected by a
horseradish peroxidase–conjugated secondary antibody
diluted in Tris-buffered saline (pH 7.4) containing 0.1%
Tween-20, and visualized with Luminata Forte Western
HRP substrate (Millipore, Billerica, MA, USA). An image
of the membrane was acquired on a LAS-4000 mini
luminescent image analyzer (Fujifilm, Tokyo, Japan). The
primary antibodies used for the analysis were as follows:
anti-phospho-VASP (Ser239) (#3114, Cell Signaling
Technology, Beverly, MA, USA), anti-phospho-VASP
(Ser157) antibody (#3111, Cell Signaling Technology),
anti-VASP (#3112, Cell Signaling Technology),
antiphospho-Smad1 (Ser463/465) / Smad5 (Ser463/465) /
Smad8 (Ser426/428) (#9511, Cell Signaling Technology),
rabbit anti-Smad1 mAb (#6944, Cell Signaling
Technology), rabbit anti-phospho-Smad2 mAb (#3108,
Cell Signaling Technology), mouse anti-Smad2 mAb
(#3103, Cell Signaling Technology), and mouse
antiGAPDH mAb (sc-32233, Santa Cruz Biotech, Dallas,
Data are expressed as means ± SEM. Between-group
comparisons were performed using the unpaired Student’s
t-test. For multiple-group comparisons, one-way ANOVA,
followed by the post-hoc Fisher’s least significant
difference test, was used. P < 0.05 was considered to be
ANP attenuated BLM-induced pulmonary fibrosis and
inflammation in mice
First, we examined the in vivo anti-fibrotic and
antiinflammatory effects of ANP on BLM-induced pulmonary
fibrosis in mice. Histological examination of the lungs in
mice after BLM administration revealed lung parenchymal
fibrotic changes in comparison with the normal control
group (Fig. 1a, b, d, e). Compared with vehicle, ANP
Fig. 1 ANP attenuates BLM-induced pulmonary fibrosis in mice. BLM was administered intratracheally to C57BL/6 mice on Day 0, and samples
were removed on Day 21. ANP or vehicle was subcutaneously infused using an osmotic mini-pump throughout the experiment. Representative
micrographs of lung tissue stained with hematoxylin–eosin (HE: upper panels) and Masson trichrome (MT: lower panels): normal control mice
(a, d), BLM-treated mice receiving vehicle (b, e), and BLM-treated mice receiving ANP (c, f). Scale bar: 500 μm. g Fibrotic area, measured using
image analysis software, and is expressed as a percentage of the whole lung field. Values represent means ± SEMs (n = 5 mice per group). *p <
0.05. NA: not assessed because of the absence of fibrotic area in normal lungs. h Body weight changes of mice after BLM administration. Values
represent means ± SEMs (in normal control group, n = 13; in BLM with vehicle group, n = 15, in BLM with ANP group, n = 14)
pretreatment significantly attenuated BLM-induced lung
fibrotic changes (Fig. 1a–f ). Quantitative assessment of
the severity of lung fibrosis in MT-stained tissue sections
demonstrated that ANP pretreatment significantly
attenuated BLM-induced lung fibrotic lesions relative to vehicle
(Fig. 1g). More than 3 days after BLM administration,
significant body weight loss was observed in the
vehicletreated group relative to the normal control group.
However, ANP pretreatment significantly attenuated
BLM-induced weight loss (Fig. 1h). To investigate the
accumulation of inflammatory cells in the lungs, we
examined the number of inflammatory cells in BAL fluid
and performed Mac3 staining of the lung. In BAL fluid,
both total and individual cell counts were significantly
elevated in the vehicle-treated group relative to the normal
control group. ANP pretreatment significantly decreased
total, macrophage, and lymphocyte cell counts relative to
vehicle (Fig. 2a-d). Quantitative assessment of the number
of inflamed cells, as determined by Mac3 staining,
demonstrated that ANP pretreatment significantly attenuated the
number of inflammatory cells in the lungs induced by
BLM relative to vehicle (Fig. 2e-h). These results indicate
A (x104cells/ml) Total cell count
8 * *
C (x103cells/ml) Neutrophils
H (Number/field) Number of Mac3-positive cells
60 * *
Fig. 2 ANP attenuates inflammation in the lungs induced by BLM administration. Numbers of total cells (a), macrophages (b), neutrophils (c), and
lymphocytes (d) in bronchoalveolar lavage (BAL) fluid on Day 7 after BLM administration. Values represent means ± SEMs (n = 5 mice per group). *p <
0.05. Lung sections from normal control mice (e), BLM-treated mice receiving vehicle (f), and BLM-treated mice receiving ANP treatment (g) obtained
7 days after BLM administration were stained with Mac-3. Representative images are shown at 200× magnification. h Mac-3–positive cells were
counted in ten high-power fields (HPF). The data are expressed as means ± SE (in normal control group, n = 4; in the other groups, n = 5. *p < 0.05)
that ANP can attenuate the fibrotic changes and
accumulation of inflammatory cells in BLM-induced pulmonary
ANP attenuated the expression of cytokines induced by
BLM in mouse lung
To evaluate the anti-inflammatory and anti-fibrotic
effects of ANP in BLM-induced lung fibrosis, we
analyzed mRNA expression changes of
proinflammatory cytokines and pro-fibrotic cytokines in
the lungs. With the exception of TGF-β and IL-1β,
expression levels of several mRNAs were significantly
elevated in the vehicle-treated group after BLM
administration in comparison with the normal control
group. ANP pretreatment significantly reduced the
expression levels of IL-6, MCP-1, TIMP1, and IL-1β
relative to vehicle (Fig. 3a, b, e, g). The gene
expression levels of bFGF and collagen 1A were lower in
the ANP-treated group than in the vehicle-treated
group, but the difference was not significant (Fig. 3c,
d). By contrast, the mRNA level of TGF-β was not
significantly altered by BLM or ANP treatment
(Fig. 3f ). These results indicate that ANP has the
potential to reduce the production of pro-inflammatory
and pro-fibrotic cytokines and collagen accumulation
associated with pulmonary fibrosis in mice.
Fig. 3 ANP attenuates the elevated mRNA levels of pro-inflammatory and pro-fibrotic cytokines in BLM-administered mice. Quantitative RT-PCR
analysis of IL-6 (a), MCP-1 (b), bFGF (c), collagen 1A (d), TIMP1 (e), TGF-β (f), and IL-1β (g) in lung tissues 21 days after BLM administration. Relative
mRNA level of each cytokine in normal control or BLM/vehicle-treated or BLM/ANP-treated mice are shown. Values represent means ± SEMs
(n = 5 mice per group). *p < 0.05
Anti-fibrotic and inflammatory effects of vascular
endothelium–specific GC-A overexpressed mice in
BLMinduced pulmonary fibrosis
Because vascular endothelial cells play a major role of
BLM-induced pulmonary fibrosis, we hypothesized that
GC-A expressed on vascular endothelial cells might be
responsible for the anti-fibrotic and inflammatory effects
of ANP. Accordingly, to determine the target of the
anti-fibrotic and inflammatory effects of ANP in
BLMinduced pulmonary fibrosis, we used vascular
endothelium–specific GC-A overexpressed mice. BLM-induced
pulmonary fibrosis was reduced in vascular
endothelium–specific GC-A overexpressed mice relative to WT
littermates (Fig. 4a-h). Quantitative histological analysis
revealed that BLM-induced fibrotic changes were
significantly reduced in vascular endothelium–specific GC-A
overexpressed mice (Fig. 4i). In BAL fluid, total and
macrophages cell counts were significantly reduced in
the overexpressed mice (Fig. 4j, k). Cell counts of
neutrophils and lymphocytes were also lower in the
overexpressed mice, but the difference was not significant
(Fig. 4l, m). Furthermore quantitative assessment of the
number of inflamed cells in Mac3 staining confirmed
that the number of inflammatory cells in the lung was
significantly lower in the overexpressed mice (Fig. 4n-r).
These results indicate that ANP exerts its anti-fibrotic
and inflammatory effects in the lung via vascular
Effects of ANP on mouse endothelial cells in TGF-β
To investigate the molecular mechanism of ANP/GC-A
signaling in endothelial cells, we established a mouse
immortalized endothelial cell line stably expressing
GCA (SVEC/GC-A). First, we observed robust
phosphorylation of VASP at Ser157 and Ser239 in SVEC/GC-A cells
following ANP administration, indicating that stable
expression of GC-A confers a high level of responsiveness
to ANP (Fig. 5a). Second, to examine the effects of ANP
on endothelial cells, we analyzed protein expression,
phosphorylation levels, and mRNA expression in SVEC/
GC-A stimulated with TGF-β and/or ANP. In response
to TGF-β, phosphorylation of Smad2 was elevated in
SVEC/GC-A cells (Fig. 5a). ANP pretreatment decreased
phosphorylation of Smad2 after TGF-β stimulation
(Fig. 5a). The phosphorylation levels of Smad1/5/8 were
similar between ANP-treated and vehicle-treated SVEC/
GC-A cells. In addition, mRNA levels of MCP-1,
collagen 1A, and CTGF were significantly elevated after
TGF-β stimulation in SVEC/GC-A cells (Fig. 5b-d).
ANP significantly decreased the expression levels of
MCP-1 and CTGF following TGF-β stimulation (Fig. 5b,
d). The mRNA level of collagen 1A was lower in
ANPtreated SVEC/GC-A cells, however the difference was
not significant (Fig. 5c). These results suggest that ANP
attenuates the TGF-β/Smad2 signaling pathway in
By comparing ANP-treated mice with vehicle mice, and
vascular endothelium–specific GC-A overexpressed with
WT mice, in a BLM-induced pulmonary fibrosis model,
we showed for the first time that ANP exerts an
antifibrotic effect on BLM-induced pulmonary fibrosis via
vascular endothelial cells. Our findings indicate that
ANP treatment represents a promising option for
preventing the progression of pulmonary fibrosis.
Previous studies showed that pulmonary inflammation
is responsible for pulmonary fibrosis in humans and
BLM-treated mice [4, 5, 16]. Several proinflammatory
cytokines, including MCP-1 and IL-6, are involved in
pulmonary inflammation and the development of
BLMinduced pulmonary fibrosis in mice [17–19]. A previous
study showed that anti-MCP-1 therapy using gene
transfection could reduce BLM-induced pulmonary fibrosis
in mice . In this study, ANP significantly attenuated
the elevated mRNA levels of MCP-1and IL-6 in
BLM-induced pulmonary fibrosis.
Activated endothelial cells secrete proinflammatory
cytokines and profibrotic mediators, which recruit and
activate inflammatory cells and fibroblasts, resulting in
collagen deposition [4, 5]. A previous study showed that
vascular endothelial cells exposed to BLM in vitro
increased the secretion of certain profibrotic mediators
. In addition, activated endothelial cells can
contribute to prolonged tissue injury by promoting the
expression of proinflammatory cytokines and adhesion
molecules, resulting in leukocyte homing and the
extravasation of cells at sites of inflammation [4, 5].
However, little is known about the benefits of targeting
vascular endothelial cells in BLM-induced pulmonary
fibrosis in vivo. In this study, we showed that ANP acts
on vascular endothelial cells, resulting in anti-fibrotic
and inflammatory effects, in BLM-induced pulmonary
TGF-β is a key signaling molecule involved in the
initiation and enhancement of tissue fibrosis . Previous
studies showed that the development of fibrosis in
various tissues including the lung is less severe in
Smad3-deficient mice than in control mice [22, 23].
Therefore, the TGF-β/Smad3 pathway plays a key role in
the mechanisms leading to fibrosis following tissue
injury. Previous studies showed that ANP/cGMP signaling
inhibits TGF-β–induced Smad2 and Smad3 nuclear
translocation in pulmonary arterial smooth muscle cells
[24, 25]. However, little is known about the effects of
ANP on vascular endothelial cells in TGF-β signaling. In
this study, we found that ANP attenuated pulmonary
M (cells/ml) Lymphocytes
Fig. 4 Anti-fibrotic and inflammatory effects of vascular endothelium–specific GC-A overexpressed mice in BLM-induced pulmonary fibrosis. BLM
was administered intratracheally into vascular endothelium–specific GC-A overexpressed (Tg) mice and WT littermates on Day 0, and lung tissues
were removed on Day 21. Representative micrographs of lung tissue stained with hematoxylin–eosin (HE: upper panels) and Masson trichrome
(MT: lower panels): WT littermates treated without BLM (a, e), vascular endothelium–specific GC-A overexpressed mice treated without BLM (b, f),
WT littermates treated with BLM (c, g), and vascular endothelium–specific GC-A overexpressed mice treated with BLM (d, h) are shown. Scale bar:
500 μm. i Fibrotic area was measured using image analysis software and is expressed as a percentage of the whole lung field. Values represent
means ± SEM (WT without BLM, n = 4; Tg without BLM, n = 3; WT with BLM, n = 5; Tg with BLM, n = 5). *p < 0.05. NA, not assessed because of the
absence of fibrotic area in normal lungs. j–m Numbers of total cells (j), macrophages (k), neutrophils (l), and lymphocytes (m) in bronchoalveolar
lavage (BAL) fluid on Day 21 after BLM administration. Values represent means ± SEM (n = 5 mice per group). *p < 0.05. Lung sections obtained
21 days after BLM administration were stained with Mac-3 in WT littermates treated without BLM (n), vascular endothelium–specific GC-A
overexpressed mice treated without BLM (o), WT littermates treated with BLM (p), and vascular endothelium-specific GC-A overexpressed mice
treated with BLM (q). Representative images are shown at 200× magnification. Macrophages were identified by Mac-3 staining. r Mac-3–positive
cells were counted in ten high-power fields (HPF), and the data are expressed as means ± SE. (n = 3–5 mice per group). *p < 0.05
Fig. 5 Effects of ANP on TGF-β signaling in mouse endothelial cells. a Western blot analysis with the antibodies indicated at left using the cell
lysates prepared from SVEC/GC-A cells stimulated with TGF-β (1 ng/ml) and/or ANP (0.1 μM) for 30 min. The blot shown is representative of three
independent experiments. b–d Quantitative RT-PCR analysis of MCP-1 (b), collagen 1A (c), and CTGF (d) in SVEC/GC-A stimulated with TGF-β
(1 ng/ml) and/or ANP (0.1 μM) for 4 h. Relative mRNA expressions in vehicle, ANP, TGF-β/vehicle, and TGF-β/ANP groups are shown. Values
represent means ± SEMs (n = 6 per group). *p < 0.05
fibrosis and inflammation induced by BLM, at least in
part, via inhibition of Smad2 phosphorylation in TGF-β
signaling. However, we could not determine the detailed
mechanisms underlying the effects of ANP treatments
on TGF-β signaling. Recent studies showed that the
most obvious involvement of endothelial cells is related
to endothelial-to-mesenchymal transition (EndoMT) in
the context of pulmonary diseases such as pulmonary
hypertension or pulmonary fibrosis [26, 27]. In addition,
we could not investigate the effects of ANP on
functional investigations including bronchoconstriction in
this study. Therefore, further studies are required to
elucidate these issues.
ANP is an endogenous peptide that has been approved
in Japan for treatment of acute heart failure since 1995,
and adverse events from its use are very rare. Therefore,
the clinical safety of ANP has already been established.
Recently, we reported that ANP not only had prophylactic
effects on postoperative cardiopulmonary complications,
but also exerted protective effect from postoperative
cancer recurrence after surgical resection of tumors [12–14].
We showed that ANP prevents cancer metastasis via
vascular endothelial cells . Therefore, ANP represents
a promising option for the safe treatment of various
In conclusion, this study is the first to show that ANP
exerts anti-fibrotic and anti-inflammatory effects in
BLM-induced pulmonary fibrosis via vascular
endothelial cells, possibly by attenuating the phosphorylation of
Smad2 in TGF-β signaling.
ANP: Atrial natriuretic peptide; GC-A: Guanylyl cyclase-A; BLM: Bleomycin;
TGF-β: Transforming growth factor-β; WT: Wild-type; BAL: Bronchoalveolar
lavage; MT: Masson trichrome; PBS: Phosphate-buffered saline; IL: Interleukin;
bFGF: Basic fibroblast growth factor; CTGF: Connective tissue growth factor;
MCP-1: Monocyte chemoattractant protein-1; TIMP1: Tissue inhibitor of
metalloproteinases type1; DMEM: Dulbecco's Modified Eagle Medium;
FCS: Fetal calf serum; SVEC: A mouse immortalized endothelial cell line;
SVEC/GC-A: SVEC cells stably expressing GC-A-FLAG.
This work was supported in part by research grants from JSPS KAKENHI Grant
Number JP26861136, Osaka Cancer Society, Japan Research Foundation for
Clinical Pharmacology, Kobayashi Foundation for Cancer Research, Mochida
Memorial Foundation for Medical and Pharmaceutical Research, Uehara
Memorial Foundation, the Senri Life Science Foundation, Kato Memorial
Bioscience Foundation, and Takeda Science Foundation to T. Nojiri.
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Conception and Design: AO, TN, KKonishi, KKangawa. Analysis and
Interpretation: AO, TN, KKonishi, KA, KKangawa. Data collection: AO, TN, TT,
KM, YK. Drafting of the manuscript and production of important intellectual
content; AO, TN, KKonishi, TT, KM, HH, JH, MM, KA, KH, KKangawa. Obtaining
funding: TN, KKangawa. All authors have approved the version of the
The authors declare that they have no competing interests.
Consent for publication
All experimental protocols described herein were approved by the Animal
Care Ethics Committee of the National Cerebral and Cardiovascular Center
Research Institute, Japan (Permit Number: 16040).
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