Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity
Sato et al. Respiratory Research
Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity
Seidai Sato 0
Shintaro Shinohara 0
Shinya Hayashi 0
Shun Morizumi 0
Shuichi Abe 0
Hiroyasu Okazaki 0
Yanjuan Chen 0
Hisatsugu Goto 0
Yoshinori Aono 0 2
Hirohisa Ogawa 1
Kazuya Koyama 0
Haruka Nishimura 0
Hiroshi Kawano 0
Yuko Toyoda 0
Hisanori Uehara 1
Yasuhiko Nishioka 0
0 Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University , 3-18-15 Kuramoto-cho, Tokushima 770-8503 , Japan
1 Department of Pathology and Laboratory Medicine, Graduate School of Health Biosciences, Tokushima University , Tokushima , Japan
2 National Hospital Organization Higashi Tokushima Medical Center , Tokushima , Japan
Background: Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear. Methods: Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin. Results: Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects. Conclusions: These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function.
Fibrocytes; Nintedanib; Pulmonary fibrosis
Platelet-derived growth factor (PDGF), fibroblast growth
factor (FGF) 1/2, and vascular endothelial growth factor
(VEGF) have been implicated in the pathogenesis of
pulmonary fibrosis [
]. Nintedanib is a tyrosine kinase
inhibitor that is specific also for PDGFRα and β, FGFR1,
2, and 3, and VEGFR1, 2, and 3 [
]. In two phase III
clinical trials (INPULSIS 1 and 2), treatment with
nintedanib for one year led to reductions in the annual
rate of decline in forced vital capacity versus placebo in
patients with idiopathic pulmonary fibrosis (IPF) [
However, the mechanisms by which nintedanib regulates
pulmonary fibrosis is not fully explored. Several studies
have reported the anti-fibrotic effects of BIBF 1000 [
and nintedanib (BIBF1120) [
]. However, the roles of
main targets, PDGFR, FGFR and VEGFR of nintedanib
have not yet been analyzed in detail.
Fibrocytes are monocyte-derived cells that are a
subpopulation of mesenchymal progenitor cells [
Fibrocytes appear to be derived from the differentiation
of CD14-positive peripheral blood mononuclear cells,
and express markers of hematopoietic cells, leukocytes,
and fibroblast products [
]. Marked increases in
circulating fibrocyte numbers and a positive correlation
between the abundance of fibroblastic foci and the
number of lung fibrocytes have been reported in patients
with IPF [
]. Moeller et al. also showed that the
percentage of CD45/collagen-1-positive fibrocytes was
increased in the peripheral blood of patients with IPF,
and proposed that the quantification of circulating
fibrocytes may allow for the prediction of early mortality in
these patients [
]. These findings strongly suggest that
fibrocytes are involved in the pathogenesis of pulmonary
fibrosis. Furthermore, we previously indicated that the
PDGF signaling pathway, which is a potential target for
nintedanib, plays a critical role in fibrocyte migration
into fibrotic lungs and contributes to fibrogenesis [
We also demonstrated that fibrocytes play a role in the
pathogenesis of pulmonary fibrosis by producing various
growth factors [
] (Abe S, et al. manuscript in
preparation). However, the effects of nintedanib on fibrocytes
Therefore, we herein focus on fibrocytes and discuss
several rationales for the anti-fibrotic properties of
nintedanib. We assess the effects of nintedanib on the
proliferation of fibroblasts induced by fibrocytes, the
differentiation of fibrocytes from monocytes, and the
migration of fibrocytes. We show that nintedanib reduces
the number of fibrocytes that infiltrate in the lungs and
mitigated fibrosis in an experimental murine model of
Detailed methods are described in the Additional file 1.
Isolation of human fibrocytes and monocytes
Human fibrocytes were isolated according to
previously described methods [
]. All procedures for
consent, sample collection, and privacy protection
were approved by the Ethics Committee of Tokushima
University Hospital. Human mononuclear cells (HMNC)
were isolated from the peripheral blood of healthy
volunteers, and cultured on fibronectin-coated dishes.
After six to seven days, adherent cells were used as
fibrocytes, the phenotype of which was confirmed by
a flow cytometric analysis. Monocytes were isolated
from HMNC with an automated magnetic cell
Nintedanib and SB431542 were obtained from Boehringer
Ingelheim GmbH & Co. KG (Biberach, Germany).
SU5416, a VEGFR-specific inhibitor, was purchased from
Abcam (Cambridge, MA). BGJ-398 and imatinib were
purchased from Chemietek (Indianapolis, IN). Bleomycin
(BLM) was purchased from Nippon Kayaku Co. (Tokyo,
Measurement of growth factors
Mediator concentrations were measured in the cell
culture supernatants of fibrocytes, monocytes, and
fibroblasts using commercial enzyme-linked immunosorbent
assay (ELISA) kits.
Fibrocytes, monocytes, and fibroblasts were lysed and
used for immunoblotting as described previously [
MRC-5 cells were cultured in the cell culture
supernatant of fibrocytes with various concentrations of
inhibitors (0–1 μM) or recombinant growth factors (FGF2:
30 ng/ml, PDGF-AA: 100 ng/ml, PDGF-BB: 100 ng/ml,
VEGF-A: 100 ng/ml) for 72 h. A [3H] thymidine
deoxyribose (3H–TdR) incorporation assay was performed as
described previously [
Differentiation assay with recombinant growth factors
HMNC were seeded in fibronectin-coated 6-well plates
with growth factor (FGF2: 30 ng/ml, PDGF-BB: 100 ng/
ml, VEGF-A: 100 ng/ml), and various concentrations of
inhibitors. Each growth factor and inhibitor was added
again every 48 h. On day 6, attached cells were stained
with Diff-Quick and counted.
Cell migration assay
Fibrocytes were added to the upper chamber of cell
culture inserts with a pore size of 8 μm in the presence
or absence of various concentrations of nhibitors (0–
100 nM). Growth factors (FGF2: 30 ng/ml, PDGF-BB:
100 ng/ml, VEGF-A: 100 ng/ml) were added to the lower
chamber. After 20-h incubation, fibrocytes that had
migrated to the bottom surface of the filter were stained
with Diff-Quick and counted [
BLM-induced pulmonary fibrosis in mice
Eight-week-old C57BL/6 male mice were purchased
from CLEA Japan (Tokyo, Japan). Mice received a single
transbronchial instillation of 7.5 mg/kg BLM on day 0.
Nintedanib at 60 mg/kg was administered daily by
gavage until day 7. Lung tissue was analyzed on day 7
via a fluorescence-activated cell sorter (FACS) analysis
and immunohistochemistry [
Paraffin-embedded lung sections were stained with
primary antibodies and then stained with
fluorescence-conjugated secondary antibodies and 4′,
6-diamidino-2-phenylindole. Fluorescence images were
captured with a confocal laser scanning microscope
and counted [
Minced lungs were digested, and the harvested cells
were stained with antibodies for CD45, CXCR4 and
collagen-1. Stained cells were analyzed using a FACScan
flow cytometer (BD Biosciences-Pharmingen, San Diego,
The significance of differences were analyzed using
Mann–Whitney U test for unpaired samples, or a
one-way ANOVA followed by a Dunnett’s test. Where
appropriate, the Kruskal-Wallis H test was applied with
Dunn’s test. P values of less than 0.05 were considered
to be significant. Statistical analyses were performed
using GraphPad Prism programme Ver. 5.01 (GraphPad
Comparison of growth factor expression among monocytes, fibrocytes, and fibroblasts
We confirmed the expression of growth factors in
fibrocytes as previously reported [
]. In the present study,
we compared their expression among monocytes,
fibrocytes, and fibroblasts. Based on the targets of
nintedanib, FGF2, PDGF-AA, PDGF-BB, VEGF-A, VEGF-B,
VEGF-C, and TGFβ-1 were examined in the different
culture supernatants using ELISA. Fibrocytes secreted
greater amounts of FGF2, PDGF-BB, and VEGF-A than
monocytes (Fig. 1a–d). Fibrocytes and fibroblasts both
secreted PDGF-AA (Fig. 1b). Only fibroblasts secreted
VEGF-C (Fig. 1e). PDGF-AB, TGFβ-1, and VEGF-B were
below the detection limit of ELISA. The expression of
FGF2 and PDGF-BB from fibrocytes was also confirmed
by an immunoblot analysis (Fig. 2). These results suggest
that fibrocytes are one of the sources of growth factors
in pulmonary fibrosis.
Fibrocytes and fibroblasts express growth factor receptors, which are the targets of nintedanib
The expression of growth factor receptors on fibrocytes,
monocytes, and fibroblasts was examined by an
immunoblot analysis. Fibrocytes expressed FGFR2 and
VEGFR1. Fibroblasts also expressed FGFR2, and strongly
expressed PDGFRα and β (Fig. 2).
Nintedanib inhibits the proliferation of lung fibroblasts induced by fibrocytes by blocking the phosphorylation of growth factor receptors on fibroblasts
In order to examine the effects of culture supernatants
of fibrocytes as well as those of nintedanib on the
phosphorylation of growth factor receptors, the expression of
all receptors and receptor phosphorylation were
examined using an immunoblot analysis. The incubation of
MRC-5 cells with the culture supernatant of fibrocytes
resulted in the phosphorylation of PDGFR, which was
inhibited by nintedanib mainly at a concentration of
100 nM or more. However, the inhibitory effects of
nintedanib were more potent on the phosphorylation of
PDGFR compared to FGFR (Fig. 3a–c). These results
indicate that growth factors produced by fibrocytes have a
biological activity to stimulate fibroblasts, which can be
inhibited by nintedanib.
Next, we investigated the effects of nintedanib on the
fibrocyte-induced proliferation of lung fibroblasts.
Fibroblast proliferation induced by the culture supernatant of
fibrocytes was inhibited by nintedanib mainly at a
concentration of 100 nM or more (Fig. 4a). The selective
inhibition of FGFR and PDGFR by their specific inhibitors,
BGJ-398 and imatinib, respectively, also inhibited
fibroblast proliferation (Fig. 4b, c), whereas the VEGFR
inhibitor SU5416 did not (Fig. 4d). These results were
supported by recombinant FGF2, PDGF-AA, and
PDGFBB also stimulating the proliferation of fibroblasts,
whereas VEGF-A did not (Fig. 4e). Taken together, these
results show that FGF and PDGF, but not VEGF are
important for the fibrocyte-mediated proliferation of
Nintedanib inhibits the differentiation of fibrocytes from
We examined the effects of nintedanib on the
differentiation of fibrocytes. We used human fibrocytes generated
from HMNC by culturing them on fibronectin-coated
6well plates for six days.
Various kinase inhibitors, including nintedanib,
BGJ398, imatinib, SU5416, and SB431542, in addition to
serum amyloid P were added to the culture medium of
human HMNC at the concentrations ranging from 0 to
1 μM (Additional file 2: Figure S1). As reported
previously, serum amyloid P inhibited the differentiation
of fibrocytes [
]. Specific inhibitors including
nintedanib, BGJ398, imatinib, and SU5416 reduced the number
of fibrocytes generated by HMNC, whereas SB431542,
which is a kinase inhibitor for the receptor of TGFβ,
In order to confirm that these results were due to the
activation of growth factor receptors, we investigated
whether the addition of the recombinant proteins of
growth factors increased the generation of fibrocytes
from HMNC. Recombinant FGF2, PDGF-BB, and
VEGF-A increased the number of fibrocytes generated
by HMNC (Fig. 5a, b, and c). In contrast, BGJ398,
imatinib, and SU5416 inhibited growth factor-induced
differentiation corresponding to each specific inhibitor
(Fig. 5a, b, and c). Nintedanib with the concentration of
100 nM or more also inhibited differentiation promoted
by all growth factors (Fig. 5a, b, and c).
In an attempt to rule out the possibility that these
results were due to the cytotoxic activity of
nintedanib against fibrocytes, we investigated the effects of
nintedanib on the viability of human fibrocytes
derived from monocytes in vitro. Nintedanib was
added to a culture of fibrocytes on day 6. The
treatment of fibrocytes with nintedanib at concentrations
up to 1 μM for six days did not decrease the number
of fibrocytes harvested on day 12 (Additional file 2:
Figure S2), indicating that the cellular toxicity of
nintedanib on fibrocytes was negligible.
Nintedanib inhibits the migration of fibrocytes induced by growth factors
We assessed the effects of kinase inhibitors on the
migration of human fibrocytes induced by various
growth factors at concentrations up to 100 nM. The
number of migrated fibrocytes markedly increased when
cells were treated with FGF2, PDGF-BB or VEGF-A.
Moreover, BGJ398, imatinib, and SU5416 inhibited
migration mediated by growth factors corresponding to
each inhibitor (Fig. 6a, b, and c). Nintedanib with the
concentration of 30 nM or more also inhibited
differentiation promoted by PDGF-BB and VEGF-A, but didn’t
inhibited differentiation promoted by FGF2 (Fig. 6a).
Nintedanib reduces the number of fibrocytes accumulating in the mouse lung with BLM-induced pulmonary fibrosis
We investigated whether nintedanib reduces the number
of fibrocytes in a mouse model of BLM-induced
pulmonary fibrosis. Mice received a single transbronchial
instillation of BLM on day 0. After the administration of
nintedanib with the concentration of 60 mg/kg by
gavage from days 0 to 7, lung tissues were harvested and
analyzed. Paraffin-embedded lung sections were stained
for S100A4/fibroblast-specific antigen-1 (FSP-1) and
CD45. Because FSP-1 is expressed not only by fibrocytes
but also by fibroblasts and endothelial cells, we counted
only strongly stained cells as FSP-1 positive cells.
Double-positive cells for S100A4/FSP-1 and CD45 in
lung sections were defined as fibrocytes as described
] (Fig. 7a). The administration of BLM
significantly increased the number of fibrocytes per
high-power field in the lungs of BLM-treated mice, while
nintedanib at 60 mg/kg significantly reduced these
numbers (Fig. 7c). We also counted the number of fibrocytes
in the lungs of BLM-treated mice using a three-color
FACS analysis (CXCR4, collagen-1, and CD45). The
percentage of fibrocytes (CXCR4+Col1+CD45+cells) among
all lung cells in BLM-treated mice was elevated on day
7, and nintedanib at 60 mg/kg was found to significantly
reduce this percentage (Fig. 7b, d). Consequently, when
nintedanib was continuously administrated until day 21,
the number of fibrotic lesions in the lungs of
BLMtreated mice was reduced (Additional file 2: Figure S3).
A quantitative histological analysis showed that the
Nintedanib (nM) 0
Fibrocyte supernatant –
Nintedanib is a tyrosine kinase inhibitor that was
recently approved for the treatment of patients with IPF in
the major parts of the world. However, the mechanisms
by which nintedanib attenuates pulmonary fibrosis have
not been fully clarified. In the present study, we
examined the effects of nintedanib on fibrocytes in order to
improve understanding of the anti-fibrotic effects of
nintedanib. Nintedanib inhibited the migration and
differentiation of fibrocytes in vitro. The activity of
fibrocytes to stimulate the proliferation of fibroblasts was also
blocked by nintedanib. In addition, treatment with
nintedanib significantly reduced the accumulation of
fibrocytes in the lungs of a BLM-induced pulmonary
fibrosis model in mice.
Fibrocytes are monocyte-derived cells that are
regarded as a subpopulation of mesenchymal progenitor
cells, and express the markers of hematopoietic cells
(CD34), leukocytes (CD11b, CD13, and CD45), and
fibroblast products (collagens I and III and fibronectin)
]. Fibrocytes have been implicated in the pathogenesis
of pulmonary fibrosis [
]. Since fibrosis is
characterized by the accumulation of activated fibroblasts and
excessive deposition of fibrotic extracellular matrix
proteins including type I collagen, fibrocytes have been
Nintedanib (nM) 0
SU5416 (nM) 0
VEGF-A (nM) –
Nintedanib (nM) 0
BGJ398 (nM) 0
FGF2 (nM) –
Nintedanib (nM) 0
Imatinib (nM) 0
PDGF-BB (nM) –
Nintedanib (nM) 0
SU5416 (nM) 0
VEGF-A (nM) –
10 100 1000 0
0 0 0 1
+ + + +
0 0 0
10 100 1000
+ + +
proposed as an important direct contributor to
pulmonary fibrosis. However, a recent study
demonstrated the negligible role of fibrocytes in the
production of collagen in a BLM-induced pulmonary
fibrosis model [
]. On the other hand, we showed
that fibrocytes are a cluster of cells that produce
various growth factors including FGF2, PDGF-BB, and
]. These findings indicate that fibrocytes
are an important cell population responsible for the
production of ligands in signaling pathways that are
targeted by nintedanib.
Nintedanib is a potent tyrosine kinase inhibitor that
targets FGFR, PDGFR, and VEGFR [
]. In the present
study, we showed that fibrocytes expressed FGFR2 and
VEGFR1 using an immunoblot analysis. We previously
demonstrated the expression of PDGFRα and β using
Fig. 7 Nintedanib regulates the pool size of fibrocytes in bleomycin (BLM)-treated mouse lungs. Normal saline (NS) or BLM-treated mice with or without
nintedanib (60 mg/kg/day) were killed on day 7. Paraffin-embedded lung sections were stained with a rabbit anti-S100A4/FSP-1 antibody (green) and
antiCD45 (red), and lung digests stained for collagen-1, CD45, and CXCR4 were examined by flow cytometry. Lung digests were also stained for isotype control
IgG labeled with FITC, phycorythrin, or phycoerythrin-cyanine. a Representative images of immunohistochemical staining in each group are shown. Arrows
indicate fibrocytes doubled stained for S100A4/FSP-1 and CD45 (scale bar = 100 μm). b After lung digests were first gated by collagen-1 as shown in above
figures, only collagen-1+ cells were examined for dual expression of CD45 and CXCR4 using the logical gates depicted as shown in below figures.
Collagen-1+ cells in lung digests were examined for the dual expression of CD45 and CXCR4 using the logical gates depicted. Collagen-1+CD45+CXCR4+
fibrocytes in lung digests from mice in each group were examined. c S100A4/FSP-1 and CD45 double-positive cells were counted in 10 random fields per
section at 20× magnification for three separate lung section. Data were analyzed using a one-way ANOVA and displayed as dot plot and their means
(n = 30 in each group). d The percentage of fibrocytes relative to all cells in lung digests were examined. Data were analyzed using the Kruskal-Wallis H test
and displayed as median and interquartile range of four separate experiments. In all graphs: *P < 0.01 versus the group treated with BLM alone
qPCR and flow cytometric analyses [
]; however, the
detection of PDGFRα and β by immunoblot analysis was
not sufficient in the present study. This may have been
due to the method used to detect the expression of
PDGFR, as PDGF proteins may stimulate the migration
of fibrocytes [
]. We also showed that fibroblasts
express FGFR2 and PDGFRα and β. Hence, fibrocytes
and fibroblasts may be putative therapeutic target cells
The supernatant of fibrocytes stimulated the
phosphorylation of tyrosine kinase receptors on fibroblasts,
and the inhibitory effects of nintedanib on these
receptors were demonstrated. Despite PDGFR of fibroblasts
was phosphorylated by addition of the culture
supernatant of fibrocytes, FGFR had been phosphorylated
regardless whether the fibrocyte supernatant was added
or not. Furthermore, although the inhibitory effects of
nintedanib on the phosphorylation of PDGFR were
observed at approximately 10–100 nM, the inhibition of
FGFR phosphorylation required 100–1000 nM. These
differences might be due to the two reason. First,
fibroblasts also produce FGF2, as shown in Fig. 1 and/or due
to the differences in the inhibitory potency. Second, The
IC50 values of nintedanib for PDGFRα and PDGFRβ
have been reported to be in the range of 41–58 nM,
whereas that for FGFR2 is 257 nM [
The activation of receptors on fibroblasts induced by
fibrocyte supernatant resulted in their proliferation.
Nintedanib and BGJ398, the FGFR inhibitor, attenuated
this proliferation at concentrations rather than 100 nM.
Although imatinib, a PDGFR inhibitor, also suppressed
the growth of fibroblasts, it required concentrations
greater than 1 μM. The IC50 value of imatinib against
PDGFR was reported to be in the range of 100 nM to
380 nM [
]. A direct comparison between imatinib and
nintedanib is difficult due to the different cell types used,
but the IC50 value of imatinib was considered to be
higher than that of nintedanib in the present study.
SU5416, a VEGF inhibitor, did not exert inhibitory
effects against cell growth, even when 1 μM was used as
the maximum concentration. Since the production of
VEGF by fibrocytes was less than that by fibroblasts, as
shown in Fig. 1, FGF and PDGF are considered to be
more important than VEGF as growth factors produced
by fibrocytes that activate the proliferation of fibroblasts.
We also investigated the effects of nintedanib on the
differentiation of fibrocytes from monocytes. In the
present study, inhibitory effects were observed not only
by nintedanib, but also by specific inhibitors for FGFR,
PDGFR, and VEGFR. We also demonstrated that several
growth factors including PDGF, FGF, and VEGF
stimulated the differentiation of fibrocytes. The differentiation
of fibrocytes is reported to be augmented by fibrogenic
cytokines such as interleukin (IL)-4 and IL-13 along with
]. However, the relationship between the
differentiation of fibrocytes and the FGF/FGFR or VEGF/
VEGFR signaling pathways has not been examined.
Monocytes have been shown to express FGFR [
], and VEGFR [
], however growth factor
receptors were not clearly detected by immunoblot on
monocytes in the present study as shown in Fig. 2.
Therefore, these growth factors may play a role in the
differentiation of fibrocytes in pulmonary fibrosis.
We showed that nintedanib inhibited the migration of
fibrocytes. In our previous study, the PDGF/PDGFR axis
was found to play a role in the migration of fibrocytes
into fibrotic lungs in vitro and in vivo [
we demonstrated that FGF and VEGF were potent
chemoattractants for fibrocytes in the present study.
Therefore, nintedanib is considered to prevent
pulmonary fibrosis by directly inhibiting the differentiation and
migration of fibrocytes. However, nintedanib could not
inhibit the migration of fibrocytes stimulated by FGF2.
As mentioned above, it is considered that this is because
the IC50 value of nintedanib against FGFR is high. The
effects of nintedanib in in vitro experiments were also
confirmed in in vivo experiments. The administration of
nintedanib significantly reduced the number of
fibrocytes in the lung tissues of mice treated with BLM to
induce pulmonary fibrosis.
The limitation of this study was that it was difficult to
examine the effect of nintedanib on fibrocyte induction in
the fibrotic phase in our model. Because in
bleomycininduced pulmonary fibrosis model in mice, the number of
fibrocytes induced to the lung begins to increase from day
7, but drastically end up decreasing on day 21 as shown in
previous report [
]. Therefore, to circumvent this
problem, the analysis of lung tissue from patients who had
nintedanib may be more informative to see the impact on
fibrocyte recruitment in future study.
In summary, the present study clearly demonstrated a
novel anti-fibrotic activity of nintedanib. Nintedanib
directly inhibited the migration and differentiation of
fibrocytes. In addition, nintedanib blocked the
receptors of pro-fibrotic growth factors which are
stimulated by mediators produced by fibrocytes (Additional
file 2: Figure S5).
Additional file 1: Detailed methods, and figure legends of Figure S1-S5.
(PDF 227 kb)
Additional file 2: Figure S1. Nintedanib inhibits the differentiation of
fibrocytes generated from HMNC. Figure S2. Nintedanib did not cause
cellular damage in fibrocytes. Figure S3. Histological examination of the
anti-fibrotic effects of nintedanib on bleomycin (BLM)-induced lung
fibrosis. Figure S4. Quantitative examination of the anti-fibrotic effects of
nintedanib on bleomycin (BLM)-induced pulmonary fibrosis. Figure S5.
Anti-fibrotic role of nintedanib in pulmonary fibrosis via the suppression
of fibrocyte activity. (PDF 884 kb)
3H–TdR: [3H] thymidine deoxyribose; BLM: Bleomycin; ELISA: Enzyme-linked
immunosorbent assay; FACS: Fluorescence-activated cell sorter;
FGF: Fibroblast growth factor; FSP-1: Fibroblast-specific antigen-1;
HMNC: Human mononuclear cells; IL: Interleukin; IPF: Idiopathic pulmonary
fibrosis; NS: Normal saline; PDGF: Platelet-derived growth factor;
TGF: Transforming growth factor; VEGF: Vascular endothelial growth factor
We thank Ms. Tomoko Oka for her technical assistance with primary cell
isolation and the members of Nishioka’s lab for their technical advice and
This work was supported by KAKENHI (20390231 and 23659434), a Grant-in-Aid
for Scientific Research (B), and Exploratory Research from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan (Y.N.), a grant to the Diffuse
Lung Diseases Research Group from the Ministry of Health, Labour and Welfare,
Japan (Y.N.) and a grant from Boehringer-Ingelheim (Y.N.).
Availability of data and materials
Essential datasets supporting the conclusions are included in this published
Conception and design: SS and YN; Analysis and interpretation: SS, SS, SH,
SM, SA, HO, YC, KK, HN, HK and HU; drafting the manuscript for important
intellectual content: SS, YA, HG, YT and YN; all authors have approved the
final version and agree to be accountable for all aspects of the work in
ensuring that questions related to the accuracy or integrity of any part of
the work are appropriately investigated and resolved.
Ethics approval and consent to participate
This study was approved by the Institutional Animal Care and Use
Committee of Tokushima University (Permission Number: 14099). In isolation
of human fibrocytes and monocytes, all procedures for consent, sample
collection, and privacy protection were approved by the Ethics Committee
of Tokushima University Hospital.
Consent for publication
Nishioka Y received a grant from Boehringer-Ingelheim. Boehringer-Ingelheim
reviewed this manuscript.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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