EW-7197, a Transforming Growth Factor-Beta Type I
Receptor Kinase Inhibitor, Ameliorates Acquired
Lymphedema in a Mouse Tail Model
Sung-Hwan Yoon, MS,1–3,* Kun Yung Kim, MD, PhD,4,5,* Zhe Wang, MD, PhD,6 Jung-Hoon Park, PhD,1,7
Sang Mun Bae, PhD,8 Sang-Yeob Kim, PhD,8,9 Ho-Young Song, MD, PhD,1,7 and Jae Yong Jeon, MD, PhD1,3
Abstract
Background: Acquired lymphedema is a common consequence of cancer surgery. Fibrosis is one of the main
causes of chronic lymphedema since it hinders lymphatic regeneration and this causes a significant decrease in
lymphatic flow and accumulation of excessive protein-rich fluid. The transforming growth factor-b1 (TGF-b1)
signaling pathway is known in a process of wound repair and fibrosis. In our study, the purpose was to evaluate
the efficacy of EW-7197, a peroral TGF-b type I receptor kinase inhibitor, in treating acquired lymphedema.
Methods and Results: For lymphedema mouse tail model, we used 10- to 12-week-old female C57BL/6 mice.
The skin was circumferentially excised, making a circular band followed by cauterization of lymphatic collecting vessels. Two groups were made in this study: control and treatment. The treatment group (n = 12)
received a solution consisting of 0.1 mL of artificial gastric juice and 20 mg/kg EW-7197 by gavage once daily.
For evaluation, tail diameter measurement, fluorescence lymphography, and immunofluorescence images were
used. EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing lymphangiogenesis and interstitial flow of the lymphatics by inhibition of the fibrosis. The differences in maximal tail
thicknesses between the control and treatment groups were statistically significant from 2 to 4 weeks after
surgery. The treatment group showed a greater number of lymphatic vessels at the surgery site than the control
group. The treatment group also showed more FITC coverage area at the surgery site.
Conclusion: EW-7197 treatment ameliorates acquired lymphedema in a mouse tail model by increasing
lymphangiogenesis and interstitial flow.
Keywords: TGF-b1 type 1 inhibitor, EW-7197, interstitial flow, fibrosis, lymphedema mouse tail model,
lymphatic flow
Introduction
Acquired lymphedema is a relatively common consequence of cancer surgery.1 The major lymphatic system
is damaged following cancer surgery with or without radiotherapy, causing a significant decrease in lymphatic flow
and accumulation of excessive protein-rich fluid. If lymphatic flow is restored by reconnecting disoriented lymphatic
vessels, the lymphedema could be successfully recovered,
but if not, chronic lymphedema occurs.2
1
Department of Biomedical Engineering Research Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul,
Republic of Korea. 2
Biomedical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
3
Department of Rehabilitation medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
4
Department of Radiology, Chonbuk National University Hospital, Jeonju-si, Republic of Korea.
5
Research Institute of Clinical Medicine, Chonbuk National University-Biomedical Research Institute, Chonbuk National University
Hospital, Jeonju-si, Republic of Korea. 6
Department of Radiology, Tianjin Medical University General Hospital, Tianjin, P.R. China.
7
Department of Radiology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
8
Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
9
Department of Convergence Medicine, University of Ulsan College of Medicine, Seoul, Republic of Korea.
*These authors contributed equally to this work.
LYMPHATIC RESEARCH AND BIOLOGY
Volume 00, Number 00, 2020
ª Mary Ann Liebert, Inc.
1
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Fibrosis is one of the main causes of chronic lymphedema,
as it hinders lymphatic regeneration.3,4 Pathological studies
of fibrosis in relation to lymphatic regeneration have been the
focus of intense investigation and new treatment strategy for
chronic lymphedema. Some of these studies have shown that
transforming growth factor-b1 (TGF-b1), one of the important regulators of fibrosis, plays a key role in lymphatic regeneration during wound repair.3–6 TGF-b1 is a member of
the TGF-b superfamily that takes an important role in homeostasis of epithelial, endothelial, and hematopoietic cells.
It promotes mesothelial-to-mesenchymal transition and results in inducing tissue fibrosis during wound repair process.7
Avraham et al.3 reported that either systemic or local TGF-b1
blockade using monoclonal antibody treatment signifi-
cantly improves lymphatic function by promoting lymphatic
regeneration and by decreasing fibrosis.3 However, to date,
there have been no lymphedema studies to assess the therapeutic effects of systemic TGF-b1 receptor inhibitor.
EW-7197, N-[[4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-
methylpyridin-2-yl)-1H-imidazol-2-yl]methyl]-2-fluoroaniline,
is a highly selective peroral TGF-b type I receptor kinase inhibitor. ALK5, one of the TGF-b1 receptors, phosphorylates
SMAD2/3 and triggers several cellular cascades involved in
wound repair and fibrosis. Son et al.8 reported that EW-7197
acts as an ATP-competitive inhibitor of ALK5 by binding to
the ATP-binding site. This results in inhibition of the TGF-b1/
Smad signaling pathway.
We hypothesized that the administration of a TGF-b type I
receptor kinase inhibitor just before or after surgery could
prevent the fibrosis process following surgery and thereby
ameliorate the resulting acquired lymphedema. Thus, the
purpose of this study was to evaluate the efficacy of EW-
7197in treating acquired lymphedema in a surgical model of
mouse tail. Since a considerable amount of lymphatic fluid is
transmitted by interstitial flow through the dermal matrix,9
both the degree of lymphatic regeneration and interstitial flow
were taken into account to evaluate the efficacy of EW-7197.
Materials and Methods
This study was approved by the committee for animal research of our institution and conformed to the U.S. National
Institutes of Health guidelines on the care and use of laboratory animals.
Mouse tail model of lymphedema
Tail lymphedema was created in 10- to 12-week-old female C57BL/6 mice (Orient Bio, Seongnam, Korea) as described previously,10 but with minor modifications. In brief,
the mice were anesthetized with an intramuscular injection
of 50 mg/kg zolazepam and tiletamine (Zoletil 50; Virbac,
Carros, France) and 10 mg/kg xylazine (Rompun; Bayer
HealthCare, Leverkusen, Germany), and the skin was circumferentially excised 18-mm distal to the base of the tail,
making a 2-mm circular band. The lymphatic collecting vessels were cauterized using Bovie cautery.
EW-7197 treatment
A total of 24 mice were divided into two groups. The
control group (n = 12) received 0.1 mL of artificial gastric
juice by gavage once daily for 4 weeks. The treatment group
(n = 12) received a solution consisting of 0.1 mL of artificial
gastric juice and 20 mg/kg EW-7197 phosphate by gavage
once daily for 4 weeks. EW-7197 phosphate was provided
by the Laboratory of Medicinal Chemistry, College of
Pharmacy, Ewha Woman’s University (Seoul, Korea). The
dosage of EW-7197 phosphate was determined based on
prior studies.8,11 All mice were maintained in a temperaturecontrolled room (22C – 2C) and supplied with food and
water ad libitum. For histologic evaluation, mice were sacrificed by carbon dioxide asphyxiation 4 weeks after surgery.
Tail diameter measurements
Digital images were obtained from the mouse tails after
surgery twice a week with use of a Canon D550 camera
mounted to its tripod. Tail diameter measurements were
made from these digital images using ImageJ imaging software (U.S. National Institutes of Health, Bethesda). Maximum tail diameter was measured at the point of greatest
diameter distal to the surgery site as described previously.12
Fluorescence lymphography
We used lysine-fixable FITC-dextran (molecular weight,
2000 kDa; Molecular Probes) fluorescence lymphography
tracer to identify lymphatic flow. Five microliters of FITCdextran tracer was injected intradermally into the distal tail
30-mm distal to the site of the surgery once a week. Digital
images were obtained with a microscope-based multispectral
imaging system, Nuance (PerkinElmer, Inc., Hopkinton,
MA) 30 minutes after the injection. The system was mounted
onto a conventional fluorescence microscope equipped with a
filter cube that comprised a standard excitation filter, dichroic
mirror, and a long-pass emission filter.
Immunofluorescence stain was used to detect lymphatic
regeneration and interstitial flow on longitudinal cross sections of mouse tail. Six mice in each group were assigned to
detect lymphatic vessel regeneration. Immunostaining was
performed using antilymphatic vessel endothelial receptor-1
(LYVE-1) to observe physical structures of lymphatic vessels and anti-a-smooth muscle actin (a-SMA) antibodies to
observe smooth muscle cells. In brief, mouse tails were
harvested 4 weeks after surgery and fixed with 4% paraformaldehyde perfusion fixative (Electron Microscopy Sciences, Hatfield, PA) overnight at 4C, followed by sequential
incubation in 15% and 30% sucrose solution. Fixed samples
were embedded in optimal cutting temperature compound,
and 5-lm cryostat sections were prepared and collected on
slides for double immunofluorescence staining. Before
staining, slides were air-dried, then fixed and permeabilized
in ice-cold acetone for 10 minutes at -20C. After fixation
and permeabilization, slides were blocked with 10% fetal
bovine serum in 1 · phosphate-buffered saline (PBS) with
0.1% Tween 20. Tissue sections were stained with primary antibodies, including rabbit anti-LYVE-1 antibody
(1:100, ab14917; Abcam, Cambridge, Cambridgeshire, United
Kingdom) and mouse-anti-a-SMA antibody (1:300, ab7817;
Abcam) overnight at 4C followed by washing with PBS with
0.1% Tween 20. After washing, secondary antibody staining
was performed using Alexa Fluor 488, Alexa Fluor 594, and
4¢-6-diamidino-2-phenylindole (DAPI) (Invitrogen, Burlington, Canada). Slides were then incubated for 1 hour at room
temperature.
2 YOON ET AL.
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Six additional mice were assigned to each group to detect
interstitial flow. FITC-dextran tracer was injected intradermally into the tip of the tail before sacrifice. Single immunofluorescence staining was performed using anti-a-SMA
antibody and Alexa Fluor 594 as described previously. Nuclei were visualized with DAPI.
Digital images were collected using a Panoramic SuperResolution Confocal microscopy system (3D HISTECH,
Budapest, Hungary).
Statistical analysis
Statistical analysis was performed using SPSS version 22.0
(SPSS, Inc., Chicago, IL). The maximum diameter of the tail
was compared using the paired t-test. Mean FITC coverage
area and mean number of vessels per high-power field were
compared using the Student’s t-test. All p-values were two
sided with statistical significance evaluated at the 0.05 alpha
level, and 95% confidence intervals were constructed to assess the precision of the obtained estimates.
Results
Tail diameter measurements
The maximum tail diameter was rapidly increased after
surgery in both groups, which lasted up to 2 weeks in the
control group and 1.5 weeks in the treatment group, respectively (Fig. 1). The differences in maximal tail diameter between the control and treatment groups were statistically
significant from 2 to 4 weeks after surgery (6.1 – 0.5 mm vs.
5.8 – 0.3 mm, p = 0.046 at 2 weeks; 5.8 – 0.5 mm vs. 5.4 –
0.2 mm, p = 0.035 at 3 weeks; 5.5 – 0.4 mm vs. 5.0 – 0.3 mm,
p = 0.030 at 4 weeks).
Fluorescence lymphography and immunofluorescence
Fluorescence lymphography showed accumulation of the
FITC-dextran tracer distal to the surgery site in both groups.
The treatment group showed fluid channels at the surgery site
4 weeks after surgery, which were not observed in any of the
mice of the control group (Fig. 2).
Regarding a-SMA expression at the surgical site, the
treatment group had lower expression than the control group
(17.1% vs. 38.5%, p < 0.001). As for lymphatic regeneration,
the treatment group showed a larger number of LYVE-1-
stained vessels at the surgery site per high-power field
(9.9 – 3.8 vs.1.6 – 1.3, p < 0.001) (Fig. 3). In terms of
FIG. 1. (A) Representative macroscopic images of changes in mouse tail diameter of the control and treatment groups 1 to
4 weeks after surgery. (B) Changes in the maximum tail diameter after surgery. Note the significant differences from 2
weeks after surgery. *p < 0.05. Color images are available online.
FIG. 2. Fluorescence lymphography was used to identify
postsurgery lymphatic flow in mouse tails. The treatment
group shows a fluid channel (arrow) in the surgery site 4
weeks after surgery, while the control group shows accumulation of the FITC-dextran tracer (2000 kDa) distal to the
surgery site with no formation of a fluid channel. Color
images are available online.
TGF-b INHIBITOR AMELIORATES ACQUIRED LYMPHEDEMA 3
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interstitial lymphatic flow, the treatment group had greater
FITC coverage area at the surgical site than the control group
(3.8% – 1.1 vs. 0.6% – 0.5, p < 0.001) (Fig. 4).
In this study, systemic TGF-b1 receptor inhibitor administration significantly decreased the degree of acquired lymphedema in a mouse tail model. The TGF-b/SMAD pathway
is a central mediator of both wound healing and fibrosis processes, and there are two possible explanations for why this
has been proposed as being highly correlated with lymphedema.3 First, stimulation of lymphangiogenesis was observed
at the surgery site in the treatment group. As seen in Figure 3, a
higher number of lymphatic vessels were observed in the
treatment group. Upregulation of LYVE-1 was specifically
observed where there was lower a-SMA expression, whereas
there was a large area of a-SMA expression in the control
group but no LYVE-1 expression. This indicates that lymphangiogenesis is achieved by suppression of a-SMA, which
inhibits the formation of lymphatic vessels. Second, increased
interstitial flow was observed at the surgery site in the treatment group. As seen in Figure 4, the lymphatic interstitial flow
was significantly higher in the treatment group according to the
amount of FITC-dextran tracer. This result may be due to the
decreased fibrosis that was represented by the lower amount of
a-SMA expression, which hinders lymphatic flow. In addition,
Figure 2 shows that lymphatic channels were re-established at
the surgery site, which helped to increase interstitial flow.
Stimulation of lymphangiogenesis can be achieved by two
major ways. First, reducing the direct inhibitory effect of
TGF-b1 on lymphatic epithelial cell proliferation can help
regeneration of lymphatic vessels.5 Avraham et al. reported
that blockage of TGF-b1 using the TGF-b monoclonal antibody accelerated lymphangiogenesis.3 They also suggested
that inhibition of the function of TGF-b decreases the expression of Th2 cytokines (IL-4 and IL-13), which are necessary for soft tissue fibrosis. Second, downregulation of the
TGF-b1 canonical pathway of fibrosis may achieve stimulation of lymphangiogenesis. Fibrosis induced by TGF-b1
causes the formation of thicker bundles of collagen matrix
that have a negative effect in lymphangiogenesis.13 Lynch
et al. reported that this collagen matrix composition change at
the surgery site hindered lymphatic regeneration.12
Apart from lymphangiogenesis, interstitial lymphatic flow
is another important transport mechanism of lymphatic fluid
clearance. We found that a substantial amount of FITCdextran tracer traveled through the interstitial space at the
surgery site in the treatment group. However, interstitial flow
was barely observed in the control group. These results
suggest that fibrosis not only inhibited lymphatic vessel regeneration but also reduced the upstream interstitial flow by
narrowing the interstitial space. In addition, the direction of
interstitial flow is a crucial factor of lymphangiogenesis. It
helps to guide the growth and organization of a developing
FIG. 3. Comparison of postsurgical lymphangiogenesis between the treatment and control groups. (A) The treatment
group shows higher expression level of LYVE-1 (arrows) and less expression level of a-SMA than the control group.
(B) The number of regenerated vessels per high-power field was significantly higher in the treatment group compared with
the control group. (C) The a-SMA coverage area was significantly lower in the treatment group compared with the control
group. Scale bar = 50 lm. *p < 0.05. a-SMA, a-smooth muscle actin. Color images are available online.
4 YOON ET AL.
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lymphatic capillary network by enhancing lymphatic endothelial cell migration and proliferation.9,14 Thus, lack of suf-
ficient interstitial flow level in fibrotic scar tissue reduces
lymphangiogenesis and limits clearance of lymphatic flow.
Our data imply that fibrosis may mediate its proedema
effects by two mechanisms: decreased lymphangiogenesis
and reduced interstitial flow. However, in most previous
studies, only the degree of lymphangiogenesis has been used
as an assessment tool for therapeutic effect. Uzarski et al.9
reported that lymphedema resolution could occur solely by
interstitial flow regardless of VEGFR-3 signaling and lymphangiogenesis. They suggested that resolution might be
more dependent upon interstitial fluid dynamics than functional lymphatic regeneration. Collectively, these findings
emphasize the current limitation of data interpretation in
studies using a mouse tail lymphedema model. For example, the proportion of lymphatic fluid transportation by
each mechanism according to the tail model would be different from that in human lymphedema. We believe that
both functional lymphatic vessel flow and interstitial flow
should be taken into consideration in assessing the therapeutic effect. Our data revealed that EW-7197 treatment increased lymphatic vessel generation and interstitial flow
simultaneously, and decreased fibroblast proliferation. Thus,
this evidence provides support for further development of
TGF-b1 inhibitor as a therapeutic agent to treat human
lymphedema.
One problem with systemic TGF-b1 inhibition is that
it could delay the wound healing process.15 We observed
leakage of the FITC-dextran tracer that lasted for 2 to 3 weeks
after surgery in the treatment group, but not in the control
group (data not shown). Considering that lymphedema usually occurs after surgery, systemic TGF-b1 inhibition could
induce complications due to delayed wound healing. Further
studies may be warranted to investigate a suitable application
strategy, including optimal timing of delivery, safe dosage,
and systemic toxicity. In addition, to avoid systemic adverse
effects, local application of Vactosertib TGF-b1 inhibitor using drugreleasing material could be recommended for further study.
The present study showed that EW-7197 ameliorates acquired lymphedema in a mouse tail model. In immunofluorescence assessment, we found that EW-7197 induced both
lymphangiogenesis and interstitial flow of the lymphatics by
inhibition of the fibrosis at the surgery site. Considering its
therapeutic effect, EW-7197 may have potential in treating
lymphedema.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by a grant (2017-478) from the
Asan Institute for Life Sciences, Asan Medical Center, Seoul,
Korea. This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MSIT) (No. 2019R1A2C1009055).
FIG. 4. Comparison of postsurgical interstitial flow between the treatment and control groups. (A) The treatment group
shows a higher flow of the FITC-dextran tracer in the interstitium of the surgery site and a lower expression of a-SMA than
those of the control group. (B) The FITC coverage area was significantly higher in the treatment group compared with the
control group. Scale bar = 50 lm. *p < 0.05. Color images are available online.
TGF-b INHIBITOR AMELIORATES ACQUIRED LYMPHEDEMA 5
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