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 col￾lecting 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 lympha￾ngiogenesis 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 conse￾quence of cancer surgery.1 The major lymphatic system
is damaged following cancer surgery with or without radio￾therapy, causing a significant decrease in lymphatic flow
and accumulation of excessive protein-rich fluid. If lym￾phatic 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 impor￾tant regulators of fibrosis, plays a key role in lymphatic re￾generation during wound repair.3–6 TGF-b1 is a member of
the TGF-b superfamily that takes an important role in ho￾meostasis of epithelial, endothelial, and hematopoietic cells.
It promotes mesothelial-to-mesenchymal transition and re￾sults 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 thera￾peutic 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 in￾hibitor. 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 re￾search of our institution and conformed to the U.S. National
Institutes of Health guidelines on the care and use of labo￾ratory animals.
Mouse tail model of lymphedema
Tail lymphedema was created in 10- to 12-week-old fe￾male C57BL/6 mice (Orient Bio, Seongnam, Korea) as de￾scribed 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 cir￾cumferentially excised 18-mm distal to the base of the tail,
making a 2-mm circular band. The lymphatic collecting ves￾sels 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 temperature￾controlled room (22C – 2C) and supplied with food and
water ad libitum. For histologic evaluation, mice were sac￾rificed 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 soft￾ware (U.S. National Institutes of Health, Bethesda). Max￾imum 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 FITC￾dextran 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 sec￾tions 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 ves￾sels 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% parafor￾maldehyde perfusion fixative (Electron Microscopy Sci￾ences, 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 pri￾mary 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, Burling￾ton, 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 intrader￾mally into the tip of the tail before sacrifice. Single immu￾nofluorescence staining was performed using anti-a-SMA
antibody and Alexa Fluor 594 as described previously. Nu￾clei were visualized with DAPI.
Digital images were collected using a Panoramic Super￾Resolution 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 as￾sess 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, respec￾tively (Fig. 1). The differences in maximal tail diameter be￾tween 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 accu￾mulation 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 adminis￾tration significantly decreased the degree of acquired lym￾phedema in a mouse tail model. The TGF-b/SMAD pathway
is a central mediator of both wound healing and fibrosis pro￾cesses, and there are two possible explanations for why this
has been proposed as being highly correlated with lymphe￾dema.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 lym￾phangiogenesis 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 treat￾ment 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 anti￾body accelerated lymphangiogenesis.3 They also suggested
that inhibition of the function of TGF-b decreases the ex￾pression of Th2 cytokines (IL-4 and IL-13), which are nec￾essary for soft tissue fibrosis. Second, downregulation of the
TGF-b1 canonical pathway of fibrosis may achieve stimu￾lation 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 FITC￾dextran 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 re￾generation 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 endo￾thelial 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 lym￾phangiogenesis. They suggested that resolution might be
more dependent upon interstitial fluid dynamics than func￾tional lymphatic regeneration. Collectively, these findings
emphasize the current limitation of data interpretation in
studies using a mouse tail lymphedema model. For exam￾ple, the proportion of lymphatic fluid transportation by
each mechanism according to the tail model would be dif￾ferent from that in human lymphedema. We believe that
both functional lymphatic vessel flow and interstitial flow
should be taken into consideration in assessing the thera￾peutic effect. Our data revealed that EW-7197 treatment in￾creased 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 usu￾ally 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 drug￾releasing material could be recommended for further study.
The present study showed that EW-7197 ameliorates ac￾quired lymphedema in a mouse tail model. In immunofluo￾rescence 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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References
1. Nguyen TT, Hoskin TL, Habermann EB, Cheville AL,
Boughey JC. Breast cancer-related lymphedema risk is
related to multidisciplinary treatment and not surgery
alone: Results from a large cohort study. Ann Surg Oncol
2017; 24:2972–2980.
2. Warren LEG, Miller CL, Horick N, Skolny MN, Jammallo
LS, Sadek BT, et al. The impact of radiation therapy on the
risk of lymphedema after treatment for breast cancer: A
prospective cohort study. Int J Radiat Oncol Biol Phys
2014; 88:565–571.
3. Avraham T, Daluvoy S, Zampell J, Yan A, Haviv YS,
Rockson SG, et al. Blockade of transforming growth factor￾beta1 accelerates lymphatic regeneration during wound
repair. Am J Pathol 2010; 177:3202–3214.
4. Clavin NW, Avraham T, Fernandez J, Daluvoy SV, Soares
MA, Chaudhry A, et al. TGF-beta1 is a negative regulator
of lymphatic regeneration during wound repair. Am J
Physiol Heart Circ Physiol 2008; 295:H2113–H2127.
5. Oka M, Iwata C, Suzuki HI, Kiyono K, Morishita Y,
Watabe T, et al. Inhibition of endogenous TGF-beta sig￾naling enhances lymphangiogenesis. Blood 2008; 111:
4571–4579.
6. James JM, Nalbandian A, Mukouyama YS. TGFbeta sig￾naling is required for sprouting lymphangiogenesis during
lymphatic network development in the skin. Development
2013; 140:3903–3914.
7. Tsauo J, Song HY, Choi EY, et al. EW-7197, an oral
transforming growth factor b type I receptor kinase inhib￾itor, for preventing peritoneal adhesion formation in a rat
model. Surgery 2018; 164:1100–1108.
8. Son JY, Park SY, Kim SJ, Lee SJ, Park SA, Kim MJ, et al.
EW-7197, a novel ALK-5 kinase inhibitor, potently inhibits
breast to lung metastasis. Mol Cancer Ther 2014; 13:1704–
1716.
9. Uzarski J, Drelles MB, Gibbs SE, Ongstad EL, Goral JC,
McKeown KK, et al. The resolution of lymphedema by
interstitial flow in the mouse tail skin. Am J Physiol Heart
Circ Physiol 2008; 294:H1326–H1334.
10. Jun H, Lee JY, Kim JH, Noh M, Kwon TW, Cho YP, et al.
Modified mouse models of chronic secondary lymphedema:
Tail and hind limb models. Ann Vasc Surg 2017; 43:288–
295.
11. Park SA, Kim MJ, Park SY, Kim JS, Lee SJ, Woo HA,
et al. EW-7197 inhibits hepatic, renal, and pulmonary fi-
brosis by blocking TGF-beta/Smad and ROS signaling.
Cell Mol Life Sci 2015; 72:2023–2039.
12. Lynch LL, Mendez U, Waller AB, Gillette AA, Guillory
RJ, 2nd, Goldman J. Fibrosis worsens chronic lymphedema
in rodent tissues. Am J Physiol Heart Circ Physiol 2015;
308:H1229–H1236.
13. Wernicke AG, Greenwood EA, Coplowitz S, Parashar B,
Kulidzhanov F, Christos PJ, et al. Tissue compliance meter
is a more reproducible method of measuring radiation￾induced fibrosis than late effects of normal tissue-subjective
objective management analytical in patients treated with
intracavitary brachytherapy accelerated partial breast irra￾diation: Results of a prospective trial. Breast J 2013; 19:
250–258.
14. Boardman KC, Swartz MA. Interstitial flow as a guide for
lymphangiogenesis. Circ Res 2003; 92:801–808.
15. Kim JH, Song HY, Park JH, Yoon HJ, Park HG, Kim DK.
IN-1233, an ALK-5 inhibitor: Prevention of granulation
tissue formation after bare metallic stent placement in a rat