European Journal of Pharmacology 938 (2023) 175422

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

RTA-dh404 decreased oxidative stress in mice ischemic limbs and

augmented efficacy of therapeutic angiogenesis by intramuscular injection
of adipose-derived regenerative cells in the limbs
Yuta Ishizaki , Ken-ichiro Sasaki *, Takahiro Yoshikawa , Takaharu Nakayoshi , Motoki Sasaki ,
Masanori Ohtsuka , Sachiko Hatada-Katakabe , Yuki Takata , Yoshihiro Fukumoto
Division of Cardiovascular Medicine, Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Although an intramuscular injection of angiogenic cells to ischemic limbs with peripheral artery disease is a
pharmacological pretreatment therapeutic option to rescue patients by augmenting neovascularization in the limbs, oxidative stress in the limbs
antioxidation may accelerate apoptosis of the injected cells and thereby reduce the therapeutic effect. In this study involving
apoptosis
mice with ischemic lower limbs, whether daily oral administration of RTA-dh404, which is an activator of nu­
cell viability
clear factor erythroid 2-related factor 2 (Nrf2) with antioxidant activity, could reduce oxidative stress in the
limbs and suppress apoptosis of adipose-derived regenerative cells (ADRCs) injected in the limbs, eventually
augmenting neovascularization in the limbs, was evaluated. The tissue expression of Nrf2 and concentrations of
total antioxidant capacity and superoxide dismutase in the mice ischemic limbs were higher in the RTA-dh404-
treated mice than in the control treated mice, and oxidative stress in the limbs of the RTA-dh404 treated mice
was decreased. The day after an intramuscular injection of human ADRCs into ischemic lower limbs of immu­
nodeficient mice, the number of apoptotic ADRCs in the ischemic limbs was decreased by approximately 25% in
the RTA-dh404-treated mice compared to the control mice. Fourteen days after cell injection, neovascularization
and the salvage ratio were increased by approximately 10% and 63%, respectively, in the ischemic limbs in the
RTA-dh404-treated mice compared to the control mice. Pretreatment of ischemic limbs by daily oral adminis­
tration of RTA-dh404 may augment the effect of therapeutic angiogenesis using an intramuscular injection of
ADRCs into the ischemic limbs.

1. Introduction adipose-derived regenerative cells (ADRCs) (Shimizu et al., 2022), into

The number of patients with peripheral artery disease, who have
atherosclerotic stenosis and/or occlusion of peripheral arteries in the
lower limbs, has been increasing (Song et al., 2019). It has been reported
that 5–10% of peripheral artery disease patients aged 50 years and older
develop critical limb ischemia within 5 years, which is defined as a
condition characterized by ischemic rest pain and/or ulceration, or
gangrene of the foot and/or toes lasting more than 2 weeks, and it is
known to be a major risk factor for lower extremity amputation (Norg­
ren et al., 2007). Augmenting neovascularization by intramuscular in­
jection of angiogenesis-promoting cells, such as bone marrow-derived
mononuclear cells (Saito et al., 2007; Matoba et al., 2008), peripheral
blood-derived mononuclear cells (Moriya et al., 2009), and
the patients’ ischemic limbs has been reported as a therapeutic option to
prevent amputation and rescue the ischemic limbs. However, in our
previous study with rats, oxidative stress in the ischemic limbs of the rats
accelerated apoptosis of intramuscularly injected angiogenic cells in the
ischemic limbs, leading to less neovascularization in the ischemic limbs
(Nakayoshi et al., 2014). Oxidative damage was reported to be increased
in ischemic limbs with progression of peripheral artery disease (Weiss
et al., 2013), suggesting that, if it were possible to reduce oxidative
stress in the ischemic limbs before intramuscular injection of the
angiogenesis-promoting cells, an increased survival rate of the injected
cells in the limbs may lead to more neovascularization of the limbs.
Bardoxolone methyl, which is a synthetic triterpenoid derived from
the natural product oleanolic acid, and its analogues are the most potent

* Corresponding author. Division of Cardiovascular Medicine, Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume,
Fukuoka, 830-0011, Japan.
E-mail address: sasaken@med.kurume-u.ac.jp (K.-i. Sasaki).

https://doi.org/10.1016/j.ejphar.2022.175422
Received 13 June 2022; Received in revised form 7 November 2022; Accepted 22 November 2022
Available online 26 November 2022
0014-2999/© 2022 Elsevier B.V. All rights reserved.
Y. Ishizaki et al. European Journal of Pharmacology 938 (2023) 175422

Table 1
Hemodynamics and blood test results of wild-type mice before and after daily administration of the control and RTA-dh404 solutions.
Administration of control solution (n=10) Administration of RTA-dh404 solution (n=10)

Before After 7 days After 14 days Before After 7 days After 14 days

BW, kg 24.2 ± 0.3 24.8 ± 0.3 26.0 ± 0.3 24.5 ± 0.3 24.8 ± 0.3 25.2 ± 0.3**
SBP, mmHg 102.2 ± 2.4 100.9 ± 3.2 97.5 ± 2.5 99.6 ± 2.4 103.6 ± 3.2 96.0 ± 2.5
HR,/min 645.2 ± 10.2 691.2 ± 7.4 661.0 ± 9.7 669.9 ± 10.2 695.4 ± 7.4 686.7 ± 9.7*

Administration of control solution (n=5) Administration of RTA-dh404 solution (n=5)

Before After 7 days After 14 days Before After 7 days After 14 days
GOT, IU/L 91.4 ± 14.3 92.6 ± 6.8 96.6 ± 7.8 95.2 ± 14.3 95.2 ± 6.8 105.2 ± 7.8
GPT, IU/L 51.2 ± 4.2 41.6 ± 4.6 57.2 ± 7.6 49.0 ± 4.2 43.6 ± 4.6 60.4 ± 7.6
LDH, IU/L 466.6 ± 33.7 532.0 ± 58.1 442.8 ± 56.8 464 ± 33.7 479.6 ± 58.1 462.4 ± 56.8
BUN, mg/dL 25.6 ± 1.6 22.7 ± 2.0 21.6 ± 2.5 23.9 ± 1.6 20.2 ± 2.0 23.5 ± 2.5
Cr, mg/dL 0.15 ± 0.03 0.23 ± 0.05 0.20 ± 0.08 0.15 ± 0.03 0.16 ± 0.05 0.23 ± 0.12

BW: body weight, SBP: systolic blood pressure, HR: heart rate. Baseline indicates the day before control or RTA-dh404 solution administration was started. Day
0 indicates the day when the hindlimb ischemia surgery was performed. All values are indicated as mean ± standard error values. **: p<0.001 vs. the values after 14
Days of control solution administration. *: p<0.05 vs. the values after 14 Days of control solution administration.

activators of nuclear factor erythroid 2-related factor 2 (Nrf2) (Wang
et al., 2014). Nrf2 is a transcription factor that binds to antioxidant
response elements, and the activated Nrf2 plays a role in decreasing
oxidative stress in tissues by producing antioxidant molecules. In
gastrocnemius tissues of patients with critical limb ischemia, Nrf2 ac­
tivity was reported to be attenuated with increasing oxidative stress in
the tissues (Islam et al., 2015). CDDO-9,11-dihydro-trifluoroethyl amide
is an analogue of bardoxolone methyl; it was previously called
RTA-dh404 and was reported to suppress oxidative stress in cultivated
cardiomyocytes (Ichikawa et al., 2009) and renal tissues (Aminzadeh
et al., 2014) by activating Nrf2 in the cells. However, with regard to
ischemic lower limb tissues, the antioxidative effect of RTA-dh404 has
not yet been reported.
Therefore, the aims of this study were to test the antioxidant effects
of RTA-dh404 and the augmentation of neovascularization in the limbs
in a hindlimb ischemia mouse model.
2. Materials and methods
2.1. Animals, substances, and cells
Eight-to-12-week-old wild-type and athymic nude mice (BALB/cA
and BALB/cA-nu/nu, respectively, CLEA Japan Inc., Tokyo, Japan) were
used. The blood pressure and heart rate of the mice were measured in
the conscious state with a tail-cuff pressure analysis system (TK370C;
UNICOM, Tokyo, Japan). A unilateral hindlimb ischemia mouse model
was surgically generated to examine ischemia-induced oxidative stress
and neovascularization in their lower limbs (Sasaki et al., 2006). The
proximal portion of the mouse femoral artery and the distal portion of
the mouse saphenous artery were occluded using an electrical coagu­
lator (Vetroson V-10 bi-polar electrosurgical unit, Summit Hill Labora­
tories, NJ, USA). The day of surgery was defined as Day 0 in this study.
RTA-dh404 was synthesized and provided by Reata Pharmaceuticals,
Inc. (Irving, TX, USA), and the RTA-dh404 suspension was made with
sesame oil (S3547, Sigma-Aldrich, MO, USA). Pretreatment by daily oral
administration of the sesame oil (i.e., control solution) or the RTA-
dh404 solution (10 mg kg− 1 day− 1) for mice was started from 7 days
before the surgery for hindlimb ischemia, and the administration of the
solutions was continued until 17 days after the surgery. Three days after
the surgery, a total of 100 μL of phosphate buffered saline with or
without human ADRCs (5×105 cells per mouse) were injected into the
ischemic adductor muscle of the athymic nude mice. The human ADRCs
were purchased (Poietics™ human adipose-derived stem cells, PT-5006,
Lonza, Basel, Switzerland).
All surgical procedures for mice were performed under anesthesia
with isoflurane (5% in 100% oxygen for induction, 1–2% in 100% ox­
ygen for maintenance) using an animal anesthesia apparatus (TK-5, Bio
Machinery, Chiba, Japan). The depth of anesthesia was monitored by
the toe pinch reflex test. When harvesting mice adductor muscle tissues,
the mice were sacrificed by cervical dislocation after deeper anesthesia
with isoflurane. Blood samples collected from the mice’s cardiac
chambers were centrifuged at 3,000 rpm for 10 min at 4 ◦ C, and the
serum samples and harvested adductor muscle tissues were immediately
stored at − 80 ◦ C or fixed with formalin for in vitro experiments. To
examine some kind of tissue damage caused by the intramuscular in­
jection of a possibly high-volume of 100 μL of phosphate buffered saline
to mice ischemic limbs using a light microscope (BX50, OLYMPUS,
Tokyo, Japan), 5 μm-thick sections were prepared from the paraffin-
fixed embedded tissue samples of the ischemic limbs for hematoxylin
and eosin staining. The blood test was performed by a commercial
laboratory (Oriental Yeast Co., Ltd., Shiga, Japan).
This study had the following experimental groups of mice with
hindlimb ischemia: (1) wild-type mice treated with control only; (2)
wild-type mice treated with RTA-dh404 only; (3) athymic nude mice
treated with control only; (4) athymic nude mice treated with RTA-
dh404 only; (5) athymic nude mice treated with control and intramus­
cular injection of human ADRCs to the ischemic lower limbs; and (6)
athymic nude mice treated with RTA-dh404 and intramuscular injection
of human ADRCs to the ischemic lower limbs. The above six groups were
defined as group WC, group WR, group C, group R, group CA, and group
RA, respectively.
The investigation conformed to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health.
The study protocol was approved by the Institutional Animal Care and
Use Committee of Kurume University School of Medicine (approval
numbers: 2016-171, 2017-098-1, 2018-167-1, 2019-090, 2020-055,
2021-048, and 2022-077).
2.2. Oxidative stress-associated assays
2.2.1. Tissue expression of reactive oxygen species
Five-μm-thick frozen sections of mice adductor muscles were made
and incubated with 2 mmol/L dihydroethidium (Invitrogen, CA, USA),
which is a fluorescence probe to detect production of reactive oxygen
species, for 30 min at 37 ◦ C in the dark. The dihydroethidium staining of
the section was assessed in 5 randomly selected high-power fields
(×200) under a fluorescence microscope (BX50, OLYMPUS, Tokyo,
Japan). Dihydroethidium fluorescence intensity was quantified with
image analysis computer software (Image-Pro Plus 6.2J, Media Cyber­
netics, Inc., FL, USA). The levels of tissue expression of reactive oxygen
species in the mice adductor muscles were expressed as the ischemic/
non-ischemic limb tissues dihydroethidium-fluorescence intensity ra­
tios (Nakayoshi et al., 2014).
2.2.2. Tissue expression of Nrf2
The stored mice muscle tissues were homogenized in a reagent

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Y. Ishizaki et al.
Fig. 1. ROS p Production of reactive oxygen species in the non-ischemic and
ischemic lower limbs of mice treated with or without RTA-dh404. (A) Repre­
sentative fluorescence microscopic images of dihydroethidium (DHE)-stained
tissues of the ischemic and non-ischemic lower limbs of mice treated with
control and RTA-dh404. The DHE is stained red. Scale bar: 100 μm. (B) Pooled
data of the DHE fluorescence intensity ratios of the mice ischemic and non-
ischemic lower limbs on Days 3 and 7. The calculated values of the ratio are
shown on the red-colored scatter plots. WC and WR indicate the experimental
groups defined as the wild-type mice treated with control only (n=10 on Day 3,
n=9 on Day 7) and the wild-type mice treated with RTA-dh404 only (n=10 on
Day 3, n=8 on Day 7), respectively. *: p<0.001 vs. group WC.
(TRIzol™ Reagent, Thermofisher Scientific, MA, USA) at 4 ◦ C, and total
ribonucleic acid was extracted with an experimental kit (Invitrogen™
PureLink™ RNA Mini Kit, Thermofisher Scientific), according to the
manufacturer’s instructions. The RNA samples (1 mg per lane) were
reverse-transcribed into complimentary deoxyribonucleic acid with an
experimental kit (High Capacity RNA-to-cDNA kit, Thermofisher Sci­
entific). A polymerase chain reaction assay was performed to examine
the gene expressions of mouse Nrf2 and 18S ribosomal ribonucleic acid,
which was previously used as the reference gene (Islam et al., 2015),
with the corresponding primer pairs (#Mm00477784_m1 and
#Mm03928990_g1, respectively, Applied Biosystems, CA, USA) using a
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European Journal of Pharmacology 938 (2023) 175422
real-time quantitative polymerase chain reaction system (StepOne™
Real-Time PCR system, Applied Biosystems).
2.2.3. Tissue concentration of total antioxidant capacity (TAC), superoxide
dismutase (SOD), and angiogenesis-related proteins
The stored mice muscle tissues were homogenized in a reagent
including Tris-buffered saline (TaKaRa, Shiga, Japan), ethyl­
enediaminetetraacetic acid (Sigma-Aldrich), and 0.05 M sucrose
(Nacalai Tesque, Kyoto, Japan) at 4 ◦ C. The tissue concentrations of TAC
and SOD in the homogenized samples were measured using commercial
enzyme-linked immunosorbent assay kits (Abcam, Cambridge, UK and
Dojindo, Kumamoto, Japan, respectively). The tissue concentrations of
angiogenesis-related proteins, angiopoietin-2, endoglin, hepatocyte
growth factor (HGF), interleukin (IL)-1β, stromal cell derived factor
(SDF)-1α, and vascular endothelial growth factor (VEGF)-A, were
measured using the multiparametric Luminex kit (Milliplex, mouse
angiogenesis/growth factor panel, Merck Millipore Corporation, Bill­
erica, MA, USA). The above measurements were performed according to
the manufacturer’s instructions.
2.3. Identification of ADRCs injected in adductor muscles
When ADRCs were intramuscularly injected into mice lower limbs,
the cells were labeled with a fluorescent red reagent Dil (CellTracker
CM-Dil C7000, Invitrogen) according to the manufacturer’s instructions.
After harvesting the adductor muscle tissues, 5-mm-thick frozen sections
of the tissues were fixed with 2% paraformaldehyde for 10 min at room
temperature. Capillary endothelial cells in the sections were stained
with a CD31 monoclonal antibody (ab56299, Abcam, Cambridge, UK)
and a fluorescent secondary antibody (Alexa Fluor 488, Invitrogen).
Deoxyribonucleic acid in the cell nuclei was stained with 4ʹ-6-dia­
midino-2-phenylindole (H-1800, Vector Laboratories, Burlingame, CA,
USA). The Dil-labeled ADRCs were examined in the stained tissues with
a fluorescence microscope (BX50, OLYMPUS).
2.4. Apoptosis assay
The tissues of adductor muscles 24 h after intramuscular injection of
Dil-labeled ADRCs were harvested, and 5-mm-thick frozen sections of
the harvested tissues were fixed with 2% paraformaldehyde for 10 min
at room temperature. The terminal deoxynucleotidyl transferase-
mediated deoxyuridine triphosphate-nick end labelling (TUNEL)-posi­
tive cells in the tissues were stained with an apoptosis detection kit (In
Situ Cell Death Detection Kit, Fluorescein, Roche, Basel, Switzerland)
according to the manufacturer’s instructions. Then, the numbers of
TUNEL-positive ADRCs and TUNEL-negative ADRCs were counted in 10
randomly selected high-power fields (×400) with the fluorescence mi­
croscope, and the number of TUNEL-positive ADRCs was divided by the
total number of ADRCs to calculate the apoptosis ratio for the injected
ADRCs (Nakayoshi et al., 2014).
2.5. Cell viability assay
Cell viability of the human ADRCs subjected to malnutrition stress,
hypoxic stress, or oxidative stress in vitro was tested. The human ADRCs
were incubated with serum-free culture medium, with a hypoxic
chamber (O2 1.0%; CO2 5.0%, N2 94%; APM-30DR, ASTEC, Fukuoka,
Japan), or with 1 mM hydrogen peroxide-added culture medium for 18 h
at 37 ◦ C (Nakayoshi et al., 2014). Hydrogen peroxide was used experi­
mentally as the chemical agent causing exogenous oxidative stress. The
incubated human ADRCs were washed twice with phosphate-buffered
saline and fixed with 1% paraformaldehyde. Cell viability was auto­
matically calculated by an integrated fluorescence microscope with dual
fluorescence channels to detect signals from total cells stained with ac­
ridine orange and dead cells stained with 4ʹ,6-diamidino-2-phenylindole
(NucleoCounter® NC-200™, chemometec, Allerod, Denmark).
Y. Ishizaki et al.
(caption on next column)
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European Journal of Pharmacology 938 (2023) 175422
Fig. 2. Anti-oxidative stress responses in the ischemic lower limbs of mice
pretreated with or without RTA-dh404. Pooled data of the (A) Nrf2/18S ribo­
somal ribonucleic acid (rRNA) gene tissue expression ratios and tissue con­
centrations of (B) total antioxidant capacity (TAC) and (C) superoxide
dismutase (SOD) in the mice ischemic limb tissues on Days 3 and 7. WC and WR
indicate the experimental groups defined as the wild-type mice treated with
control solution only (panel A, n=5 on Day 3, n=5 on Day 7; panel B, n=9 on
Day 3, n=9 on Day 7; panel C, n=11 on Day 3, n=8 on Day 7) and the wild-type
mice treated with RTA-dh404 solution only (panel A, n=6 on Day 3, n=5 on
Day 7; panel B, n=10 on Day 3, n=10 on Day 7; panel C, n=11 on Day 3, n=8
on Day 7), respectively. *: p<0.05 vs. group WC, #: p<0.001 vs. group WC, and
§
: p<0.01 vs. group WC.
2.6. Neovascularization assays
2.6.1. Blood flow in the lower limbs
Fourteen days after the intramuscular injection of phosphate-
buffered saline with or without human ADRCs into ischemic limbs of
the athymic nude mice, which corresponded to Day 17, the mice were
placed on a heating pad (FHP-300S, TGK, Tokyo, Japan) at 37 ◦ C to
minimize variations in room temperature, and blood perfusion of the
ischemic and non-ischemic lower limbs was measured with a laser
Doppler blood flow imager (moorLDI-Mark 2, Moor Instruments Ltd.,
Devon, UK). Calculated blood perfusion was expressed as the ischemic/
non-ischemic lower limb blood flow ratio (Nakayoshi et al., 2014).
2.6.2. Capillary and smooth muscle cell densities in adductor muscles
After the blood flow analysis, the ischemic and non-ischemic
adductor muscle tissues were harvested to make 5-μm-thick frozen
sections of the tissues, followed by immunohistochemical staining of
capillary endothelial cells and smooth muscle cells. The smooth muscle
cells were stained with an alpha-smooth muscle actin monoclonal
antibody (A5228, Sigma-Aldrich) and a fluorescent secondary antibody
(Alexa Fluor 647, Jackson ImmunoResearch Inc., West Grove, PA, USA).
The numbers of CD31-positive capillaries (less than 20 μm in diameter),
alpha-smooth muscle actin-positive conductance vessels (20–100 μm in
diameter), and myofibers in 10 randomly selected high-power fields
(×200) of the same transverse sections were counted with the fluores­
cence microscope. The numbers of CD31-positive capillaries and alpha-
smooth muscle actin-positive conductance vessels were divided by the
number of myofibers to calculate capillary density (Nakayoshi et al.,
2014) and conductance vessel density in the tissue, respectively.
2.7. Statistical analysis
Differences in the baseline values of the mice’s body weight, systemic
blood pressure, pulse rate, and blood test results were assessed between
group WC and group WR using the Wilcoxon rank-sum test. Changes in
the values during solution administration were examined in the two
groups by logistic regression analysis. The difference in the results of the
apoptosis assay was compared between group C and group R using the
Wilcoxon rank-sum test. Differences in the results of other assays among
four groups were analyzed using the Steel-Dwass post hoc test. Signifi­
cance was assumed at a value of p<0.05. Data were analyzed using JMP
Pro 16.0 (SAS Institute, Cary, NC, USA).
3. Results
3.1. Hemodynamics, blood tests, and hematoxylin and eosin staining
The mice’s bodyweight, systolic blood pressure, heart rate, and
serum concentrations of GOT, GPT, LDH, BUN, and creatinine were
measured in group WC and group WR before, 7 days after, and 14 days
after the start of daily administration of the control and RTA dh404
solutions. Although the body weights and heart rates after 14 days of
treatment with the control and RTA-dh404 solutions were slightly, but
Y. Ishizaki et al. European Journal of Pharmacology 938 (2023) 175422

Fig. 3. Representative fluorescence microscopic images showing distributions

of ADRCs injected 24 hours earlier in mouse ischemic limb tissue. The images
are merged with 3 images for detecting 4ʹ-6-diamidino-2-phenylindole stained
blue for nuclei in the tissue, CD31 antibody stained green for capillary endo­
thelial cells in the tissue, and red colored Dil-labeled ADRCs existing in the
tissue. The small image separately placed at the upper left of another larger
image is the magnified image focused on the center of the whole image.

significantly, higher in group WR than in group WC, there were no

differences in other variables between the two groups (Table 1). On
hematoxylin and eosin staining of mice ischemic lower limbs with or
without intramuscular injection of 100 μL of phosphate buffered saline,
there was no difference in the muscle condition between the two groups
(Supplementary Fig. 1).

3.2. Oxidative stress in ischemic limb tissues

On Days 3 and 7, the ischemic/non-ischemic limb tissue

dihydroethidium-fluorescence intensity ratios were less in group WR
than in group WC (Fig. 1A, B). Nrf2/18S ribosomal ribonucleic acid gene
expression ratios (Fig. 2A) and ischemic limb tissue concentrations of
TAC and SOD (Fig. 2B, C) in the ischemic limb tissues were greater in
group WR than in group WC.
3.3. Distribution of ADRCs injected in ischemic limb tissues
In group CA and group RA, the Dil-labeled ADRCs that were intra­
muscularly injected into the ischemic limbs on Day 3 were observed
around existing vessels together with inflammatory cells on Day 4
(Fig. 3). However, the Dil-labeled ADRCs were not found at all from Day
5 in a considerable number of tissue sections.
3.4. Viability of ADRCs injected in ischemic limb tissues and subjected to
experimental stress in vitro
On Day 4, the percentage of TUNEL-positive ADRCs (i.e., apoptotic
ADRCs) in the ischemic limb tissues was lower in group RA than in group
CA (Fig. 4). In addition, the percentage of viable ADRCs in vitro was
lower in ADRCs subjected to oxidative stress than in those subjected to
malnutrition and hypoxic stress (Fig. 5).
3.5. Neovascularization in ischemic lower limbs
On Days 3, 5, 7, and 17, there were no differences in ischemic/non-
ischemic lower limb blood flow ratios (Supplementary Table 1, Fig. 6A),
capillary densities in the ischemic limbs (Supplementary Table 2,
Fig. 6B), and conductance vessel densities in the ischemic limbs
Fig. 4. Apoptotic ADRCs in ischemic lower limbs of mice treated with or
without RTA-dh404. The images above the box and red-colored scatter plots are
representative fluorescence microscopic photos of the Dil-labeled ADRCs on
Day 4 (i.e., 24 hours after intramuscular injection of ADRCs in the ischemic
lower limbs of mice), corresponding each to the below box and red-colored
scatter plots showing pooled data of the apoptosis ratio for the injected
ADRCs. CA and RA indicate the experimental groups defined as the athymic
nude mice treated with control solution and intramuscular injection of human
ADRCs to the ischemic lower limbs (n=11) and the athymic nude mice treated
with RTA-dh404 solution and intramuscular injection of human ADRCs to the
ischemic lower limbs (n=11), respectively. The red cells and green cells indi­
cate the Dil-labeled ADRCs and terminal dUTP nick-end labeling (TUNEL)-
positive cells (i.e. apoptotic cells), respectively. *: p<0.05 vs. group C.
(Supplementary Table 3, Fig. 6C) between group C and group R. In
group CA, the blood flow ratios on Days 5, 7, and 17, the capillary
densities on Days 5, 7, and 17, and the conductance vessel densities on
Day 17 were higher than those in group C. Furthermore, the increased
capillary densities on Days 7 and 17 and conductance vessel densities on
Day 17 seen in group CA were augmented in group RA.
3.6. Autoamputation rate of ischemic lower legs
The mice in group C and group R showed a high rate of ischemic
gangrene in the ischemic lower legs, and the gangrene eventually caused
autoamputation of toes, heels, and lower legs with progression of
ischemia (Fig. 7). The autoamputation rate of the lower legs in group RA
on Day 17 was very low, and the rate was significantly lower than that in
group CA.

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Y. Ishizaki et al.
Fig. 5. Viability of the human ADRCs subjected to stressful conditions in vitro.
Pooled data of the percentages of cell viability of non-treated ADRCs defined as
group N (ADRCs in no stress, n=13), serum free medium-treated ADRCs defined
as group M (ADRCs in malnutrition stress, n=11), low oxygen-treated ADRCs
defined as group H (ADRCs in hypoxic stress, n=11), and H2O2-treated ADRCs
defined as group O (ADRCs in oxidative stress, n=9). *: p<0.001 vs. group N, #:
p<0.005 vs. group M, and §: p<0.005 vs. group H.
3.7. Tissue levels of angiogenesis-related proteins in ischemic limbs
On Day 7, tissue concentrations of angiopoietin-2, endoglin, IL-1β,
and VEGF-A in ischemic limbs were significantly higher in group RA
than in group C (Fig. 8). As regards IL-1β, the tissue concentration was
significantly higher in group RA than in group CA.
4. Discussion
In the present study, we tested a pharmacological strategy, which
was considered a comparatively easy one, for preventing oxidative
stress-induced apoptosis of angiogenic cells injected in ischemic skeletal
muscle tissues by reducing the oxidative stress in the tissues with an
analogue of bardoxolone methyl called RTA-dh404. This is the first
report to demonstrate the antioxidative efficacy of RTA-dh404 in the
ischemic skeletal muscle tissues of mice.
Oral administration of bardoxolone methyl, at a daily dose of 20 mg,
was reported to delay the progression of chronic kidney disease in pa­
tients with diabetes mellitus via reduction of oxidative stress in the
kidney tissue (Chin et al., 2018). The daily dosage of bardoxolone
methyl for a patient with a weight of 50 kg is equivalent to 0.4 mg kg− 1
day− 1. In the present study, oxidative stress in mice ischemic limb tis­
sues was suppressed in mice that were administered RTA-dh404 into the
stomach through the mouth using a tube at a daily dose of 10 mg kg− 1
day− 1. Although RTA-dh404 is an analogue of bardoxolone methyl, the
dosage might be high for mice. However, it was impossible to measure
the blood concentration of RTA-dh404 or bardoxolone methyl. Never­
theless, the RTA-dh404 treated mice did not show harmful changes in
weight, hemodynamics, or blood test results for liver and kidney func­
tions with 14-day treatment, suggesting that the daily dosage of
RTA-dh404 was safe for mice. Thus, the treatment of mice with
RTA-dh404 at a dosage of 10 mg kg− 1 day− 1 safely succeeded in
reducing oxidative stress in their ischemic lower limbs.
The successful antioxidation pretreatment of ischemic limb tissues of
mice with RTA-dh404 was achieved with increased tissue expression of
Nrf2, an activator that produces antioxidant molecules and tissue
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European Journal of Pharmacology 938 (2023) 175422
concentrations of TAC and SOD, which are antioxidant molecules in the
tissues. Although intramuscularly injected ADRCs were not found
ischemic limb tissues two days after cell injection, the number of
apoptotic ADRCs in the tissues with oxidative stress was significantly
decreased in the tissues one day after the cell injection compared to
other ischemic limb tissues of untreated mice. Ischemic tissue with
decreased oxygenated blood supply is subjected to a variety of stresses,
such as malnutrition stress, hypoxic stress, and oxidative stress. ADRCs
actually have a high susceptibility to oxidative stress-induced apoptosis
compared with malnutrition stress- and hypoxic stress-induced
apoptosis in vitro. Even though intramuscularly injected ADRCs in
ischemic tissue might achieve the antioxidative efficacy of RTA-dh404
in the tissue for one day only, the ADRCs escaped oxidative stress-
induced apoptosis in the ischemic tissue. The surviving ADRCs in the
tissue would contribute to augmenting neovascularization in the tissue.
Nevertheless, it was not possible to find any ADRCs in mice ischemic
limbs 2 days after the intramuscular injection of ADRCs to the ischemic
limbs. All of the injected ADRCs might have been phagocytized by
phagocytic cells within 48 h after playing a role in stimulating angio­
genesis to increase capillaries in the ischemic tissue. A step-by-step
mechanism of the neovascularization effect on mice ischemic limbs
despite the rapid disappearance of the injected ADRCs from the ischemic
tissue could have been presented as follows: (1) increased capillaries led
to increased blood flow, shear stress, and wall strain in capillaries; (2)
these increases stimulated arteriogenesis, angiogenesis, and capillary
arterialization followed by vessel growth; (3) the increased shear stress
stimulated nitric oxide synthesis in endothelial cells, thereby providing
nitric oxide in the ischemic tissue; and (4) increased nitric oxide
augmented the blood-flow-mediated arteriogenesis and angiogenesis, as
well as collateral artery development in the mice ischemic limbs. In the
present study, such acute neovascularization effect was probably
brought to the mice ischemic limbs up to 7 days after tissue ischemia
occurred (Fig. 6), resulting that the amputation ratio of the mice
ischemic limbs 14 days after the injection of ADRCs was reduced by
approximately 72% (Fig. 7). Despite that the antioxidative effect of RTA-
dh404 on ischemic skeletal muscle tissue was contributed to the
reduction in the number of apoptotic ADRCs in mouse ischemic limbs by
only approximately 25% (Fig. 4), it enabled several ADRCs in the
ischemic limbs to survive, thereby enhancing the acute neo­
vascularization effect enough to salvage the ischemic limbs. Neverthe­
less, whether the antioxidant effect of RTA-dh404 on ischemic tissue in
the mouse model is strong enough to be applied in humans is unknown
because the levels of oxidative stress in the ischemic limbs are different
between the acute limb ischemia mouse model and peripheral artery
disease patient with chronic limb ischemia.
ADRCs have been reported to release various growth factors and
chemokines that promote angiogenesis, such as VEGF, HGF, and SDF-1
(Murohara, 2009). In the present study, tissue concentrations of HGF
and SDF-1α were not different among the experimental groups, whereas
those of angiopoietin-2, endoglin, IL-1β, and VEGF-A were higher in the
ischemic lower limbs of mice in group RA than in the other groups.
Angiopoietin-2 is an important proangiogenic factor (Scholz et al.,
2015), which also has a central role in regulating vessel maturation and
stabilization (Augustin et al., 2009). Vessel maturation was examined by
alpha-smooth muscle actin-positive conductance vessel density in the
present study. Endoglin, a proliferation-associated and
hypoxia-inducible protein abundantly expressed in angiogenic endo­
thelial cells, is involved in endothelial cell adhesion, proliferation,
migration, and angiogenesis (Nassiri et al., 2011]. It has another role as
a cell adhesion molecule in mature and progenitor endothelial cells
interacting with vascular mural cells (Rossi et al., 2019). VEGF-A
modulates angiogenesis prominently through receptor tyrosine kinase
vascular endothelial growth factor receptor-2 (Abhinand et al., 2016).
IL-1β mediates angiogenesis by upregulating the expression of VEGF in
vascular smooth muscle cells (Fahey and Doyle, 2019), as well as by
mobilizing progenitor endothelial cells (Heinisch et al., 2022) in a
Y. Ishizaki et al.
(caption on next column)
7
European Journal of Pharmacology 938 (2023) 175422
Fig. 6. Neovascularization in the ischemic lower limbs of mice after treat­
ments. (A) Representative laser Doppler blood flow (LDBF) images of the
ischemic and non-ischemic lower limbs of mice (right panels) and pooled data
of the limb ischemic/non-ischemic blood flow ratio (left panel) on Day 17. C, R,
CA, and RA indicate the experimental groups defined as the athymic nude mice
treated with control solution only (n=10), athymic nude mice treated with
RTA-dh404 solution only (n=10), athymic nude mice treated with control so­
lution and intramuscular injection of human ADRCs to the ischemic lower limbs
(n=10), and athymic nude mice treated with RTA-dh404 solution and intra­
muscular injection of human ADRCs to the ischemic lower limbs (n=10),
respectively. Representative fluorescence microscopic photos of (B) CD31-
positive endothelial capillaries stained green and (C) alpha-smooth muscle
actin (α-SMA)-positive conductance vessels stained red in the mice ischemic
lower limbs (right panels) and pooled data of the capillary density of the mice
ischemic limbs (left panel) on Day 17. The mice lower limbs indicated by the
white arrows correspond to the right limbs with ischemia. The red to white
color and the dark blue color on the image indicate high and low perfusion
signals, respectively. Scale bar: 100 μm. Panel A: **: p<0.001 vs. group C, *:
p=0.001 vs. group C, §: p<0.05 vs. group C, #: p<0.05 vs. group CA. Panel B:
**: p<0.005 vs. group C, *: p=0.005 vs. group C, §: p<0.05 vs. group C, ##:
p<0.001 vs. group CA, #: p<0.05 vs. group CA. Panel C: *: p<0.05 vs. group C,
#
: p<0.05 vs. group C, ##: p<0.005 vs. group C.
VEGF-dependent manner (Fahey and Doyle, 2019). Angiogenesis at the
tissue level was examined by capillary density in the present study.
Although the above 4 proteins might be released from or expressed on
not only ADRCs, but also skeletal muscle cells, endothelial cells, and
various inflammatory cells in ischemic lower limb tissues, an increase in
the number of variable ADRCs in the tissues of group RA might, at least
in part, contribute to the increased tissue concentrations of the 4 pro­
teins in the tissues. Taken together, a series of actions by which the 4
proteins augmented angiogenesis and subsequently matured angiogenic
endothelial cells in the ischemic lower limb tissues might result in the
differences in the ischemic/non-ischemic lower limb blood flow ratios,
capillary densities, and conductance vessel densities among the 4
experimental groups.
Vitamin C, vitamin E, and beta carotene, which are antioxidants,
Fig. 7. Autoamputation rates of ischemic lower legs of mice with surgically-
induced hind limb ischemia 17 days earlier. C, R, CA, and RA indicate the
experimental groups defined as the athymic nude mice treated with control
solution only (n=11), athymic nude mice treated with RTA-dh404 solution only
(n=10), athymic nude mice treated with control solution and intramuscular
injection of human ADRCs to the ischemic lower limbs 14 days earlier (n=15),
and athymic nude mice treated with RTA-dh404 solution and intramuscular
injection of human ADRCs to the ischemic lower limbs 14 days earlier (n=15).
*: p<0.001 vs. group C, §: p<0.05 vs. group CA.
Y. Ishizaki et al. European Journal of Pharmacology 938 (2023) 175422

Fig. 8. Tissue concentrations of angiogenesis-related proteins in mice

ischemic lower limbs. The concentrations of angiopoietin-2, endoglin, he­
patocyte growth factor (HGF), interleukin (IL)-1β, stromal cell derived
factor (SDF)-1α, and vascular endothelial growth factor (VEGF)-A are shown
in panels A, B, C, D, E, and F, respectively. C, R, CA, and RA indicate the
experimental groups defined as the athymic nude mice treated with control
solution only (n=10), athymic nude mice treated with RTA-dh404 solution
only (n=10), athymic nude mice treated with control solution and intra­
muscular injection of human ADRCs to the ischemic lower limbs (n=10),
and athymic nude mice treated with RTA-dh404 solution and intramuscular
injection of human ADRCs to the ischemic lower limbs (n=10). *: p<0.01
vs. group C, §: p<0.05 vs. group C, #: p<0.05 vs. group R, and ¶: p<0.05 vs.
group CA.

8
Y. Ishizaki et al.
were expected to reduce cardiovascular disease by preventing oxidative
stress-induced tissue damage; however, supplementation with them
failed to prevent cardiovascular disease in large-scale human studies
(Cook et al., 2007; Sesso et al., 2008). Another antioxidant, acetylcys­
teine, has also failed to prevent contrast-associated acute kidney injury
due to oxidative stress in the tissue (Weisbord et al., 2018). Bardoxolone
methyl was reported to show Nrf2-associated antioxidant efficacy in vivo
(Ichikawa et al., 2009; Aminzadeh et al., 2014), whereas there were no
reports of the antioxidative efficacy of vitamin C, vitamin E, beta caro­
tene, or acetylcysteine in vitro or in vivo. Nonetheless, one limitation to
the present study was that the antioxidative effect for mice ischemic
limb tissues by daily oral treatment with vitamin C, vitamin E, beta
carotene, or acetylcysteine was not tested.
In addition, there were other limitations to the present study. First, it
is unclear whether the RTA-dh404-induced antioxidation of mice
ischemic skeletal muscle tissue is achieved by oral administration of
substance dosages less than and more than 10 mg kg− 1 day− 1. The dose-
dependent effect of RTA-dh404 is also unclear. Second, the in vivo ani­
mal experiments were performed with healthy mice with acute limb
ischemia, differing from the clinical situation in peripheral artery dis­
ease patients with chronic limb ischemia. Third, data of hemodynamics,
blood test, and reactive oxygen species production in the non-ischemic
and ischemic lower limbs in athymic nude mice with or without daily
administration of the control and RTA-dh404 solutions were lacking,
even though we previously showed that the morphology, hemody­
namics, reactive oxygen species production, and angiogenesis in the
ischemic lower limbs of athymic nude rats were similar to those of wild-
type rats (Nakayoshi et al., 2014). Fourth, whether the antioxidant effect
of RTA-dh404 for mice ischemic limb tissues augmented neo­
vascularization in the ischemic limbs following the intramuscular in­
jection of ADRCs in a case in which daily oral administration of
RTA-dh404 was started on Day 0 or after was not tested.
5. Conclusions
Pretreatment of ischemic limbs by daily oral administration of RTA-
dh404 may be a potential strategy to augment the effect of therapeutic
angiogenesis using intramuscular injection of ADRCs to the ischemic
limbs by reducing oxidative stress in the limbs via RTA-dh404-induced
antioxidation and prevention of oxidative stress-induced apoptosis of
the injected ADRCs.
Declarations of competing interest
The authors declare that they have no competing interests.
CRediT authorship contribution statement
Yuta Ishizaki: Formal analysis, Investigation, Data curation, Writing
– original draft. Ken-ichiro Sasaki: Conceptualization, Methodology,
Validation, Formal analysis, Investigation, Writing – original draft,
Visualization, Supervision, Project administration, Funding acquisition.
Takahiro Yoshikawa: Investigation, Data curation, Funding acquisi­
tion. Takaharu Nakayoshi: Investigation. Motoki Sasaki: Investiga­
tion, Funding acquisition. Masanori Ohtsuka: Investigation. Sachiko
Hatada-Katakabe: Investigation, Funding acquisition. Yuki Takata:
Investigation. Yoshihiro Fukumoto: Resources, Writing – review &
editing, Project administration.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper
9
European Journal of Pharmacology 938 (2023) 175422
Data availability
The authors are unable or have chosen not to specify which data has
been used.
Acknowledgements
This work was supported by the Japan Society for the Promotion of
Science KAKENHI [grant numbers 18K08089, 16K19433, 19K17539,
19K17540].
The authors would like to thank Colin J. Meyer and Joel W. Proksch
of Reata Pharmaceuticals, Inc. for their kind provision of RTA-dh404.
The authors would also like to thank Katsue Okuizumi, Mai Yama­
moto, Mami Nakayama, Miho Nakao, and Miyuki Nishigata of the Car­
diovascular Research Institute, Kurume University, for their excellent
technical support in RT-PCR, ELISA, and tissue staining.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2022.175422.
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