Biomedicine & Pharmacotherapy 167 (2023) 115504

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Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha

Angiogenesis inhibitor-specific hypertension increases the risk ofdeveloping aortic dissection
Kaito Tsujinaka a, b, Yuki Izawa-Ishizawa a, c, *, Koji Miyata a, Toshihiko Yoshioka a, b,
Kohei Oomine a, Honoka Nishi a, Masateru Kondo a, b, Syuto Itokazu a, Tatsumi Miyata a,
Takahiro Niimura a, d, Maki Sato a, Fuka Aizawa a, b, Kenta Yagi a, d, Masayuki Chuma e,
Yoshito Zamami f, Mitsuhiro Goda a, b, Keisuke Ishizawa a, b, da

Department of Clinical Pharmacology and Therapeutics, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
Department of Pharmacy, Tokushima University Hospital, Tokushima, Japanc
Department of General Medicine, Taoka Hospital, Tokushima, Japand
Clinical Research Center for Developmental Therapeutics, Tokushima University Hospital, Tokushima, Japane
Department of Hospital Pharmacy & Pharmacology, Asahikawa Medical University & University Hospital, Asahikawa, Japanf
Department of Pharmacy, Okayama University Hospital, Okayama, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Angiogenesis inhibitors
Aortic dissection
Real-world database
Hypertension
FDA Adverse Event Reporting System

Aortic dissection is an adverse event of angiogenesis inhibitors; however, the association between the drugs andaortic dissection is unclear. Therefore, we investigated if and how angiogenesis inhibitors increase the onset of
aortic dissection using pharmacologically-induced aortic dissection-prone model (LAB) mice, cultured endo­
thelial cells, and real-world databases, which is a novel integrated research approach. Disproportionality analysis
was performed and calculated using the reporting odds ratio as a risk signal using a worldwide database of
spontaneous adverse events to estimate the risk of adverse events. Angiogenesis inhibitors, but not otherhypertension-inducing drugs, showed significant risk signals for aortic aneurysms and dissection. A retrospective
cohort analysis using JMDC, a medical receipt database in Japan, showed that the history of atherosclerosis anddyslipidemia, but not hypertension, were significantly associated with the onset of aortic dissection during
angiogenesis inhibitor medication administration. For in vivo studies, sunitinib (100 mg/kg/day) was admin­
istered to LAB mice. Sunitinib increased systolic blood pressure (182 mmHg vs. 288 mmHg with sunitinib; p＜0.01) and the incidence of aortic dissection (40% vs. 59% with sunitinib; p = 0.34) in mice. In vivo and in vitro
studies revealed that sunitinib increased endothelin-1 expression and induced endothelial cell damage evaluatedby intracellular- and vascular cell adhesion molecule-1 expressions. The increased risk of developing aortic
dissection with angiogenesis inhibitors is associated with the development of drug-specific hypertension via
endothelial cell damage and endothelin-1 expression. Our findings are invaluable in establishing safer anticancer
therapies and strategies to prevent the development of vascular toxicity in high-risk patients.

1. Introduction
Aortic dissection is a dynamic condition in which the aortic wall is
detached in two layers at the tunica media level, forming a false lumen

with blood flow or hematomas within the aortic wall. Aortic dissection isa lethal disease, with sudden onset and death [1]. Aortic dissection iscaused by hemodynamic loading against a background of tunica media
weakening over time [2]. The etiology of tunica media lesions may be

Abbreviations: Ang II, angiotensin II; BAPN, β-aminopropionitrile; eNOS, endothelial nitric oxide synthesis; ET-1, endothelin-1; FAERS, FDA Adverse Event
Reporting System; G-CSF, granulocyte colony-stimulating factor; ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin-6; KC, keratinocyte-derived chemo­kines; L-NAME, Nω-nitro-l-arginine methyl ester; MCP-1, monocyte chemoattractant protein 1; ROR, reporting odds ratio; SNT, sunitinib; VEGF, vascular endothelial
growth factor; VCAM-1, vascular cell adhesion molecule 1.
* Correspondence to: Department of Clinical Pharmacology and Therapeutics, Tokushima University Graduate School of Biomedical Sciences, 3-18-15 Kuramoto,Tokushima 770-8503, Japan.
E-mail address: yuki.ishizawa@gmail.com (Y. Izawa-Ishizawa).
https://doi.org/10.1016/j.biopha.2023.115504
Received 15 July 2023; Received in revised form 30 August 2023; Accepted 12 September 2023
Available online 16 September 2023
0753-3322/© 2023 The Author(s).
Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Biomedicine & Pharmacotherapy 167 (2023) 115504

due to decreased elastic lamina and cross-linking fibers between elastic
fibers because of hypertension or inherited connective tissue diseases
such as Marfan’s syndrome [3,4]. Endothelial cell dysfunction may play
an essential role in the onset of aortic dissection, adding to the medial
fragility and hemodynamic loading [5–7].
Angiogenesis inhibitors are anticancer drugs that target vascularendothelial growth factor (VEGF), including anti-VEGF antibodies, antiVEGF receptor antibodies, and VEGF tyrosine kinase inhibitors. These
anticancer drugs are widely used for various carcinomas, includingcolorectal, lung, and breast cancer. Major side effects of angiogenesis
inhibitors are hypertension, renal failure, neurotoxicity, and appetite
loss. A meta-analysis showed a significant increase in blood pressure in
8% of bevacizumab-treated patients [8]. The cardiovascular toxicity ofangiogenesis inhibitors is caused by their effects on cardiomyocytes,
endothelial cells, and pericytes [9,10]. Depleting the VEGF pathway by
inhibiting the VEGF receptor induces vascular endothelial cell apoptosis
and may lead to acute arterial thrombotic events [11,12]. VEGF
signaling inhibition also decreases nitric oxide production, a vasodi­
lator, leading to increased vascular resistance and hypertension [13].
Endothelin-1 (ET-1) is associated with angiogenesis inhibitor-induced
hypertension and renal failure [14]. Thus, various mechanisms havebeen demonstrated for cardiovascular toxicities associated with angio­
genesis inhibitors.
Aortic dissection in patients using angiogenesis inhibitors is a rare
but severe side effect [15]. The U.S. Food and Drug Administration(FDA) and other agencies issued a warning in 2018 regarding the risk ofaortic dissection. However, how angiogenesis inhibitors cause aortic
dissection has not been clarified, nor have preventive methods been
established. In the present study, we demonstrate the risk and causes ofaortic dissection induced by angiogenesis inhibitors.
We approached this research problem using a novel research meth­odology that we have recently established, which entails the integrationof a large-scale medical information database analysis and in vivo and invitro experiments. A large-scale medical information database, that is,“a real world database”, is useful for researching emergency diseases,
rare diseases, and the side effects that occur infrequently like aortic
dissection. Many cases of aortic dissection happen suddenly and develop
rapidly. Therefore, it is difficult to design a clinical study. This is one of
the reasons why aortic dissection has not been studied well to date. The
real-world database reflects the clinical situation and allows forcomprehensive analysis of a vast number of aortic dissection cases. On
the other hand, database analysis only proposes hypotheses and does notprove interrelationships. Therefore, we established a new research
approach in which hypotheses obtained from databases are elucidated
by basic pharmacological studies. In this study, we utilized thepharmacologically-induced aortic dissection prone model mice, which
we established, to clarify the pathogenesis of angiogenesis inhibitorinduced aortic diseases.
2. Methods
2.1. Data analysis using the FDA adverse event reporting system
The FDA Adverse Event Reporting System (FAERS) is a voluntary
adverse event reporting system operated by the U.S. FDA. Of the
14,524,065 spontaneous adverse events report data from the firstquarter of 2004 to second quarter of 2020, duplicate reports were
excluded according to FDA recommendations, and the remaining
12,190,284 reports were used for analysis (Fig. 1). The Navicat for
SQLite database 3.33.0 was used to process the data. The types ofadverse events were obtained from the International Council for Har­
monisation of Technical Requirements for Pharmaceuticals for Human
Use (ICH) International Glossary of Pharmaceutical Terms, Medical
Dictionary. The term “aortic dissection” was defined and extracted using

Fig. 1. Flow diagram of the study design. FAERS: FDA Adverse Event Reporting System.
2

K. Tsujinaka et al.

Biomedicine & Pharmacotherapy 167 (2023) 115504“10002895/aortic dissection/Aortic dissection,” based on the ICH
Medical Dictionary for Regulatory Activities/J ver. 23.1. The risk of an
adverse event was assessed by calculating the reporting odds ratio(ROR) and 95% confidence interval (CI) as previously reported [16].
Briefly, patients were classified into four groups: (A) patients who
received a drug and reported aortic dissection as a side effect, (B) pa­
tients who received a drug and did not report aortic dissection as anadverse reaction, (C) patients who did not receive a drug and reported
aortic dissection as an adverse reaction, and (D) patients who did notreceive a drug and did not report aortic dissection as an adverse reac­tion. The number of reports in each group was calculated based on theabove classification, and the ROR and 95% CI were calculated according
to the following formula:
ROR

=
=

2.4. Experimental animals and treatment administration
The experimental animals were 8-week-old male C57BL/6 J mice
(CLEA Japan Inc. Tokyo, Japan). Mice were acclimated to obtain similar
mean body weight and blood pressure in each group, and allowed to eat
and drink freely through the experimental period.
For the sunitinib (SNT) alone study, mice were divided into fourgroups; the control, 4 mg/kg/day SNT (SNT4), 40 mg/kg/day SNT(SNT40), and 100 mg/kg/day SNT (SNT100), and received 1% car­boxymethyl cellulose (CMC, Wako, Osaka, Japan) or SNT (Selleck
Chemicals, Houston, TX, USA) suspended in 1% CMC at concentrationsof 4, 40, or 100 mg/kg/day, orally via a feeding needle. Body weight
was measured daily, and blood pressure was measured weekly using a
tail-cuff plethysmography (BP-98AL, Softron, Tokyo, Japan). Vascular
toxicity was assessed 4 weeks after the start of treatment as explained
under the following section.
In the SNT study in the aortic dissection-prone mouse model (LAB
model), LAB and SNT+LAB groups received Nω-nitro-L-arginine methyl
ester (L-NAME; Sigma-Aldrich, St. Louis, MO, USA) (10 mg/kg/day)orally in drinking water for 4 weeks starting at 8 weeks of age [18].
Angiotensin II (Ang II; 1000 ng/kg/min) and β-aminopropionitrile(BAPN; 150 mg/kg/day) (both from Sigma-Aldrich, St. Louis, MO, USA)
were administered continuously for 1 week using an osmotic pump
(MINI-OSMOTIC PUMP MODEL 2002; Alzet, Muromachi Kikai Co.)
from 3 weeks later starting with L-NAME administration. The SNT+LAB
group was administered SNT (100 mg/kg day) dissolved in 1% CMC
continuously for 4 weeks starting at 8 weeks of age. The onset of aortic
dissection and vascular pathology were assessed 1 week after osmotic
pump implantation.

A/B, 95% CIC/D√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
1 1 1 1exp (ln (ROR) ± 1.96
+ + + )A B C D

A, B, C, and D represent the number of reports in each group, and ln isthe natural logarithm.
Drugs for which A, B, C, and D exceeded 10 and the ROR was > 1
were defined as those with an increased risk of developing aortic
dissection, and a marker for dissection development was detected. Sta­tistical analysis was performed using R version 4.2.1 for Windows.
2.2. Data analysis using JMDCThis retrospective cohort analysis used the Diagnosis Procedure
Combination (DPC) database, a nationwide Japanese inpatient databaseprovided by JMDC (JMDC Inc. Tokyo, Japan) as previously reported[17]. This database comprises medical fee schedules (receipt data) and
DPC data for patients who visited medical institutions contracted by
JMDC. As of August 2022, JMDC had contracts with 620 DPC andnon-DPC hospitals. The database includes a tabulation of major di­agnoses, comorbidities at admission, complications during hospitaliza­
tion, and treatment information such as medications, procedures, andrehabilitation. Cases in the receipt database from April 2014 to October
2020, including angiogenesis inhibitors (bevacizumab, aflibercept,
ramucirumab, sorafenib, regorafenib, lenvatinib, sunitinib, pazopanib,
axitinib, cabozantinib and vandetanib by component name), wereanalyzed, excluding patients younger than 18 years and those withoutsmoking information (Fig. 1). The incidence of aortic aneurysm or
dissection (“aortic aneurysm and dissection” International Statistical
Classification of Diseases and Related Health Problems (ICD) 10 code:I71) within 1 year of receiving an angiogenesis inhibitor was used as an
objective factor. The following were used as objective factors: age 60
years or older, sex, smoking status, history of anthracyclines (doxoru­
bicin, amrubicin, daunorubicin, epirubicin, pirarubicin, idarubicin, andachlorubicin by component name), history of hypertension (ICD-10: I10,
I15, I 674, P29), atherosclerosis (ICD-10: I70), dyslipidemia (ICD-10:
L94, M30, M31, M32, M33, M34, M35, M36), and connective tissue
disorders (ICD-10: E78). The Mann–Whitney U test was used forcontinuous variables, and Fisher’s exact test for categorical variables.
Multivariate analysis was performed using multiple logistic analyses.
Statistical analysis was performed using R version 4.2.1 for Windows.

2.5. Blood and tissue collection
After the measurement of body weight, mice were anesthetized byintraperitoneal administration with a three-drug mixture of 1 mg/mL
medetomidine hydrochloride (Nippon Zenyaku Kogyo Co., Ltd.,
Fukushima, Japan), 5 mg/mL midazolam (Sandoz K.K. Tokyo, Japan),and 5 mg/mL butorphanol tartrate (Meiji Seika Pharma Co., Ltd. Tokyo,
Japan), mixed with saline. Blood was collected from the orbital venous
plexus of the mice and centrifuged at 1000 ×g for 10 min in a tube toseparate serum. The systemic circulation was perfused with 25–50 mL ofsaline, and tissues including aorta, heart, and kidney were harvested.
Tissues were weighed and frozen at − 80 ◦ C or fixed using 4% para­
formaldehyde (Wako) and embedded in paraffin. The aortic diameter
was measured using ImageJ v.1.37 (National Institutes of Health,Bethesda, Maryland, USA).
2.6. Histology
Section (5 µm thick) were subjected to Elastica van Gieson (EVG)
staining (Merck, Darmstadt, Germany) according to the manufacturer’s
instructions. The development of aortic dissection was determined byfalse lumen formation under EVG staining. Elastin degradation was
graded as follows: grade 1, intact and well-organized elastic lamina;
grade 2, elastic lamina with some interruption and breakage; grade 3,
severe elastin fragmentation or loss; grade 4, aortic rupture [19].

2.3. Ethical considerations

2.7. Real-time PCR

Due to the anonymity of the data, the obligation to obtain informed
permission was waived to perform database analysis. All animal pro­cedures were performed per the guidelines of the Animal ResearchCommittee of the University of Tokushima Graduate School which
complied with ARRIVE 2.0.

RNA was extracted from aortic homogenates collected following SNT
monotherapy and the LAB model using the RNeasy® Fibrous Tissue Mini
Kit (QIAGEN, Hilden, Germany) and from cultured cells using the
RNeasy® Plus Mini Kit (QIAGEN). The extracted RNA was dissolved in
RNase-free water, and the concentration was measured using a Nano­drop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Reverse
transcription was then performed using a PrimeScript RT reagent Kit(Takara Bio, Shiga, Japan). Quantitative analysis of gene expression
3

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Biomedicine & Pharmacotherapy 167 (2023) 115504

levels was performed by the relative quantification method using theMyiQ2 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA)and THUNDERBIRD® SYBR qPCR Mix (TOYOBO LIFE SCIENCE, Osaka,
Japan) was used to prepare the PCR reaction solution. The primer se­quences used are listed in Supplementary Table 1. All primers werepurchased from Eurofins Genomics (Tokyo, Japan).

Table 1
Risk signal for aortic dissection with angiogenesis inhibitors and other drugs
which cause hypertension in FAERS.
Drug AAD without
Drug A /total,n (%)

Angiogenesis inhibitors

2.8. Multiplex assay

Bevacizumab

3048/
12,114,970(0.025)
Lenvatinib
3130/
12,181,388(0.026)
Sorafenib
3119/
12,171,499(0.026)
Sunitinib
3096/
12,155,257(0.025)
Other representative drugs whichcause hypertension
Epinephrine
3138/
12,157,254(0.026)
Norepinephrine
3143/
12,186,139(0.026)
Celecoxib
3126/
12,091,998(0.026)
Cyclosporine
3119/
12,108,911(0.026)
Dexamethasone
3126/
12,004,712(0.026)
Methylpredonisolone
3126/
12,128,410(0.026)
Predonisolone
3059/
12,065,172(0.025)

Serum concentrations of granulocyte colony-stimulating factor (GCSF), CCL11/eotaxin, monocyte chemoattractant protein 1 (MCP-1),
interleukin-6 (IL-6), CXCL10/IP-10 (IP-10), keratinocyte-derived che­mokines (KC), and vascular endothelial growth factor A (VEGF-A) wereanalyzed using the MILLIPLEX® kit (Merck) using Luminex®200TM(Merck) according to the manufacture’s instruction.
2.9. Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchasedfrom Lonza K.K. (Tokyo, Japan). The culture medium was EGM-2 Bullet
Kit (Lonza K.K.) in 4% fetal bovine serum at 37 ◦ C and 5% CO2 until the
passages of 3–8. Cells were stimulated with SNT (0.1, 1, 3, 5, 10 µM) for
24 h when they became confluent.
2.10. Statistical analysis
Data are expressed as the mean ± standard error (S.E.) for eachvalue. Statistical analysis was performed using R version 4.2.1 forWindows. One-way analysis of variance was performed for comparisonsof three or more groups. Tukey’s test was performed as a post hoc
analysis. The Kruskal–Wallis test was also used for ordinal scale data.
Two-tailed p < 0.05 values were considered statistically significant.
3. Results
3.1. Disproportionality analysis using the FAERS database

AD withDrug A/total, n(%)

ROR (95%
CI)

P value*

97/
75,314(0.13)
15/8896(0.17)

5.12(4.19–6.27)< 0.01

6.57(3.96–10.92)< 0.01

26/
18,785(0.14)
49/
35,027(0.14)

5.41(3.67–7.96)< 0.01

5.50(4.15–7.29)< 0.01

7/33,030(0.021)0.82(0.39–1.72)

0.732

2/4145(0.048)1.87(0.47–7.49)

0.29

19/
98,286(0.019)26/
81,373(0.032)
19/
185,572(0.010)19/
61,874(0.031)
86/
125,112(0.069)0.75(0.48–1.17)

0.231

1.24
(0.84–1.83)

0.272

0.39
(0.25–0.62)< 0.011.19(0.76–1.87)

0.449

2.71
(2.19–3.36)< 0.01

Footnotes: AD; aortic dissection, ROR; reporting odds ratio, CI; confidential
intervals,*
Fisher’s exact test

Of the 12,190,284 adverse event cases, 3145 cases of aortic dissec­
tion were reported (Fig. 1). The reported aortic dissection rates weresignificantly higher in the patients receiving angiogenesis inhibitors,
including bevacizumab (ROR, 5.12; 95% CI, 4.19–6.27), SNT (ROR,
5.50; 95% CI, 4.15–7.29), sorafenib (ROR, 5.41; 95% CI 3.67–7.96), andlenvatinib (ROR, 6.57 95% CI 3.96–10.92) compared to the patients notreceiving these drugs (Table 1). All cases were statistically significant;therefore, angiogenesis inhibitors were strongly suggested to be asso­
ciated with aortic dissection onset.

using angiogenesis inhibitors tended to be men over 60 years of age(Table 2, Supplementary Fig. 1 A). Of these, 258 (1.24%) developed anaortic aneurysm or dissection within 1 year of the first angiogenesis
inhibitor administration (Supplementary Fig. 1B). Higher percentages ofthe patients were 60 years or older, male, and had a history of hyper­
tension, atherosclerosis, or dyslipidemia in the group of the patientswith the aortic aneurysm or dissection compared to the group without
these aortic diseases (Table 3). Only atherosclerosis and dyslipidemia

3.2. Drugs that are risk factors for drug-induced hypertension do notnecessarily cause aortic dissection
Among the representative drugs which may induce hypertension as
an adverse drug event, significant ROR signals for aortic dissection wereobserved in prednisolone (ROR, 2.71; 95% CI, 2.19–3.36), but not in
epinephrine (ROR, 0.82; 95% CI, 0.39–1.72), norepinephrine (ROR,
1.87; 95% CI, 0.47–7.49), cyclosporine (ROR, 1.24; 95% CI, 0.84–1.83),
methylprednisolone (ROR, 1.19; 95% CI, 0.76–1.87), and celecoxib(ROR, 0.75; 95% CI, 0.48–1.17). In the case of dexamethasone, signifi­
cant protective signals were observed (ROR, 0.39; 95% CI, 0.25–0.62)(Table 1). Therefore, blood pressure elevation alone cannot cause drugassociated aortic dissection.

Table 2
Number of patients using each angiogenesis inhibitor by JMDC.

3.3. Retrospective cohort analysis using the JMDC receipt database
Of the 4,105,013 cases accumulated from April 2014 to November
2020, 20,828 cases with angiogenesis inhibitors were subjected toanalysis (Fig. 1). Bevacizumab users were the most (37.6%). Patients

Drug

Number of patients (%)

Bevacizumab
Aflibercept
Ramucirumab
Sorafenib
Regorafenib
Lenvatinib
Sunitinib
Pazopanib
Axitinib
Cabozantinib
Vandetanib

10,702 (51.4)
4325 (20.8)
3437 (16.5)
1174 (5.6)
1160 (5.6)
1086 (5.2)
537 (2.6)495 (2.4)510 (2.4)
17 (0.1)
0 (0.0)Footnotes: Total number of patients using angiogenesis inhibitors
were 20,828. Some patients received more than one angiogenesis
inhibitor.
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Biomedicine & Pharmacotherapy 167 (2023) 115504

Table 3
Patient background of patients using angiogenesis inhibitors with different onset of aortic dissection in JMDC.
VariableAge (min,max)Over 60 years old
Male
Smoking
History of Anthracycline use
Hypertension
Atherosclerosis
Dyslipidemia
Connective tissue disorders

Angiogenesis inhibitors user
non-AD/AA (n = 20,570)
70 (19,90)
16,870 (82%)
12,224 (59%)
11,439 (56%)
959 (4.7%)
9464 (46%)
673 (3.3%)4603 (22%)
265 (1.3%)

AD/AA (n = 258)
73 (32,90)
223 (86%)
174 (67%)154 (60%)
15 (5.8%)
138 (54%)
17 (6.6%)
77 (30%)2 (0.78%)

Univariate analysis
P value< 0.001a
0.072b
0.009b0.207b
0.371b
0.017b0.007b
0.005b0.777bMultivariate analysis
OR (95%CI)

P value

1.39 (0.92–2.1)
1.31 (0.94–1.82)
1.13 (0.84–1.54)
1.62 (0.90–2.93)
1.19 (0.89–1.58)
1.81 (1.04–3.14)
1.46 (1.07–1.99)
0.31 (0.0044–2.25)

0.12
0.11
0.42
0.11
0.24
0.036
0.017
0.25

Footnotes: AD; aortic dissection, AA; aortic aneurysm, OR; odds ratio, CI; confidential intervals,aMann–Whitney U test,b
Fisher’s exact test

were significantly associated with the onset of aortic aneurysm or
dissection in patients prescribed angiogenesis inhibitors. In contrast, ahistory of hypertension was not significantly associated with aortic
aneurysm onset or dissection. These findings suggest that hypertension
before angiogenesis inhibitor administration may not influence aortic
aneurysm or dissection development (Table 3).

of SNT on endothelial cells. The gene expressions for ICAM-1, VCAM-1,
ET-1, and VEGFR2 significantly increased concentration-dependently.
Moreover, eNOS expression was decreased by SNT stimulation in aconcentration-dependent manner (Fig. 3C). Therefore, ICAM-1, VCAM1, and eNOS expressions indicated that SNT induced endothelial
damage.
In summary, SNT increases the likelihood of developing aortic
dissection via ET-1-related hypertension and endothelial dysfunction,
but does not exacerbate systemic inflammation.

3.4. SNT alone does not cause vascular injury in mice
To confirm the relationship between angiogenesis inhibitor andaortic dissection, in vivo studies were performed using sunitinib (SNT).
First, the single SNT administration was examined in mice. Systolicblood pressure (SBP), body, heart, and kidney weights were unchanged
after SNT administration (4, 40, and 100 mg/kg/day) after 4 weeks(Supplementary Figure 2 A, Supplementary Table 2). No elastin degra­
dation was observed at any dose following EVG staining of aortic sec­
tions (Supplementary Figure 2B). Thus, SNT alone, up to 100 mg/kg/
day, did not worsen the general condition, increase SBP, or form
vascular injuries, including aortic dissection in mice.

4. Discussion
We elucidated the background factors and mechanisms underlying
aortic dissection development induced by angiogenesis inhibitors using
a novel approach that combines real-world databases, in vivo, and invitro studies. The pathogenesis mechanism may involve impaired
vasoconstrictive capacity resulting from elevated ET-1, decreased eNOS,
and vascular endothelial cell damage with increased ICAM-1 and VCAM1 expressions. Elevated blood pressure after angiogenesis inhibitor use,
but no history of hypertension, before treatment initiation had a greater
impact on the risk of developing aortic dissection. Furthermore, drugs
that increase blood pressure do not necessarily increase the risk ofdeveloping aortic dissection or aneurysms, which are adverse events
specific to angiogenesis inhibitors.
In our study, SNT administration aggravated vascular injury in LAB
mice. The expressions of inflammatory markers were elevated in pa­
tients who developed aortic dissection [20]. Similar to what is observedin human patients, an inflammatory response may be involved in theaortic dissection development in the LAB model [5,18]. Therefore, wefirst hypothesized that enhancing inflammatory responses might be oneof the mechanisms of SNT-induced vascular injury. However, G-CSF,
IL-6, KC, and eotaxin serum concentrations were unchanged, and IP-10
and MCP-1 were somewhat decreased by SNT administration. The effectof SNT on these inflammation-related molecules is controversial, with
reports of exacerbation[21] and suppression [22]. The effect of SNT may
depend on the base inflammation level, but in our LAB model, SNT
showed a suppressive trend. SNT-associated aortic dissection was notdependent on inflammatory response in mice.
Aortic dissection is known to be associated with endothelial
dysfunction [6,7], and the LAB model is unique because it allows for ahigh rate of aortic dissection based on endothelial damage caused by
L-NAME [5]. Although the effects of SNT on the expressions of endo­
thelial cell-specific genes were not apparent in mice aorta (Fig. 3), sig­
nificant increases in ICAM-1 and VCAM-1, and a decrease in eNOS by
SNT, were observed in cultured HUVECs, consistent with previous re­ports [23,24]. These results indicate that SNT-induced vascular endo­
thelial damage in the aorta may be involved in developing aortic
dissection. Moreover, ET-1 expression increased in the aorta of
SNT-treated LAB mice and significantly increased in HUVECs (Fig. 3B
and C). Although systemic ET-1 elevation does not correlate with blood

3.5. Sunitinib aggravates vascular injury in LAB mice
As SNT had no toxicity on control mice up to 100 mg/kg/day, theeffects of SNT on the aortic dissection onset were examined at 100 mg/
kg/day. Consequently, neither LAB nor SNT affected body and kidneyweight (Fig. 2A and B). SNT did not aggravate cardiac hypertrophy, as
observed in LAB mice (Fig. 2C). However, SBP showed a significant
increase in the SNT group compared to in the LAB group at week 4(p < 0.01), 1 week after adding Ang II (Fig. 2D). The incidence of aortic
dissection increased in the SNT-treated mice (59%) compared to the LAB
group (40%). In the SNT group, 16% of mice died from aortic rupture,
but no mice died in the LAB group (Table 4, Fig. 2E). Although aortic
diameter enlargement was not observed in the LAB group, the diameter
was significantly increased in the SNT+LAB group compared to in thecontrol group (Fig. 2F). The elastin degradation score evaluated using
EVG staining was also significantly higher in the SNT+LAB group thanin the LAB group (Fig. 2G).
In LAB mice, the serum concentrations of inflammatory cytokines, GCSF, IL-6, KC, eotaxin, and IP-10 were significantly increased (Fig. 3A).
These increases in serum concentration were significantly decreased (IP10 and MCP-1) or unchanged (G-CSF, IL-6, KC, and eotaxin) after SNT
administration (Fig. 3A). Thus, systemic inflammation was suppressedby SNT administration in LAB mice.
The gene expressions for endothelial cell specific molecules, such as
ICAM-1, VCAM-1, ET-1, eNOS, and VEGFR2, in the aortae were exam­ined. No statistically significant changes were observed in all parame­ters, but SNT increased these endothelial cell-derived gene expressions
compared to those in the LAB group (Fig. 3B).
Cultured HUVECs were stimulated with 0, 0.1, 1, 3, 5, and 10 µM
SNT for 24 h, and gene expressions were examined to clarify the impact
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Biomedicine & Pharmacotherapy 167 (2023) 115504

Fig. 2. Effects of oral administration of sunitinib on the aortic dissection mouse model (LAB). (A) Body weight was monitored weekly until 4 weeks after starting
sunitinib administration. (open circles: control; double circles: LAB group; black squares: LAB + SNT group). (B) Relative kidney weight is expressed as a ratio of
kidney weight to 100 g body weight. (C) Relative heart weight was expressed as a ratio of heart weight to 100 g body weight. (D) Systolic blood pressure (SBP) wasmonitored from week 0 which indicates starting time of sunitinib (SNT) administration until week 4. (open circles: control group; double circle: LAB group; blacksquare: LAB+SNT group). (E) Representative images of Elastica van Gieson’s staining (bar: 10 µm). (F) The enlargement rate of aortic diameter was calculated by theratio of maximum to minimum diameter. (G) Average values of elastin degradation score estimated under EVG staining. Values are expressed as mean ± SEM fromn = 19–20, and statistically analyzed using Tukey’s test (a-d and f) or Kruskal–Wallis test followed by Dunn’s test (g). *p < 0.05, * *p < 0.01 vs control. †p < 0.05,††
p < 0.01 vs LAB. n.s: not significant.

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Biomedicine & Pharmacotherapy 167 (2023) 115504

Figure 2) and aggravated SBP only after Ang II loading in the LAB model.
Therefore, SNT likely increases blood pressure and may worsen vascularlesions if the patients or mice are initially at risk for blood pressure
elevation.
Therefore, we tested the background factors for angiogenesis
inhibitor-associated vascular injury in clinical situations. Higher per­centages of patients with a history of hypertension in the group with theaortic aneurysm or dissection compared to the group without these
aortic diseases were observed. However, no significant differences were
found for hypertension. Therefore, the risk factor for developing arterial
dissection by angiogenesis inhibitors may involve increased bloodpressure after drug administration, regardless of whether the patient had
hypertension before drug use. In contrast, a significant relevance be­
tween the onset of aortic dissection and atherosclerosis or dyslipidemia
was observed. These database analyses and the results of in vivo ex­
periments suggest that worsening hypertension after angiogenesis in­hibitor use plays a vital role in developing aortic dissection.
As shown in Table 1, there was no increased risk from other drugs
that may induce hypertension as a drug adverse event. This result in­
dicates that hypertension was not a necessary and sufficient condition to
induce aortic aneurysms and dissection. Thus, the mechanism of aortic
dissection development caused by drug-induced hypertension may be anangiogenesis inhibitor-specific event.
Animal experiments showed a marked increase in blood pressure

Table 4
Incidence of aortic dissection and death from aortic rupture.
AD
RuptureControl, n (%)

LAB, n (%)

SNT+LAB, n (%)

P value*

0/20 (0)0/20 (0)8/20 (40)0/20 (0)11/19 (59)3/19 (16)

0.34
0.11

Footnotes: Values are shown as n (%). Data are statistically analyzed by chisquared test. AD, aortic dissection; LAB, L-NAME+AngII+BAPN treated group;
SNT+LAB, SNT 100 mg/kg per day+L-NAME+Ang II+BAPN treated group.
*
LAB vs SNT+LAB by Fisher’s test.

pressure elevation under normal conditions [25], ET-1 elevation isthought to be essential in angiogenesis inhibitor-induced hypertension[26]. Therefore, SNT-exacerbated hypertension in LAB mice (Fig. 2D)
was determined to be due to increased ET-1 expressions. A decrease innitric oxide production capacity and an increase in ET-1 may be
involved in the SNT-induced increase in blood pressure [27]. In the LAB
model, SNT significantly aggravated SBP at the 4-week point, which waselevated after Ang II loading, consistent with the ET-1 increase. Thismodel required a rapid change in blood pressure after Ang II loading to
induce aortic dissection development. The degree of blood pressure
variability exacerbation was the most crucial factor for the increased
incidence of aortic dissection with SNT. SNT administration alone did
not affect SBP in mice up to 100 mg/kg/day for 4 weeks (Supplementary

Fig. 3. The effects of sunitinib on the inflammatory response and endothelial cell damage in LAB mice or in cultured human umbilical vein endothelial cells(HUVECs). (A) Serum concentrations of G-CSF, IL-6, KC, EOTAXIN, IP-10, and MCP-1 in LAB mice with our without sunitinib (SNT) were analyzed using the
LUMINEX assay. (B) Gene expressions for ICAM-1, VCAM-1, ET-1, eNOS, and VEGFR2 in aortas from LAB mice with or without SNT were determined using real-timereverse transcription-polymerase chain reaction. (C) Gene expressions for VCAM-1, ICAM-1, ET-1, eNOS, and VEGFR2 in HUVECs stimulated by SNT at indicatedconcentrations for 24 hr. Data represent the mean ± SEM from n = 9–10 (for A and B), or 4 (for C). Values are statistically analyzed by Tukey’s test. *p < 0.05,* *p < 0.01 vs control group, †p < 0.05, ††p < 0.01 vs LAB.
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Biomedicine & Pharmacotherapy 167 (2023) 115504

induced by angiogenesis inhibitors when loaded with Ang II and BAPN.
In addition, database analysis revealed that a history of atherosclerosisor dyslipidemia was highly associated with the development of aortic
dissection, independent of a history of hypertension. Therefore, vasculardamage from such diseases may be the underlying pathology that ex­
acerbates the increase in blood pressure induced by angiogenesis in­hibitors. In the present study, we demonstrated the importance ofangiogenesis inhibitor specifically induced hypertension in the onset of
aortic dissection. However, our study also has several limitations. The
database analysis of aneurysms and dissections included only the aorta
but not peripheral areteries such as carotid, cerebral, and coronary ar­teries. In addition, ascending and descending dissections have not been
distinguished neither in silico or in vivo studies. With regard to in vivo
study, the direct association between VEGF inhibition and aortic
dissection were not elucidated. Therefore, further basic and clinical in­vestigations are desired.

Yuki Izawa-Ishizawa reports financial support was provided by Taiho
Pharmaceutical, Inc. Keisuke Ishizawa reports financial support wasprovided by Japan Society for the Promotion of Science. Kaito Tsujinaka
reports financial support was provided by Japan Society for the Pro­
motion of Science. Yuki Izawa-Ishizawa reports financial support wasprovided by Japan Society for the Promotion of Science.
Acknowledgments
We acknowledge the expert assistance by the Support Center for
Advanced Medical Sciences, Tokushima University Graduate School of
Biomedical Sciences in technical support for basic experiments. English
writing support was provided by Editage of Cactus Communications K.K.
(Tokyo, Japan).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2023.115504.

5. Conclusion
Aortic dissection that develops after administering angiogenesis in­hibitors might be related to elevated blood pressure associated with ET-1
and vascular endothelial damage, indicating that this phenomenon
might be specific to angiogenesis inhibitors. This concept was proven byboth a real-world database and basic pharmacological research withcertainty. It is important to keep in mind that regardless of the bloodpressure prior to initiation of administration, patients are at risk ofdeveloping aortic dissection if their blood pressure increases after
initiation of angiogenesis inhibitor administration. Since it has beenshown that endothelin-1-induced blood pressure elevation may play animportant role in the development of dissection, it is expected thatstrategies to prevent adverse events targeting endothelin-1 will beestablished in the future. Thus, our findings are quite meaningful toestablish safer anticancer therapies using angiogenesis inhibitors andstrategies to prevent the development of vascular toxicity in high-riskpatients with atherosclerosis and dyslipidemia, but not hypertension.

References[1] P.S. Mody, Y. Wang, A. Geirsson, N. Kim, M.M. Desai, A. Gupta, J.A. Dodson, H.
M. Krumholz, Trends in aortic dissection hospitalizations, interventions, andoutcomes among medicare beneficiaries in the United States, 2000-2011, Circ.
Cardiovasc Qual. Outcomes 7 (6) (2014) 920–928.
[2] B.O. Aicher, J. Zhang, S.C. Muratoglu, R. Galisteo, A.L. Arai, V.L. Gray, B.K. Lal, D.
K. Strickland, A.A. Ucuzian, Moderate aerobic exercise prevents matrix
degradation and death in a mouse model of aortic dissection and aneurysm, Am. J.
Physiol. Heart Circ. Physiol. 320 (5) (2021) H1786–H1801.
[3] V. Marque, P. Kieffer, B. Gayraud, I. Lartaud-Idjouadiene, F. Ramirez, J. Atkinson,
Aortic wall mechanics and composition in a transgenic mouse model of Marfan
syndrome, Arterioscler. Thromb. Vasc. Biol. 21 (7) (2001) 1184–1189.
[4] E. Bossone, K.A. Eagle, Epidemiology and management of aortic disease: aortic
aneurysms and acute aortic syndromes, Nat. Rev. Cardiol. 18 (5) (2021) 331–348.
[5] Y. Izawa-Ishizawa, M. Imanishi, Y. Zamami, H. Toya, T. Nagao, M. Morishita,
K. Tsuneyama, Y. Horinouchi, Y. Kihira, K. Takechi, Y. Ikeda, K. Tsuchiya,
M. Yoshizumi, T. Tamaki, K. Ishizawa, Development of a novel aortic dissection
mouse model and evaluation of drug efficacy using in-vivo assays and database
analyses, J. Hypertens. 37 (1) (2019) 73–83.
[6] L. Pan, Z. Lin, X. Tang, J. Tian, Q. Zheng, J. Jing, L. Xie, H. Chen, Q. Lu, H. Wang,
Q. Li, Y. Han, Y. Ji, S-nitrosylation of plastin-3 exacerbates thoracic aortic
dissection formation via endothelial barrier dysfunction, Arterioscler. Thromb.
Vasc. Biol. 40 (1) (2020) 175–188.
[7] M. Toral, A. de la Fuente-Alonso, M.R. Campanero, J.M. Redondo, The NO
signalling pathway in aortic aneurysm and dissection, Br. J. Pharm. 179 (7) (2022)
1287–1303.
[8] M.M. An, Z. Zou, H. Shen, P. Liu, M.L. Chen, Y.B. Cao, Y.Y. Jiang, Incidence andrisk of significantly raised blood pressure in cancer patients treated with
bevacizumab: an updated meta-analysis, Eur. J. Clin. Pharm. 66 (8) (2010)
813–821.
[9] J.M. Duran, C.A. Makarewich, D. Trappanese, P. Gross, S. Husain, J. Dunn, H. Lal,
T.E. Sharp, T. Starosta, R.J. Vagnozzi, R.M. Berretta, M. Barbe, D. Yu, E. Gao,
H. Kubo, T. Force, S.R. Houser, Sorafenib cardiotoxicity increases mortality after
myocardial infarction, Circ. Res 114 (11) (2014) 1700–1712.
[10] S. Lee, T.T. Chen, C.L. Barber, M.C. Jordan, J. Murdock, S. Desai, N. Ferrara,
A. Nagy, K.P. Roos, M.L. Iruela-Arispe, Autocrine VEGF signaling is required for
vascular homeostasis, Cell 130 (4) (2007) 691–703.
[11] J. Herrmann, E.H. Yang, C.A. Iliescu, M. Cilingiroglu, K. Charitakis, A. Hakeem,
K. Toutouzas, M.A. Leesar, C.L. Grines, K. Marmagkiolis, Vascular toxicities ofcancer therapies: the old and the new–an evolving avenue, Circulation 133 (13)(2016) 1272–1289.
[12] R.M. Touyz, S.M.S. Herrmann, J. Herrmann, Vascular toxicities with VEGF
inhibitor therapies-focus on hypertension and arterial thrombotic events, J. Am.
Soc. Hypertens. 12 (6) (2018) 409–425.
[13] E.S. Robinson, E.V. Khankin, S.A. Karumanchi, B.D. Humphreys, Hypertension
induced by vascular endothelial growth factor signaling pathway inhibition:mechanisms and potential use as a biomarker, Semin Nephrol. 30 (6) (2010)
591–601.
[14] K.M. Mirabito Colafella, K.B. Neves, A.C. Montezano, I.M. Garrelds, R. van Veghel,R. de Vries, E. Uijl, H.J. Baelde, A.H. van den Meiracker, R.M. Touyz, A.H.
J. Danser, J. Versmissen, Selective ETA vs. dual ETA/B receptor blockade for theprevention of sunitinib-induced hypertension and albuminuria in WKY rats,
Cardiovasc Res 116 (10) (2020) 1779–1790.
[15] N. Niwa, T. Nishiyama, C. Ozu, Y. Yagi, S. Saito, Acute aortic dissection in a patientwith metastatic renal cell carcinoma treated with axitinib, Acta Oncol. 54 (4)(2015) 561–562.

Sources of Funding
This work was supported by Taiho Pharmaceutical, Inc. as collabo­
rative research, and by grants from Grants-in-Aid for Scientific Researchof Japan Society for the Promotion of Science, KAKENHI, Grant Number,
JP22H04283 (K.T.), JP22K06863 (Y.I.-I.), and JP21H02646 (K.I.).
CRediT authorship contribution statement
Kaito Tsujinaka: Conceptualization, Methodology, Formal analysis,
Investigation, Writing-original draft, Funding acquisition. Yuki IzawaIshizawa: Conceptualization, Methodology, Resources, Data Curation,
Writing-Review and Editing, Visualization, Supervision, Funding
acquisition. Koji Miyata: Investigation, Validation. Toshihiko Yosh­ioka: Investigation. Kohei Omine: Investigation. Honoka Nishi:
Investigation. Masateru Kondo: Investigation. Syuto Itokazu: Inves­tigation. Tatsumi Miyata: Investigation. Takahiro Niimura: Method­
ology, Software, Validation. Maki Sato: Validation. Fuka Aizawa:
Methodology, Visualization. Kenta Yagi: Methodology, Formal anal­
ysis. Masayuki Chuma: Methodology, Formal analysis. Yoshito
Zamami: Software, Resources. Mitsuhiro Goda: Methodology, Re­
sources, Supervision. Keisuke Ishizawa: Resources, Supervision, Proj­
ect administration, Funding acquisition. supervised this project. All
authors critically reviewed and revised the manuscript draft andapproved the final version for submission.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­lationships which may be considered as potential competing interests:
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K. Tsujinaka et al.

Biomedicine & Pharmacotherapy 167 (2023) 115504
regulating inflammation-related factors with suppressing beta-cateinin/c-Myc
pathway and re-balancing Bcl-6 and Caspase-3, Int Immunopharmacol. 90 (2021),
107128.
[23] P. Nowak-Sliwinska, J.R. van Beijnum, C.J. Griffioen, Z.R. Huinen, N.G. Sopesens,
R. Schulz, S.V. Jenkins, R.P.M. Dings, F.H. Groenendijk, E.J.M. Huijbers,
V. Thijssen, E. Jonasch, F.A. Vyth-Dreese, E.S. Jordanova, A. Bex, R. Bernards, T.
D. de Gruijl, A.W. Griffioen, Proinflammatory activity of VEGF-targeted treatment
through reversal of tumor endothelial cell anergy, Angiogenesis 26 (2) (2023)
279–293.
[24] Y. Liu, L.L. Tang, C. Liang, M.M. Wu, Z.R. Zhang, Insulin resistance and pellino-1
mediated decrease in the activities of vasodilator signaling contributes to sunitinibinduced hypertension, Front Pharm. 12 (2021), 617165.
[25] Y.Y. Kisanuki, N. Emoto, T. Ohuchi, B. Widyantoro, K. Yagi, K. Nakayama, R.
M. Kedzierski, R.E. Hammer, H. Yanagisawa, S.C. Williams, J.A. Richardson,
T. Suzuki, M. Yanagisawa, Low blood pressure in endothelial cell-specificendothelin 1 knockout mice, Hypertension 56 (1) (2010) 121–128.
[26] M.H. Kappers, F.M. Smedts, T. Horn, J.H. van Esch, S. Sleijfer, F. Leijten,
S. Wesseling, H. Strevens, A.H. Jan Danser, A.H. van den Meiracker, The vascularendothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like
syndrome with activation of the endothelin system, Hypertension 58 (2) (2011)
295–302.
[27] H.C. Liu, X.T. Zhou, Y.S. Zheng, H. He, X.Q. Liu, PK/PD modeling based on NO-ET
homeostasis for improving management of sunitinib-induced hypertension in rats,
Acta Pharm. Sin. 41 (5) (2020) 719–728.

[16] Y. Zamami, T. Niimura, N. Okada, T. Koyama, K. Fukushima, Y. Izawa-Ishizawa,
K. Ishizawa, Factors associated with immune checkpoint inhibitor-related
myocarditis, JAMA Oncol. 5 (11) (2019) 1635–1637.
[17] Y. Zamami, T. Niimura, T. Koyama, Y. Shigemi, Y. Izawa-Ishizawa, M. Morita,
A. Ohshima, K. Harada, T. Imai, H. Hagiwara, N. Okada, M. Goda, K. Takechi,
M. Chuma, Y. Kondo, K. Tsuchiya, S. Hinotsu, M.R. Kano, K. Ishizawa, Search fortherapeutic agents for cardiac arrest using a drug discovery tool and large-scale
medical information database, Front Pharm. 10 (2019) 1257.
[18] M. Kondo, Y. Izawa-Ishizawa, M. Goda, M. Hosooka, Y. Kagimoto, N. Saito,
R. Matsuoka, Y. Zamami, M. Chuma, K. Yagi, K. Takechi, K. Tsuneyama,
K. Ishizawa, Preventive effects of quercetin against the onset of atherosclerosisrelated acute aortic syndromes in mice, Int J. Mol. Sci. 21 (19) (2020).
[19] M. Imanishi, Y. Izawa-Ishizawa, T. Sakurada, Y. Kohara, Y. Horinouchi, E. Sairyo,
Y. Zamami, K. Takechi, M. Chuma, K. Fukushima, Y. Ikeda, H. Fujino,
M. Yoshizumi, K. Tsuchiya, T. Tamaki, K. Ishizawa, Nitrosonifedipine, aphotodegradation product of nifedipine, suppresses pharmacologically induced
aortic aneurysm formation, Pharmacology 102 (5–6) (2018) 287–299.
[20] H. Liu, Z. Luo, L. Liu, X. Yang, Y. Zhuang, G. Tu, G. Ma, Y. Zhang, J. Zheng, D. Zhu,
C. Wang, Inflammatory biomarkers to predict adverse outcomes in postoperative
patients with acute type A aortic dissection, Scand. Cardiovasc J. 54 (1) (2020)
37–46.
[21] M.E. Asker, A.A. Abdelmeged, M.A. Shaheen, R.G. Eissa, Sunitinib displays
pulmonary fibrosis in experimental rats: Role of IL-17A dependent pathway, Int
Immunopharmacol. 119 (2023), 110173.
[22] L. Chen, P. Xu, Q. Xiao, L. Chen, S. Li, J.M. Jian, Y.B. Zhong, Sunitinib malate
inhibits intestinal tumor development in male Apc(Min/+) mice by down-

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