Cilostazol Promotes the Migration of Brain Microvascular Endothelial Cells
Sae-Won Lee1,2*, Jung Hwa Park1,2 and Hwa Kyoung Shin1,2,3*
1Korean Medical Science Research Center for Healthy-Aging, Pusan National University, Yangsan, Gyeongnam 626-870, Korea 2Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam 626-870, Korea
3Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, Gyeongnam 626-870, Korea
Received September 1, 2016 /Revised November 2, 2016 /Accepted November 3, 2016
Cilostazol is known to be a selective inhibitor of phosphodiesterase III and is generally used to treat stroke. Our previous findings showed that cilostazol enhanced capillary density through angiogenesis after focal cerebral ischemia. Angiogenesis is an important physiological process for promoting re-vascularization to overcome tissue ischemia. It is a multistep process consisting of endothelial cell pro-liferation, migration, and tubular structure formation. Here, we examined the modulatory effect of cil-ostazol at each step of the angiogenic mechanism by using human brain microvascular endothelial cells (HBMECs). We found that cilostazol increased the migration of HBMECs in a dose-dependent manner. However, it did not enhance HBMEC proliferation and capillary-like tube formation. We used a cDNA microarray to analyze the mechanisms of cilostazol in cell migration. We picked five candidate genes that were potentially related to cell migration, and we confirmed the gene expression levels by real-time PCR. The genes phosphoserine aminotransferase 1 (PSAT1) and CCAAT/enhancer binding protein β (C/EBPβ) were up-regulated. The genes tissue factor pathway inhibitor 2 (TFPI2), retinoic acid receptor responder 1 (RARRES1), and RARRES3 were down-regulated. Our observations suggest that cilostazol can promote angiogenesis by promoting endothelial migration. Understanding the cilostazol-modulated regulatory mechanisms in brain endothelial cells may help stimulate blood vessel formation for the treatment of ischemic diseases.
Key words : Brain microvascular endothelial cell, expression microarray, ischemic disease, motility, therapeutic angiogenesis
*Corresponding authors
*Tel : +82-51-510-8476, Fax : +82-51-510-8437
*E-mail : [email protected] (Hwa Kyoung Shin)
*Tel : +82-51-510-8434, Fax : +82-51-510-8437
*E-mail : [email protected] (Sae-Won Lee)
This is an Open-Access article distributed under the terms of
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ISSN (Print) 1225-9918ISSN (Online) 2287-3406
Journal of Life Science 2016 Vol. 26. No. 12. 1367~1375 DOI : http://dx.doi.org/10.5352/JLS.2016.26.12.1367
Introduction
Angiogenesis is the formation of new blood vessels from
pre-existing vessels, and is a multistep process consisting of
endothelial cell proliferation, migration, and tubular struc-
ture formation [1, 4]. When quiescent endothelial cells are
activated by various stimuli, activated endothelial cells de-
grade extracellular matrix, proliferate, and migrate to the site
of stimuli. Angiogenesis is completed by the formation and
organization of a new lumen, namely, in a tube structure.
Angiogenesis is involved in embryonic vasculature for-
mation and is a central process in cancer progression, which
means that tumor growth can be effectively inhibited by an-
ti-angiogenic therapy [1, 5]. Conversely, angiogenesis is also
an important physiological process for promoting revascula-
rization to overcome tissue ischemia. Vascular endothelial
growth factor (VEGF) restores ischemic renal injury through
angiogenesis [20]. Pioglitazone promotes blood flow recov-
ery and capillary density through VEGF upregulation [2].
Administration of plant extract in cases of ischemic stroke
reduced infarct damage and enhanced angiogenesis via the
angiogenic factors VEGF and angiopoietin-1 and 2 [6]. In
addition, the transplantation of bone marrow mononuclear
cells into ischemic myocardium augmented collateral vessel
formation and improved functional outcome [11]. Implanta-
tion of peripheral mononuclear cells into ischemic hindlimb
prevented limb amputation by inducing neovascularization
[35]. Therefore, therapeutic angiogenesis holds promise for
treatment of ischemic diseases.
Cilostazol, a selective type III phosphodiesterase inhibitor,
is known to be an anti-platelet agent with vasodilatory activ-
ity, and to increase intracellular concentrations of cyclic ad-
enosine monophosphate (cAMP) [11]. In addition to these
vasodilator and anti-platelet effects, cilostazol also possesses
anti-inflammatory and antioxidant properties [15, 26]. Cilo-
1368 생명과학회지 2016, Vol. 26. No. 12
stazol showed protective effects against cerebral ischemic in-
jury via inhibition of apoptotic and oxidative cell death [15].
Interestingly, cilostazol possesses neovascularization effects.
Cilostazol increased the production of hepatocyte growth
factor in vascular smooth muscle cells, which enhanced an-
giogenesis in hind limb ischemia [29]. In addition, increased
vasculature post-cilostazol administration was observed in
mdx dystrophic skeletal muscle [10]. Cilostazol promoted
endothelial nitric oxide production in human aortic endothe-
lial cells, as well as endothelial tube formation [9]. Cilostazol
reduced infarct volume and preserved motor and cognitive
function in a rat stroke model. These effects were mediated
by promoting angiogenesis through pericyte proliferation
[24]. Cilostazol has been reported to promote neovasculari-
zation and the expression of angiogenic factors in the hippo-
campus in a mouse forebrain ischemia model [30]. Cilostazol
also enhanced integrin-dependent homing of endothelial
progenitor cells to sites of ischemia in a forebrain ischemia
model [14]. Herein, we investigated which angiogenic mech-
anisms are regulated by cilostazol by using human brain mi-
crovascular endothelial cells.
Materials and Methods
Cell culture and drugs
Human brain microvascular endothelial cells (HBMECs;
passages 5-7, ACBRI) were cultured in complete medium
[M199 medium containing 20% fetal bovine serum (FBS;
Gibco), basic fibroblast growth factor (bFGF; 3 ng/ml;
Millipore), heparin (10 U/ml) and 1% penicillin/streptomy-
cin (both from Gibco) [17]]. Cilostazol (donation from
Otsuka Pharmaceutical Co. Ltd) was dissolved in dimethyl
sulfoxide (10 mM stock solution).
In vitro tube formation assay
Formation of capillary-like tubular structure was analyzed
by tube formation assay. Briefly, growth factor-reduced
Matrigel (200 μl, BD Biosciences) was placed into 24-well
culture plates and polymerized for 30 min at 37°C. HBMECs
(1×104 cells/well) were seeded onto Matrigel-polymer and
incubated in M199 containing 1% FBS with or without cil-
ostazol (30 μM). Every hour up to 26 hr, the tubular structures
were photographed with an Olympus TH4-200 microscope.
BrdU incorporation assay
Cell proliferation was measured with a 5-bromo-2'-deoxy-
uridine (BrdU) incorporation assay. HBMECs (3,000 cells/
well) were seeded in 96-well culture plates and cultured in
a complete medium for 24 hr. The cells were then treated
with cilostazol in basal medium (M199 medium containing
1% FBS) for 72 hr. Cells were labeled with BrdU and in-
cubated with anti-BrdU antibody conjugated with perox-
idase according to the instructions for the Cell Proliferation
ELISA, BrdU (calorimetric) (Roche). After removing anti-
body conjugate and washing, the substrate solution was
added for 20 min and the reaction was stopped by adding
a stop solution. The absorbance at 450 nm was measured
(SpectraMax 190 microplate reader, Molecular device).
Cell migration assay
HBMECs were seeded on 60-mm culture dishes and cul-
tured up to 95% confluence. After scratching with a razor
blade 5-mm in width, the cultures were washed with se-
rum-free medium twice and further incubated in M199 me-
dia containing 1% FBS and 1 mM thymidine. Appropriate
concentrations of cilostazol were added to HBMECs, and mi-
gration was allowed for 16 hr. Cells were rinsed with se-
rum-free medium, fixed with absolute methanol for 2 min,
and stained by Giemsa’s staining solution (Kanto). Cells
were photographed with an Olympus TH4-200 microscope.
Migration activity was quantitated by counting the number
of cells that moved beyond the reference line (the injury line
by the razor blade).
RNA preparation, labeling, and microarray analysis
Total RNA from HBMECs was extracted using Trizol
(Invitrogen Life Technologies, Carlsbad, USA) according to
the manufacturers’ protocol. For quality control, RNA purity
and integrity were evaluated using denaturing gel electro-
phoresis, as well as analysis of the OD 260/280 ratio and
using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Palo Alto, USA). Total RNA was amplified and purified us-
ing the Ambion Illumina RNA amplification kit (Ambion,
Austin, USA) to yield biotinylated cRNA according to the
manufacturer’s instructions. Labeled cRNA samples (750 ng)
were hybridized to each Illumina Human HT-12 expression
(V4) bead array for 16-18 hr at 58°C according to the manu-
facturer's instructions (Illumina, Inc., San Diego, USA).
Detection of array signal was carried out using Amersham
fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences,
Little Chalfont, UK) following the bead array manual.
Arrays were scanned with an Illumina Bead Array Reader
Journal of Life Science 2016, Vol. 26. No. 12 1369
Table 1. Sequences of primers for real-time PCR
Gene name Primer sequence Size (bp) Gene Bank ID
PSAT1(Forward) 5’-TGCCCAGAAGAATGTTGGCT-3’
(Reverse) 5’-TCCAGAACCAAGCCCATGAC-3’177 NM_058179.3
C/EBPβ(Forward) 5’-CGACGAGTACAAGATCCGGC-3’
(Reverse) 5’-TGCTTGAACAAGTTCCGCAG-3’186 NM_005194.3
TFPI2(Forward) 5’-GCCAACAGGAAATAACGCGG-3’
(Reverse) 5’-AGAAATTGTTGGCGTTGCCC-3’149 NM_006528.3
RARRES1(Forward) 5’-GCGCTACAACCCAGAGTCTT-3’
(Reverse) 5’-TCGATGAGCCGTGTACAAGTT-3’126 NM_206963.1
RARRES3(Forward) 5’-TCTGGCTCCTCCAAGTGAGT-3’
(Reverse) 5’-CCAACCATCTCCTTCGCAGA-3’198 NM_004585.2
confocal scanner according to the manufacturer's instructions.
Array data export, processing, and analysis were performed
using Illumina BeadStudio v3.1.3 (Gene Expression Module
v3.3.8). All data analysis and visualization of differentially
expressed genes was conducted using GeneSpring 7.3 soft-
ware (Agilent Technologies., Inc.).
Verification of gene expression with real-time PCR
The Moloney Murine Leukemia Virus-reverse tran-
scriptase (Promega, Madison, WI, USA) was used to produce
cDNA from 2 μg of total RNA according to the manu-
facturer’s recommendations. Real-time PCR was performed
using a Rotor-Gene Q real-time PCR system (Qiagen,
Düsseldorf, Hilden, Germany) with SYBR Green PCR Master
Mix (Qiagen, Düsseldorf, Hilden, Germany) using the pri-
mers listed in Table 1. The results were normalized to 18S
rRNA gene expression. All experiments were performed in
triplicate and repeated at least three times, and threshold
cycles (Ct) were used to quantify the mRNA expression of
the target genes.
Western blot analysis
Total protein from HBMECs was isolated as standard
techniques, separated by 12% sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE), and transferred
onto a nitrocellulose membrane (Amersham Biosciences,
Piscataway, NJ). Primary antibody against PSAT1 (Thermo
Fisher scientific Inc., Waltham, USA) was applied to nitro-
cellulose membrane, followed by secondary antibody con-
jugated with horseradish peroxidase. Anti-α-tubulin anti-
body (Sigma, St. Louis, USA) was used as an internal control.
The intensity of chemiluminescence was measured using an
ImageQuant LAS 4000 apparatus (GE Healthcare Life
Sciences, Uppsala, Sweden). Band intensity was quantified
using ImageJ (NIH).
Statics
All data are expressed as the mean ± the standard devia-
tions (SD). The statistical differences between the groups
were compared using the unpaired t-test or the one-way
analysis of variance (ANOVA). P-values≤0.05 were considered
to indicate statistically significant results.
Results
Cilostazol increased brain endothelial migration
Cilostazol has been known to enhance neovascularization
after focal ischemia [30]. We observed the effect of cilostazol
at each step of the angiogenic mechanism using human brain
microvascular endothelial cells (HBMECs). We first con-
ducted a wound migration assay to analyze endothelial mo-
tility in response to cilostazol (Fig. 1). Treatment with cil-
ostazol significantly increased HBMEC migration compared
to the control basal medium group in a dose-dependent
manner (Fig. 1B).
Endothelial proliferation and capillary-like tube
formation was not affected by cilostazol
We next analyzed the effect of cilostazol on HBMEC pro-
liferation (Fig. 2A). In contrast to the migration assay, cil-
ostazol did not enhance HBMEC proliferation compared to
basal medium alone. HBMECs cultured in complete medium
were used as a positive control, and they showed enhanced
proliferation (Fig. 2A). We then tested if cilostazol could
stimulate vessel-like tube formation in HBMECs (Fig. 2B).
Tube formation capacity was not different between cil-
ostazol-treated and basal medium-treated cells.
1370 생명과학회지 2016, Vol. 26. No. 12
A
B
Fig. 1. Cilostazol promoted HBMEC migration. (A) A HBMEC migration assay was performed 16 hr after cilostazol treatment.
After Giemsa’s staining, cells were photographed (Magnification, x400). (B) A quantitative graph of three independent experi-
ments with duplicates is shown. **p<0.01 and ***p<0.001 compared to HBMECs cultured in control basal medium.
A
B
Fig. 2. Cilostazol did not affect the proliferation
and tube formation of brain endothelial
cells. (A) HBMEC proliferation was as-
sessed by a BrdU incorporation assay at
72 hr after cilostazol treatment (n=3,
**p<0.01). Complete medium: M199 me-
dium containing 20% FBS and growth
factors; basal medium: M199 medium
containing 1% FBS. (B) Cilostazol (30 μm)
did not affect the formation of capil-
lary-like tube networks. Magnification:
×400.
Journal of Life Science 2016, Vol. 26. No. 12 1371
Table 2. Selected genes by us involving known migration-related genes
Gene name Description Synonyms Differential expressiona
PSAT1Homo sapiens phosphoserine
aminotransferase 1 (PSAT1)MGC1460; PSA; EPIP; PSAT Up-regulated
C/EBPβHomo sapiens CCAAT/enhancer binding
proteinβ (C/EBPβ)
CRP2; TCF5; LAP; IL6DBP;
C/EBP-beta; NF-IL6; MGC32080Up-regulated
TFPI2Homo sapiens tissue factor pathway
inhibitor 2 (TFPI2)TFPI-2; PP5; FLJ21164 Down-regulated
RARRES1Homo sapiens retinoic acid receptor
responder (tazarotene induced) 1 (RARRES1)TIG1 Down-regulated
RARRES3Homo sapiens retinoic acid receptor
responder (tazarotene induced) 3 (RARRES3)MGC8906; HRASLS4; TIG3; RIG1 Down-regulated
aBetween cilostazol (30 μM)-treated HBMECs and control HBMECs as described.
Gene regulation by cilostazol in brain endothelial
cells
A microarray analysis was performed for control HBMECs
and cilostazol-treated HBMECs to investigate the genes in-
fluenced by cilostazol. Compared with control HBMEC cells,
there were 107 genes in the expression profile changed
(>1.5-fold DEGs) following cilostazol treatment, with 15
genes up-regulated and 92 genes down-regulated. We
searched the roles of these genes in previous reports and
selected 5 candidate genes that were known to be possibly
involved in cell migration (Table 2). We confirmed the candi-
date gene expression of 2 up-regulated genes (PSAT1, C/
EBPβ) and 3 down-regulated genes (TFPI2, RARRES1 and
RARRES3) by real-time PCR (Fig. 3 and Fig. 4). In addition,
PSAT1 expression was confirmed by western blot analysis,
which was upregulated by cilostazol treatment (Fig. 3C).
Discussion
Endothelial migration is one of the most intensively stud-
ied processes in angiogenesis. The migration process is regu-
lated by multiple signaling pathways that can act in a differ-
ent manner on different blood vessels, and this signaling
selectivity is thought to contribute to the modulation of the
simultaneous formation of separate blood vessels [31, 33].
We found that cilostazol treatment stimulated brain endo-
thelial motility (Fig. 1), and several genes known to be asso-
ciated with cell migration were modulated by cilostazol:
PSAT1 and C/EBPβ were up-regulated and TFPI2, RARRES1
and RARRES3 were down-regulated (Table 2, Fig. 3, Fig. 4).
Phosphoserine aminotransferase 1 (PSAT1) is an enzyme
involved in serine biosynthesis. PSAT1 has some important
metabolic functions such as PSAT1 deficiency results in in-
tractable seizures and acquired microcephaly [8], and re-
duced expression in diabetic mice [36]. However, there was
no previous information regarding PSAT1 expression and
function in endothelial migration. Increased expression of
PSAT1 has been reported to be associated with cancer meta-
stasis [19, 27, 34]. PSAT1 overexpression promoted esoph-
ageal squamous cell carcinoma metastasis, and PSAT1 stim-
ulates the expression of snail, which has been reported as
a key regulator of cell migration and epithelial-mesenchymal
transition [13, 19]. In addition, PSAT1 inhibition reduced the
invasion and metastasis of cancer cells [34]. From these pre-
vious reports, we assumed that cilostazol-mediated PSAT1
up-regulation may have a role in endothelial migration.
Expression of CCAAT/enhancer binding protein β (C/
EBPβ) increases in cilostazol-treated HBMECs (Fig. 3). C/
EBPβ is a member of a transcription factor family that takes
part in the cell migration process [3, 12, 18]. Induction of
C/EBPβ in muscle stem cells enhanced their migration and
contributed to muscle repair in dystrophic muscle [12].
C/EBPβ is an important mediator in endothelial migration
during pathological angiogenesis [18]. In addition, hep-
atocyte growth factor-mediated motility upregulation of hu-
man melanocyte depended on C/EBPβ [3]. Therefore, the
upregulation of C/EBPb by cilostazol in HBMECs may play
an important role in endothelial migration.
Among the down-regulated genes by cilostazol (Fig. 4),
tissue factor pathway inhibitor 2 (TFPI2) has been well char-
acterized to be involved in tumor angiogenesis and pro-
gression. Reduced TFPI2 expression correlated with angio-
genesis in pancreatic carcinomas [37], TFPI2 knock-down
promoted the migration of glioma cells [7], and TFPI2 over-
expression inhibited the migration of trophoblast cells [38].
In addition, the overexpression of TFPI2 decreased matrix
1372 생명과학회지 2016, Vol. 26. No. 12
A
B
C
Fig. 3. Quantitative real-time PCR confirmation of the up-regulated candidate genes by cilostazol treatment in HBMECs. Real-time
PCR analysis of (A) phosphoserine aminotransferase 1 (PSAT1) mRNA and (B) CCAAT/enhancer binding protein β (C/EBPβ)
mRNA. RNAs from cilostazol (30 μM)-treated HBMECs and control HBMECs were used. Three independent experiments
with triplicates were performed. **p<0.01, ***p<0.001. (C) Cilostazol upregulated the PSAT1 protein level. Total protein (20
μg) was subjected to 12% SDS-PAGE, followed by western blotting using anti-PSAT1. Quantification graphs (n=3, ***p<0.001).
A B C
Fig. 4. Quantitative real-time PCR confirmation of the down-regulated candidate genes by cilostazol treatment in HBMECs. Real-time
PCR analysis of (A) tissue factor pathway inhibitor 2 (TFPI2), (B) retinoic acid receptor responder 1 (RARRES1) and (C)
retinoic acid receptor responder 3 (RARRES3) mRNA. RNAs from cilostazol (30 μM)-treated HBMECs and control HBMECs
were used. Three independent experiments with triplicates were performed. **p<0.01, ***p<0.001.
metalloproteinases (MMPs), which play critical roles in the
degradation of extracellular matrix, and in cell migration
during metastasis [16, 25]. Another down-regulated gene by
cilostazol were retinoic acid receptor responder 1 (RARRES1)
and RARRES3 (Fig. 4). RARRES1 was initially identified as
a novel retinoic acid receptor regulated gene [22], and has
been reported as a potential tumor suppressor gene down-
regulated in tumors [23, 28]. Inhibition of RARRES1 in-
Journal of Life Science 2016, Vol. 26. No. 12 1373
creased prostate cancer cell invasion [23]. RARRES3 has been
reported to associate with metastasis, suppressing the meta-
stasis of colorectal and breast cancers [21, 32]. Although
there is no published data of RARRES1 and RARRES3 in
endothelial migration, reports about how reduction of
RARRES1 or RARRES3 increased tumor metastasis suggest
that reduction of RARRES1 or RARRES3 by cilostazol may
be one of the regulatory mechanisms for HBMEC motility
stimulation by cilostazol.
Taken together, the expression of the PSAT1, C/EBPβ,
TFPI2, RARRES1, and RARRES3 genes modulated by cil-
ostazol treatment in HBMECs may result in increased brain
endothelial cell migration ability, thus promoting the angio-
genesis process. Thus, the clarification of the cilostazol-
modulated molecular mechanisms involving these genes
will be helpful for angiogenesis-based therapeutics for the
treatment of ischemic diseases.
Acknowledgement
This work was supported by a 2-Year Research Grant of
Pusan National University.
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Journal of Life Science 2016, Vol. 26. No. 12 1375
록:Cilostazol에 의한 뇌 내피세포의 세포이동 증진 효과연구
이세원1,2*․박정화1,2․신화경1,2,3*
(1부산대학교 건강노화 한의과학 연구센터, 2부산대학교 한의학전문대학원 한의과학과, 3부산대학교 한의학전문대학원 경락구조의학부)
Cilostazol은 phosphodiesterase III의 선택적 저해제로 알려져 있으며, 뇌졸중 치료에 일반적으로 사용되고 있
다. Cilostazol을 처리한 경우, 국소 뇌허혈이 발생한 후에 혈관신생을 통해서 혈관형성이 향상된다는 것을 본 연
구자들이 발표하였다. 혈관신생은 조직의 허혈상태를 극복하기 위해서 혈관재생을 촉진하는 중요한 과정으로써,
혈관내피세포의 증식, 이동, 모세관구조 형성의 다단계 과정으로 구성되어 있다. 이에 본 연구에서는 인간 뇌혈관
내피세포를 이용하여 cilostazol이 혈관신생의 각 단계들에 어떤 영향을 미치는지 조사하였다. Cilostazol은 농도
의존적으로 뇌혈관내피세포의 이동성을 촉진하였으나, 뇌혈관내피세포의 증식과 모세관구조 형성에는 영향을 미
치지 않았다. Cilostazol이 세포이동을 조절하는 기전을 분석하기 위해서 cDNA microarray를 수행하였고, 세포이
동에 관련성이 있는 5종의 후보 유전자들을 선택하여 real-time PCR을 통해 해당 유전자의 발현을 검증하였다.
Cilostazol에 의해서 발현양이 조절되는 유전자들로써, phosphoserine aminotransferase 1 (PSAT1)와 CCAAT/en-
hancer binding protein β (C/EBP β)은 발현이 증가하였고, tissue factor pathway inhibitor 2 (TFPI2), retinoic
acid receptor responder 1 (RARRES1), RARRES3는 발현이 감소하였다. 이상의 결과를 통해서 cilostazol이 혈관
내피세포의 이동을 촉진하여 혈관신생을 향상시킬 수 있음을 제안할 수 있으며, 뇌혈관내피세포에 대한 cilostazol
의 조절기전에 대해서 더욱 상세히 규명을 한다면 혈관형성을 통하여 허혈성 질환을 치료할 수 있는 유용한 정보
가 될 것으로 기대한다.
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