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 : julie@pusan.ac.kr (Hwa Kyoung Shin)

*Tel : +82-51-510-8434, Fax : +82-51-510-8437

*E-mail : brainsw@gmail.com (Sae-Won Lee)

This is an Open-Access article distributed under the terms of

the Creative Commons Attribution Non-Commercial License

(http://creativecommons.org/licenses/by-nc/3.0) which permits

unrestricted non-commercial use, distribution, and reproduction

in any medium, provided the original work is properly cited.

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.