ORIGINAL ARTICLE

The suppressive effect of sphingosine 1-phosphate onmonocyte-endothelium adhesion may be mediated by therearrangement of the endothelial integrins a5b1 and avb3

S . AOKI ,*� Y . Y ATOMI , * T . SH IMOSAWA ,* H. YAMASHITA ,� J . K I TAYAMA,� N . H . T S U NO , §

K . TAKAHASHI§ and Y . OZAK I�*Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo; �Department of Laboratory

Medicine, University of Yamanashi Faculty of Medicine, Yamanashi; �Department of Surgical Oncology, Graduate School of Medicine, The

University of Tokyo, Tokyo; and §Department of Transfusion Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

To cite this article: Aoki S, Yatomi Y, Shimosawa T, Yamashita H, Kitayama J, Tsuno NH, Takahashi K, Ozaki Y. The suppressive effect of

sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins a5b1 and avb3.

J Thromb Haemost 2007; 5: 1292–1301.

Summary. Background: Sphingosine 1-phosphate (S1P),

known to play important roles in vascular biology, is a bioactive

lysophospholipid mediator that maintains endothelial integrity

via its cell-surface receptors (S1Ps). In this in vitro study, we

aimed to examine the role of S1P in monocyte-endothelium

adhesion,which is an important event in thepathophysiologyof

atherosclerosis. Methods and Results: S1P pretreatment of

human umbilical vein endothelial cells (ECs), but not U937

cells, effectively suppressed U937-EC adhesion independently

from the expression of adhesion molecules, namely ICAM-1,

VCAM-1, and E-selectin. This S1P-induced suppressive effect

was inhibited by the blockage of S1P1 and S1P3 receptors and

the specific inhibitors of Gi protein, Src family proteins,

phosphatidylinositol 3-kinase, and Rac1, indicating involve-

ment of these key downstream pathways. Moreover, the RGD

peptide and antibodies, which neutralize adhesion via a5b1 andavb3, effectively inhibited U937-EC adhesion with a degree

similar to S1P pretreatment. Both an adhesion assay and flow-

cytometric analysis demonstrated that U937 cells adhered

through integrins a5b1 and avb3 expressed on the apical

surface of monolayer ECs, and S1P shifted the localization

of these integrins from the apical surface to the basal surface.

Conclusions:From the present results, we propose that S1Pmay

contribute to the maintenance of vascular integrity and the

regulation of atherogenesis through the rearrangement of

endothelial integrins.

Keywords: integrin, sphingosine 1-phosphate, vascular endo-

thelial cells.

Introduction

As interactions of leukocytes with endothelial cells (ECs) are

physiologically requisite responses to acute and chronic

inflammation, immune surveillance, and the healing process

to vascular injury, unregulated leukocyte–endothelial inter-

actions mediate pathological situations such as inflammatory

tissue injury, thrombosis and other pathologic sequelae [1].

Moreover, ECs rendered dysfunctional by inflammatory and

oxidative mediators recruit circulating monocytes and T-

lymphocytes that accelerate pathological conditions [2]. This

process is an initial and important step in atherogenesis,

which is mediated by the expression of endothelial adhesion

molecules, including E-selectin, intercellular adhesion mole-

cule-1 (ICAM-1), and vascular cell adhesion molecule-1

(VCAM-1) [1,2].

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator,

induces a variety of cellular responses depending on the

expression of the subtype of five G protein-coupled S1P

receptors (S1P1-5), while S1P can also act as an intracellular

messenger in some cases [3–7]. S1P stimulates several functions

such as survival [8,9], migration [10–12], and nitric oxide (NO)

synthesis [9,13] in ECs. It has also been demonstrated that S1P

dynamically regulates the vascular barrier system through

cytoskeletal rearrangement [14–17] and adherens junction

assembly [18,19] in ECs. Moreover, the migration of vascular

smooth muscle cells is markedly inhibited by S1P [12,20,21].

These bodies of evidence suggest the possible anti-atherogenic

properties of S1P.

In spite of the reported anti-atherogenic effects of S1P,

this bioactive lipid has also been shown to induce the

expression of adhesion molecules including E-selectin,

Correspondence: Yutaka Yatomi, Department of Clinical Laboratory

Medicine, Graduate School of Medicine, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

Tel.: + 81 3 5800 8721; fax: + 81 5689 0495; e-mail: yatomiy-

lab@h.u-tokyo.ac.jp

Received 7 May 2006, accepted 26 March 2007

Journal of Thrombosis and Haemostasis, 5: 1292–1301

� 2007 International Society on Thrombosis and Haemostasis

ICAM-1, and VCAM-1 on ECs [22–27], leading to the

enhanced interaction with monocytic cells in vitro [24]. In

contrast to these pharmacological studies, we have observed

that the stimulatory effect of S1P on endothelial adhesion

molecule expression via S1P receptors is much weaker than

that of tumor necrosis factor-a (TNF-a) [25]. Bolick et al.

[28] have also reported that 100 nM S1P did not modify the

expression of the adhesion molecules in ECs and prevented

TNF-a-mediated monocyte-EC adhesion ex vivo and in vitro,

although the precise mechanisms remain unclear. Moreover,

a recent study revealed that S1P can either stimulate or

inhibit the expression of adhesion molecules in ECs and the

adhesion of monocytic cells to ECs; under TNF-a-stimula-

ted conditions S1P can be inhibitory but it acts as a

stimulant under TNF-a-unstimulated conditions [27]. There-

fore, the role of S1P on monocyte-endothelial adhesion, an

initial step for atherogenesis, and whether S1P is anti-

atherogenic or pro-atherogenic are still controversial.

In the present study, we report that S1P-pretreated ECs

suppress monocyte adhesion, which may be as a result of

S1P induction of integrins a5b1 and avb3 redistribution on

monolayer ECs from the luminal (apical) surface to the

matrix-adherent (basal) surface. This integrin shift is a novel

finding of a S1P effect that may solve the seemingly

contradictory findings of S1P on adhesion molecule expres-

sion and adhesion itself.

Materials and methods

Materials

The materials and their suppliers are as follow: S1P, sphing-

osine (Sph), platelet-activating factor (PAF), lysophosphat-

idylcholine (LPC), and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)

pyrazolo[3,4-d]pyrimidine (PP2) (Biomol, Plymouth Meeting,

PA, USA); lysophosphatidic acid (LPA), bovine gelatin (GN),

bovine collagen (COL), fibronectin (FN) from human plasma,

laminin (LN) from human placenta, pertussis toxin (PTX), 2-

(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002;

LY), NG-nitro-L-arginine methyl ester (L-NAME), and NG-

nitro-L-arginine (L-NNA) (Sigma, St. Louis, MO, USA); (R)-

phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-

ethyl] ester (VPC23019; VPC) (Avanti Polar Lipids, Alabaster,

AL, USA); the cell-permeable pyrimidine compound

NSC23766 (NSC) and the MEK inhibitor PD98056 (Calbio-

chem, San Diego, CA, USA); thrombin (Mochida Pharma-

ceutical Co., Tokyo, Japan); recombinant human TNF-a(Genzyme, Cambridge, MA, USA); human interleukin-1b (IL-

1b) (Boehringer Mannheim Biochemica, Mannheim,

Germany); FITC-conjugated mouse anti-human ICAM-1

monoclonal antibody (mAb) (R&D Systems, Minneapolis,

MN, USA); PE-conjugated mouse anti-human ICAM-1 mAb,

FITC-conjugated mouse anti-human E-selectin mAb, and PE-

conjugated mouse anti-integrin a5 or av mAb (Chemicon

international Inc., Temecula, CA, USA); mouse immunoglob-

ulin control unlabeled or labeled with FITC and PE andmouse

anti-human integrin avb3 mAb (BD PharMingen, San Diego,

CA, USA); mouse anti-human integrin a5b1 mAb (DakoCy-

tomation, Carpinteria, CA, USA); Gly-Arg-Gly-Asp-Ser

(GBGDS; Peptide Institute, Osaka, Japan). The pyrazolopy-

ridine derivative JTE-013 [12] was a gift from the Central

Pharmaceutical Research Institute, Japan Tobacco Incorpor-

ation, Osaka, Japan.

Cell culture

Human umbilical vein endothelial cells (HUVECs) and human

peripheral blood CD14+ monocytes were obtained from

Sanko Junyaku (Tokyo, Japan). HUVECs were routinely

plated onto 0.2% gelatin-coated dishes, and cultured in

Dulbecco�s modified Eagle medium (DMEM; Gibco BRL,

Gaithersburg,MD,USA) supplemented with 20% fetal bovine

serum (FBS; ICN Biomedicals, Aurora, OH, USA),

10 ng mL–1 of recombinant basic fibroblast growth factor

(bFGF; PeproTech EC, Ltd, London, UK), penicillin G

(100 U mL–1) (Gibco BRL), and streptomycin sulfate

(100 lg mL–1) (Gibco BRL). HUVECs were serum starved

in DMEM containing 0.1% bovine serum albumin (BSA;

essentially fatty acid-free, Sigma) for at least 1 h before

stimulation. We confirmed that HUVEC reactivity toward

S1P was almost fully recovered under the serum removal

conditions, using intracellular Ca2+ mobilization as an indica-

tor for the S1P-induced response; this bioactive lipid is a potent

intracellular Ca2+ mobilizer. Furthermore, we confirmed that

the concentration-dependent effects of S1P on the HUVEC/

U937 adhesion after 1 h serum starvation were similar whether

HUVECs had been treated with normal serum or charcoal-

stripped serum (data not shown). U937 cells and Jurkat T cells

were obtained from IFO, and cultured in RPMI-1640 (Gibco

BRL) containing 10% FBS, penicillin G (100 U mL–1), and

streptomycin sulfate (100 lg mL–1). All cells were maintained

in a humidified incubator at 37 �C under an atmosphere of 5%

CO2 and 95% air.

Preparation of peripheral blood monocytes

Human venous blood was obtained from healthy adult

volunteers, using 3.8% sodium citrate (9 vol. of blood to 1

vol. of sodium citrate) as the anticoagulant. Peripheral

blood mononuclear cells were isolated by centrifugation on

the LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway),

according to the manufacturer�s instructions, and after being

washed twice with PBS, were suspended in RPMI-1640

medium containing 0.1% BSA. The cell suspension was

seeded on a 10-cm petri dish and cultured at 37 �C for

30 min to allow the cells to adhere. Non-adherent cells were

discarded by gentle washing, the remaining cells were

harvested by scraping in the presence of 0.2% EDTA

(ethylenediaminetetraacetic acid, Sigma) and suspended in

RPMI-1640 containing 0.1% BSA. This study was approved

by the institutional review board and informed consent was

obtained from all participants.

Sphingosine 1-phosphate in endothelial function 1293

� 2007 International Society on Thrombosis and Haemostasis

Adhesion assay

A 24-well plate was coated with 10 lg mL–1 solutions of

each extracellular matrix (ECM), namely GN, COL, LN,

and FN, in PBS for 24 h at 4 �C, followed by incubation

with PBS containing 1% BSA for 1 h to block non-specific

binding. On the other hand, HUVECs were cultured until

subconfluence in gelatin-coated 24-well plates and then

serum starved in DMEM containing 0.1% BSA for 1 h.

U937 cells (1 · 106 cells mL–1) were incubated in RPMI-

1640 medium containing 0.1% BSA and 5 lmol mL–1 of

the fluorescent dye, calcein-AM solution (Dojindo, Kuma-

moto, Japan), for 30 min at 37 �C. Calcein-labeled U937

cells were washed twice with PBS and then resuspended in

DMEM containing 0.1% BSA. After HUVECs and/or

calcein-labeled U937 cells were pretreated with the indicated

concentrations of S1P or other lipids at 37 �C for the

indicated periods, calcein-labeled U937 cells (2.5 · 105) were

seeded onto matrix-coated plates or on HUVECs, and the

plates incubated at 37 �C for 30 min. After gentle washing

to remove non-adherent cells, the adherent cells were lyzed

with 0.1% of Triton X-100 (Sigma) in PBS (500 lL per

well) and the fluorescence measured with a HITACHI

F-4600 spectrophotofluorometer (excitation 485 nm, emis-

sion 535 nm) (Hitachi, Tokyo, Japan). The number of

adherent cells/well was calculated by comparing the amount

of fluorescence to a standard curve of calcein activity/cell

and expressed as the percentage of adherent cells/well.

The effect of S1P on the interaction of ECs with the ECMs

Monolayers of HUVECs were cultured in 96-well plates coated

with GN and FN until subconfluence. The cells were serum

starved inDMEMcontaining 0.1%BSA for 1 h andwere then

pretreated with the indicated concentrations of S1P at 37 �Cfor 4 h. After beingwashed twice with PBS, the PBS containing

0.02% EDTA was added to the HUVECs and incubated for

10 min at 37 �C. After gentle washing to remove the EDTA-

detached cells, the ECM-bound cells were stained with calcein-

AM (5 lmol mL–1) for 30 min. The number of adherent ECs

was determined by measuring the fluorescence intensity at

525 nm in a microscope photometer (Terrascan VP; Miner-

vatech, Tokyo, Japan).

Flow-cytometric analysis of EC adhesion molecules expression

Flow cytometry was used to determine the expression of

adhesion molecules, namely VCAM-1, ICAM-1, and E-

selectin, on HUVECs. HUVECs were resuspended in DMEM

containing 0.1% BSA at 1 · 106 cells mL–1, and incubated at

room temperature (RT) for 15 min with the FITC-labeled

antibody (1:100 dilution) to the respective adhesion molecules.

Samples were washed twice with PBS, fixed with 3% parafor-

maldehyde (PFA) in PBS for 20 min at RT, and subsequently

analyzed with a fluorescence-activated cell sorter (FACS;

Becton Dickinson, San Jose, CA, USA).

Flow-cytometric analysis of the surface localization of

integrins on EC

Subconfluent monolayers of HUVECs cultured in a 6-cm petri

dish were incubated with or without S1P and the indicated

inhibitors in DMEM containing 0.1% BSA for 4 h. The EC

monolayer was subsequently treated with PE-labeled antibod-

ies (1:200 dilution) to integrins for 15 min, which allowed

antibodies to bind to antigens expressed on the apical surface

but not the basal surface of HUVECs. The cells were then

washed twice with PBS, and harvested by treatment with

0.01% trypsin and 0.02% EDTA. Harvested cells were then

washed with PBS, divided into two aliquots, one of which was

treated again with the same antibody (1:200 dilution) for

15 min, which allowed the access of antibodies to all the

antigens on the cell surface, and the other was treated with PE-

labeled control IgG (1:200 dilution) for 15 min. The cells in all

samples were fixed with 3% PFA in PBS for 20 min at RT and

then analyzed immediately with the FACS.

Statistics

Data were expressed as the mean ± SD (n = 3, from separate

experiments) or from a single representative experiment out of

three. When indicated, the statistical significance of differences

was determined using the method described in each figure

legend; P < 0.05 was considered to be significant.

Results

The suppressive effect of S1P on monocyte-EC adhesion

The effect of S1P on the adhesion of themonocyte-like cell line,

U937 cells, to ECs was evaluated. The adhesion rates of U937

cells (2 · 105) were 0.73% ± 0.02%, 1.20% ± 0.17%,

1.03% ± 0.24%, and 8.05% ± 0.73%, respectively for GN,

COL, LN, andFN.GNwas the ECM towhich the adhesion of

U937 cells was the weakest, and it was chosen as the ECM for

the culture of ECs. The adhesion rate of U937 cells to ECs

cultured on GN-coated plates was 7.95% ± 0.74%. Interest-

ingly, S1P concentration-dependently suppressed the adhesion

of U937 cells to ECs by a direct effect on the ECs, but not on

the U937 cells (Fig. 1A). This suppressive effect of S1P on ECs

was not specific for the adhesion of U937 cells, but also for

Jurkat T cells and peripheral blood monocytes (Fig. 1B).

Notably, the S1P-induced attenuation of the adhesion to ECs

was more pronounced with monocytes than the other two cell

types tested. Furthermore, among the bioactive lipids tested,

only S1P suppressed U937 cell-EC adhesion, and it was in

concentration- and time-dependent manners (Fig. 1C,D).

Next, to explore the mechanisms involved in S1P-induced

suppression of U937-EC adhesion, the changes in the expres-

sion levels of endothelial adhesion molecules, namely ICAM-1,

VCAM-1, and E-selectin, were analyzed. The results indicated

that S1P failed to cause down-regulation of the expression

levels of those adhesion molecules (Fig. 1E).

1294 S. Aoki et al

� 2007 International Society on Thrombosis and Haemostasis

The suppression of U937-EC adhesion by S1P in the presence

of inflammatory mediators

Next we examined the effect of S1P on U937 adhesion to ECs

pretreated with inflammatory mediators, namely thrombin,

TNF-a, and IL-1b to induce the expression of adhesion

molecules. The adhesion of U937 cells to stimulated ECs

increased 2.2, 7.4, and 8.3 times, respectively for thrombin,

TNF-a, and IL-1b, compared with that of unstimulated ECs.

Interestingly, S1P also significantly attenuated the adhesion of

U937 cells to these stimulated ECs (Fig. 2A). Furthermore, we

investigated the effect of S1P on the expression of adhesion

molecules induced by TNF-a or IL-1b in ECs. S1P caused a

slight down-regulation of the expression levels of ICAM-1 and

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Fig. 1. Sphingosine 1-phosphate (S1P) suppresses the adhesion of monocytes to endothelial cells (ECs). (A) U937 cells (closed circles), ECs (open

triangles), or both U937 and ECs (closed squares) were treated as described in the Methods section. Treatment of ECs, but not U937 cells, resulted in a

dose-dependent decrease inU937-EC adhesion. (B) The adhesion ofU937 cells (open circles), human peripheral bloodCD14+monocytes (closed squares),

and Jurkat T cells (closed triangles) to S1P-treated ECs. S1P treatment dose-dependent decreased the adhesion of each cell type to ECs. (C and D) The

effect of various lipidmediators onU937-EC adhesionwas examined according to the lipid concentration (C) and the pretreatment time (D). S1P treatment

(open circles) specifically caused a dose- and time-dependent decrease in U937-EC adhesion. On the other hand, lysophosphatidic acid (open triangles),

lysophosphatidylcholine (closed squares), and platelet-activating factor (crosses) dose- and time-dependently increased U937-EC adhesion, while Sph

(closed diamonds) failed to affect it. In (D), all lipids were employed at 1 lM. In (A–D), the data represent the percentage adhesion compared with control

untreated cells. (E) The effect of S1P on the expression of endothelial E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1

was analyzed by flow cytometry. Light gray peaks represent the untreated cells labeled with control IgG, the dark gray peaks and black line peaks are the

untreated cells and S1P-treated cells, respectively, labeled with the antibody to each adhesionmolecule. S1P treatment only slightly increased the expression

of these adhesion molecules on ECs.

Sphingosine 1-phosphate in endothelial function 1295

� 2007 International Society on Thrombosis and Haemostasis

VCAM-1, which were markedly increased by TNF-a and

IL-1b (Fig. 2B).

Effect of various inhibitors on S1P-mediated suppression of

the U937 adhesion to ECs

To investigate the involvement of NO in and the signaling

pathways of the suppressive effect of S1P on the monocyte-

EC adhesion, we tested several inhibitors of NO synthase,

S1P receptors, and intracellular signal molecules known to be

associated with S1P signaling. The NO synthase inhibitors,

for example, L-NAME and L-NNA, weakly but significantly

reversed the suppressive effect of S1P (Fig. 3A). This

indicates that NO, which is an important anti-atherogenic

mediator released from HUVECs challenged with this

bioactive lipid [9,13], is involved in the S1P-induced suppres-

sion of U937-EC adhesion at least partly. On the other hand,

this S1P-induced suppressive effect was inhibited by

VPC23019, an antagonist of S1P1 and S1P3 [29], but not

JTE-013, an antagonist of S1P2 [12] (Fig. 3B). Furthermore,

this suppressive effect of S1P was inhibited by PTX, PP2,

LY294002, and NSC23766 [30], the specific inhibitors of Gi

protein, Src family proteins, phosphatidylinositol 3-kinase

(PI3-K), and Rac1, respectively, but not PD98056 and

Y27632, the specific inhibitors of ERK1/2 and Rho-associ-

ated protein kinase (ROCK), respectively (Fig. 3B).

S1P-induced a suppressive effect on U937 cell adhesion to

TNF-a- (see Fig. 7A) and IL-1b-treated ECs were also

inhibited by the above inhibitors (data not shown), except for

PD98056 and Y27632.

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ICAM-1 VCAM-1

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Fig. 2. Sphingosine 1-phosphate (S1P) suppresses U937-EC adhesion independently of the expression of E-selectin and intercellular adhesion molecule-1

(ICAM-1). (A) The effect of S1P on the adhesion of U937 cells to cytokine-stimulated endothelial cells (ECs). ECs were treated with S1P at the indicated

concentrations without (closed circles) or with the inflammatory cytokines, namely tumor necrosis factor-a (TNF-a) at 10 U mL–1 (open squares),

interleukin-1b (IL-1b) at 10 U mL–1 (closed diamonds), or thrombin at 1 U mL–1 (open triangles), for 4 h. U937-EC adhesion was dose-dependently

suppressed by S1P, independent of each cytokine. (B) Flow-cytometric analysis of the effect of S1P on the expression of endothelial E-selectin (a and b),

ICAM-1 (c and d), and vascular cell adhesionmolecule-1 (VCAM-1) (e and f) in the presence of TNF-a (a, c, and e) or IL-1b (b, d, and f). Light gray peaks

represent cytokine- and S1P-untreated cells, dark gray peaks are cytokine-treated, S1P-untreated cells, and the black line peaks, the cytokine- and

S1P-treated cells. S1P treatment slightly down-regulated the expression of ICAM-1 and VCAM-1, but not E-selectin on cytokine-treated ECs.

1296 S. Aoki et al

� 2007 International Society on Thrombosis and Haemostasis

Inhibition of U937-EC adhesion by S1P may be dependent on

the regulation of integrins a5b1 and avb3

GRGDS is known to inhibit protein-integrin binding by

interacting with the RGD motif, a common motif recognized

by some integrins including a5b1 and avb3 [31]. GRGDS

successfully inhibited U937-EC adhesion, and the GRGDS-

inhibitedU937-ECadhesionwasnotadditionally suppressedby

S1P (Fig. 4A). Similar results were obtained with antibodies

against integrins a5b1 and avb3, which also inhibited the bindingof the respective integrins to the targetmolecules (Fig. 4B).That

is, each of these antibodies or both combined caused partial

inhibition of U937-EC adhesion, and the inhibition rate was

consistent with that caused by S1P (Fig. 4B).

Next, the changes in the expression levels or the localization

of integrins a5b1 and avb3 on the ECmembrane were evaluated

by flow cytometry. S1P did not affect the total expression level

of integrins a5b1 and avb3 on the cell surface (data not shown).

Then, the effect of S1P on the distribution of integrin a5 on the

EC membrane was evaluated. To detect the apical cell surface

integrin a5, ECs as a monolayer, for example, adherent to the

ECMwere treated with the PE-labeled antibody to integrin a5,and analyzed by flow cytometry after harvesting by trypsini-

zation. To detect total (apical plus basal) integrin a5, ECs werestained as a cell suspension, and analyzed similarly. Interest-

ingly, S1P treatment caused a dose-dependent decrease in the

expression of integrin a5 on the apical cell surface of ECs, but

not in that of total one (Fig. 5A,B). It is suggestive of a

redistribution of the integrin a5 from the apical to the basal

surface of monolayer ECs although the mechanism by which

S1P induces this redistribution remains to be solved. The

suppression of integrin a5 expression by S1P could be

completely reversed by LY294002 and NSC23766. The similar

effect of S1P on the integrin av was observed (data not shown).

Furthermore, S1P-mediated integrin redistribution was

confirmed by examining the matrix-attachment strength of

monolayer ECs using EDTA to inhibit the endothelial integrin

function. Although only a few ECs remained attached to the

ECM-coated plate in the presence of EDTA, S1P treatment

dose-dependently increased the number of ECs remaining

attached (Fig. 6), which was inhibited by VPC23019,

LY294002, and NSC23766 (Fig. 6).

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Fig. 3. The reversibility of sphingosine 1-phosphate (S1P)-induced decrease of U937-EC adhesion by inhibitors of nitric oxide (NO) synthase and the

intracellular signaling pathway of S1P. (A) Human umbilical vein endothelial cells were pretreated with the NO synthase inhibitors L-NAME (2 mM) and

L-NNA (0.1 mM) for 4 h, and then S1P (1 lM) inhibition of U937-EC adhesion were assessed. (B) The effects of a variety of inhibitors of the S1P signaling

pathway, namely VPC23019 (VPC) (10 lM, 30 min), JTE-013 (10 lM, 10 min), PTX (500 ng mL–1, 12 h), PD98056 (PD) (10 lM, 30 min), PP2 (5 lM,1 h), Y27632 (20 lM, 30 min), LY294002 (LY) (10 lM, 30 min), and NSC (10 lM, 2 h), on the decreased U937-EC adhesion induced by S1P (1 lM) wereevaluated. VPC and PP2 significantly but not completely reversed the effect of S1P, and PTX, LY, andNSC almost completely reversed the suppression of

U937-EC adhesion caused by S1P. P < 0.05 and ##P < 0.01, compared with non-treated control; *P < 0.05 and **P < 0.01, compared with S1P-

treated control, by two-way ANOVA.

Sphingosine 1-phosphate in endothelial function 1297

� 2007 International Society on Thrombosis and Haemostasis

These findings were also confirmed using inflammatory

cytokine-stimulated ECs. The suppressive effect of S1P on the

adhesion of U937 cells to TNF-a-stimulated ECs could be

almost completely reversed by LY294002 and NSC23766

(Fig. 7A). Similarly, although S1P treatment did not affect the

total surface expression of integrin a5 on TNF-a-stimulated

ECs (data not shown), its expression level on the apical

surface, for example, the non-adhered one, was significantly

decreased (Fig. 7B). Similar results were obtained for integrin

av (data not shown).

Discussion

The vascular endothelium plays important roles in keeping the

integrity of the vascular system, and dysfunction of the EC is

related to vascular diseases such as arteriosclerosis and

thrombosis. S1P, which is a component found in blood

plasma, has been shown to serve several vascular functions

such as endothelial barrier enhancement, anti-apoptosis, and

NO synthesis as an anti-atherogenic factor for ECs, whereas

some reports show that S1P synergistically stimulates the

expression of thrombin-induced tissue factor (TF), the initiator

of the blood coagulation cascade, in ECs [32]. Thus, S1P seems

to exert both physiologic and pathophysiologic effects on

vascular ECs.

In the present study, we found that S1P (with its concen-

trations below 1 lM) is able to suppress the adhesion of U937

cells to ECs in a concentration-dependentmanner, and it is by a

direct effect on ECs but not on U937 cells (Fig. 1A). It is

known that monocytes and T-lymphocytes interact with

vascular ECs and transmigrate to the intimal layer as the

initial step in the atherogenic process. Interestingly, S1P was

also effective in suppressing the adhesion of peripheral blood

monocytes and Jurkat T cells, but the monocyte-EC adhesion

was the one most prominently suppressed by S1P, compared

with U937 and Jurkat T cells (Fig. 1B). Furthermore, the other

bioactive lipids existing in the blood plasma were also tested,

but only S1P was able to suppress the U937-EC adhesion

concentration and time dependently (Fig. 1C,D). From these

results, we propose that the suppressive effect of S1P on

monocyte-EC adhesion may be one of the protective functions

of S1P against atherosclerosis in vascular ECs. On the other

hand, high concentrations of S1P have been shown to up-

regulate the expression of the endothelial adhesion molecules

[22–24,26,27]. However, in the present study, the expression

levels of adhesion molecules, namely E-selectin, ICAM-1, and

VCAM-1, were only merely modified even by S1P at 1 lM(Fig. 1E), which is compatible with previous reports [25,28].

Our results indicate the possible novel mechanism of mono-

cyte-EC adhesion and its regulation by S1P independent of

endothelial adhesion molecules.

S1P suppressed the adhesion of U937 cells to ECs stimulated

with inflammatory mediators, such as thrombin, TNF-a, andIL-1b, to induce the expression of adhesion molecules (Fig. 2).

This finding is consistent with two recent reports [27,28]. It has

been revealed that S1P-induced NO reduced the TNF-a-stimulated expression of adhesion molecules in ECs [27]. In the

present study, S1P only slightly attenuated the TNF-a- and IL-

1b-induced expression of ICAM-1 and VCAM-1, and the

extent of the effect was small compared with the suppressive

effect of S1P on U937-EC adhesion. We hypothesized that

there are twomechanisms in the S1P-induced suppressive effect

on the U937-EC adhesion; one is the effect of NO induced by

S1P, and the other is a novel mechanism independent of

endothelial adhesion molecules, as we demonstrated here.

Subsequently, we investigated the EC signaling pathway

involved in the S1P-induced suppression of monocyte-EC

adhesion independent of the endothelial adhesion molecules.

This suppressive effect of S1P was reversed by the inhibitor

against S1P1 and S1P3. In fact, ECs reportedly express S1P1and S1P3 (the former being more abundant), but not S1P2 [18].

120

140

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60

40

20

0

80

100

60

40

20

00 10 100 1000

Sph-1-P (nM)

Sph-1-P

U93

7-E

C a

dhes

ion

(% o

f con

trol

)

U93

7-E

C a

dhes

ion

(% o

f con

trol

)

–

Control Control lgG Anti-α5β1 Anti-α5β1Anti-αvβ3+Anti-αvβ3

– – – –+ + + + +

A

B

Fig. 4. The roles of integrins a5b1 and avb3 on the U937-EC adhesion and

their suppression by sphingosine 1-phosphate (S1P). (A) Endothelial cells

(ECs) were pre-incubated with indicated concentrations of S1P (4 h) and

with (open triangles) or without (closed circles) the GRGDS peptide

(1 mM, 10 min), and then allowed to adhere to U937 cells. S1P dose-

dependently inhibited the adhesion of U937 cells to control ECs without

GRGDS, but GRGDS itself caused a significant inhibition of the U937-

EC adhesion; S1P treatment did not cause further inhibition. (B) U937

cells and ECs were pretreated for 30 min with control IgG (1:50 dilution)

to block the antibody interaction with the Fc receptors, and then ECs were

incubated with the specific antibodies against integrins a5b1 or avb3 (1:50).When treated with anti-a5b1, the control adhesion (without S1P) was

greatly inhibited. Accordingly, the suppressive effect of S1P was less

obvious. On the other hand, the effect of anti-avb3 was marginal. When

both antibodies were simultaneously added, the control adhesion was

further inhibited; the suppressive effect of S1P was not observed.

**P < 0.01, compared with the value for control IgG by two-way ANOVA.

1298 S. Aoki et al

� 2007 International Society on Thrombosis and Haemostasis

Moreover, the S1P effect was blocked by the inhibitors of Gi

protein, PI3-K, Rac1, and Src family protein but not by

ERK1/2 and ROCK inhibitors. Several reports have indicated

that the PI3-K/Rac1 pathway via Gi-coupled S1P1 and S1P3ligation in ECs is important in enhancing the endothelial

barrier function by adherens junction assembly [18] and

cytoskeletal rearrangement [14,17]. The PI3-K/Rac1 pathway

via Gi-coupled S1P1 is also involved in NO synthase activation

and in fact, the suppressive effect of S1P on the monocyte-EC

adhesion was reversed by the NO synthase inhibitors, but the

effect was only partial (Fig. 3A). Furthermore, it has also been

reported that the Rac and Src activation by S1P mediates the

formation of the actin cortical ring and the peripheral

redistribution of the focal adhesion (FA) complex [15,16].

These data lead us to hypothesize that S1P suppresses

monocyte-EC adhesion by the remodeling and peripheral

translocation of the actin and FA complex in ECs. Among the

FA complex, we focused on integrins a5b1 and avb3, whichwork as the FN and vitronectin receptors, respectively. The

expression levels of these integrins on ECs were not affected by

S1P treatment (data not shown). When EC monolayers were

pretreated with GRGDS or blocking antibodies to these

integrins, U937-EC adhesion was inhibited to a similar degree

as the S1P treatment, and no additive effect of S1P treatment

was observed (Fig. 4). These results suggest that integrins a5b1and avb3 expressed on ECs may be involved in the interaction

with U937 cells and in the suppressive effect of S1P on U937-

EC adhesion, although the possibility cannot be ruled out that

the integrin blockage exerts a strong effect that no other agent

can further cause an additional effect. Furthermore, we showed

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Exp

ress

ion

of in

tegr

in α

5

MF

I (%

of c

ontr

ol)

Fluorescence intensity Fluorescence intensity Fluorescence intensity

Fluorescence intensityFluorescence intensity

a) Control

d) 1000 nM Sph-1-P + LY

020

4060

8010

0

020

4060

8010

0

020

4060

8010

0

020

4060

8010

0

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104100

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100

80

60

40

20

0

101 102 103 104

200

4060

8010

0

e) 1000 nM Sph-1-P + NSC

c) 1000 nM Sph-1-Pb) 100 nM Sph-1-P

0 100 1000 1000 1000Sph-1-P (nM)

Inhibitors ––– LY NSC

####

****

B

A

Fig. 5. Sphingosine 1-phosphate (S1P) causes the redistribution of integrin a5 on the cell membrane of endothelial cells (ECs). The effect of S1P on the

surface distribution of integrin a5 on ECs was indirectly determined using flow-cytometry. (A) Representative flow-cytometric graphs of ECs pre-incubated

in the absence (a) or presence of S1P (b–e) with LY (10 lM) (d) or NSC (10 lM) (e). PE-labeled specific antibodies against integrin a5 and PE-labeled

control mAb of unrelated specificity were used. The light gray peak (a only) represents the negative control, and dark gray peaks represent the integrin a5expression on the apical surface of ECs. The dotted lines (b–e) represent the total integrin a5 expression on the endothelial surface of S1P-untreated ECs,

and the solid lines, on S1P-treated ECs. The bold lines represent the integrin a5 expression on the apical surface of S1P-treated ECs. (B) The mean

fluorescence intensity of the integrin a5 expression on the apical surface of ECs was determined and expressed as the mean ± SD of the percentage of the

control. #P < 0.01, compared with non-treated control; **P < 0.01, compared with S1P-treated control, by one-way ANOVA.

Sphingosine 1-phosphate in endothelial function 1299

� 2007 International Society on Thrombosis and Haemostasis

that S1P shifted integrins a5 and av, which are expressed on the

apical surface, to the basal surface of ECs through the PI3-K/

Rac1 pathway. As a consequence, S1P-treated monolayer ECs

could strengthen their attachment to ECMs (Fig. 6). S1P-

induced redistribution of endothelial integrins via the PI3-K/

Rac1 pathway was also observed in monolayer ECs stimulated

by inflammatory mediators (Fig. 7). We propose that S1P

induces dynamic rearrangement of integrins and thus mediates

the suppression of U937-EC adhesion, which also plays an

important role, at least partly, in vascular barrier functions. On

the other hand, we failed to observe the redistribution of

adhesion molecules in monolayer ECs both in the absence and

presence of the inflammatory mediators, namely TNF-a and

IL-1b.The physiological roles of S1P on the adhesion molecule

expression and subsequently monocyte adhesion to the vascu-

lar endothelium remain controversial. Several reports have

shown that high concentrations of S1P (1–20 lM) strongly

induce the expression of adhesion molecules on ECs

[23,24,26,27], resulting in monocyte interactions; the concen-

trations of S1P in human blood plasma and serum are thought

to be approximately 300 nM and 1 lM, respectively [33–35]. Onthe other hand, our present results indicated that S1P, with its

concentrations below 1 lM, suppressed the adhesion of mono-

cytes and T-lymphocytes to the endothelium dependently on

the rearrangement of endothelial integrins but not adhesion

molecules. Therefore, we suggest that S1P naturally prevents

this initial event of atherosclerosis, at least partly by the

rearrangement of integrins a5b1 and avb3.In sum, we clarified the physiological effect of S1P. S1P

suppresses monocyte-EC adhesion by the rearrangement of

endothelial integrins a5b1 and avb3. We showed that S1P

mediated redistribution of integrins from the apical to the basal

surface of monolayer ECs probably by the remodeling and

cortical assembly of actins and FA complexes through the PI3-

K/Rac1 and Src-dependent pathways. However, the proteins

counterparts on monocytes, which interact with endothelial

integrins, remain to be characterized, as does the intimate

association between the redistribution of integrins, and the

rearrangementofactinsandFAcomplexes remain tobeproved.

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific

Research from the Ministry of Education, Culture, Sports,

Science and Technology, Japan. The authors would also like to

thank the Central Pharmaceutical Research Institute, Japan

Tobacco Incorporation for donating JTE-013.

Bou

nd c

ells

(% o

f con

trol

)

80

60

40

20

00 10 100 1000 1000 1000 1000Sph-1-P (nM)

Inhibitors VPC LY–––– NSC

####

**

**

**

Fig. 6. Effect of sphingosine 1-phosphate (S1P) on the interaction of

endothelial cells (ECs) with the extracellular matrix (ECMs). ECM

(GN + FN)-adherent EC monolayers, untreated or treated with S1P, in

the presence or absence of the indicated inhibitors, were treated with

ethylenediaminetetraacetic acid and, after removal of the non-adherent

cells, the percentage of adherent cells was determined. Compared with the

negative control (black bar), consisting of S1P-untreated ECs in the ab-

sence of inhibitors, the adhesion of S1P-treated ECs (white bars) was

stronger, in a dose-dependent manner. The effect of S1P on EC–ECM

interaction was inhibited by VPC (10 lM), LY (10 lM), andNSC (10 lM).#P < 0.01, compared with non-treated control; **P < 0.01, compared

with S1P-treated control, by one-way ANOVA.

140

120

100

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7-E

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dhes

ion

(% o

f con

trol

)M

FI (

% o

f con

trol

)E

xpre

ssio

n of

inte

grin

α5

80

60

40

20

0Sph-1-P

Inhibitors

– + + + +

–

+ + +––

–– –

LY

TNF-α

TNF-α

NSC

LY NSC

– – –

–

–

Sph-1-PInhibitors

140

120

100

80

60

40

20

0

#

**

##

**

A

B

Fig. 7. Sphingosine 1-phosphate (S1P) suppresses tumor necrosis factor-a(TNF-a)-induced U937-EC adhesion through the induction of the redis-

tribution of integrin. (A) S1P (1 lM) also inhibited the adhesion of U937

cells to 10 U mL–1 TNF-a-treated endothelial cells (ECs), and this inhibi-

tory effect could be reversed bybothLY (10 lM) andNSC(10 lM). (B)Theexpression of integrin a5 on the apical surface of TNF-a-treated ECs was

also significantly reduced by S1P, and the effect could be almost completely

reversed by LY (10 lM) and NSC (10 lM). P < 0.05 and ## P < 0.01,

compared with TNF-a-treated control; *P < 0.05, compared with TNF-

a + S1P, by two-way ANOVA (A) and one-way ANOVA (B).

1300 S. Aoki et al

� 2007 International Society on Thrombosis and Haemostasis

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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� 2007 International Society on Thrombosis and Haemostasis