Endogenous prostaglandin D<sub>2</sub> synthesis inhibits e-selectin generation in human umbilical vein endothelial cells

doi:10.4236/health.2011.35053

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Vol.3, No.5, 304-311 (2011)

doi:10.4236/health.2011.35053

Health

Endogenous prostaglandin D2 synthesis inhibits

e-selectin generation in human umbilical vein

endothelial cells

Hideyuki Negoro1*, Hiroyuki Kobayashi2, Yoshio Uehara3

1Harvard Medical School, Internal Medicine, Boston, USA; *Corresponding Author: [email protected]

2 Department of Hospital Administration, Graduate School of Medicine, Juntendo University, Tokyo, Japan;

3University of Tokyo Internal Medicine, Tokyo, Japan.

Received 14 March 2011; revised 25 April 2011; accepted 29 April 2011.

ABSTRACT

We examined the role of prostaglandin D2 (PGD2)

in the formation of E-selectin following inter-

leukin-1 (IL-1) stimulation in human umbilical

vein endothelial cells (HUVEC) transfected with

lipocaline-type PGD2 synthase (L-PGDS) genes.

HUVEC were isolated from human umbilical

vein and incubated with 20 U/mL IL-1 and vari-

ous concentrations of authentic PGD2. The iso-

lated HUVEC were also transfected with L-PGDS

genes by electroporation. The L-PGDS-trans-

fected HUVEC were used to investigate the role

of endogenou s PGD2 in IL-1-stimulated E-selectin

biosynthesis. We also used an anti-PGD2 anti-

body to examine whether an intracrine mecha-

nism was involved in E-selectin production.

PGD2 and E-selectin levels were determined by

radio-immunoassay and enzyme-immunoassay,

respectively. E-selectin mRNA was assessed by

real-time RT-PCR. IL-1-stimulated E-selectin

production by HUVEC was dose-dependently

inhibited by authentic PGD2 at concentrations

greater than 10−6 mol/L. L-PGDS gene-trans-

fected HUVEC produced more PGD2 than HU-

VEC transfected with the reporter gene alone.

IL-1 induced increases in E-selectin production

in HUVEC transfected with the reporter genes

alone. However, this effect was significantly

attenuated in the case of IL-1 stimulation of

HUVEC transfected with L-PGDS genes, and

accompanied by an apparent suppression of

E-selectin mRNA expression. Neutralization of

extracellular PGD2 by anti-PGD 2- sp ecifi c antibody

influenced neither E-selectin mRNA expression

nor E-selectin biosynthesis. HUVEC transfected

with L-PGDS genes showed increased PGD2

synthesis. This increase was associated with

attenuation of both E-selectin generation and

E-selectin mRNA expression. The results sug-

gest that endogenous PGD2 decreases E-se-

lectin synthesis and E-selectin mRNA expres-

sion, probably through an intracrine mecha-

nism.

Keywords: Prostaglandin; E-Selectin; P GDS;

Endothelial Cell

1. INTRODUCTION

Adhesion molecules play an important role in the de-

velopment and progression of atherosclerosis. The hy-

pothesis proposed by Ross and its modifications have

been generally accepted as a mechanism of atherosclero-

sis where adhesion of circulating monocytes and lym-

phocytes to vascular endothelium presumably initiates a

series of events toward atherosclerosis [1]. Cellular ad-

hesion molecules mediate the adhesion, margination, and

transendothelial migration of circulating mononuclear

cells from the blood stream to the extravascular com-

partment to have an important part in the progression of

atherosclerotic plaque [2]. Recent studies have eluci-

dated further that to anchor leukocytes onto the endothe-

lial cells, the adhesion molecules expressed on the sur-

face of endothelial cells necessitated to bind to their

ligands expressed on leukocytes. Endothelial cells are

stimulated by inflammatory agents to express selectins,

such as endothelial-leukocyte adhesion molecule-1

(E-selectin), which interact with carbohydrate ligands on

leukocytes, and to express immunoglobulin superfamily

proteins, such as vascular cell adhesion molecule-1

(VCAM-1). The adhesion molecules expressed on the

endothelial cells include VCAM-1, intracellular adhe-

sion molecule (ICAM)-1, P-selectin and E-selectin [3].

Selectins, including E-selectin and P-selectin, are in-

H. Negoro et al. / Health 3 (2011) 304-311

305305

volved in the first step of leukocyte adhesion at sites of

inflammation or injury. Selectins are characterized by

rolling and tethering of leukocytes to the endothelial

surface, to platelets or to other leukocytes [4]. In fact,

E-selectin is demonstrated to occur in atherosclerotic

lesions in the coronary artery of humans [5]. Therefore,

E-selectin is believed to be a key factor for the develop-

ment of immune-mediated cardiovascular injury.

Intrestngly, we have recently reported that PGD2 at-

tenuates inducible nitric oxide generation in vascular

smooth muscle cells [6]. Endogenous prostaglandin D2

synthesis reduces plasminogen activator inhibitor-1 gen-

eration following cytokines stimulation in bovine endo-

thelial cells [7]. Especially, PGD2 is synthesized in vas-

cular components of atheromatous lesions including

endothelial cells, macrophages, platelets, and mast cells

[8] and lipocalin-type PGD2 synthase (L-PGDS) is

demonstrated to occur in atheromatous lesions in the

cardiovascular system [9]. These data strongly suggest

that L-PGDS/PGD2 is upregulated in response to im-

mune-related vascular lesions and in turn, the increase of

L-PGDS/PGD2 is exerted to attenuate the progression of

the arterial remodeling.

Taken together, we proposed the hypothesis that PGD2

regulates E-selectin expression in endothelial cells,

thereby contributing to leukocyte adhesion, an integral

component of the development of vascular injury. How-

ever, there had been few data investigating the crosstalk

between endogenous L-PGDS/PGD2 system and the

adhesion molecule expression by cytokines. In the pre-

sent study, in order to test our hypothesis that L-PGDS/

PGD2 protects the vascular wall against immune-related

vascular injury, we examined the relationship between

endogenous PGD2 and E-selectin expression by endo-

thelial cells and attempted to reveal its intracellular

mechanism mediated by PGD2 using L-PGDS gene-

transfected endothelial cells in culture. We also exam-

ined whether the increases in intracellular PGD2 synthesis

influenced E-selectin mRNA expression and E-selectin

biosynthesis observed following interleukin-1b (IL-1)

stimulation.

2. MATERIALS AND METHODS

2.1. Materials

Eicosanoids and related compounds were purchased

from Funakoshi chemicals (Tokyo, Japan). Arachidonic

acid was purchased from Sigma (St. Louis, MO, USA).

Recombinant murine IL-1 was purchased from R&D

Systems (Minneapolis, MN, USA). Radioactively la-

beled materials were purchased from Amersham (Tokyo,

Japan). A 3-kb gene for rat brain PGDS [(5Z, 13E)-

(15S)-9a, 11a -epidoxy-15-hydroxyprosta-5,13-dienoate

D-isomerase, EC 5.3.99.2] was isolated from a rat ge-

nomic DNA library by plaque hybridization with cDNA

for the PGDS enzyme, as described in our previous

studies [10]. A 3-kb BamHI fragment of rat PGDS,

which belongs to the lipocalin family, was inserted into a

pcD2 plasmid containing the SV40 promoter, along with

a polyA signal at the XhoI site (Invitrogen, Carlsbad, CA,

USA) [11]. The b-galactosidase gene with a cytomega-

lovirus (CMV) promoter at an XbaI site was inserted

into the pBluescript 2 KS+ plasmid (Stratagene, La Jolla,

CA, USA).

2.2. Cell Culture

Human umbilical vein endothelial cells (HUVEC)

were harvested enzymatically as described previously

[12]. They were maintained in medium 199 (GIBCO

BRL, Gaithersburg, MD), containing Hepes, heparin

(1%), endothelial cell growth factor (50 mg/ml),

L-glutamin (1%), antibiotics, and 5% fetal bovine serum

(FBS). When the cells reached confluence, they were

replanted onto low pyrogen fibronectin at 20,000 cells/

cm2. HUVEC which were isolated from a confluent

monolayer of polygonal cells. The cells expressed von

Willbrand factor as determined by their content of spe-

cific mRNA and immunoreactive protein. Cellular vi-

ability was assessed by Trypan blue exclusion.

2.3. Effect of Exogenous PGD2 on

E-Selectin Expression in Endothelial

Cells

Cultures of HUVEC were treated with 20 U/ml IL-1,

according to the previous study, in the presence of vari-

ous concentrations of PGD2 to be incubated for 18 h.

Thereafter, the HUVEC were washed three times with

FBS-free Dulbecco’s phosphate-buffered saline (D-PBS;

Gibco) and the cells were re-incubated in 1 ml of fresh

D-PBS for 2 h. Subsequently, the cells and culture su-

pernatants were used in various assays.

2.4. Transfection of L-PGDS Genes into

HUVEC

HUVEC were transfected with L-PGDS genes using

the Shimadzu GTE-10 electroporation device (Gene

Transfer Equipment-10, Shimadzu Co., Ltd., Kyoto,

Japan). This equipment transiently increases the perme-

ability of plasma membranes of HUVEC, thereby facili-

tating translocation of genes into the cytoplasm. Briefly,

the cells were washed three times with FBS-free D-PBS

and 10 mg of pcD2-rat PGDS in 0.5 ml of fresh D-PBS

were added to each well [13]. A transient electrical cur-

rent was applied onto HUVEC growing in the culture

dishes, using a 35-mm round electrode (Model FTC-

H. Negoro et al. / Health 3 (2011) 304-311

306

33D3, Shimadzu Co., Ltd., Kyoto, Japan), after which

the cells were incubated for 30 minutes.

Following transfection, 2 ml of DMEM containing

10% FBS was added to the HUVEC and the dishes were

incubated for an additional 24 hours. Endogenous PGD2

production was stimulated by adding 10–6 mol/l arachi-

donic acid for 24 hours. Fresh medium containing 10–6

mol/l arachidonic acid was then added and E-selectin

mRNA expression and E-selectin expression were

stimulated for 18 hours by the addition of 20 U/ml IL-1.

At the end of this incubation period, the HUVEC were

washed three times with FBS-free D-PBS and incubated

in 1 ml of fresh D-PBS for 2 hours. Finally, the cells and

culture supernatants were collected for analysis.

For comparison, HUVEC were transfected with b-

galactosidase genes (b-gal) by electroporation to deter-

mine the efficacy of gene transfection. Three days after

the transfection, HUVEC were stained with X-gal [14],

and b-galactasidase expression was measured. Transfec-

tion efficacy was estimated as the ratio of the X-gal

stained area to the sectional area of the HUVEC.

2.5. Neutralizing Extrinsic PGD2 Released

from HUVEC

We attempted to neutralize PGD2 using an anti-PGD2-

specific antibody in order to investigate the effects of

PGD2 released from L-PGDS-transfected HUVEC. We

estimated the amount of anti-PGD2 antibody required to

completely neutralize the secreted PGD2 using the

Scatchard analysis. The binding affinity was 0.0051

ml/pg, and the Bmax (maximal binding capacity) was 25

pg/l [15]. Therefore, 1 liter of antibody had the capacity

to bind 25 pg PGD2. Taking into account of these results,

we used 200 ml of the antibody to inhibit the receptor-

mediated actions of PGD2 in HUVEC under our culture

conditions. The anti-PGD2 antibody was raised in our

laboratories using PGD2-conjugated thyroglobulin and

Freund’s complete adjuvant. The antibody cross-reacted

0.003% with thromboxane B2, 0.01% with prostaglandin

E2 (PGE2), 0.009% with prostaglandin F2a (PGF2a),

0.008% with 6-keto-PGF1a and 0.01% with arachidonate

[16]. Antibody activity was confirmed by suppression of

intracellular cyclic AMP (cAMP) following PGD2

stimulation via PGD2 receptor, which acts as a second

messenger for PGD2 signal transduction [17].

2.6. Eicosanoid Radioimmunoassay

Eicosanoids were determined in culture media using

the direct radioimmunoassay method described previ-

ously [16]. Briefly, 0.1 ml of sample, 0.1 ml of [3H] ei-

cosanoid (5000 dpm) and 0.1 ml of the diluted antibody

were mixed and incubated at 4˚C for 24 hours. To sepa-

rate bound from free [3H] eicosanoid, 0.1 ml of dex-

tran-coated charcoal in a 50 mmol/l phosphate buffer at

pH 7.4 containing 0.1% gelatin and 100 mmol/l NaCl

was added to the ice-chilled assay mixture. The mixture

was vortexed and centrifuged at 3000 rpm for 5 min at

4˚C. The supernatant was assayed for [3H] eicosanoids

bound to the antibody. Radioactivity was determined

using an automatic liquid scintillation counter. The

properties of the anti-6keto-PGF1a antibody was de-

scribed previously [15,16]. The cross-reactivity and its

properties of the anti-PGD2 antibody was detailed above.

The low cross-reactivity of each antibody made it feasi-

ble to measure directly the eicosanoid in media.

2.7. E-Secletin and E-Secletin mRNA

Measurements

We measured E-selectin expression by commercially

available cell surface enzyme immunoassay as described

previously (R&D Systems, Inc., USA).

Cultured E-selectin transcripts were detected using the

real-time reverse transcription-polymerase chain reaction

(real-time RT-PCR) method using real-time RT-PCR

machine as described previously [18].

2.8. Statistical Analysis

All values are expressed as the mean ± SE. The dif-

ferences between values were assessed by an one-way

ANOVA and Duncan’s multiple range test using the

STATISTICA program (StatSoft, Tulsa, OK, USA) on a

Gateway G6-400 computer system (Gateway Inc., N

Sioux City, SD, USA) running the Windows 98 operat-

ing system. P values less than 0.05 were considered sta-

tistically significant.

3. RESULTS

3.1. Effect of Exogenous PGD2 on

E-Selectin Expression in Endothelial

Cells

Stimulation of endothelial cells with IL-1 significantly

increased E-selectin expression. The increase was re-

duced in a dose-dependent manner by the addition of

PGD2 at concentrations ranging from 10–7 to 10–4 mol/l

(Figure 1).

3.2. Gene Transfection and Eicosanoid

Generation in HUVEC

We transfected HUVEC with L-PGDS genes in order

to increase endogenous PGD2 formation. PGD2 was as-

sayed using radioimmunoassay. The basal levels of PGD2

in reporter-gene-transfected HUVEC maintained in

H. Negoro et al. / Health 3 (2011) 304-311

307307

Figure 1. Effect of exogenous PGD2 on E-selectin generation in

endothelial cells. Stimulation of endothelial cells with IL-1 sig-

nificantly increased E-selectin generation (left two columns).

The increase was reduced in a dose-dependent manner by the

addition of PGD2 at concentrations ranging from 10–7 to 10–4

mol/l. All experiments were performed three different times with

at least six replicates. Statistical differences were analyzed by

one-way ANOVA and Duncan’s multiple range tests. *P < 0.01

vs the value at 0 mol/L PGD2.

arachidonate-free media were as low as 51.5 ± 2.2

pg/106 cells/2 hours, and the addition of 10–6 mol/l ara-

chidonate had no effect on PGD2 synthesis. In contrast,

L-PGDS gene-transfected HUVEC showed an increase

(178.0%) in PGD2 generation even in arachidonate-free

media, as compared with control HUVEC carrying re-

porter genes alone. Furthermore, 10–6 mol/l arachidonate

markedly stimulated PGD2 biosynthesis by 640.7%, as

compared with control HUVEC carrying vector genes

alone (Figure 2(a)). Thereafter, PGI2 was assayed as

6-keto-PGF1a using radioimmunoassay. Basal levels of

prostacyclin (PGI2), the major eicosanoid synthesized in

HUVEC, were 1488 ± 66 pg/106 cells/2 hours in cells

having reporter genes alone under arachidonate-free

conditions. The PGI2 generation was a markedly in-

creased by 327% when the cells were stimulated with

10–6 mol/l arachidonate (Figure 2(b)). L-PGDS gene

transfection did not influence PGI2 synthesis in HUVEC

maintained under either arachidonate-free or arachido-

nate-stimulated conditions, as compared to HUVEC

transfected with reporter genes. These data clearly sug-

gest that the L-PGDS genes alter the phenotype of HU-

VEC so that the recombinant cells acquire the capacity

to produce PGD2 in response to arachidonate stimula-

tion.

3.3. Effect of L-PGDS Gene Transfection on

E-Selectin Expression in HUVEC

Using the HUVEC having L-PGDS genes, we invest-

(a)

(b)

Figure 2. PGD2 and PGI2 generation in endothelial cells. PGD2

was assayed using radioimmunoassay. 10–6 mol/l arachidonate

did not stimulate PGD2 generation in cells having reporter

genes alone (two columns to the left in Graph a). However,

cells carrying the L-PGDS genes acquired the ability to

produce PGD2 with or without arachidonate stimulation (two

columns to the right in Graph a). (Graph a). PGI2 was assayed

as 6-keto-PGF1a using radioimmunoassay. The addition of 10–6

mol/L arachidonate to the cultures increased PGI2 generation;

however, there were no differences in PGI2 production between

PGDS(–), AA(+) and PGDS(+), AA(+) lines. (Graph b). Statis-

tical differences were assessed by Student’s t-test (n = 6). *P <

0.01. N.S. represents not statistically significant.

tigated the effects of endogenous PGD2 on the expres-

sion of E-selectin with or without IL-1 stimulation. The

E-selectin expression was significantly increased upon

IL-1 stimulation in endothelial cells transfected with

transporter genes. This increase in E-selectin with or

without IL-1 stimulation was significantly blunted in

HUVEC transfected with L-PGDS genes that produced

indeed endogenous PGD2 in response to arachidonate

stimulation (Figure 3).

H. Negoro et al. / Health 3 (2011) 304-311

308

Figure 3. Effect of PGD2 synthase gene transfection on

E-selectin expression in endothelial cells. Under normal (un-

stimulated) conditions, endothelial ells bearing L-PGDS genes

(IL-1(–), PGDS(+)) expressed less E-selectin than those bear-

ing reporter genes alone (IL-1(–), PGDS(–)). E-selectin ex-

pression increases observed following IL-1 stimulation were

significantly attenuated in cells carrying L-PGDS genes in

response to arachidonic acid stimulation (IL-1(+), PGDS(+),

AA(+)), though there was no attenuation of E-selectin expres-

sion in cells carrying L-PGDS genes but not stimulated by

arachidonic acid (IL-1(+), PGDS(+), AA(–)), compared to

endothelial cells having only reporter genes (IL-1(+), PGDS

(–)). The experiment was carried out in the presence of 10–6

mol/l arachidonate. Statistical differences were assessed by

Student’s t-test (n = 6). *P < 0.01.

3.4. Intracellular Effects of PGD2 on

E-Selectin Expression

In order to assess the role of PGD2 receptor-mediated

signal transduction in E-selectin expression, we studied

changes in cAMP, a second messenger of PGD2 signal

transduction in HUVEC transfected with transporter

gene alone. Intracellular cAMP was unaffected by

L-PGDS transfection per se. However, intracellular cAMP

was increased by exogenous PGD2 stimulation [19]. This

response was completely abrogated by addition of

anti-PGD2 antibody to the media, the amount of which

was more than the concentrations sufficient to neutralize

PGD2 in the media (Figure 4(a)). Using such a dose of

anti-PGD2 antibody enough to inhibit the receptor-me-

diated cyclic AMP rising, we investigated contribution

of PGD2 receptor-mediated signal transduction to the

PGD2-mediated E-selectin expression, and determined

E-selectin expression in the supernatants following neu-

tralization with an anti-PGD2-specific antibody. The ad-

dition of anti-PGD2 antibody to the media did not influ-

ence E-selectin expression following IL-1 stimulation in

the endothelial cells transfected with L-PGDS genes

(Figure 4(b)).

(a)

(b)

Figure 4. Effect of endogenous PGD2 on E-selectin expression

in endothelial cells. cAMP levels were measured to assess the

antibody inhibition of PGD2 receptor-mediated signal trans-

duction in the cells with transporter gene alone (Graph a). 10-5

mol/l PGD2 greatly increased cAMP formation in the cells in

the absence of anti-PGD2 antibody (anti-PGD2(–)). This in-

crease was completely abolished to the basal levels by the ad-

dition of anti-PGD2 antibody in the media. Using such a dose

of anti-PGD2 antibody, we examined the effects of neutraliza-

tion of PGD2 in media on E-selectin expression (Graph b).

E-selectin expression following IL-1 stimulation was signify-

cantly reduced in endothelial cells having L-PGDS genes and

without anti-PGD2 antibody (PGDS(+), anti-PGD2(–)), com-

pared to endothelial cells with reporter genes alone (PGDS(–),

anti-PGD2(–)). The reduction in E-selectin expression was

unaffected when PGD2 in the culture was neutralized with an

anti-PGD2-specific antibody (PGDS(+), anti-PGD2(+)). These

studies were carried out in the presence of 10–6 mol/l arachi-

donate. Statistical differences were assessed by Student’s t-test.

*P < 0.01. N.S. represents not statistically significant.

These results clearly indicated that anti-PGD2-specific

antibody inhibited the PGD2 receptor-mediated signal

transduction and that PGD2-mediated reduction in

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309309

E-selectin expression was not due to the PGD2 receptor-

mediated events.

3.5. L-PGDS Genes and Expression of

E-Selectin mRNA

We demonstrated that L-PGDS gene transfection onto

HUVEC brought about increases in PGD2 formation and

decreases in E-selectin expression using real-time

RT-PCR (Figure 5). The expression of E-selectin mRNA

following IL-1 stimulation was significantly less in the

HUVEC carrying L-PGDS genes. Expression was nor-

malized to β-actin. Values are expressed as fold change

compared to untreated controls.

4. DISCUSSION

Both PGD2 and adhesion molecules play a very im-

portant part in the process of atherosclerosis. In the pre-

sent study, we demonstrated that PGD2 regulates E-se-

lectin generation in HUVEC following IL-1stimulation.

More interestingly, we demonstrated that endogenous

PGD2 production in HUVEC transfected with L-PGDS

genes brought about a decrease of E-selectin expression.

This effect was observed even if the PGD2-mediated

cAMP increase was reversed to basal levels using

anti-PGD2-specific antibody. These findings strongly

suggest that PGD2 exerts inhibitory effects on E-selectin

expression via an intracellular mechanism as well as the

well-known receptor-mediated mechanism [20].

In this context, recent studies have demonstrated that

the orphan nuclear receptor of peroxysome proliferator-

activated receptor (PPAR)-g, a member of the nuclear

receptor superfamily of ligand-dependent transcription

factors, binds to PGD2 metabolites and thereby regulates

adipocyte differentiation and glucose homeostasis [21,

22].

PGD2 is converted quickly to PGJ2, delta 12-PGJ2,

and 15-deoxy-delta12,14 PGJ2 in plasma [23]. 15-deoxy-

delta12,14 PGJ2 inhibits inhibitor of kB kinase that

phosphorylates another inhibitor of kB after activation

with cytokines and also affects the DNA-binding do-

mains of nuclear factor-kB (NF-kB) subunits [24]. Be-

cause the genes involved in E-selectin expression in-

clude the NF-kB binding site in its promoter regions [25],

it is presumable that PGD2 and its metabolites inhibit

cytokine induced E-selectin expression, at least in part,

through inhibition of NF-kB translocation.

It is reported that E-selectin mRNA and E-selectin are

much expressed in vascular lesions such as atherosclero-

sis [5]. Different cell lines like macrophages, platelets or

lymphocytes work together to secrete cytokines in re-

sponse to the inflammatory events, and in turn, the se-

creted cytokines stimulate the endothelial cells, thereby

Figure 5. Effect of PGD2 synthase gene transfection on

E-selectin mRNA. Changes in E-selectin mRNA levels were

investigated using real-time RT-PCR. The expression of

E-selectin mRNA following IL-1 stimulation was significantly

less in the HUVEC carrying L-PGDS genes. Expression was

normalized to β-actin. Values are expressed as fold change

compared to untreated controls. Statistical differences were

assessed by Student’s t-test. *P < 0.01.

increasing E-selectin mRNA and E-selectin expression

in the atherosclerotic lesions. In fact, it is well postulated

that L-PGDS is highly expressed in the stenotic lesions

and in the lipid core of advanced atherosclerotic plaques

in patients with stable angina [9]. Moreover, PGD2 re-

duces inducible nitric oxide synthase formation. These

actions of PGD2 and metabolites on vasoactive sub-

stances regulated by cytokines are in favor of vascular

protection against vascular injury [6].

The endothelial cells exhibited a striking increase in

PGI2 generation, but not PGD2, following arachidonate

stimulation, suggesting that these cells have a large ca-

pacity to synthesize PGI2, but not PGD2. In spite of this,

L-PGDS gene transfection greatly enhanced PGD2 syn-

thesis in EC. This was particularly apparent when eico-

sanoid expression was stimulated with its precursor,

arachidonate. The alteration of phenotype of these cells

reduced E-selectin mRNA expression and consequently

E-selectin expression. This genetic procedure would

provide a new strategy against vascular lesions. These

activities have relevance to the in vivo situation, and

remain to be clarified.

In conclusion, the introduction of PGD2 synthase

genes into endothelial cells increased endogenous PGD2

generation. This brought about a reduction in E-selectin

expression and a decrease in E-selectin mRNA expres-

sion following IL-1 stimulation. The inhibitory effects of

PGD2 on E-selectin expression were due to an intracrine

mechanism rather than to any receptor-mediated events.

Since suppression of the E-selectin system is postulated

H. Negoro et al. / Health 3 (2011) 304-311

310

to be protective, an increase in endogenous PGD2 syn-

thesis might represent a novel strategy to prevent car-

diovascular injury in humans.

5. ACKNOWLEDGEMENTS

The authors acknowledge Yukari Kawabata and Chieko Henmi for

technical assistance.

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