J. Biomedical Science and Engineering, 2010, 3, 550-555
doi:10.4236/jbise.2010.36077 Published Online June 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online June 2010 in SciRes. http://www.scirp.org/journal/jbise
Gene silencing of E-selectin block recruitment of endothelial
progenitor cell to vascular endothelium under flow
Sunil Sharma, Masayuki Yoshida
Life Science and Bioethics Research Center, Tokyo Medical and Dental University, Tokyo, Japan.
Received 16 February 2010; revised 20 March 2010; accepted 25 March 2010.
ABSTRACT
Short interfering RNA (siRNA) is a powerful tech-
nique that can suppress gene expression in a variety
of cells including mammalian cells. Endothelial pro-
genitor cells (EPCs) are bone marrow—derived
haematopoietic progenitor cells that have been im-
plicated in vasculogenesis. We demonstrated for the
first time that gene silencing of endothelial E-selectin
using siRNA transfection in human umbilical vein
endothelial cells (HUVECs) causes inhibition of EPC
adhesion under flow conditions. Fluorescence im-
munobinding assay analysis showed that significant
reduction of E-selectin surface expression in HU-
VECs (activated with IL-1β (10 U/mL) for 4 h)
transfected with siRNA against E-selectin, but not in
HUVECs transfected with LacZ siRNA (control). An
EPC adhesion assay under flow conditions (shear
stress = 1.0 dyne/cm2) then demonstrated that HU-
VECs transfected with E-selectin siRNA supported
significantly less adhesion of EPCs than those HU-
VECs treated with control siRNA and no siRNA after
activation by IL-1β (p < 0.05). Our experiments have
shown the importance of E-selectin in EPC adhesion
to HUVECs and the potential utility of gene silencing
of E-selectin in EPC recruitment.
Keywords: Endothelial Progenitor Cell; E-Selectin; Rnai
1. INTRODUCTION
The vascular endothelium is a vital border zone between
the circulatory system and the tissues it supplies. It is a
monolayer of endothelial cells (EC) that has pivotal roles
in coagulation, inflammation, vasodilatation and vaso-
constriction through substances such as nitric oxide and
prostaglandins [1].
Recently identified vascular progenitor cells from
bone marrow (BM) and non-BM origin have been shown
to contribute to neovessel formation [2]. Upon inflam-
matory conditions, the vascular endothelium was acti-
vated to capture progenitor cells in regions where endo-
thelium regeneration is needed.
Though ischemia is believed to be the physiological
stimulus for EPC mobilisation from the bone marrow,
the mechanisms of EPC recruitment are less well under-
stood.
Little is understood concerning the molecular mecha-
nisms of EPC adhesion to endothelial cells. In contrast,
extensive studies have been conducted regarding leuko-
cyte-endothelial interactions. Previous experiments have
demonstrated that P-selectin and E-selectin are involved
in leukocyte rolling, whilst intercellular adhesion mole-
cule-1 (ICAM-1) is associated with leukocyte firm adhe-
sion [3]. The molecular players involved in leukocyte
transmigration have been found to be molecules such as
platelet–endothelial-cell adhesion molecule-1 (PECAM-1),
junctional adhesion molecules (JAMs), vascular endothe-
lial-cadherin (VE-cadherin) and CD99 [4].
Chavakis et al had implicated roles for β2-integrins in
the homing and revascularisation of EPCs [5]. Vajcokzy
et al had carried out in vivo experiments with mice to
demonstrate that E-selectin is involved in adhesion of
embryonic EPCs [6].
We recently reported an important role of endothelial
E-selectin in mediating endothelial progenitor cell (EPC)
recruitment [7].
In this study, we used a flow chamber system to
mimic the in vivo circulation. The major advantage of
this set-up is that conditions can be much more tightly
controlled, so that the effect of individual molecules can
be better ascertained than with an in vivo procedure.
RNA inhibition has allowed us to silence genes for a
variety of purposes. Short interfering RNA (siRNA) is a
useful technique to define the function of a given mole-
cule [8].
In this study we look at the role of E-selectin in EPC
adhesion to endothelial cells using double stranded short
interfering RNA (siRNA), in conjunction with the in
vitro flow assay system. Knowledge of this study will
allow us to better understand the process of EPC re-

S. Sharma et al. / J. Biomedical Science and Engineering 3 (2010) 550-555 551
cruitment to endothelial cells, which will give greater
insight into the process of vasculogenesis and its clinical
implications.
2. MATERIALS AND METHOD
2.1. Cell Culture and Reagents
Human umbilical vein endothelial cells (HUVECs) were
cultured on 0.1% gelatin-coated tissue culture dishes as
described previously [9,10]. The following antibodies
were used in this study; H18/7 [11] (anti-human
E-selectin mAb), Hu5/3 [12] (anti-human ICAM-1
mAb). Recombinant human IL-1β was a gift from Bio-
gen Inc (Cambridge, MA). The HL60 cell lines were
obtained from the American Type Culture Collection and
cultured in RPMI-1640 containing 10% FCS. For use in
the flow-chamber apparatus, HUVECs were plated onto
22 mm fibronectin-coated glass coverslips as has been
described previously [13,14].
Preparation of endothelial progenitor cells was previ-
ously described in detail [7].
In brief, 25 mL of the blood was collected from hu-
man volunteers, to which 15 mL Histopaque®-1077 so-
lution (Sigma-Aldrich Japan, KK) was added. After cen-
trifugation for 20 minutes at 1600 rpm, the upper layer
was collected and diluted in Dulbecco’s phosphate buff-
ered saline, then add 2 mM EDTA (DPBSE, Sigma) and
centrifuged at 2000 rpm for 5 minutes. The residual pel-
let was resuspended in 10 mL ammonium chloride solu-
tion (Stem Cell Technologies Inc) to lyse red blood cells
and centrifuged at 1000 rpm for 10 minutes. The pellet
was resuspended in Endothelial Cell Basal Medium
(EBM-2, Clonetics®) and cultured in each well of a C-6
plate (coated with fibronectin). Media exchange of the
EPC culture was carried out after 4 days, and EPCs were
used for flow assay after 7 days. All experimental pro-
cedures were approved by the Experimental Research
Review Committee of the Tokyo Medical and Dental
University (No. 0090014).
2.2. Flow Assay
The parallel-plate flow chamber that was used in this
study has been described previously in more detail
[15,16]. In summary, the chamber was composed of 2
aluminium steel plates separated by a 200 µm thick si-
lastic gasket, and the flow channel was formed by re-
moval of a 5 × 20 mm rectangular section from the gas-
ket. Defined levels of flow were applied to the HUVEC
monolayer by drawing the flow media (see above)
through the channel with a syringe pump (model 44
Harvard Apparatus). A plastic heating plate (Tokai Hit
Co) was attached to the stage of an inverted microscope
(IX50, Olympus) to maintain the temperature at 37℃.
Using this set-up, the channel flow could be modelled as
a two-dimensional fully developed laminar flow with a
simple parabolic velocity profile.
HUVEC monolayers on coverslips were stimulated
for 4 h using IL-1β (10 U/mL), mixed with RPMI and
1% FBS, then positioned in the flow chamber. The
monolayers were perfused for 5 minutes with perfusion
medium (flow media), then examined carefully to estab-
lish the monolayer as confluent. HL60 cells or EPCs
were then diluted in the flow media to 1 × 106 cells/mL.
The cells were then drawn through the chamber at con-
trolled flow rates to achieve wall shear stresses of 1.0
and 2.0 dyne/cm2 for 10 minutes.
2.3. Fluorescent Immunobinding Assay (FIA)
HUVEC monolayers in 96-well plates were incubated on
ice with the indicated primary antibody in RPMI/1%
FCS at 10 μg/mL for 45 minutes. These wells were then
washed three times with RPMI/1% FCS, after which
they were incubated with a FITC-conjugated goat
anti-mouse polyclonal F(ab’)2 antibody (Caltag Labora-
tories) diluted 1:100 in DPBS on ice. After 45 minutes,
the wells were washed with DPBS/20% FCS twice, fol-
lowed by teo washes with DPBS only. Cells were lysed
with 0.01% NaOH in 0.1% SDS. The fluorescence was
then measured using a CytoFluor II (Perspective Bio-
systems) fluorescent plate reader set at 485 (excitation)/
535 (emission).
2.4. siRNA Transfection of E-Selectin
Short interfering RNAs (siRNAs) were designed to tar-
get the coding sequence of human E-selectin cDNA. The
target sequences were directed as described previously8.
Briefly, the sequences were targeted to the single-strand
region consistent with the predicted secondary RNA
structure and sequences of the form (AA/CA)N19 with
GC contents of less than 70% were isolated from this
region. Nineteen RNA nucleotides followed by TT/TG
were selected, then chemically synthesised, and finally
gel-purified.
In order to anneal the single stock strands of RNAi
(JbioS) 20 μL 50 μM sense, 20 μL 50 μM antisense and
10 μL 10 mM MgCl2 in DEPC-PBS was mixed together,
giving a dsRNA concentration of 20 μM. This was then
heated at 95℃ for 2 minutes, 70℃ for 1 minute, 20℃ for
30 minutes, and then 4℃ before being stored at –80℃.
Solutions of 120 µL Optimem® (Invitrogen), 6 µL dou-
ble-stranded siRNA for E-selectin (siE-01) (or control
using LacZ (scE-01) or no siRNA (i.e. Lipofectin alone)),
120 µL Optimem® (Invitrogen) and 5 µL Lipofectin® (In-
vitrogen) were prepared, and left for 30 minutes then
combined and again left for 30 minutes. Optimem® was
then added to make up a total volume of 1.2 mL. This
solution was then added to HUVEC monolayers pre-
washed with Optimem®, then incubated for 4 h at 37℃,

552 S. Sharma et al. / J. Biomedical Science and Engineering 3 (2010) 550-555
after which the HUVEC media was changed to EBM-2.
The cells were used 24 h after transfection.
2.5. Western Blot
Western blot analysis was performed using lysates pre-
pared from HUVEC, as described previously [7]. An
equal amount of protein (10 μg) from each condition
was subjected to 5-20% SDS-PAGE. I mmunoreactive
proteins were detected using an enhanced chemilumi-
nescence (ECL) kit (Amersham Bioscience).
2.6. Statistical Analysis
Results are presented as mean ± standard deviation as
indicated. Two-tailed Student’s t tests were performed
using Microsoft Excel to analyse data. Probability values
represent the results of these t tests with a value of p <
0.05 considered statistically significant.
3. RESULTS
3.1. Activation of HUVEC with IL-1β
First, we tried an adhesion assay using monocytic HL60
cells over an activated HUVEC monolayer to validate
our assay system. As shown in Figure 1, we found a
time-dependent increase of HL60 rolling and adhesion
when stimulated with IL-1β (10 U/mL), with a peak ac-
tivation after 4 h stimulation. 2 h IL-1β stimulation led
to a dramatic increase in adhesion (13.3 ± 9.92 cells/HPF,
p < 0.0005) and rolling (4.8 ± 3.34, p < 0.0005) of HL60
cells when compared to those HUVECs without activa-
tion (adhesion, 1.8 ± 1.78; rolling, 0 ± 0). 4 h IL-1β
stimulation led to a further increase in adhesion (25.8 ±
12.1 cells/HPF, p < 0.00001) and rolling (3 ± 2.83, p <
0.03) of HL60 cells. IL-1β stimulation for 6 h caused an
increase in adhesion of HL60 cells greater than that with
2 h IL-1β stimulation but not as significant as that with 4
h IL-1β stimulation, when compared to the control sam-
ple (6 h, 16.5 ± 4.90 cells vs 0 h, 1.8 ± 1.78 cells, p =
5.62 × 10-8). We then checked the surface expression of
adhesion molecules after IL-1β activation using fluores-
cent immunobinding assay (FIA), as shown in Figure 2.
To check the validity of the FIA analysis, intensity of
negative controls (no primary antibody) and positive
controls (W6/32, anti-HLA Class I antibody) were ex-
amined (negative control: 0 h, 4.8 ± 3.19 RFU vs 4 h,
5.6 ± 3.29 RFU, p = 0.71; positive control: 0 h,17 ± 2
RFU vs 4h, 21.2 ± 2.59 RFU, p = 0.02). E-selectin ex-
pression (detected using H18/7 Ab) was significantly
upregulated after 4 h IL-1β stimulation compared to no
stimulation (4 h, 25.2 ± 1.79 RFU vs 0 h, 9 ± 2.92 RFU,
p = 5.52 × 10-6). ICAM-1 expression (detected using
Hu5/3 Ab) was also significantly upregulated after 4 h
IL-1β stimulation (4 h, 23.4 ± 3.05 RFU vs 0 h, 9.2 ±
0.84 RFU, p = 8.23 × 10-6).
3.2. Adhesion of EPC to Activated HUVEC
under Flow
Based on the preliminary experiment using HL-60 cells
(a)
(b)
Figure 1. IL-1β induces a time-dependent rolling and adhesion
of HL60 cells to HUVEC. (a) Representative micrographs
taken from video recording during the flow assay. HL60 cells
(white particles) were perfused over a HUVEC monolayer
(grey background) that had been subjected to activation by
IL-1β (50 U/mL) for four different time periods; (b) The num-
ber of rolling and adherent HL60 cells were measured as de-
scribed in Methods. (* = p < 0.05 vs 0 h IL-1β); data based on
10 fields of observation for each condition.
Figure 2. Cell surface expression of E-selectin and ICAM-1 in
HUVEC after IL-1β stimulation. The expression intensity of
each of the adhesion molecules was determined using a fluo-
rescent immunobinding assay as described in Methods. (* = p
< 0.05 vs corresponding 0 h IL-1β); data based on 5 repeats for
each condition.

S. Sharma et al. / J. Biomedical Science and Engineering 3 (2010) 550-555 553
(Figure 1), we used a condition of IL-1β (10 U/mL) for
4 h in our following experiments. When EPCs, cultured
ex vivo for 7 days, were perfused over activated HU-
VEC monolayer, adhesion of EPCs was dramatically
increased compared to the control HUVEC monolayer
(4 h, 4.4 ± 1.50 cells vs 0 h, 1.3 ± 0.9 cells, p = 8.97 ×
10-5), as shown in Figure 3. There was however no roll-
ing of EPCs observed both in the control and 4 h IL-1β
stimulation.
3.3. Gene Silencing of E-Selectin in HUVEC
Having demonstrated that 4 h of IL-1β stimulation was
optimal for adhesion, we looked at the effects of silenc-
ing E-selectin, using siRNA transfection, on the rolling
and adhesion of EPCs. siRNA transfection on HUVEC
monolayers was carried out 24 h prior to the adhesion
assay under flow conditions (the time point was deter-
mined to optimize the effect of siRNAs in HUVECs, as
previously described [8]). We utilized FIA assay to dem-
onstrate the level of E-selectin in these cells. As demon-
strated in Figure 4, positive controls (W6/32, anti-HLA
Class I antibody) exhibited no significant difference be-
tween siRNA treatment groups, enhancing the validity of
these results. However, there was a significant reduction
of E-selectin surface expression in the E-selectin siRNA
—treated HUVECs when compared to the LacZ siRNA
—treated HUVECs after 4 h IL-1β stimulation (E-selectin,
27.3 ± 5.40 RFU vs LacZ, 45.5 ± 7.37 RFU, p = 0.013)
or no siRNA group (E-selectin, 27.3 ± 5.40 RFU vs no
siRNA, 52.5 ± 10.4 RFU, p = 0.010).
3.4. Effect of Gene Silencing of E-Selectin on
EPC Adhesion to HUVEC under Flow
Figure 5 showed results of the flow assay using IL-1-
activated HUVECs after siRNA transfection. There was
Figure 3. The number of rolling and adherent EPC to ac-
tivated HUVEC were measured as described in Methods.
(* = p < 0.05 vs 0 h IL-1β); data based on 10 fields of
observation for each condition. EPCs were perfused over
a HUVEC monolayer (grey background) that had been
subjected to activation by IL-1β (50 U/mL) for two dif-
ferent time periods (0 h IL-1β and 4 h IL-1β).
Figure 4. Cell surface expression of E-selectin (H18/7) and
positive control (W6/32) in HUVEC after siRNA silencing.
The expression intensity of each of the adhesion molecules was
determined using a fluorescent immunobinding assay as de-
scribed in Methods. (* = p < 0.05 vs corresponding 0hr IL-1β);
data based on 5 repeats for each condition.
Figure 5. The number of rolling and adherent EPC to activated
HUVEC after siRNA transduction. Graph showing the effect of
E-selectin siRNA transfection on rolling and adhesion of EPCs
compared to control siRNA transfection and no siRNA treat-
ment. (* = p < 0.05 vs control); data based on 10 fields of ob-
servation for each condition.
a significant decrease in EPC adhesion in HUVECs
transfected with E-selectin siRNA compared to those
treated with LacZ siRNA (controls) (E-selectin siRNA,
2.7 ± 0.64 cells vs LacZ siRNA, 5.1 ± 1.22 cells, p =
5.74 × 10-5). In comparison, there was no significant
difference in rolling between those HUVECs transfected
with E-selectin siRNA and the control HUVECs (E-
selectin siRNA, 0.2 ± 0.4 cells vs LacZ siRNA, 0.5 ±
0.67 cells, p = 0.26). There was also a significant de-
crease in EPC adhesion in HUVECs treated with
E-selectin siRNA compared to those treated with no
siRNA (Lipofectin® only) (E-selectin siRNA 2.7 ± 0.64
cells vs no siRNA, 7 ± 1.26 cells, p = 3.74 × 10-8).
4. DISCUSSION
In this project, we have demonstrated that 1) 4 h of in-

554 S. Sharma et al. / J. Biomedical Science and Engineering 3 (2010) 550-555
cubation with IL-1β induces adhesion of HL60 cells to
HUVEC under flow; 2) 4 h of incubation with IL-1β
induces expression of E-selectin and ICAM-1 in HU-
VEC; 3) 4 h of incubation with IL-1β induces adhesion
of EPCs to HUVEC under flow; 4) siRNA against
E-selectin reduces endogenous expression of E-selectin
in HUVEC; 5) gene silencing of E-selectin resulted in
reduction of EPC adhesion to HUVEC. These lines of
findings point a predominant role for this molecule in
neovascularisation. In previous work from our labora-
tory using siRNA transfection carried out by Nishiwaki
et al (2003), we successfully silenced endogenous E-
selectin expression in vascular endothelium using siRNA
transfection, and demonstrated that this inhibition caused
a significant reduction in leukocyte adhesion [8]. Current
results compliment this work and emphasise the efficacy
of siRNA in the field of vascular biology.
We have also reinforced the work of Nishiwaki et al
(using antibodies to downregulate E-selectin) and Va-
jkoczy et al (using an in vivo method to silence E-
selectin expression) in demonstrating that E-selectin is
important in EPC adhesion by using a different method
(siRNA transfection) [6,7].
Using siRNA transfection to inhibit E-selectin expres-
sion, as demonstrated by our experiments, may have
implications for its role in angiogenesis. Bischoff has
reported that E-selectin is involved in angiogenesis
through a series of experiments [17]. One of these ex-
periments showed that antibodies directed against E-
selectin or sialylated fucosylated oligosaccharides (stru-
ctures to which E-selectin binds) inhibit the formation of
capillary-like tubes in vitro [18]. The presence of E-
selectin in human infantile haemangioma tumours (en-
dothelial cell tumour of capillary blood vessels), as well
as in tissues with on going growth of microvessels (such
as human placenta and neonatal foreskin) has been
shown in vivo [19]. Yu et al had also shown that endo-
thelial progenitor cells are present in infantile haeman-
gioma; 11 of 12 proliferating haemangioma specimens
contained EPCs (determined by flow cytometry using
CD133 and CD34 as markers for EPCs) [20]. This raises
the possibility of using our method of silencing E-
selectin expression in treating tumour angiogenesis, by
preventing localised EPC adhesion to the endothelium,
thus inhibiting angiogenesis in the tumour, reducing its
growth. As described previously, we found that there
was no significant reduction in rolling after E-selectin
down-regulation. This could have been because of the
low level of EPC rolling in general, as demonstrated in
Figure 3. On the other hand, the binding characteristics
of E-selectin (E-selectin has a greater role in adhesion
than rolling) may have some effects. This speculation
has been supported by the results from Mazo, et al.’s
experiments [21]. They demonstrated that blocking E-
selectin with antibodies resulted in a reduction of haema-
topoietic progenitor cells (HPC) rolling of 32% com-
pared to a reduction in HPC rolling of 58% when
P-selectin was inhibited (measured using intravital mi-
croscopy). Therefore the other selectins (P- and L-
selectins) are probably more important for HPC rolling.
A limitation of this experiment is that we have only
demonstrated that gene silencing of E-selectin expres-
sion reduces EPC adhesion in vitro, but not in vivo.
Therefore future work could be aimed at seeing if siRNA
transfection has similar effects in vivo. It would also be
interesting to try similar experiments using siRNA trans-
fection against other adhesion molecules such as ICAM-
1 or P-selectin, and look at the effects on EPC recruit-
ment.
In conclusion, we have demonstrated that E-selectin—
short interfering RNA inhibits endothelial progenitor cell
recruitment to vascular endothelium under flow condi-
tions.
5. ACKNOWLEDGEMENTS
Grateful thanks to Dr Naokazu Nakamura, Michiyo Deushi, Noriko
Nitta and Mariko Tani for their experimental help and advice, Profes-
sor Peter Sugden for his help and advice throughout the course of the
project.
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