ORIGINAL ARTICLE

Decidual NK Cell-Derived Conditioned Medium (dNK-CM)

Mediates VEGF-C Secretion in Extravillous Cytotrophoblasts
Genevieve D. M. Eastabrook1,2, Yuxiang Hu1,2, Rusung Tan2,3, Jan P. Dutz2,4, Colin D. MacCalman1,2 ,
Peter von Dadelszen1,2
1
Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, Canada;
2
The Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada;
3
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada;
4
Department of Dermatology and Skin Science, University of British Columbia, Vancouver, BC, Canada

Keywords,
Decidual natural killer cells, extravillous
cytotrophoblasts, Janus kinase, p38, signal
transducers and activators of transcription,
VEGF-C
Correspondence
Genevieve Eastabrook, Department of
Obstetrics and Gynaecology, University of
British Columbia, 2H30-4500 Oak Street,
Vancouver, BC V6H 3N1, Canada.
E-mail: geastabrook@cw.bc.ca
 Deceased.
Submitted August 8, 2011;
accepted September 13, 2011.
Problem
The regulatory mechanisms involved in VEGF-C secretion by tropho-
blasts during placentation are poorly understood. We investigated
whether or not decidual natural killer cell conditioned medium (dNK-
CM) stimulated VEGF-C secretion in the extravillous cytotrophoblast
(EVT) cell line HTR8 ⁄ SVneo.
Method of Study
The effects of dNK-CM and recombinant IFN-c on VEGF-C induction by
HTR8 ⁄ SVneo were studied in the absence or presence of IFN-c or its
receptor blocking antibodies, p38 inhibitor (SB202190), JAK inhibitor
(JAK inhibitor-1, JI-1), and on STAT1 knockdown HTR8 ⁄ SVneo. VEGF-C
was quantified by ELISA. FACS was used to investigate the phosphoryla-
tions of Tyr701 or Ser727 of STAT1 on stimulated HTR8 ⁄ SVneo.

Citation
Eastabrook GDM, Hu Y, Tan R, Dutz JP,
MacCalman CD, von Dadelszen P. Decidual NK
cell-derived conditioned medium (dNK-CM)
mediates VEGF-C secretion in extravillous
cytotrophoblasts. Am J Reprod Immunol 2012;
67: 101–111
doi:10.1111/j.1600-0897.2011.01075.x
Results
dNK-CM facilitated VEGF-C secretion by HTR8 ⁄ SVneo. IFN-c and IFN-
cR1 or IFN-cR2 blocking antibodies reduced both dNK-CM- and IFN-c-
induced VEGF-C secretion. Phosphorylations on Tyr701 or Ser727 of
STAT1 were elevated upon stimulation. Secretion of VEGF-C was
reduced by treatment with SB202190, JI-1, or STAT1 knockdown by
siRNA.
Conclusion
VEGF-C production by trophoblasts is regulated by soluble factors
secreted by dNK through p38 and JAK-STAT1 pathways.

and spiral artery remodeling. CTB expression of

Introduction
During the first and second trimesters of pregnancy,
the invasive cytotrophoblasts (CTB) display a
unique repertoire of substances that influence vascu-
logenesis and angiogenesis. Evidence indicates that
VEGF-C, along with its receptors, plays a critical role
in trophoblast invasion, endovascular differentiation,
American Journal of Reproductive Immunology 67 (2012) 101–111
ª 2011 John Wiley & Sons A/S
VEGF-A, VEGF-C, placental growth factor (PlGF),
and VEGFR-1 is accompanied by downregulation of
VEGFR-2 expression.1,2 These cells are able to
respond to the signals from endothelial cells and par-
ticipate in endothelial cell-guided tubule formation.3
At term, CTB express VEGF-A and PIGF but express
little VEGF-C and VEGFR-2.1,2 They fail to respond
101
 EASTABROOK ET AL.
to signals from endothelial cells and do not partici-
pate in endovascular remodeling, displaying
restricted migratory and interactive properties.3 The
regulation of VEGF-C expression in CTB is not fully
understood, but merits further investigation both in
normal and pathological placentation.
Expression of both VEGF-C and VEGFR-3 is
decreased in placentas from pregnancies affected by
intrauterine growth restriction (IUGR), suggesting
that abnormal levels of VEGF-C and VEGFR-3 may
contribute to the abnormal villous development.4 An
imbalance between pro- and anti-angiogenic factors
is involved in the pathogenesis of pre-eclampsia.
Soluble fms-like tyrosine kinase-1 (sFLt-1), a splice
variant of the VEGFR-1 receptor lacking transmem-
brane and cytoplasmic domains, acts as a potent
anti-angiogenic factor by binding to both VEGF and
PlGF, thus preventing their interaction with their
endogenous receptors.5 Recent evidence also sug-
gests that intracellular VEGF is decreased in the
peripheral NK cells of women with pre-eclampsia as
compared with normal pregnant controls.6
During the first trimester, extravillous cytotropho-
blast (EVT) are in direct contact with dNK, a popula-
tion of maternal lymphocytes with low cytotoxicity
as compared with peripheral blood NK cells. How-
ever, dNK are capable of secreting numerous cyto-
kines, chemokines, and growth factors, an
observation previously reported7–10 and also recently
described by our own group.11 These factors influ-
ence EVT by regulating their invasion and vascular
growth.12–15 IFN-c is one of the critical cytokines
produced by dNK. In mice, IFN-c regulates the
expression of >0.5% of the mouse genome, includ-
ing genes involved in variety of cell functions16,17
Furthermore, 90% of the IFN-c found in early to
mid-gestation decidua basalis is derived from dNK
cells.18 Specific examples of IFN-c-regulated genes
include inducible nitric oxide synthase (iNOS), endo-
thelial NOS (eNOS), and endothelin-1.16 IFN-c has
been shown to both enhance and reduce VEGF-C
secretion depending on the cell lines studied.18,19
However, there is a dearth of evidence pertaining to
the role of decidual natural killer cells conditioned
medium (dNK-CM) or IFN-c in the regulation of
VEGF-C secretion by CTB.
IFN-c activates Janus kinases (JAKs) through
binding to the cell surface receptors IFN-c receptor-1
(IFN-cR1) or IFN-c receptor-2 (IFN-cR2). This bind-
ing causes the rapid phosphorylation of Tyr701 of
the latent cytoplasmic transcription factor STAT1.
102
Phospho-Tyr701 STAT1 forms homodimers which
translocate to the nucleus and bind to r-activating
sequences (GAS) in IFN-c-responsive promoters.20,21
The second phosphorylation site, Ser727 on STAT1,
is also activated by IFN-c.20,21 A variety of serine
kinases have been reported to phosphorylate Ser727,
including phosphatidylinositol 3-kinase ⁄ AKT,19
Ca ⁄ calmodulin-dependent kinase II,22 protein
2+
kinase C isoform,23 and MAPK.24 In addition, p38 is
involved in LPS- and UV light-induced STAT1
Ser727 phosphorylation in macrophages25,26 and
HeLa cells treated with IFN-c.27 STAT1 requires
phosphorylation on Tyr701 and Ser727 to achieve
full transcriptional and anti-pathogenic activity.28
Our objective was to determine whether dNK-
derived soluble factors were able to directly influ-
ence VEGF-C secretion by EVT, both in primary cell
cultures and in the EVT cell line HTR8 ⁄ SVneo. In
addition, we aimed to determine which factor(s)
contained in dNK-CM contributed to this effect.
Here, we report that dNK-CM was able to induce
VEGF-C production in both primary CTB and the
EVT cell line HTR8 ⁄ SVneo. IFN-c contained in the
CM was a major player in this process, as both IFN-c
blocking antibody and IFN-cR1 and IFN-cR2 block-
ing antibodies significantly reduced this effect. dNK-
CM and IFN-c phosphorylated both Tyr701 and
Ser727 of STAT1. p38 MAPK inhibitor, JAK inhibi-
tor, and STAT1 siRNA decreased dNK-CM- or IFN-c-
mediated VEGF-C secretion.
Materials and methods
Preparation of dNK-CM
Following informed consent, placental tissues were
collected from healthy women undergoing elective
terminations of pregnancy at gestational ages
between 6 and 10 weeks. Ethics approval was
obtained from the Children’s and Women’s Research
Ethics Board. dNK isolation and dNK-CM prepara-
tion were performed as described previously.12,13 In
brief, specimens were thoroughly washed with PBS,
minced into fragments of approximately 1 mm3, and
digested for 1 hr at 37C in RPMI 1640 medium with
1.5 mg ⁄ mL collagenase and 2 mg ⁄ mL hyaluronidase
(Sigma-Aldrich, Oakville, ON, Canada). Cell suspen-
sions were washed and plated in dishes with
2 ng ⁄ mL IL-15 (R&D Systems, Minneapolis, MN,
USA), and after an overnight incubation at 37C,
non-adherent cells were harvested from the culture
American Journal of Reproductive Immunology 67 (2012) 101–111
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dishes. The cell suspension obtained was then sepa-
rated by Ficoll-Hypaque gradient 1077 (Amersham
Biosciences, Baie d‘Urfe, QB, Canada). Subsequently,
NK cells were enriched with human NK cell enrich-
ment mixture following the manufacturer’s instruc-
tions (StemCell Technologies, Vancouver, BC,
Canada). On average, 6 · 105 enriched dNK per
gram of decidual tissue were isolated. Previously, we
have confirmed the purity of this cell population
using flow cytometry.12
Decidual natural killer cells were cultured and
maintained in vitro with DMEM ⁄ F12 supplemented
with 10% FBS and 10 ng ⁄ mL IL-15 for approxi-
mately 1 month. The conditioned medium (CM)
from different passages of dNK and from different
subjects were collected, pooled, and stored at )80C
in advance of the assays. The concentrations of
VEGF-C and IFN-c contained in the dNK-CM were
quantified.
Culture of Primary CTB and HTR8 ⁄ SVneo cells
The isolation and culture of primary CTB were
described previously.13 In brief, CTB-containing cell
suspension was obtained from villi by 0.1% trypsin
digestion, purified by 40% percoll density centrifuga-
tion and further enriched by removing non-adherent
cells after overnight attachment. The CTB obtained
contained approximately 80–90% cytokeratin posi-
tive cells. No further purification was carried out for
the initial assays, and the CTB were grown for
4–5 days before the assay was set up. The primary
CTB were only used for initial assays, to determine
whether they were capable of VEGF-C secretion
after stimulation with either IFN-c or dNK-CM.
HTR8 ⁄ SVneo cells were a gift provided by Dr.
Charles H. Graham (Queen’s University, Kinston,
ON, Canada).29 Cells were routinely maintained in
RPMI-1640 medium containing 5% FBS and were
passaged every 2–3 days. HTR8 ⁄ SVneo cells were
used throughout the study.
Generation of VEGF-C-Containing Supernatants
from Trophoblasts and VEGF-C Measurement by
ELISA
HTR8 ⁄ SVneo and primary CTB were seeded into 24-
well plates at 100,000 cells ⁄ well in DMEM ⁄ F12 sup-
plemented with 10% FBS. On the following day, the
media were removed and replaced with fresh
DMEM ⁄ F12 supplemented with 10% FBS. Cells
American Journal of Reproductive Immunology 67 (2012) 101–111
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dNK-CM MEDIATES VEGF-C SECRETION IN EVT
were then exposed to dNK-CM at 1:2–1:8 dilutions
for a total of 48 hr. At the end of incubation, super-
natants were collected and saved at )80C for
VEGF-C measurement. The concentration of VEGF-C
was measured with Quantikine ELISA kit (R&D
Systems) following the manufacturer’s instructions.
The results were expressed as normalized % using
the following formula: normalized % = OD [assay
group]*100 ⁄ OD [ctrl group], where ctrl group =
100%.
To determine whether or not IFN-c contained in
the supernatants contributed to VEGF-C induction,
anti-IFN-c blocking antibody (mouse IgG2a; R&D
Systems) was pre-incubated with dNK-CM at a con-
centration of 10 lg ⁄ mL for 1 hr. Antibody-treated or
control dNK-CM were then used to stimulate
HTR8 ⁄ SVneo cells. IFN-cR1 and IFN-cR2 blocking
antibodies (Goat IgG; R&D Systems) were incubated
with HTR8 ⁄ SVneo at a concentration of 10 lg ⁄ mL
for 1 hr. dNK-CM was then used to stimulate anti-
IFN-cR1- or anti-IFN-cR2-treated or untreated
HTR8 ⁄ SVneo cells for a total of 48 hr. The experi-
ments were completed as above.
Recombinant IFN-c (R&D Systems), at a range of
0.8–100 ng ⁄ mL, was tested on both CTB and
HTR8 ⁄ SVneo cells for VEGF-C induction. IFN-c
blocking antibody or IFN-cR1 and IFN-cR2 antibod-
ies were tested on HTR8 ⁄ SVneo cells as well. Other
than for the initial titration, IFN-c at 20 ng ⁄ mL con-
centration was used throughout the study.
To determine the involvement of p38 and JAK-
STAT1 pathways in the induction of VEGF-C by
dNK-CM and IFN-c, HTR8 ⁄ SVneo cells were treated
with 3.1–12.5 lm of SB202190 (Biomol Interna-
tional, Plymouth Meeting, PA, USA) or 1.2–5 lm of
JI-1 (EMD, Mississauga, ON, Canada) for 1 hr before
exposure to dNK-CM or IFN-c. The experiments
were completed as above.
FACS Staining
Phosphorylation of Tyr701 and Ser727 of STAT1
were performed by flow cytometry as described with
minor modifications.30 HTR8 ⁄ SVneo cells maintained
with 5% FBS containing PRMI1640 were harvested
and divided into tubes, with 1 · 106 cells ⁄ mL per
tube. Cells were treated with either dNK-CM or IFN-c
for different periods of time, or cells were pre-treated
with anti-IFN-cR1 or IFN-cR2 antibodies at a con-
centration of 10 lg ⁄ mL for 1 hr followed by dNK-
CM or IFN-c treatment for 1 hr. At the end of the
103
 EASTABROOK ET AL.

treatment, treated and untreated cells were fixed by
adding 37% formaldehyde solution directly into the
cell suspension to obtain a final concentration of
1.5% formaldehyde. Cells were incubated for
20 min and pellets were then collected by centrifu-
gation. Cells were permeabilized by 100% ice-cold
methanol for 10 min. Following permeabilization,
cells were either stained immediately or saved at
)20C for future staining.
Cells were washed with PBS containing 1% BSA
(staining solution) before staining procedures. Cells
were incubated with 1:100 diluted rabbit anti-
human Tyr701 or Ser727 of STAT1 (Cell Signalling
Technology (CST), Pickering, ON, Canada) for 2 hr
at room temperature, then washed with staining
solution three times. FITC-conjugated goat anti-rab-
bit IgG (BD PharMingen, Mississauga, ON, Canada)
at 1:100 dilution was added to cells and incubated
for 30 min. Cells were then washed and subjected to
FACS analysis in a FACScan flow cytometer (BD
Biosciences, Mississauga, ON, Canada).
Transfection of STAT1 siRNA into HTR8 ⁄ SVneo
cells
X-tremeGENE siRNA transfection reagent (Roche,
Mississauga, ON, Canada) was used for transfection.
The cytotoxicity of the reagent was detected by its
serial dilutions, and transfection efficiency was deter-
mined by fluorescein-conjugated control siRNA
(CST) monitored under fluorescent microscope. The
condition which resulted in no cytotoxicity but with
100% transfection efficiency was chosen to transfect
target siRNA. ON-TARGETplus STAT1 siRNA and
non-targeting control siRNA were purchased from
Dharmacon (Lafayette, CO, USA). STAT1 siRNA (but
not control siRNA) at a concentration as low as
12.5 nm showed >95% STAT1 mRNA reduction as
detected by real-time PCR (data not shown). After
assay optimization, HTR8 ⁄ SVneo were transfected
with 25 or 50 nM STAT1 siRNA or control siRNA.
Twenty-four hours after transfection, the transfected
cells were exposed to dNK-CM or IFN-c for an addi-
tional 48 hr. The culture supernatants were then
collected and VEGF-C quantification was performed
as described above.
Statistics
Because our data was not normally distributed,
groups were compared using non-parametric Krus-
kal–Wallis anova, with Dunn’s post-test, as appropri-
ate. graphpad prism 4.0 software (GraphPad Software
Inc., San Diego, CA, USA) was used for all analyses.
Statistical significance was set at P < 0.05.
Results
dNK-CM Increases CTB and HTR8 ⁄ SVneo
Secretion of VEGF-C
Cytotrophoblasts and HTR8 ⁄ SVneo grown in
DMEM ⁄ F12 with 10% FBS produced basal levels of
VEGF-C. dNK-CM significantly increased VEGF-C

(a) (b)

Fig. 1 dNK-CM facilitated VEGF-C induction in both cytotrophoblasts (CTB) (a) and HTR8 ⁄ SVneo (b). CTB and HTR8 ⁄ SVneo cells were stimulated
with serial dilutions of dNK-CM. The culture supernatants were collected and VEGF-C concentrations were measured. The results were expressed
as normalized % with following formula: normalized % = OD [assay group]*100 ⁄ OD [control medium group], where control medium group = 100%.
The dNK-CM significantly increased VEGF-C secretions from both CTB and HTR8 ⁄ SVneo. The data shown (median) were from one representative
experiment. Dotted line and P-value: Kruskal–Wallis non-parametric ANOVA. Dunn’s multiple post hoc test: *P < 0.05 compared with control (a, b).

American Journal of Reproductive Immunology 67 (2012) 101–111

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 dNK-CM MEDIATES VEGF-C SECRETION IN EVT

secretion by both CTB (Fig. 1a) and the EVT cell line
HTR8 ⁄ SVneo, seeded on bare plates (Fig. 1b) in a
dose-dependent manner. Previously, it has been
shown that HTR8 ⁄ SVneo cells produce VEGF-C
when seeded on Matrigel.13 dNK-CM at 1:2 dilution
was used for the remainder of the study, as dNK-CM
at this dilution induced maximal VEGF-C secretion
among the tested concentrations. HTR8 ⁄ SVneo cells
were chosen for completion of the entire study.
dNK-CM harvested from short-term culture of dNK
(48 hr after dNK collection from decidua) was simi-
larly capable of VEGF-C induction. However, long-
term dNK-CM from long-term culture was used
throughout the study.
Previously, it has been reported that dNK express
VEGF-C.31 Our data confirmed VEGF-C mRNA
expression as detected by real-time PCR and VEGF-C
secretion by dNK (data not shown). While the VEGF-
C concentration was <5 pg ⁄ mL in dNK-CM, it was
>300 pg ⁄ mL in the supernatants from untreated
HTR8 ⁄ SVneo. Thus, the impact of VEGF-C existing in
dNK-CM as compared with the quantity produced by Fig. 2 dNK-CM-mediated VEGF-C induction on HTR8 ⁄ SVneo was
blocked by both IFN-c and IFN-cR1 or IFN-cR2 neutralizing antibodies.
HTR8 ⁄ SVneo was negligible. VEGF-C concentration
dNK-CM was pre-treated with IFN-c neutralizing antibody and then
was even greater after dNK-CM stimulation. Previous
used for stimulation on HTR8 ⁄ SVneo. Alternately, cells were pre-trea-
evidence has shown that IFN-c increased18 or ted with IFN-cR1 or IFN-cR2 blocking antibodies and were then
decreased VEGF-C secretion19 in different cell lines. exposed to dNK-CM. Data demonstrated that both IFN-c neutralizing
Our next step was to determine whether or not IFN-c antibody and IFN-cR1 or IFN-cR2 antibodies partially, but significantly,
contained in dNK-CM contributed to VEGF-C induc- reduced dNK-CM-mediated VEGF-C secretion. The data shown (median)
tion by HTR8 ⁄ SVneo exposed to dNK-CM. were from one representative experiment. Dotted line and P-value:
Kruskal–Wallis non-parametric ANOVA. Dunn’s multiple post hoc test:
*P < 0.05 compared with control; **P < 0.01 compared with control.
IFN-c Neutralizing Antibody and IFN-cR1 or
IFN-cR2 Neutralizing Antibodies Reduce
dNK-CM-Mediated VEGF-C Secretion from the antibodies tested above (Fig. 3c) but not by
HTR8 ⁄ SVneo cells mouse IgG2a or goat IgG isotype controls.
dNK-CM was pre-treated with anti-IFN-c blocking
antibody for 1 hr before addition to HTR8 ⁄ SVneo IFN-c Can Activate STAT1 Tyr701 and Ser727
cells. Alternately, HTR8 ⁄ SVneo cells were pre-treated
with anti-IFN-cR1 or IFN-cR2 blocking antibodies IFN-c-induced STAT1 Tyr701 activation acts through
and then exposed to dNK-CM. Our results demon- JAK.20,21 STAT1 Ser727 activation may or may not
strated that both IFN-c blocking antibody and be dependent on p38 MAPK.27,32,33 Other serine
IFN-cR1 or IFN-cR2 blocking antibodies were able to kinases are also proposed to be involved.22–24,34 We
partially, but significantly, reduce VEGF-C secretion; sought to determine whether dNK-CM or IFN-c was
mouse IgG2a or goat IgG isotype controls had no able to increase phosphorylations of Tyr701 and
effect (Fig. 2). As measured by conventional sand- Ser727 of STAT1 on HTR8 ⁄ SVneo.
wich ELISA, the dNK-CM in our system contained
approximately 3.5 ng ⁄ mL IFN-c. Recombinant IFN-c dNK-CM and IFN-c Mediate Phosphorylations of
in the range of 0.8–100 ng ⁄ mL was able to increase Tyr701 and Ser727 of STAT1 on HTR8 ⁄ SVneo
VEGF-C secretion in both CTB (Fig. 3a) and
HTR8 ⁄ SVneo (Fig. 3b); the VEGF-C-inducing ability Phosphorylations of Tyr701 and Ser727 of STAT1
of IFN-c on HTR8 ⁄ SVneo cells was ameliorated by on HTR8 ⁄ SVneo occurred very quickly following

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 EASTABROOK ET AL.

(a) (b)

(c)

Fig. 3 Recombinant IFN-c enhanced VEGF-C secretions from both cytotrophoblasts (a) and HTR8 ⁄ SVneo (b). IFN-c-mediated VEGF-C induction on
HTR8 ⁄ SVneo was blocked by both IFN-c and IFN-cR1 or IFN-cR2 neutralizing antibodies (c). Cells were stimulated with various concentrations of
IFN-c and the supernatants were collected for VEGF-C measurement. For antibody blocking assays, the methods were the same as using dNK-CM
as a stimulator as described above. The data (median) showed that VEGF-C induction was increased by IFN-c (a, b) and was blocked by antibodies
tested (c). Dotted line and P-value: Kruskal–Wallis non-parametric ANOVA. Dunn’s multiple post hoc test: *P < 0.05 compared with control (a, b), or
IFN-c (c); **P < 0.01 compared with IFN-c (c).

exposure to either dNK-CM or IFN-c. After 10 min
treatment with dNK-CM or IFN-c, both Tyr701 and
Ser727 phosphorylations were increased signifi-
cantly. The signals remained at high level at the
observation times 1hr (Fig. 4a–d) and 6 hr (data
not shown). The high basal levels of phosphoryla-
tion of Tyr701 and Ser727 on HTR8 ⁄ SVneo were
observed in the medium control containing 10%
FBS.
Decidual natural killer cells conditioned medium
or IFN-c-induced phosphorylations of Tyr701 and
Ser727 on HTR8 ⁄ SVneo were reduced by IFN-cR1
and IFN-cR2 blocking antibodies (Fig. 5a–d). This
effect was especially significant for IFN-c-induced
Tyr701 and Ser727 phosphorylations (Fig. 5c,d), and
dNK-CM-induced Tyr701 phosphorylation (Fig. 5a),
106
but was seen to a lesser extent on dNK-CM-induced
Ser727 phosphorylation (Fig. 5b).
To confirm the involvement of p38 and JAK-
STAT1 pathways in VEGF-C induction, SB202190,
JI-1, and STAT1 siRNA were introduced to the
study.
SB202190, JI-1, and STAT1 siRNA Reduce dNK-
CM- or IFN-c-Mediated VEGF-C Secretions on
HTR8 ⁄ SVneo
HTR8 ⁄ SVneo were pre-treated with SB202190 at
concentrations ranging from 3.1 to 12.5 lm, and JI-1
at concentrations ranging from 1.2 to 5 lm for 1 h.
The untreated and treated HTR8 ⁄ SVneo cells were
then exposed to dNK-CM or IFN-c. The results
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 dNK-CM MEDIATES VEGF-C SECRETION IN EVT

(a) (b)

(c) (d)

Fig. 4 Both dNK-CM and IFN-c activated STAT1 Tyr701 and Ser727 phosphorylations (a–d). dNK-CM- or IFN-c-treated HTR8 ⁄ SVneo were fixed, per-
meabilized, and then conducted for FACS staining. The cells were incubated with anti-human Tyr701 or Ser727 of STAT1 followed by incubating
with FITC-conjugated secondary Ab. The results showed the high basal level phosphorylations of Tyr701 and Ser727 on control cells; both dNK-CM
(a, b) and IFN-c (c, d) were able to enhance Tyr701 and Ser727 phosphorylations.

showed that SB202190 and JI-1 decreased both IFN-c-
and dNK-CM-induced VEGF-C secretion in a dose-
dependent manner (Fig. 6a,b). For SB202190, this
effect was statistically significant only at the highest
concentrations of the inhibitor when stimulated with
either IFN-c or dNK-CM. Likewise, for JI-1, the
inhibitory effect was statistically significant for
IFN-c-induced VEGF-C secretion at the two high-
est concentrations (5 and 2.5 lm, respectively).
Although a dose-dependent inhibition of dNK-CM-
induced VEGF-C secretion was observed, this did not
reach statistical significance.
To study whether or not STAT1 knockdown would
affect VEGF-C secretion, HTR8 ⁄ SVneo were trans-
fected with STAT1 siRNA or control siRNA at various
concentrations. Twenty-four hours after transfection,
cells were exposed to IFN-c or dNK-CM. The data
demonstrated that VEGF-C secretion was partially,
but significantly, reduced in STAT1 siRNA transfect-
ed cells but not in control siRNA transfected cells
(Fig. 7).
Discussion
VEGF-C plays important roles in angiogenesis and
lymphangiogenesis. Endothelial cells are a major
source of VEGF-C. Invasive trophoblasts1–3 and
dNK31 also produce VEGF-C, findings which have
been previously reported and have been confirmed
in this study. However, we have found that VEGF-C
is found at very low, to undetectable, levels in our
dNK-CM.11
Trophoblasts may acquire their endothelial pheno-
type through their production of VEGF-C and other
angiogenic factors.2 Previously, we reported that
dNK-CM facilitated the formation of capillary tube

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 EASTABROOK ET AL.

(a) (b)

(c) (d)

Fig. 5 dNK-CM- and IFN-c-mediated Tyr701 and Ser727 phosphorylations on HTR8 ⁄ SVneo were blocked by IFN-cR1 or IFN-cR2 antibodies (a–d).
HTR8 ⁄ SVneo were pre-treated with anti-IFN-cR1 or IFN-cR2 antibodies, followed by either dNK-CM or IFN-c stimulation. At the end of the treat-
ment, the cells were fixed, permeabilized, and then stained for FACS analysis. IFN-cR1 and IFN-cR2 antibodies showed greatest inhibition of dNK-
CM-induced (a) and IFN-c-induced Tyr701 (c) phosphorylation, and to a lesser extent, dNK-CM-induced (b) and IFN-c-induced (d) phosphorylation.

and network structures on thick-layer Matrigel both
in primary CTB and in HTR8 ⁄ SVneo, accompanied
by a higher level of VEGF-C secretion as compared
to VEGF-A.13 In this study, we found that dNK-CM
was able to facilitate VEGF-C secretion by both pri-
mary CTB and the EVT cell line, HTR8 ⁄ SVneo
(Fig. 1a,b) in the absence of Matrigel.
IFN-c contained in the dNK-CM appeared to con-
tribute significantly to VEGF-C secretion by CTB and
HTR8 ⁄ SVneo, as both IFN-c neutralizing antibody
and IFN-cR1 or IFN-cR2 antibodies were able to
partially, but significantly, block dNK-CM-induced
VEGF-C secretion (Fig. 2). dNK-CM contained approxi-
mately 3.5 ng ⁄ mL IFN-c. However, the VEGF-C stimu-
latory ability of dNK-CM was greater than that of
the equivalent concentration of recombinant IFN-c
alone (Figs 1 and 3). Furthermore, the ability of
recombinant IFN-c to promote VEGF-C secretion
was completely blocked by IFN-c blocking antibody
or IFN-cR1 and IFN-cR2 blocking antibodies
(Fig. 3c). Thus, the data indicate that in addition to
IFN-c, other soluble factors contained in the dNK-
CM contribute to the stimulation of VEGF-C secre-
tion. For example, we observed that TNF-a in
dNK-CM increased VEGF-C production, and its effect
was reduced by TNF-a blocking antibody. Recombi-
nant TNF-a also had the ability to induce VEGF-C
secretion by HTR8 ⁄ SVneo (data not shown).
Both Tyr701 and Ser727 phosphorylations of
STAT1 are required for full STAT1 transcriptional
and anti-pathogenic activity.20,21 Both Tyr701 and
Ser727 of STAT1 were quickly phosphorylated upon
exposure to either dNK-CM or IFN-c in the study.
After 10 min of exposure, elevated phosphorylations

American Journal of Reproductive Immunology 67 (2012) 101–111

108 ª 2011 John Wiley & Sons A/S
 dNK-CM MEDIATES VEGF-C SECRETION IN EVT

(a) (b)

Fig. 6 Both dNK-CM- and IFN-c-mediated VEGF-C inductions were reduced by SB202190 (a) and JI-1 (b) treatment. HTR8 ⁄ SVneo were pre-treated
with SB202190 or JI-1, and then exposed to either dNK-CM or IFN-c for VEGF-C inductions. The results showed that both SB202190 and Janus
kinase inhibitor-1 decreased dNK-CM- or IFN-c-induced VEGF-C secretions. Dotted line and P-value: Kruskal–Wallis non-parametric ANOVA. Dunn’s
multiple post hoc test with SB202190 (a): *P < 0.05 compared with control; **P < 0.01 compared with ctrl; ***P < 0.05 compared with IFN-c;
#
P < 0.05 compared with CM. Dunn’s multiple post hoc test with JI-1 (b): *P < 0.05 compared with control; **P < 0.01 compared with control;
***P < 0.01 compared with IFN-c; #P < 0.05 compared with IFN-c.

Fig. 7 Both dNK-CM- and IFN-c-induced VEGF-C secretions were reduced by siRNA-mediated STAT1 knockdown on HTR8 ⁄ SVneo. Cells were pre-
transfected with STAT1 siRNA and non-targeting control siRNA and were then stimulated with either dNK-CM or IFN-c for VEGF-C inductions. The
results demonstrated that STAT1 siRNA, but not non-targeting control siRNA, partially but significantly blocked dNK-CM- and IFN-c-mediated VEGF-C
inductions. The data shown (median) were from one representative experiment. Dotted lines and P-values: Kruskal–Wallis non-parametric ANOVA.

were observed (Fig. 4). High levels of phosphoryla-
tion were maintained up to 6 hr after treatment
(data not shown). HTR8 ⁄ SVneo showed a high basal
level of Tyr701 or Ser727 phosphorylation under
control conditions (DMEM supplemented with 10%
FBS). Clearly, detectable elevations of both Tyr701
and Ser727 phosphorylation were detected using
FACS after both dNK-CM and IFN-c treatments
(Fig. 4a–d). High basal levels of Tyr701 and Ser727
phosphorylation explain the basal level VEGF-C
secretion from HTR8 ⁄ SVneo, indicating the existence
of VEGF-C-inducing factors in FBS. Elevated Tyr701

American Journal of Reproductive Immunology 67 (2012) 101–111

ª 2011 John Wiley & Sons A/S 109
 EASTABROOK ET AL.
and Ser727 phosphorylation induced by dNK-CM or
IFN-c were reduced by IFN-cR1 or IFN-cR2 blocking
antibodies (Fig. 5a–d), indicating specific ligand stim-
ulation by IFN-c. The reduction in both dNK-CM-
induced Tyr701 (Fig. 5a) and IFN-c-induced Tyr701
(Fig. 5c) phosphorylation was seen to a greater
extent than the reduction in dNK-CM-induced
(Fig. 5b), and particularly IFN-c-induced Ser727
phosphorylation (Fig. 5d). dNK-CM-induced Ser727
activation may be acting through alternative means
aside from IFN-c receptors. TNF-a, another dNK-pro-
duced pro-inflammatory cytokine, has also been pro-
posed in mediating p38 activation.35,36
It had been shown that Tyr701 was activated by
JAK, with the most likely candidate being JAK2.20,21
JI-1 resulted in significant inhibition of VEGF-C
secretion from HTR8 ⁄ SVneo (Fig. 6b), indicating the
involvement of JAK kinase in the process. STAT1
Ser727 activation may or may not rely on
p38.27,32,33 The involvement of other kinases has
been proposed.22,23,34 The p38 MAPK inhibitor
SB202190 was able to significantly inhibit VEGF-C
secretion from HTR8 ⁄ SVneo (Fig. 6a), implying that
p38 MAPK participated in dNK-CM- or IFN-c-medi-
ated VEGF-C responses. However, whether or not
p38 mediates STAT1 Ser727 phosphorylation has yet
to be determined.
HTR8 ⁄ SVneo cells transfected with STAT1 siRNA
at concentration as low as 12.5 nm were able to
knock down STAT1 mRNA by >95%. This effect was
not seen with control siRNA (data not shown).
VEGF-C secretion from HTR8 ⁄ SVneo cells was also
reduced by STAT1 siRNA (Fig. 7). However, as much
as 50 nm STAT1 siRNA was not able to block VEGF-
C secretion completely. Other STAT1-independent,
IFN-c-induced transcription factors such as STAT3
and STAT5,37,38 NF-kB39 and AP-140,41 are also likely
activated, as previously reported in other systems.
In summary, we have shown that maternally
derived dNK cells are capable of influencing fetal-
derived trophoblast secretion of angiogenic factors,
including VEGF-C. As a result, dNK may influence
trophoblast endovascular differentiation and uterine
artery remodeling. IFN-c secreted by dNK plays an
important role in VEGF-C induction in EVT. Both
the p38 MAPK and the conventional JAK-STAT1
pathways are involved in dNK-CM- and IFN-c-medi-
ated VEGF-C secretions by HTR8 ⁄ SVneo. Other
STAT1-independent, IFN-c-induced transcription fac-
tors may also contribute and warrant further investi-
gation.
110
Acknowledgements
We thank the CARE Program, BC Women’s Hospital
and Health Centre, for their assistance in gaining
access to reproductive tissue, and Dr. Geoff Ham-
mond and Dr. Peter Leung for access to laboratory
equipment. The study was funded by a pilot grant
from the Infection and Immunity Program, Child
and Family Research Institute (CFRI). YH is funded
through grants from the Canadian Institutes for
Health Research, from whom PvD receives salary
support. PvD receives salary support from CFRI.
PvD, JPD, and RT receive salary support from the
Michael Smith Foundation for Health Research.
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