Post on 29-Mar-2023
ORIGINAL ARTICLE
Heparin promotes soluble VEGF receptor expression in humanplacental villi to impair endothelial VEGF signaling
S . DREWLO,*� K . LEVYTSKA ,* M. SOBEL ,�� D. BACZY K ,* S . J . LYE*� and J . C . P . K INGDOM*��*Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital; �Maternal-Fetal Medicine Division,
Department of Obstetrics and Gynecology, Mount Sinai Hospital; and �Department of Obstetrics and Gynecology, University of Toronto,
Toronto, Canada
To cite this article: Drewlo S, Levytska K, Sobel M, Baczyk D, Lye SJ, Kingdom JCP. Heparin promotes soluble VEGF receptor expression in human
placental villi to impair endothelial VEGF signaling. J Thromb Haemost 2011; 9: 2486–97.
Summary. Background: Severe preeclampsia is characterized
by hypertension, renal injury and placental dysfunction.
Prothrombotic disorders are discovered in 10–20% of women
with preeclampsia, providing the rationale for prescribing low-
molecular-weight heparin (LMWH) in future pregnancies.
Heparin has diverse molecular actions and appears to reduce
the recurrence risk of preeclampsia in women without pro-
thrombotic disorders. The placenta-derived anti-angiogenic
splice-variant protein soluble vascular endothelial growth factor
(VEGF) receptor-1 (sFLT1) is strongly implicated in the
pathogenesis of the underlying endothelial dysfunction. As
the placental syncytiotrophoblast is the principal source of
sFLT1, we tested the hypothesis that heparin suppresses
placental sFLT1 secretion.Methods and Results:First trimester
placental villi exposed to LMWH (0.25–25 IU mL)1) in an in
vitro explant model significantly increased the expression and
release of sFLT1 by the syncytiotrophoblast into culturemedia,
reducing phosphorylation of FLT1 and KDR receptors in
cultured human umbilical vein endothelial cells. This response
was significantly diminished in placental villi from healthy term
pregnancies. Placental villi from severely preeclamptic pregnan-
cies had a higher baseline sFLT1 release, compared with first
trimester placental villi and did not respond to LMWH
treatment. LMWH promoted villous cytotrophoblast prolifer-
ation (BrdU incorporation) and impaired syncytial fusion-
differentiation, causing syncytiotrophoblast apoptosis (by cas-
pase 3&7 activity and TUNEL staining) and necrosis (ADP/
ATPratio).Conclusion:LMWHpromotes sFLT1synthesis and
release from first trimester placental villi in a manner similar to
that of severely preeclamptic placental villi, which antagonizes
VEGF signaling in endothelial cells. These effects in part are
mediated by an interaction between heparin and the cytotroph-
oblasts that regenerates the overlying syncytiotrophoblast
responsible for sFLT1 secretion into the maternal blood.
Keywords: heparin, human placental villi, preeclampsia, solu-
ble FLT1.
Introduction
Preeclampsia (PE) is a potentially life-threatening hypertensive
disorder of pregnancy characterized by vascular dysfunction
and systemic inflammation involving the brain, liver and
kidneys of the mother [1]. Most cases that occur near term are
relatively mild and are associated with a variety of maternal
risk factors, while the more severe forms of preeclampsia begin
during the second trimester and are associated with severe
placental dysfunction, extreme preterm birth and perinatal
handicap or death, and an increased risk of cardiovascular
disorders [2]. The disease may be palliated, to advance
gestation, using antihypertensive drugs, but can only be
effectively reversed by removal of the placenta at delivery.
Pathologic analysis of placentas from severely preeclamptic
women delivered preterm demonstrates high rates of infarction
of placental villi [3], and maternal vascular thrombosis of spiral
arteries within the decidua. Women with severe preeclampsia
have high circulating levels of soluble splice variant forms of the
FLT1 receptor (sFLT1), that bind vascular endothelial and
placental growth factors (VEGF and PLGF) as well as the
soluble Endoglin (sENG) that binds TGF-beta [4]. Disrupted
signaling of these ligands at the endothelial surface synergis-
tically contributes to endothelial cell dysfunction characterized
by elevated systemic vascular resistance [5]. Severely pre-
eclamptic placentas strongly express sFLT1 [6] and the levels of
both sFLT1 and sENG in the maternal circulation decline
rapidly after delivery in association with the clinical resolution
of hypertension, hepatic dysfunction and proteinuria [7].
Experimental evidence in rodent models strongly implicates
sFLT1 in disease pathogenesis [8]. In human pregnancy, sFLT1
is secreted by the healthy first trimester placenta and is
augmented by hypoxia in in vitro models [9].
Correspondence: Sascha Drewlo, Samuel Lunenfeld Research
Institute, Mount Sinai Hospital, 25 Orde Street, Room 6-1020,
Toronto, ON M5T 3H7, Canada.
Tel.: +1 416 586 8322; fax: +1-416 586 8565.
E-mail: Drewlo@lunenfeld.ca
Received 5 July 2011, accepted 26 September 2011
Journal of Thrombosis and Haemostasis, 9: 2486–2497 DOI: 10.1111/j.1538-7836.2011.04526.x
� 2011 International Society on Thrombosis and Haemostasis
Given the associations between severe disease, maternal
vascular thrombosis and maternal thrombophilia, several trials
have explored the potential role of prophylactic low-molecular-
weight heparin (LMWH) in preventing preeclampsia. They did
not, however, include the analyses of the placenta at delivery to
determine the likely mechanism of action [10]. Despite being a
more potent anticoagulant than low-dose aspirin, LMWH had
only a limited effect on disease progression and no clinical study
has thus far explored the effect of heparin on the placental
production of anti-angiogenic proteins such as sFLT1.
Low-molecular-weight heparin appears to exert a variety of
non-anticoagulant actions, including the ability to promote
prolonged survival in metastatic cancer patients [11]. The
concept of a non-anticoagulant action of heparin in the
placenta is supported by several pieces of evidence. First,
heparin is required as a co-factor for fibroblast growth factor 4
(FGF4) in the maintenance of rodent trophoblast stem cells
[12]. Second, the FGF receptor 2 (FGFR2) signaling via the
FGF4/heparin is expressed in a subset of villous cytotropho-
blasts that proliferate within floating first trimester villi [13].
Third, the villous cytotrophoblasts within these explanted villi
proliferate in response to FGF4/heparin treatment [13]. Lastly,
heparin affects proliferation and differentiation in trophoblast-
derived cell lines [14,15].
We tested the hypothesis that LMWH interacts with both
normal and pathologic human placental villi in a non-antico-
agulant manner to favorably affect maternal vascular function.
Our findings, however, indicate that LMWH disrupts the
physiological function of the villous trophoblast compartment,
significantly increasing sFLT1 release and impairing VEGF
signaling in endothelial cells in a manner that can be reversed
in vitro by exogenousVEGF.Our findings challenge thewidely-
held view that heparin prevents complications of pregnancy
attributable to placental dysfunction and illustrates the impor-
tance of understanding the interactions of this complex
naturally-derived macromolecule with the human placenta.
Materials and methods
Placental villous tissue cultures
Placental tissue was obtained from elective social terminations
via the Mount Sinai Hospital Biobank with research ethics
board approval (MSH 10-0128-E). Individual clusters of
floating villi (15–35 mg pieces) from placentas in the first
(week 8–12) and early second (week 13–15) trimesters of
pregnancy were dissected in sterile cold PBS (Multicell,
Woonsocket, RI, USA) and cultured as previously described
[16]. Pathological samples and controls were classified into the
following groups: (i) pure severe early-onset intrauterine
growth restriction (IUGR); (ii) pure preeclampsia (PE); (iii)
mixed preeclampsia/IUGR (PE-IUGR); and (iv) healthy term
controls (for details see Table 1 and Supporting Information).
Samples were treated as described above. Experiments used
floating villi with an intact syncytiotrophoblast. The media was
pre-incubated under culture conditions at 37 �C, in 5% CO2
and in 8% pO2 for at least 1 h with and without increasing
concentrations of LMWH (see below). The oxygen tension was
chosen to mimic the early development of the uteroplacental
circulation in the transition to the second trimester [17]. Tissue
explants were cultured for a maximum period of 72 h, at which
point the media was collected and stored at )80 �C for further
analysis. Experimental controls were time 0 h, vehicle (saline)
and non-treatment controls. Vehicle and non-treatment con-
trols were not significantly different; in all experiments data
from vehicle controls are shown unless otherwise stated.
Healthy term placentas were used as third trimester control
tissues (see Table 1).
Heparin Dalteparin sodium LMWH (Fragmin, Pfizer
Canada Inc., Kirkland, Canada) of the same lot in
concentrations of 0.25, 2.5 and 25 IU mL)1 in saline was
used throughout all experiments [18].
Table 1 Clinical characteristics of patients with severe early onset preeclampsia (PE), preeclampsia with IUGR (PE-IUGR) and IUGR and term controls
for pathologic studies
Term IUGR PE-IUGR PE
Maternal age (years) 34.6 ± 2 311 ± 5.5 35.7 ± 6.3 34.8 ± 5
GA (weeks) 38.3 ± 1.4a 31.6 ± 5 33.7 ± 2.5 33.6 ± 3.3
Delivery (c/s %) 100 70 83 83
Systolic blood pressure (mmHg) 124.5 ± 10.3 138.5 ± 10.7 159.5 ± 17.9b 168.5 ± 12.5b
Diastolic blood pressure (mmHg) 77.8 ± 3.2 85.2 ± 6.5 104.3 ± 11.4 97 ± 5.6
Birth weight (g) 4114 1009c 1477c 2012
Umb art
Normal flow
0 0 4/6 2/6
Umb art
ARED flow
0 7/7 2/6 1/6
Umb art flow
Not recorded
7/7 0/7 0 3/6
n 7 7 6 6
Data presented as mean + SD for maternal age and gestational age (GA). Birth weight (BW) is shown as the % in the given range (% in brackets).
Letters (a, b, c) indicate significant differences from other values in the same category, P < 0.01. Differences in maternal age, gestational age and
blood pressures were determined using ANOVA with post-hoc Bonferroni correction for multiple comparisons. Umb Art, umbilical artery; ARED,
absent or reversed end diastolic flow.
LMWH controls sFLT1 release in placenta 2487
� 2011 International Society on Thrombosis and Haemostasis
BrdU labeling Culture media was substituted with BrdU
(3 days) solution to label total mitotic activity of cells (BrdU
kit; Hoffmann-LaRoche, Mississauga, ON, Canada). After
3 days, explants were fixed and wax-embedded. Sections were
stained according to the manufacturer�s instructions. A
minimum of six explants per LMWH concentration with 10
random pictures per treated explant (40 ·) were used to count
the number of BrdU positive and negative trophoblast nuclei
per field of view, comprising proliferating cytotrophoblast and
syncytially-fused nuclei.
qRT-PCR
RNAwas extracted fromplacental explants using the TRIZOL
(Invitrogen, Burlington, Canada) method combined with the
Qiagen RNA Plus Purification kit (Qiagen, Toronto, Canada).
RNA quality was verified by using a nanodrop machine
(VWR). RNA (1 lg) was reverse-transcribed according to the
manufacturer�s instructions (Applied Biosystems Canada,
Streetsville, ON, Canada). Real-time PCR was performed on
an Eppendorf qPCR System (ABI) GCM1 [16] and sFLT1
primers (FW5¢-3¢; TCA GGC TCG GAG GAG ATG; RV;
CAA ACG TGC ACC AAG TCG GC) were designed using
PRIMER EXPRESS 2.0 software (Applied Biosystems, Foster
City, CA, USA). The Comparative CTMethod (ABI technical
manual) was used to analyze the real-time PCR. The expression
of GCM1 and FLT1 genes was normalized to the geometric
mean of SDHA (succinate dehydrogenase complex, subunit A,
flavoprotein) and TBP (TATA box binding protein) genes [16]
and expressed as fold change relative to non-silenced (NS)
control (n = 7).
Protein analysis Protein was extracted from snap frozen
tissue samples by adding the appropriate volume of RIPA
buffer or where appropriate PBS with 0.1% Nonident and
standard protein inhibitor cocktails (Thermo Fisher Scientific,
Rockfort, IL, USA). Soluble supernatants were used for
protein quantification (Bradford-reagent) (BioRad, Missis-
sauga, Canada). (A) DuoSet or (B) Quantikine ELISA kits
(R&D Systems, Burlington, Canada) were used for VEGF (B),
sFLT1 (VEGF receptor-1; A) and PLGF (B), total as well as
phospho FLT1 (A), KDR (VEGF receptor-2; A) and
sEndoglin (B) to quantify proteins with a TecanTM Infinite
photometer (Tecan, Mannedorf, Switzerland). Human
chorionic gonadotrophin (hCG) was measured in culture
media using the Delfia System from Perkin Elmer (Wallac,
Roxbury, MA, USA) on a Victor 2 machine. Data were
normalized to wet weight or protein amount.
Cycloheximide treatment Placental explants were treated
withheparin (0–25 IU mL)1),withandwithout 50 lmol L)1 of
cycloheximide (ribosome inhibitor) (Sigma, Oakville, Canada)
to distinguish the effects of LMWHtreatment on sFLT1 release
from placental villi (pre-existing vs. de-novo synthesis). After
28 h of culture, explants and media were collected and sFLT1
levels assessed using ELISA (as described above).
TUNEL assay staining in paraffin-embedded tissue sections
of explants was performed by the Toronto Centre of Pheno-
genomics using the Hoffmann LaRoche kit. The ATP/ADP
ratio in the tissue was determined with the ApoGlow
(Promega, Madison, WI, USA) bioluminescent enzyme kit
and was used to estimate the dominant cell fate (apoptosis vs.
necrosis). The activity of initiator caspases 3/7 was assessed by
using CaspaGlow fluorescent peptide based assay from Pro-
mega according to the manufacturer�s recommendations. Free
placental DNA was isolated from the culture media using the
QIAmpDNABloodMini Kit (Qiagen, Toronto, Canada) and
separated on agarose gels and stained with SybrSafeTM
(Invitrogen, Burlington, Canada) and photographed.
Standard Western blotting was carried out with a BioRad
system; 50 lg of protein was SDS-poly-acrylamide gel sepa-
rated and blotted on a PVDFmembrane, which was blocked in
5% Blotting-Grade Blocker (BioRad, Missisasuga, Canada),
washed with (0.1%,v/v)Tween Tris-buffer saline and incubated
with Cytokeratin-7 antibody (DAKO Canada, Burlington,
Canada) overnight (1:5000). Membranes were washed, incu-
bated with anti-rabbit-HRP (1:4000) antibody for 1 h and
reaction was detected using ECL-reagent (GE HealthCare,
Baie d�Urfe, QU, Canada).
VEGF receptor phosphorylation assays
HUVEC cells were cultivated in six well plates in growth factor
reduced media until 85% confluency. Cells were starved in
growth factor-depleted media for 8 h to induce dephosphor-
ylation of the VEGFR1&2 receptors. Cells were then treated
with conditioned media from either LMWH-treated explants
or controls. The phosphorylated forms of VEGFR1&2 were
quantified using ELISA (R&D Systems) (50 lg total protein)
and the ratio of total receptor to phosphorylated receptor was
determined.
Histology and immuno-histochemistry
Paraformaldehyde-fixed placental explants were wax-embed-
ded for hematoxylin and eosin histology and immuno-histo-
chemistry. Antibody concentrations and antigen retrieval
conditions (citrate buffer) were performed according to the
manufacturer�s recommendations (R&D Systems; total FLT1
AF321; 1:300) and stained as previously described [16].
Statistical analysis
All experiments were performed as a minimum of six biological
and technical triplicates unless otherwise stated. Where appli-
cable, data have been normalized in individual experiments
relative to the internal control/vehicle treatment to account for
inter-tissue variation. One-way ANOVA was used to analyze
multiple groups with post-test analysis (Bonferroni) where
applicable.Dataarepresentedasmeanwithstandarderror (SE).
PRISM�5.0 software (GraphPad, La Jolla, CA, USA) was used
and P values £ 0.05 were considered significant (*P < 0.05,
2488 S. Drewlo et al
� 2011 International Society on Thrombosis and Haemostasis
**P < 0.01, ***P < 0.001). (An extended �Material and
methods� section is available in the Supporting Information.)
Results
LMWH-induced sFLT1 release declines with gestational age
but persists in severely preeclamptic placental villi
First and second trimester tissues showed similar data and were
therefore grouped in all experiments as the early pregnancy
group. We compared the release of sFLT1 obtained from early
pregnancy (8–15 weeks) with healthy term and pathological
samples (Fig. 1, Table 1). Baseline release of sFLT1 in explants
cultured at 8% pO2 without LMWH declined significantly
from early pregnancy (8–15 weeks) (803 ± 99.1 pg mg)1;
n = 38) to term pregnancy (152 ± 20.9 pg mg)1 tissue;
n = 7). Intermediate secretion levels were found in IUGR
(538 ±120.8 pg mg)1; n = 7) and PE-IUGR groups
(753 ± 210.5 pg mg)1; n = 6) while the highest release of
sFLT1 was found in pure severe PE (1623 ±313 pg mg)1;
n = 6) (P = 0.006 for comparison with early pregnancy
group) (Fig. S1; Table 2). The introduction of LMWH signif-
icantly increased sFLT1 release in the early pregnancy group
(up to 2199 ± 64.9 pg mg)1) and the term pregnancy group
(up to 285 ± 21.5 pg mg)1) (data normalized to untreated
controls; Fig. 1B,C). The sFLT1 levels in the early pregnancy
group exposed to LMWH were as high as those found at
baseline in preeclamptic subjects (Table 2, Figs. S1 and S2).
In the mixed PE-IUGR groups (Fig. 1E), LMWH did not
alter relative sFLT1 secretion. The pure PE and IUGR groups
showed a trend towards a reduction of sFLT1 release in
response to increasing LMWH concentrations (Fig. 1D,F).
2500 300 8–15 weeksexplants
200
100
0
*
***
**
*
*
*
****
*** ***
2000 sFLT-1 baselineexpression
1500
1000
500
0
250 Term expalnts
200
150
100
50
0
250 Severe preeclampsia(PE) explants
200
150
100
50
0
250
200
150
100
50
0
Severe IUGR250
200
150
100
50
0
Mixed severePE with IUGR
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Term IUGR PE-IUGR PE8–15 weeks
sFLT
1 re
leas
e(p
g m
L–1
mg
tissu
e–1 )
Rel
ativ
e sF
LT1
rele
ase
vs c
ontr
ol
Rel
ativ
e sF
LT1
rele
ase
vs c
ontr
olR
elat
ive
sFLt
1re
leas
e vs
con
trol
Rel
ativ
e sF
LT1
rele
ase
vs c
ontr
ol
Rel
ativ
e sF
LT-1
secr
etio
n vs
con
trol
A B
DC
E F
Fig. 1. sFLT1 release into the culture media of untreated and cultured explants from first trimester (n = 38), term (n = 7), pure PE (n = 6), PE with
IUGR (n = 6) and pure IUGR (n = 7) tissue is shown in (A). All groups showed significantly higher sFLT1 baseline expression compared with the term
control group. Additionally, PE secreted significantly higher amounts compared with the early pregnancy group (A). Relative changes of sFLT1 release in
response to LMWH in each group are presented in (B–F). Early pregnancy showed the highest significant response (3-fold) to LMWH, with sFLT1 levels
as high as those found in the PE group (1634–2199 vs.1672–2744 pg mg)1 tissue; Table 2). Term explants showed a moderate but significant induction of
sFLT1 release into the culture media of up to 95% (152–285 pg mg)1 tissue (C)). Severe pure PE and pure IUGR explants demonstrated an LMWHdose-
dependent trend towards a reduction in sFLT1 release (D/F). This effect was significant in the IUGR group when 25 IU mL)1 were used in the culture
media, resulting in a mean sFLT1 reduction of 43.8%. The IUGR-PE group showed no significant sSFLT1 changes in response to LMWH ((E) Table 2).
LMWH controls sFLT1 release in placenta 2489
� 2011 International Society on Thrombosis and Haemostasis
This observation was significant at 25 IU mL)1 in the IUGR
group, with a mean reduction of 43.8% (P < 0.05).
LMWH interaction with placental villi increased PLGF release
but impaired VEGF signaling in HUVECs
To investigate the biological significance of the above-described
phenomenon, we used ELISA to quantify the release of disease-
relevant proteins such as of sENG, VEGF and PLGF from
floating explants into the media in response to increasing
concentrations of LMWH (72 h, n = 12 experiments, all
conducted in triplicates) (Fig. 2). Low-dose LMWH had no
effect on sENG release while the highest dose significantly
reducedsENGrelease (P < 0.01;Fig. 2A).FreeVEGFwasnot
detectable, whereas PLGF increased significantly in response to
both2.5and25 IU mL)1 concentrationsofLMWH(P < 0.05;
Fig. 2B).Assuminga2:1 stoichiometric interactionbetween two
sFLT1 molecules and one molecule of either VEGF or PLGF,
these data predict a net anti-angiogenic response in the media
from floating early pregnancy villous explants (n = 12;
Fig. 2C). Interestingly, the switch in the ratio of sFLT1/PLGF
has been proposed as a screening test in maternal blood to
predict severe preeclampsia in the second trimester [19].
To characterize the mechanism of LMWH-induced sFLT1
release and distinguish the release of sequestered sFLT1 from
de-novo synthesis [20] we performed qRT-PCR for sFLT1
mRNA in parallel with sFLT1 immuno-histochemistry (for
total FLT1) in explants exposed to LMWH for 24–72 h of the
culture conditions (Fig. 3). sFLT1 mRNA increased signifi-
cantly about 3-fold across all LMWH concentrations
(P < 0.05) (Fig. 3A). This observation was time and dose
dependent. Parallel immuno-histochemical analysis using an
antibody directed to total FLT1 showed progressively increas-
ing staining of the outer syncytiotrophoblast layer, indicating
accumulation of the FLT1 protein (Fig. 3B–E). Collectively
these data indicate that LMWH promotes expression of both
total and sFLT1 and the release of the sFLT1 splice variant
and is consistent with the ELISA data demonstrating increased
release of the protein into the media. To further validate these
data we treated explants in parallel with cycloheximide for 28 h
to inhibit de-novo protein synthesis in the tissue. The effects of
cycloheximide treatment on sFLT1 release were measured
(Fig. S3). LMWH combined with cycloheximide significantly
increased sFLT1 levels in the culture media compared with the
cycloheximide without LMWH, suggesting that heparin solu-
bilizes pre-existing sFLT1, as recently shown by Sela et al. [20].
However, the dominant effect is due to denovo protein
synthesis, because the amount of sFLT1 in the heparin group
was significantly higher compared with matched cyclohexi-
mide-treated samples.
Table 2 sFLT1 secretion levels of normal and pathological villi in response to lowmolecular weight heparin (LMWH) (±= SEM).Note: normalized data
are shown in Fig. 2
sFLT1 (pg mg)1 tissue) Control 0.25 IU mL)1 2.5 IU mL)1 25 IU mL)1 n
Term tissue 152 ± 21 285 ± 22 248 ± 25 239 ± 27 7
IUGR 538 ± 87 404 ± 87 331 ± 28 332 ± 78 7
PE + IUGR 753 ± 210 848 ± 168 1064 ± 465 996 ± 334 6
PE 1623 ± 313 1672 ± 350 2744 ± 114 1860 ± 657 6
8–15 weeks 619 ± 130 1634 ± 139 2198 ± 55 2199 ± 88 38
250
200
150
100
50
0
0
25
10
5
15
20
0
20
40
60
80
Control
sFLT1PLGF
0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
**
***
*
**
*** ***
****
Sol
uble
end
oglin
rel
ease
(pg
mL–
1 m
g tis
sue–
1 )P
LGF
rel
ease
(pg
mL–
1 m
g tis
sue–
1 )P
LGF
and
sF
LT1
rele
ase
(fM
ole)
A
B
C
Fig. 2. Release of pro-angiogenic placental growth factor (PLGF) and
the anti-angiogenic protein soluble Endoglin (sENG) from first trimester
placental villi in response to LMWH are presented in (A) and (B)
(n = 12). LMWH repressed significantly sENG release at 25 IU mL)1
(A). PLGF release was significantly increased in cultures containing 2.5
and 25 IU mL)1 LMWH (B). Assuming a stochiometric interaction (2:1)
between sFLT1 and VEGF or PLGF, the magnitude of the rise in sFLT1
is predicted to antagonize PLGF-directed signaling (C). Free VEGF was
undetectable in all concentrations of LMWH, suggesting low expression
and/or complete binding by secreted sFLT1 (data not shown).
2490 S. Drewlo et al
� 2011 International Society on Thrombosis and Haemostasis
To explore net biologic implications of our findings on
endothelial cells exposed to these pro/anti-angiogenic factors in
the media, (representing maternal blood), we determined the
influence of conditioned media from explants cultured in the
absence/presence of increasing concentrations of LMWH on
the phosphorylation of the endothelial cell receptors of VEGF
and PLGF, VEGFR1&2 (Fig. 4). CulturedHUVEC cells were
serum-starved for 8 h to return them to baseline phosphory-
lation status for the VEGFR1&2 receptors. Explant-condi-
tioned media significantly reduced the phosphorylation status
of both the VEGFR1&2 receptors in a dose-dependent manner
(Fig. 4A,B). This finding is consistent with the significant
increases in sFLT1 mRNA expression (Fig. 3) and protein
release (Fig. 2A, Fig. S3). To show that sFLT1was responsible
for the reduced receptor phosphorylation of VEGFR1&2 in
theHUVEC culture, VEGFwas successfully used in saturating
concentrations to reverse this effect. Additionally, secreted
sFLT1 was removed using anti-sFLT1 immuno-precipitation
to show the critical role of sFLT1 in the receptor phosphor-
ylation process (Fig. 4) [21]. Taken together, these data indicate
that LMWH interacts with first trimester floating villi in a
manner that is predicted to antagonize the actions of VEGF
and PLGF at the level of the maternal vascular endothelium in
vivo.
LMWH promotes villous cytotrophoblast proliferation
and syncytial fusion
As heparin is a co-factor for fibroblast growth factor 4 (FGF4),
which maintains trophoblast stem cells [12] and acts as a
Rel
ativ
e m
RN
AsF
LT1
expr
essi
on0
100
200
300A
B
D
C
E
6 h
24 h
72 h
**
*
*
*
*
*
Control
Control
0.25 IU mL–1
0.25 IU mL–1
2.5 IU mL–1
2.5 IU mL–1
25 IU mL–1
25 IU mL–1
Fig. 3. LMWH promotes sFLT1 expression in first trimester explants. RNA was extracted from first trimester placentae (n = 6) previously exposed
to different LMWH concentrations and harvested at various time points (6, 24 and 72 h) to assess expression levels of sFLT1. Primers were designed
to detect sFLT1 splice variants. sFLT1 mRNA expression levels were time and dose dependent (A). Immuno-histochemical analysis of total FLT1
localization, shows a progressive increase in total FLT1 staining in the syncytiotrophoblast (B–E), consistent with both the mRNA expression data (A) and
the release of sFLT1 into the media (Fig. 1).
LMWH controls sFLT1 release in placenta 2491
� 2011 International Society on Thrombosis and Haemostasis
mitogen in human placenta via the FGFR2 receptor [13], we
assessed the mitogenic response of villous cytotrophoblasts
within first trimester placental villi to LMWH. Floating
explants were exposed to increasing concentrations (0.25–
25 IU mL)1) of LMWH in 8% oxygen for 72 h of culture in
the presence of BrdU. Cytotrophoblast proliferation and
subsequent syncytial fusion was estimated by counting BrdU-
positive (villous cytotrophoblast and syncytiotrophoblast)
nuclei. Representative BrdU-labeled explants are shown in
Fig. 5(B–E). BrdU incorporation into dividing villous cytot-
rophloblasts that underwent syncytial fusion during the
experimental period was significantly increased in response to
higher doses of LMWH (Fig. 5A). During the culture period,
BrdU-positive nuclei resulting from syncytial fusion are seen to
enter the syncytiotrophoblast layer (Fig. 5B,E). These findings
suggest that LMWH promotes the turnover of the villous
trophoblast compartment.
Because sFLT1 synthesis is significantly increased in the
outer syncytiotrophoblast in response to LMWH, we also
measured the trophoblast transcription factor, glial cell miss-
ing-1 (GCM1) [16], during the 72-h culture period in 8% pO2.
qRT-PCR analysis demonstrated a significant (> 2-fold)
increase in mRNA expression of this differentiation marker
at 72 h in response to the lowest dose of LMWH, which is
analogous to prophylactic heparin doses used in pregnancy
(Fig. 6A). In parallel, a significant elevation in human chori-
onic gonadotropin (hCG) release into the media at the lowest
dose of LMWH was observed (Fig. 6B), suggesting increased
syncytial fusion and syncytiotrophoblast differentiation neces-
sary to promote hCG synthesis by lower dose LMWH.
As opposed to pathologic necrosis, apoptosis is a regulated
physiologic event during the maturation of the syncytiotroph-
oblast (for detailed review see [22]) (Fig. 6). The progression of
apoptosis was monitored using the caspase 3/7 and TUNEL
assays. The lowest (0.25 IU mL)1) doses of LMWH signifi-
cantly increased the relative caspase 3/7 ratio in explant extracts
at 72 h, consistent with augmented syncytial fusion, while the
highest concentrations of LMWHhad no effect compared with
control conditions (n = 7) (Fig. 6C). Physiologic apoptosis
and pathologic necrosis in response to LMWH were distin-
guished by analyzing the ratio of low energy ADP and high
energyATP using an enzyme-based assay (Fig. 6D).Generally,
healthy tissue has low amounts of ADP and high levels of ATP,
resulting in ratios below 0.5, as was seen under control
conditions (n = 6). Apoptosis results in higher levels of ADP
and ADP/ATP ratios of 0.5–1, which was observed during
0.25 IU mL)1 LMWH treatment. High ADP/ATP ratios
(> 1) indicate high levels of ADP (low ATP), which are
indicative of tissue necrosis, which was seen in explants exposed
to 2.5 IU mL)1 LMWH (Fig. 6D). The ADP/ATP ratio in
explants exposed to 25 IU mL)1 LMWH was significantly
increased, but remained in the apoptosis range (Fig. 6D).
Corresponding TUNEL-stained sections are shown in
Fig. 6(E–H) and demonstrate an increase in TUNEL-positive
syncytiotrophoblast nuclei in explants exposed to 25 IU mL)1
LMWH.
To confirm this switch from regulated physiologic apoptosis
to apo-necrosis we monitored the release of laddered free fetal
DNA into the media, indicating completion of the apoptotic
cascade in the syncytiotrophoblast. We found a reduced release
of laddered DNA in the media in response to higher doses of
LMWH (Fig. 7C). A key component of syncytiotrophoblast
physiology is the active release of epithelial cytokeratin-7
positive (CK-7) micro-particles into maternal blood [23]. We
monitored accumulated total protein release into the media
during exposure to LMWH (from 24 to 72 h) and performed
Western blotting for CK-7 on samples normalized for protein
in control conditions and in 2.5 IU mL)1 LMWH. Incremen-
tal LMWH exposure resulted in a substantial 6-fold reduction
Contro
l
0.25
IU m
L–1 +
100
ng m
L–1
VEGF
0.25
IU m
L–1
2.5
IU m
L–1
25 IU
mL–
1
0.25
IU m
L–1
IP-s
FLT
Contro
l
2.5
IU m
L–1 +
100
ng V
EGF
0.25
IU m
L–1
2.5
IU m
L–1
25 IU
mL–
1
0.25
IU m
L–1
IP-s
FLT
200
150
100
50
0
150
100
50
0
* ***
* ****
Rel
ativ
e ph
osph
oryl
atio
n of
FLT
1/ V
EG
FR
-1 c
ompa
red
to n
o he
p co
ntro
l
Rel
ativ
e ph
osph
oryl
atio
n of
KD
R/ V
EG
FR
-2 c
ompa
red
to n
o he
p co
ntro
l
A
B
Fig. 4. LMWH-conditioned media from first trimester placental villi
attenuates the phosphorylation of both VEGFR1 (FLT1) and VEGFR2
(KDR) receptors in HUVEC cells and is reversed by VEGF and sFLT1
immuno-precipitation. All concentrations of LMWH in early pregnancy
conditioned media significantly attenuated receptor phosphorylation of
both receptors VEGFR1 (A) and VEGFR2 (B) in HUVECs. These effects
were dose dependent for each receptor. sFLT1 specificity was demon-
strated by a significant restoration of phosphorylation for both receptors
by either the addition of a saturating level of exogenous VEGF
(100 ng mL)1) or the immuno-precipitation of endogenous sFLT1
(secreted in response to LMWH) with anti-sFLT1 coated protein A/G
beads (n = 5). These data demonstrate that LMWH-induced release of
sFLT1 from first trimester explants is capable of disrupting VEGF
signaling in endothelial cells.
2492 S. Drewlo et al
� 2011 International Society on Thrombosis and Haemostasis
in total protein release (Fig. 7A). Western blotting of explant
media from first trimester explants (and one healthy term
control explant) showed reduction of CK-7 in the secreted
protein (Fig. 7B). Collectively, these data indicate that lower
doses of LMWH, equivalent to levels that the placental villi
would be exposed to in pregnancy, induce syncytial fusion,
hCG secretion and apoptotic turnover. By contrast, supra-
physiologic doses of LMWHshift towards syncytiotrophoblast
necrosis with loss of energy-dependent protein shedding
(micro-particle) and laddered DNA release into the media.
Discussion
In this studyweemployedafloatingvillousexplantmodel [16] to
explore the hypothesis that heparin is capable of exerting non-
anticoagulant actions in the developing human placenta. Our
data demonstrate that LMWH interacts with first trimester
placental villi in a manner that is predicted to have negative
effects upon the maternal vascular endothelium. We showed a
significant increase in villous cytotrophoblast proliferation and
syncytiotrophoblast differentiation in response to low levels
of LMWH. Equivalent to or above maximal doses of LMWH
used in pregnancy appeared to induce either focal necrosis or
apoptosis of the syncytiotrophoblast. These changes due to
LMWHpromoted the releaseofpreformed/sequestered sFLT1,
as recently described by Sela et al. [20], but, more importantly,
also augmented the expression of sFLT1.
At present, the underlying mechanisms by which alterations
in villous trophoblast compartment physiology induce sFLT1
synthesis are unknown. Heparin is known to have a wide
variety of non-anticoagulant effects [24]. In vitro, heparin
participates as a co-factor in the FGF4-directed maintenance
of trophoblast cell lines and stem cells via symmetric cell
proliferation, thus preventing terminal differentiation
[12,14,15]. We previously demonstrated that in combination,
FGF4 and unfractionated heparin induce a similar pattern of
symmetric proliferation of villous cytotrophoblasts in first
trimester human placental villi [13]. Here, we explored the
effects of low-molecular-weight heparin across clinically-rele-
vant concentrations on human placental villi in vitro. We
previously demonstrated that FGF4 and heparin evoke a
mitogenic response in first trimester placental villi resulting in
the accumulation of proliferating villous cytotrophoblasts at
the expense of syncytiotrophoblast formation [13]. Present
experiments, however, were conducted with lower doses of
LMWH and without FGF4, reflecting the in vivo pregnancy
situation. In addition to our earlier work, we now demonstrate
an increased accumulation of BrdU-positive nuclei in the
No
hepa
rin2.
5 IU
mL–
1
25 IU
mL–
10.
25 IU
mL–
1
0
50
100
150
200* *
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Rel
ativ
e pr
olife
ratio
n In
dex
(Brd
U-p
ositi
ve/to
tal
CT
nuc
lei c
ontr
ol ti
ssue
–1)
20.0 µm 20.0 µm
20.0 µm20.0 µm
A
B C
D E
Fig. 5. LMWH promotes cytotrophoblast proliferation and syncytial fusion as assessed by BrdU incorporation over a time period of 72 h (B–E). Shown
are the BrdU-positive (brown) CT nuclei and SCT that entered the syncytium by cell-cell fusion during the culture period. The proliferation index was
determined as a percentage of positive vs. total CT and SCT nuclei. The summarized data (A) show a significant increase in CT proliferation and syncytial
fusion (*) in response to both 2.5 IU mL)1 and 25 IU mL)1 LMWH (77.6 ± 5.3 vs. 57 ± 3.3 non-treatment controls [n = 5]).
LMWH controls sFLT1 release in placenta 2493
� 2011 International Society on Thrombosis and Haemostasis
syncytiotrophoblast, an upregulation of GCM1 mRNA
expression and an elevated hCG secretion into the media.
These data suggest that LMWH can stimulate the asymmet-
rical mitosis in villous cytotrophoblasts, promoting the daugh-
ter GCM1-positive cells to undergo syncytial fusion. In
comparison to the highest dose of LMWH above therapeutic
dosing levels, we detected no effect on GCM1 mRNA
expression or hCG secretion, but observed biochemical
evidence of explant necrosis and increased apoptosis in the
syncytiotrophoblast via TUNEL labeling. Furthermore, we
0
100
200
300
0.0
0.5
1.0
2.0
Necrosis
Apoptosis
1.5
0
50
100
150
200
Rel
ativ
e G
CM
1 m
RN
Aex
pres
sion
(%
)
Rel
ativ
e hC
Gs
(%)
0
100
200
300
400
Rel
ativ
e ca
spas
e 3/
7 a
ctiv
ity
Apo
ptos
is v
s N
ecro
sis
(A
DP
/AT
P r
atio
)
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control 0.25 IU mL–1
0.25 IU mL–1
2.5 IU mL–1
2.5 IU mL–1
25 IU mL–1
Control
Control
0.25 IU mL–1
2.5 IU mL–1
25 IU mL–1
25 IU mL–1
* *
***
**
A B
C
E
G H
F
D
Fig. 6. High-dose LMWH (25 IU mL)1) impairs syncytial fusion and promotes syncytiotrophoblast apoptosis and necrosis. Syncytiotrophoblast for-
mation was monitored by mRNA expression of the transcription factor GCM1 required for syncytial fusion. 0.25 IU mL)1 LMWH significantly
promoted GCM1 transcription (A) (n = 7) and release of hCG into the media (B) (n = 8). These data suggest accelerated syncytial fusion. Energy-
dependent apoptosis is a physiological part of the SCT turnover. This process may be aborted by pathologic signals, such as restricted syncytial fusion,
resulting in focal necrosis. Apoptosis progression in response to LMWHwas monitored in several ways. Relative activities of the execution caspase 3 and
initiator caspase 7 were measured in explant protein extracts and compared with control explants. 0.25 IU mL)1 LMWH increased the caspase 3/7 ratio
(n = 7), indicating accelerated apoptosis in line with accelerated differentiation and elevated hCG secretion (C). ADP/ATP levels were assessed to
distinguish functional apoptosis from necrosis (D). Apoptosis results in higher levels of ADP (ratio 0.5) while high levels (> 1) indicate necrosis. Healthy
tissue has low ADP ratio (< 0.5). In comparison to control explants, 2.5 IU mL)1 LMWH (necrotic) and 25 IU mL)1 LMWH (apoptotic/necrotic)
significantly increased explant ADP/ATP ratios (n = 6). Corresponding TUNEL assays were performed on paraffin sections to visualize late stage
apoptotic/apo-necrotic events in the treated explants (E–H). Increased TUNEL labeling was noted in 2.5 and 25 IU mL)1 LMWH.
2494 S. Drewlo et al
� 2011 International Society on Thrombosis and Haemostasis
observed reduced explant release of cytokeratin-7 micro-
particles and loss of physiologic DNA laddering that occurs
during normal syncytiotrophoblast apoptosis. Therefore very
high levels of LMWH may be capable of disrupting, rather
than promoting, normal syncytiotrophoblast formation.
We did not investigate in detail the shedding of placental
cellular debris into the culture wells, because our focus was on
the active release and displacement of soluble pro- and anti-
angiogenic factors into the media. We are aware that sFLT1
associates with nano-particles released by both the placenta
and by the maternal immune system and might play a
significant role in sFLT1 trafficking [25].
To determine if LMWH could modulate the risk of
preeclampsia by altering the release of proteins that participate
in the regulation of angiogenesis, we studied their release into
the culture media of the explants. Pro-angiogenic placental
growth factor (PLGF) increased proportionally in small
amounts across the LMWH range, whereas free VEGF was
not detected in the media. The anti-angiogenic soluble receptor
of the transforming growth factor-beta soluble endoglin (sEng)
was unaffected by lower doses of LMWH and was reduced
under highest LMWH concentration exposure. By contrast,
sFLT1 significantly increased in response to all LMWH
concentrations, up to levels found in culture media of
preeclamptic villi. The biologic relevance of this finding was
demonstrated by significant reduction in VEGF-induced
receptor phosphorylation of VEGF-R1 and VEGF-R2 in
endothelial cells following LMWH treatment. This signal
disruption was specific as it could be reversed by exogenous
VEGF and the depletion of sFLT1 by immuno-precipitation.
We predict that conditioned media from severely preeclamptic
villi would show the same response, because they had nearly
identical baseline and LMWH-induced sFLT1 release
responses. Similar baseline responses of both sera and placental
explants have been demonstrated in preeclamptic villi [21,26].
It has been shown that PLGF, VEGF and sFLT1 each bind
to heparin. A recent paper by Sela et al. showed that preformed
surface bound sFLT1 can be displayed by heparin from the
explant surface [20]. However, these authors did not look for
de novo expression of sFLT1 nor did they employ immuno-
histochemistry to detect FLT1 expressed in the villi. By
contrast, our data suggest that both mechanisms (release of
preformed sFlt1 and promoted expression of sFlt1) occur when
placental villi are exposed to LMWH.
Due to the high affinity of heparin to multiple proteins [1],
it is possible that heparin disturbs the equilibrium of several
growth factors and other molecules at the syncytiotrophoblast
surface. The present experiments were confined to a com-
monly-used LMWH in pregnancy. The potential of unfrac-
tionated heparin (UFH) to induce similar changes is
unknown. However, we recently demonstrated differences
between LMWH and UFH in their ability to modulate
angiogenesis in conditioned media from first trimester villous
explants. High-dose LMWH, but not UFH, inhibited angio-
genesis, while at lower concentrations, relevant to clinical
practise, both forms of heparin promoted angiogenesis [27].
Here, under LMWH treatment conditions, both the first
trimester and the term explants doubled their baseline sFLT1
secretion, though the elevation in the term group was
significantly below the levels observed in all other groups.
Under control conditions, early pregnancy tissue showed
baseline sFLT1 expression, which was as high as that found in
the PE-IUGR group and rose under treatment conditions to
levels found in severe PET. These data indicate that first
trimester tissue is by nature anti-angiogenic (high sFLT1).
High and similar levels of sFlt1 secretion in severely
preeclamptic villi may represent a failure of the villi to
differentiate and subsequently repress sFlt1, as is demon-
strated in term placental villi. This is a novel hypothesis and is
an example of the first trimester origins of severe preeclamp-
sia. In comparison to the PE villi response, LMWH reduced
sFLT1 release in severe IUGR villi. We believe these divergent
data may be due to the depletion of proliferating villous
cytotrophoblasts observed in severe IUGR [28], because these
cells are the target for LMWH-induced cell signaling, for
example as a co-factor for the mitogen FGF4 [29]. LMWH is
likely to indirectly promote sFLT1 synthesis by a disrupted
outer syncytiotrophoblast layer. Symmetrical mitosis in high-
dose LMWH will result in reduced syncytial fusion. Because
0.08A
B
C
0.06
0.04
Tota
l pro
tein
sec
retio
n(µ
g µL
–1 m
g tis
sue–
1 )
0.02
0.00Control
7 w– +
– – –+ + +
– + – + – +
CK-7
8 w
8 wMarker
12 w
12 w
38 w
38 w
0.25 IU mL–1 2.5 IU mL–1 25 IU mL–1
***
***
Fig. 7. Effect of LMWHon protein release and apoptotic shedding. Total
protein release into the media declined significantly in response to 2.5 and
25 IU mL)1 LMWH (n = 12/group) (A). The SCT layer is cytokeratin-7
(CK7) positive and actively secretes proteins/micro-particles into maternal
blood. LMWH (2.5 IU mL)1, indicated with +) reduced CK-7 presence
in protein extracts from the explant media, including an additional term
explant (38 weeks) (normalized to tissue weight) (B). Physiologic SCT
turnover is characterized by shedding of apoptotic or laddered fetal DNA,
whichwas reduced in response to 2.5 IU and 25 IU mL)1 (shown, indicated
with +) LMWH compared with untreated samples (indicated with )),indicating disruption of regulated apoptosis in favor of apo-necrosis (C).
LMWH controls sFLT1 release in placenta 2495
� 2011 International Society on Thrombosis and Haemostasis
in a randomized control trial setting LMWH actually reduces
the risk of severe preeclampsia [30], by implication other
factors must override an anti-angiogenic signal by sFLT1 to
prevent preeclampsia from becoming manifest. One explana-
tion is that the normal decidua, rich in pro-angiogenic
proteins [31], is able to compensate for high sFLT1 release
by first and early second trimester placental villi. By contrast,
in severe preeclampsia, where there is often considerable
decidual bed pathology [32], this compensatory mechanism is
lost, resulting in an overriding systemic action of sFLT1.
Another possible explanation is that the actions of sFLT1 are
much more complex than mere inhibition of VEGF receptor
phosphorylation. New data indicate that sFLT1 plays a
critical role in the local coordination of vessel sprouting [33], a
mechanism highly relevant to mediating the increase in
vascular capacitance that characterizes normal pregnancy.
Recent clinical trials failed to show prevention of recurrent
miscarriages independent of the status of thrombophilia by
LMWH treatment [34–36]. Pilot studies suggest a beneficial
effect of LMWH on perinatal outcome, justifying the need for
larger studies [10]. Recently, LMWH has been shown in recent
clinical trials to reduce the reoccurrence of preeclampsia, but
surprisingly no placental pathology was reported [30]. The lack
of detailed molecular placental pathology in any of the current
clinical trials is the main reason for our lack of knowledge
about the in vivo effects of LMWH in pregnancy.
Our observation that high-dose LMWH elevates circulating
sFLT1 and causes syncytiotrophoblast dysfunction in vitro
would predict that women exposed to high doses of LMWH in
pregnancy, for example to prevent thrombosis with a mechan-
ical heart valve, would experience higher rates of placental
complications. However, recent audits of such pregnancies do
not support this speculation [37,38]. One explanation for this
paradox may be that our acute in vitro findings differ from the
chronic in vivo situation where, for example, heparin-induced
release of preformed sFlt1 is not likely to be sustained. Sela
et al. recently reported a 4-fold increase in circulating sFLT1 in
women chronically exposed to LMWH [20]. Presumably,
women receiving LMWH during healthy pregnancies for
maternal reasons, such as a mechanical heart valve, have
normal placental function and therefore evoke additional
mechanisms to mediate systemic vasorelaxation, for example
by generating carbon monoxide via syncytiotrophoblast-
derived heme-oxygenase-1 (HO-1) [39]. Conversely, women
receiving LMWH in an attempt to prevent severe preeclampsia
are more likely to have underlying placental dysfunction, in
particular abnormalities of the syncytiotrophoblast covering
the placental villi that secrete sFlt1 [40].
In conclusion, we present evidence that LMWH interacts
with human placental villi, altering its physiology to signifi-
cantly increase the release of sFLT1 and impair VEGF
signaling in endothelial cells in vitro. This non-anticoagulant
effect of LMWH probably resides at the level of the villous
cytotrophoblast turn-over and is most pronounced in the first
trimester placenta. Because the phenomenon declines in normal
pregnancy, but is sustained in severe preeclampsia, we conclude
that severe preeclampsia is a disease conferred by the failure of
human placental villi to repress a tendency to secrete large
amounts of sFLT1 during early development. Use of LMWH
in this context should be carried out with caution based on our
findings, because it could exacerbate sFlt1-mediated maternal
hypertension and/or induce further placental dysfunction via
its adverse effects upon syncytiotrophoblast integrity. The
molecular actions of LMWH in the human placenta are
complex and deserve further study.
Acknowledgements
The authors thank the donors and the Biobank Program of the
CIHR Group in Development and Fetal Health (CIHR no
MGC-13299), the Samuel Lunenfeld Research Institute and
theMSH/UHNDepartment of Obstetrics and Gynecology for
the human specimens used in this study. The study was
supported by the PSI (No. 11-02 to JK) andRose TornoChair,
Mount Sinai Hospital, to JK and a Molly Towell Perinatal
Research Foundation Fellowship to SD.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
Supporting Information
Additional Supporting Informationmay be found in the online
version of this article:
Data S1. Material and Methods.
Figure S1. sFLT1 secretion of term, 8–15 weeks, IUGR, PE
mixed with IUGR, and PE placental villi (pg mL)1*mg tissue).
Figure S2. Total FLT1 was stained in tissues incubated with or
without LMWH (0.25 IU mL)1).
Figure S3. �De-novo� synthesized and preformed release of
sFLT1 from first trimester explants in response to heparin
treatment.
Please note:Wiley-Blackwell are not responsible for the content
or functionality of any supporting materials supplied by the
authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
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