Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial...

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: [email protected]

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


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


**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


2.5 IU mL–1

25 IU mL–1


2.5 IU mL–1

25 IU mL–1


2.5 IU mL–1

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).



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


2.5 IU mL–1

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).



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).



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.



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* *


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]).



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

)


2.5 IU mL–1

25 IU mL–1


2.5 IU mL–1

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.



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).



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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Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial...

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