Original Research

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Metformin as a prevention and treatment for preeclampsia:

effects on soluble fms-like tyrosine kinase 1 and soluble
endoglin secretion and endothelial dysfunction
Fiona C. Brownfoot, MBBS; Roxanne Hastie, BBiomed Sc; Natalie J. Hannan, B Sci, PhD;
Ping Cannon, B Sci; Laura Tuohey, BSci; Laura J. Parry, B Sci, PhD; Sevvandi Senadheera, B Sci PhD;
Sebastian E. Illanes, MBBS, MSc; Tuuhevaha J. Kaituu-Lino, PhD1; Stephen Tong, MBBS, PhD1

BACKGROUND: Preeclampsia is associated with placental ischemia/

hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble
endoglin into the maternal circulation. This causes widespread endothelial
dysfunction that manifests clinically as hypertension and multisystem organ
injury. Recently, small molecule inhibitors of hypoxic inducible factor 1a have
been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin
secretion. However, their safety profile in pregnancy is unknown. Metformin
is safe in pregnancy and is also reported to inhibit hypoxic inducible factor
1a by reducing mitochondrial electron transport chain activity.
OBJECTIVE: The purposes of this study were to determine (1) the
effects of metformin on placental soluble fms-like tyrosine kinase 1 and
soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion
are regulated through the mitochondrial electron transport chain, and
(3) to examine its effects on endothelial dysfunction, maternal blood
vessel vasodilation, and angiogenesis.
STUDY DESIGN: We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion
from placenta, endothelial cells, and placental villous explants. We used
succinate, mitochondrial complex II substrate, to examine whether the
effects of metformin on soluble fms-like tyrosine kinase 1 and soluble
endoglin secretion were mediated through the mitochondria. We also
isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron
transport chain activity using kinetic spectrophotometric assays.
Endothelial cells or whole maternal vessels were incubated with metformin
to determine whether it rescued endothelial dysfunction induced by either
tumor necrosis factor-a (to endothelial cells) or placenta villous
explanteconditioned media (to whole vessels). Finally, we examined the
effects of metformin on angiogenesis on maternal omental vessel explants.

Cite this article as: Brownfoot FC, Hastie R, Hannan NJ,

et al. Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1
and soluble endoglin secretion and endothelial dysfunction. Am J Obstet Gynecol 2016;214:356.e1-15.
0002-9378/free
Crown Copyright 2016 Published by Elsevier Inc. All rights
reserved.
http://dx.doi.org/10.1016/j.ajog.2015.12.019

RESULTS: Metformin reduced soluble fms-like tyrosine kinase 1 and

soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The
reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin
secretion was rescued by coadministration of succinate, which suggests
that the effects of metformin on soluble fms-like tyrosine kinase 1 and
soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors
rotenone and antimycin reduced soluble fms-like tyrosine kinase 1
secretion, which further suggests that soluble fms-like tyrosine kinase 1
secretion is regulated through the mitochondria. Mitochondrial electron
transport chain activity in preterm preeclamptic placentas was increased
compared with gestation-matched control subjects.
Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular
cell adhesion molecule 1 that was induced by tumor necrosis factorea
(vascular cell adhesion molecule 1 is an inflammatory adhesion molecule
up-regulated with endothelial dysfunction and is increased in preeclampsia).
Placental conditioned media impaired bradykinin-induced vasodilation;
this effect was reversed by metformin. Metformin also improved whole
blood vessel angiogenesis impaired by fms-like tyrosine kinase 1.
CONCLUSION: Metformin reduced soluble fms-like tyrosine kinase 1
and soluble endoglin secretion from primary human tissues, possibly by
inhibiting the mitochondrial electron transport chain. The activity of the
mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced
vasodilation in omental arteries, and induced angiogenesis. Metformin has
potential to prevent or treat preeclampsia.
Key words: electron transport chain, metformin, preeclampsia, soluble

endoglin, soluble fms-like tyrosine kinase 1

reeclampsia is a serious pregnancy

complication that globally is
responsible for >100 maternal and 400
perinatal deaths each day.1-7 An important
step in the pathophysiologic condition
may be placental ischemia/hypoxia,8-15
which leads to the release of soluble fmslike tyrosine kinase 1 (sFlt-1)16-18 and soluble endoglin (sENG)19 into the maternal
circulation.17,20-22 These cause endothelial
dysfunction that leads to multisystem

356.e1 American Journal of Obstetrics & Gynecology MARCH 2016

organ injury.23-35 There are no treatments

to arrest disease progression; expectant
management and delivery remain the only
treatment options.1,25,36-39 A medication
that is safe in pregnancy, that reduces
placental sFlt-1 and sENG secretion, that
rescues endothelial dysfunction, and that is
angiogenic may be effective in the treatment or prevention of preeclampsia.
There is interest in the use of drugs
that inhibit hypoxic inducible factor 1a
(HIF1a) to treat preeclampsia.9,40,41
HIF1a is up-regulated with ischemia/

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hypoxia42 and facilitates sFlt-1 secretion.43,44 Therefore, drugs that block
HIF1a activity may decrease sFlt-1
secretion. Indeed, the HIF1a inhibitors
YC-140 and ouabain41 have been shown
to reduce sFlt-1 secretion from placental
tissues. However, the safety prole of
YC-1 and ouabain in pregnancy are not
known, and they are still in clinical trials
for use in pulmonary hyperplasia45,46
and cancer,47,48 respectively.
This prompted us to explore repurposing a medication that is thought to be
safe in pregnancy that inhibits HIF1a
and lead us to investigate metformin.49
Metformin is an oral hypoglycemic
agent that is used to treat gestational
diabetes mellitus.49-51 Recently, metformin has been reported to reduce
breast52,53 and prostate54 cancer metastasis and prolong survival. This discovery renewed interest in the further
exploration of its mechanism of action; it
recently was shown to inhibit HIF1a by
blocking complex I of the mitochondrial
electron transport chain.53,55,56 Therefore, we hypothesized that metformin
may reduce sFlt-1 secretion in women
with preeclampsia.
Metformin has been reported to have
vasoprotective properties; epidemiologic
studies have shown that it reduces
cardiovascular morbidity in patients
with polycystic ovarian syndrome26 and
diabetes mellitus.57,58 This has been
attributed to its ability to reduce vascular
cell adhesion molecule 1 (VCAM-1),59,60
which is a molecule that is expressed on
the luminal surface of blood vessels in
the presence of inammation and is
increased in preeclampsia.61 Metformin
has also been reported to induce vasodilation of diabetic rat vessels.62

Objective
This study had 3 objectives: (1) to assess
the effects of metformin on sFlt-1 and
sENG secretion from primary placental
and endothelial cells/tissues and to
investigate whether these effects are
mediated through mitochondrial electron transport chain inhibition; (2) to
assess whether mitochondrial electron
transport chain activity positively regulates sFlt-1 secretion and if preterm
preeclamptic placenta have increased

OBSTETRICS
mitochondrial electron transport chain
activity; and (3) to assess whether metformin can reduce endothelial dysfunction, induce vasodilation, and stimulate
angiogenesis in human omental arteries.

Materials and Methods

Patient population
We performed functional experiments in
which we administered metformin
to human tissues and assessed its effects
on sFlt-1 and sENG secretion, mitochondrial electron transport chain function, and endothelial dysfunction. To
perform our experiments, we examined
tissues from placenta and blood vessels.
We collected several types of placental
tissues. We isolated human umbilical
vein endothelial cells (HUVECs)63 and
primary villous cytotrophoblast cells64
from the placenta and umbilical cord
that had been collected from patients at
term. We also collected placental villous
explants from patients with severe early
onset proteinuric preeclampsia (delivered at
34 weeks gestation by cesarean
delivery) as dened by the American
College of Obstetricians and Gynecologists (ACOG) guidelines.65 Placental
biopsy specimens were taken from 4
random placental sites as recommended
by the Co-Lab consortium.66 There
was hypertension and proteinuria (>300
mg of protein in a 24-hour urine
collection) in all cases. The placental
villous explants were prepared, as
previously described.63,67
To assess mitochondrial electron
transport chain activity, we obtained a
placental biopsy specimen from 23
women with severe preterm proteinuric
preeclampsia (dened by ACOG 2013
guidelines65) and from 25 gestationally
matched normotensive control subjects
who had a preterm delivery without evidence of signicant chorioamnionitis or
maternal comorbidities (Supplementary
Table). All the women who were diagnosed with preeclampsia had proteinuria. All placental samples were collected
from women who underwent a cesarean
delivery. Mitochondrial electron transport chain activity studies were performed, as previously described.68,69
We also collected omental tissue from
patients who had elective term cesarean

Original Research

delivery and dissected omental arteries,

as previously described.70 These omental
arteries were used to assess the effects of
metformin on vasodilation (by pressure
myography) and angiogenesis (omental
artery explant assay), as described later.
This study was approved by The
Mercy Health Human Research Ethics
Committee (Institutional review board
number R11/34; approved on the
November 12, 2014); all women gave
written informed consent.

In vitro experiments that

assessed metformin and
mitochondrial electron transport
chain activity inhibitors and
substrates on sFlt-1 and sENG
production and endothelial
dysfunction
HUVECs were plated at 24,000 cells/cm2
between passages 2 and 4 and cultured
at 37 C in 20% O2 and treated for 24
hours. Primary villous cytotrophoblast
cells were plated at 24,000 cells/cm2,
incubated overnight to ensure larger
viable villous cytotrophoblasts had
adhered, washed to remove apoptotic
mononuclear syncytiotrophoblast fragments, then treated for 24 hours at
500,000 cells/cm2 and treated for 48 hours
at 37 C in 8% O2 to assess sFlt-1 and
sENG secretion, respectively. Placental
villous explants of approximately 20-mg
tissue per well (dried weight) were
treated for 72 hours at 37 C in 8% O2.
HUVECs, primary villous cytotrophoblast cells, and placental villous
explants were treated with 0, 1, 2, and 5
mmol/L metformin (Sigma Chemical
Company, St. Louis, MO); HUVECs and
primary villous cytotrophoblast cells
were treated with 0, 0.5, and 1 mmol/L
metformin  25 mmol/L succinate
(mitochondrial electron transport chain
substrate; Sigma Chemical Company).
Primary villous cytotrophoblast cells
were also treated with mitochondrial
electron transport chain inhibitors
rotenone (Sigma Chemical Company) at
0, 0.625, 1.25, 2.5, and 5 mmol/L or
antimycin (Sigma Chemical Company)
at 0, 0.156, 0.31, 0.63, and 1.25 mmol/L.
Endothelial dysfunction was induced
in (1) HUVECs that used a constant dose
of 10ng/mL tumor necrosis factor a

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(TNFa; Sigma Chemical Company),

(2) whole omental arteries with the use
of 25% conditioned placental villous
explant media (this media is collected
24 hours after being cultured with
placental villous explants that were obtained from normal pregnancies at
term), and (3) omental artery explants
that used 250 ng/mL sFlt-1 at 37 C at
20% O2. Metformin was administered
simultaneously to HUVECs at 0, 1, 2,
or 5 mmol/L for 24 hours, to whole
omental arteries at 0 and 5 mmol/L for
3 hours, and omental artery explants at
1 mmol/L for 120 hours.

Measurement of sFlt-1, sENG, and

VCAM-1, sFlt-1 e15a and i13
Conditioned media were collected, and
RNA was extracted with the RNeasy mini
kit (Qiagen, Valencia, CA) from functional experiments and HUVEC endothelial dysfunction assay. Enzyme-linked
immunosorbent assay for sFlt-1 and
sENG was performed with the DuoSet
VEGF R1/Flt-1 kit (R&D Systems by
Bioscience, Waterloo, Australia) and a
DuoSet Human Endoglin CD/105 ELISA
kit (R&D Systems), respectively. RNA
was quantied with the Nanodrop ND
1000 spectrophotometer (NanoDrop
Technologies Inc, Wilmington, DE).
RNA (0.2 mg) was converted to complementary DNA with the use of Applied
Biosystems high capacity cDNA reverse
transcriptase kit (Life Technologies,
Mulgrave, Australia). Sybr gene expression assay for sFlt-1 e15a and sFlt-1 i13
(Geneworks, South Australia, Australia)
was performed,71 and a taqman gene
expression assay was used for VCAM-1
(Life Technologies).

Assessment of the effect of

metformin on whole omental
artery vasodilation
Treated whole omental arteries were
mounted on a pressure myograph organ
bath (Living Systems Instrumentation,
Burlington, VT). Incremental doses
of 0.01 nmol/L to 1mmol/L bradykinin
(Auspep, West Melbourne, Australia)
were
infused,
and
vasodilation
was assessed with video microscopy
(Diamtrak Software, Adelaide, SA,
Australia).72

Assessment of the effect of

metformin on angiogenesis with
the use of omental artery explants
Omental artery explants (0.5-mm rings)
were stained with calcein AM (Merck
Millipore, Darmstadt, Germany) and
imaged at 40 magnication with the
EVOS FL microscope (Life Technologies); outgrowth was assessed with image
J (http://imagej.nih.gov/ij/).73

Statistical analysis
Technical triplicates were performed
for each experiment, with a minimum of
3 biologic replicates for each in vitro
study (samples from different patients
were used for each biologic replicate).
When 2 groups were analyzed, a t-test
(parametric) or a Mann-Whitney test
(nonparametric data) was used. When
3 groups were compared, a 1-way
analysis of variance test (parametric) or
a Kruskal-Wallis test (non-parametric)
was used. Statistical analysis was done
with GraphPad Prism 6 software
(GraphPad Software, La Jolla, CA). All
data are expressed as mean  SEM;
probability values of <.05 were considered signicant.
Detailed methods are included in the
supplementary Methods section.

Results
Metformin reduces sFlt-1 secretion
from primary endothelial cells and
placental tissues
We assessed the effects of metformin on
sFlt-1 secretion from endothelial and
placental tissues because they are its main
tissue source. Administering metformin
dose-dependently reduced sFlt-1 secretion
from endothelial cells (HUVECs; Figure 1,
A) and primary cells that were isolated
from placenta (villous cytotrophoblast
cells; Figure 1, B). At the highest doses,
metformin reduced endothelial and
placental cell secretion by 53% and 63%,
respectively. Metformin also reduced sFlt1 secretion from placental villous explants
that were obtained from 4 women who
had been diagnosed with preterm preeclampsia (delivery required <34 weeks
gestation; Figure 1, C).
We investigated the effect of metformin on messenger RNA (mRNA)
expression of different sFlt-1 variants in

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the cells, or placental villous explant
tissues. sFlt-1 i13 is the most abundant
sFlt-1 variant in endothelial cells.74
Metformin dose-dependently reduced
sFlt-1 i13 mRNA expression in endothelial cells (Figure 1, D). sFlt-1 e15a is
the predominant variant expressed in
human placenta.74 Metformin reduced
sFlt-1 e15a mRNA expression in primary
villous cytotrophoblasts cells (Figure 1,
E) and placental villous explants
(Figure 1, F) that were obtained from
women with preterm preeclampsia.
Thus, we conclude that metformin reduces sFlt-1 isoform expression and sFlt1 secretion in endothelial and placental
cells/tissues, including placental villous
explants from patients diagnosed with
preterm preeclampsia.

Metformin reduces sENG secretion

from primary endothelial and
placental tissues
We next investigated the effects of metformin on sENG secretion from primary
endothelial cells and placental cells/
tissues. Metformin dose-dependently
reduced sENG secretion from HUVECs
(Figure 2, A) and primary villous cytotrophoblast cells (Figure 2, B). Metformin induced a trend towards a reduction
in sENG secretion from preterm preeclamptic placental villous explants at
3 doses, but none of these decreases were
signicant (Figure 2, C).

Metformin reduces sFlt-1 and sENG

secretion by inhibiting the
mitochondrial electron transport
chain
Given that metformin inhibits mitochondrial electron transport chain activity
by blocking complex I,53,55,56 we examined whether the decrease in sFlt-1
secretion was mediated through mitochondrial electron transport chain inhibition. Succinate is a substrate for complex
II of the mitochondria, downstream of the
effect of metformin blockade. Thus, if
metformin was reducing sFlt-1 secretion
by directly blocking complex I, then succinate should restore electron ow and
rescue this effect. Indeed, succinate
rescued a reduction in sFlt-1 secretion that
was induced by endothelial cells (Figure 3,
A) and primary villous cytotrophoblasts

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Original Research

FIGURE 1

FIGURE 2

Effect of metformin on soluble fms-like tyrosine kinase 1 secretion and

isoforms e15a and i13 expression in endothelial cells and placental tissue

Effect of metformin on soluble

endoglin secretion from endothelial
cells and placental tissue

Metformin (0, 1, 2, 5 mmol/L) dose-dependently reduced soluble fms-like tyrosine kinase 1 secretion
from A, endothelial cells, B, villous cytotrophoblast cells, and C, preterm preeclamptic placental
villous explants. Metformin reduced endothelial cell expression of D, sFlt-1 i13 isoform, E, villous
cytotrophoblast cells, and F, preterm preeclamptic placental villous explant messenger
RNA expression of sFlt-1 e15a. The single asterisk indicates P < .05; the double asterisks indicate
P < .01; the triple asterisks indicate P < .0001; the quadruple asterisks indicate P < .00001.
sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol
2016.

cells (Figure 3, B). Succinate also rescued

a decrease in sENG secretion that
was induced by metformin in primary
HUVECs (Figure 3, C) and villous cytotrophoblast cells (Figure 3, D). These data
raise the possibility that the effects of
metformin on sFlt-1 and sENG secretion
are mediated through its effects on the
mitochondria.

Inhibition of the mitochondrial

electron transport chain reduces
sFlt-1 secretion from primary
villous cytotrophoblasts cells
To our knowledge, the concept that the
mitochondria regulate sFlt-1 secretion is
novel. To obtain further evidence that
this is the case, we examined whether
other mitochondrial electron transport

Metformin (0, 1, 2, and 5 mmol/L) reduced

soluble endoglin secretion from A, endothelial
cells and B, villous cytotrophoblast cells. Metformin did not change soluble endoglin secretion from C, preterm preeclamptic placental
villous explants. The single asterisk indicates
P < .05; the double asterisks indicate P < .01;
the quadruple asterisks indicate P < .00001.
sENG, soluble endoglin.
Brownfoot et al. Metformin decreases sFlt-1 and sENG,
improves endothelial function, and is angiogenic. Am
J Obstet Gynecol 2016.

chain inhibitors reduce sFlt-1 secretion.

Administering rotenone, another complex I inhibitor, to primary villous
cytotrophoblast cells reduced sFlt-1
secretion by 65% (Figure 4, A). Antimycin, a complex III inhibitor, also
reduced st-1 secretion by 75%
(Figure 4, B). These doses of rotenone
and antimycin did not induce cell death

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FIGURE 3

FIGURE 4

Effect of metformin and mitochondrial electron transport chain complex I

activity on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion
from endothelial cells and villous cytotrophoblast cells

Mitochondrial electron transport

chain inhibitors and sFlt-1
secretion

The effect of metformin (0.5 and 1 mmol/L) on soluble fms-like tyrosine kinase 1 secretion from
A, endothelial cells and B, villous cytotrophoblast cells is likely mediated through mitochondrial
electron transport chain complex 1 inhibition because the effect is rescued in the presence of the
mitochondrial electron transport chain substrate, succinate (25 mmol/L). Furthermore, metformin
also reduced soluble endoglin secretion likely through mitochondrial electron transport chain
complex 1 inhibition from C, endothelial cells (0.5, 1 mmol/L) and D, villous cytotrophoblast cells
(1 mmol/L) because succinate (25 mmol/L) rescued its effect on secretion. The single asterisk indicates P < .05; the double asterisks indicate P < .01; the triple asterisks indicate P < .0001; the
quadruple asterisks indicate P < .00001.

A, Rotenone (0, 0.62, 1.25, 2.5, and 5 mmol/L),

a complex 1 mitochondrial electron transport
chain inhibitor, and B, antimycin (0, 0.16, 0.31,
0.63, and 1.25 mmol/L), a complex III mitochondrial electron transport chain inhibitor, both
significantly reduce soluble fms-like tyrosine
kinase 1 secretion from villous cytotrophoblast
cells. The single asterisk indicates P < .05; the
double asterisks indicate P < .01; the triple
asterisks indicate P < .0001.
sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG,
improves endothelial function, and is angiogenic. Am
J Obstet Gynecol 2016.

sENG, soluble endoglin; sFlt-1, soluble fms-like tyrosine kinase 1.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol
2016.

(MTS assay and calcein stain; data not

shown). These studies provide further
evidence that the mitochondria is
involved in the regulation of sFlt-1
secretion.

Mitochondrial electron transport

chain activity is up-regulated in
preterm preeclamptic placenta
Given the mitochondria appears to positively regulate sFlt-1 and sENG secretion,

we hypothesized that preeclamptic placentas might have increased mitochondrial electron transport chain activity. We
therefore compared mitochondrial electron transport chain activity in preterm
preeclamptic placentas (n 23) and
normotensive gestation matched preterm
control placentas (n 25; Supplementary
Table contains baseline characteristics).
We observed an increase in mitochondrial electron transport chain activity in

356.e5 American Journal of Obstetrics & Gynecology MARCH 2016

the preeclamptic placentas for all 4

complexes, and this was signicant
for complex II (Figure 5). Therefore,
mitochondrial electron transport chain
activity may be increased in preterm
preeclamptic placenta.

Metformin reduces VCAM-1

expression on endothelial cells
Endothelial dysfunction is associated
with increased VCAM-1 expression in
the endothelium.61,75 VCAM-1 is an
adhesion molecule that is expressed
on the luminal surface of blood vessels

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Original Research

FIGURE 5

FIGURE 6

Mitochondrial electron transport chain activity in preterm preeclamptic

placenta compared with gestationally matched controls

Effect of metformin on endothelial

cell vascular cell adhesion
molecule 1 expression

Inflammatory cytokine tumor necrosis factor a

increased endothelial cell expression of vascular
cell adhesion molecule 1 and was significantly
reduced with increasing doses of metformin
(0, 1, 2, and 5 mmol/L). The single asterisk
indicates P < .05; the triple asterisks indicate
P < .0001.
TNFa, tumor necrosis factor a; VCAM 1, vascular cell adhesion
molecule 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG,
improves endothelial function, and is angiogenic. Am
J Obstet Gynecol 2016.

Activity of all (A-D) complexes in the mitochondrial electron transport chain was increased in
preeclamptic placenta (n 23), compared with preterm placental controls (n 25), and was
significant for complex II (B). The single asterisk indicates P < .05.
CS, citrate synthase.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol
2016.

and can cause an inammatory mesh

and snare circulating blood cells.
Preeclampsia is also associated with
increased circulating TNFa,76 which is
a proinammatory cytokine that upregulates VCAM-1.61,75 We therefore performed an in vitro assay for which
we added TNFa to HUVECs, which
signicantly up-regulated VCAM-1
expression in endothelial cells (Figure 6).
The administration of metformin
signicantly reduced TNFa-induced
VCAM-1 expression, which suggests that
it may have effects to decrease endothelial dysfunction.

Metformin induces vasodilation in

maternal vessels that are isolated
from the omentum
We next set up an assay to mimic the fact
that placental factors that are released

into the maternal circulation have a

role in inducing whole blood vessel
dysfunction. We incubated human
omental arteries in either conditioned
placental culture media or normal culture media (not incubated with placental
tissue). The vessels that were incubated
in normal culture media dilated 100%
in response to bradykinin (endogenous
vasodilator), but vessels that were
incubated in placental culture media
exhibited a signicant impairment in
vasorelaxation (40% less; Figure 7, A).
This suggests that placental factors
seem to impair vasorelaxation in our
assay.
When metformin was coadministered
to vessels that were cultured with the
placental culture media, this impairment
of vasodilation was reversed, and the
bradykinin-mediated relaxation did not

differ from the normal culture media

control (Figure 7).

Metformin enhances angiogenic

sprouting from omental vessel
explants
Reduced angiogenesis is thought to
contribute to placental hypoxia and
contribute to the development of preeclampsia.23,77 We explored whether
metformin could rescue the inhibition of
angiogenesis that is caused by sFlt-1. We
devised a human angiogenesis omental
ring explant assay that was based on
a mouse aorta explant model.73 We
obtained omental biopsy specimens at
cesarean delivery, isolated the omental
vessels, dissected them into small
explants, and cultured them with or
without sFlt-1. We found a signicant
reduction in angiogenic sprouting from
the vessels in the presence of sFlt-1
(Figure 8). However, metformin rescued sFlt-1-induced inhibition of
angiogenic sprouting (Figure 8).
A PowerPoint presentation that
summarizes these data is included in
supplementary materials.

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Comment
Primary findings of the study
Metformin reduces sFlt-1 and sENG
secretion from primary human tissues,
possibly by inhibiting the mitochondrial
electron transport chain. Second,
mitochondrial electron transport chain
activity positivity regulates sFlt-1 secretion, and mitochondrial electron transport chain activity is increased in
preterm preeclamptic placenta. By
using assays to replicate the endothelial
and vascular dysfunction that may be
occurring in preeclampsia, we found
that metformin reduces endothelial
dysfunction, improves vasodilation, and
is angiogenic. Collectively, our results
suggest that metformin has potential to
prevent or treat preeclampsia.

FIGURE 7

Effect of metformin on whole blood vessel dilation

sFlt-1 secretion is regulated

through the mitochondrial
electron transport chain; metformin
possibly blocks this pathway
This is the rst report to show that
sFlt-1 secretion is regulated through the
mitochondrial electron transport chain.
Using 2 inhibitors, we showed that
blocking complex I or III signicantly
reduced sFlt-1 secretion.
We have generated circumstantial
evidence that suggests that metformin
inhibits sFlt-1 and sENG secretion by
inhibiting complex I of the mitochondrial electron transport chain. The
administration of succinate (a substrate
for complex II of the mitochondrial
electron transport chain) rescued the
metformin-induced reduction in sFlt-1
secretion but had no effect on sFlt-1
secretion when administered alone.
However, a limitation of this experiment
is that succinate is also thought to stabilize HIF1a directly.78 Furthermore,
metformin is known to have other
intracellular targets, such as activating
AMP-activated protein kinase.79 Another
consideration is whether metformin may
be inhibiting syncytialization to reduce
sFlt-1 secretion. We do not believe this is
likely for the following reasons: (1)
metformin had the same effect on sFlt-1
secretion from endothelial cells, which
do not fuse; (2) we observed a reduction
in sFlt-1 secretion from intact placental
villous explants, and (3) there was a

Whole omental blood vessels that were cultured with placental villous explant media and were
compared with those blood vessels that were cultured with normal media have impaired relaxation to
the vasodilator bradykinin (1 mmol/L). Metformin (5 mmol/L) reversed the effect of placental villous
explant media on vasodilation and improved relaxation to levels that were seen in those vessels
cultured in normal media. The single asterisk indicates P < .05.
pEC50, half maximal effective concentration.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol
2016.

reduction in sFlt-1 mRNA from primary

trophoblasts that indicated a direct effect
on the transcriptional machinery of the
cell. We note that we have not demonstrated conclusively that metformin is
decreasing sFlt-1 secretion via its effects
on the mitochondria.

Mitochondrial electron transport

chain activity is increased in
preterm preeclamptic placenta
We found an increase in activity in all
electron transport chain complexes in
preterm preeclamptic placentas, which
was signicant for the activity of complex
II. There are 2 other reports that
have examined mitochondrial electron
transport chain activity. One report
was concordant with our results, also
reporting an increase in complex II

356.e7 American Journal of Obstetrics & Gynecology MARCH 2016

activity in preeclamptic placenta,80 while

the other concluded mitochondrial electron transport chain activity was
decreased.81 Both reports examined only
6 preeclamptic placentas that were obtained predominately from term pregnancies (10 of the 12 placentas were
at term).80,81 Our study is the rst to
examine preterm preeclampsia specifically, and we investigated a considerably
larger cohort of samples compared with
those previous reports. 80,81
Given that our data suggests that
preeclamptic placentas have increased
electron transport chain activity, we
speculate that enhanced mitochondrial
electron transport chain activity in
preeclampsia may have a role in the
increased sFlt-1 and sENG secretion seen
in this disease.82

ajog.org

OBSTETRICS

Original Research

FIGURE 8

Effect of metformin on angiogenesis

Omental whole vessel rings cultured in the presence of soluble fms-like tyrosine kinase 1 have reduced vessel outgrowth, which is restored with
the addition of metformin (1 mmol/L). The white arrows point at vessel outgrowth. Representative micrographs are shown by the asterisk that indicates
P < .05
sFlt-1, soluble fms-like tyrosine kinase 1.
Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016.

The therapeutic implication of our

ndings that the mitochondria regulates
sFlt-1 and sENG secretion is that
screening other mitochondrial electron
transport chain inhibitors may identify
new
therapeutic candidates for
preeclampsia.

Metformin rescues endothelial

dysfunction and impaired
vasodilation specific to
preeclampsia
VCAM-1 is an inammatory protein
that is up-regulated in preeclampsia

and associated with endothelial

dysfunction.61 Metformin previously
has been shown to reduce endothelial
cell inammatory gene and protein
expression83,84 and serum VCAM-1
concentrations in patients with diabetes
mellitus59 and impaired glucose tolerance.60 We demonstrated that, in the
presence of TNFa that is an inammatory cytokine that is up-regulated in
preeclampsia,76 metformin reduced
VCAM-1 expression in endothelial cells.
Metformin has been shown to
vasodilate mouse and rat vessels that

are affected by insulin resistance85 and

diabetes mellitus,62 respectively. We
developed an assay to assess whether
metformin vasodilates maternal vessels
that are incubated in placentalconditioned media. Indeed, metformin
appeared to enhance the vasodilatory
response to bradykinin. We propose
that this technique could be used
more widely to assess the effect of
small molecules on vasodilation in the
context of pregnancy. Experiments to
determine whether this effect was
dependent on an intact endothelium

MARCH 2016 American Journal of Obstetrics & Gynecology

356.e8

Original Research

OBSTETRICS

were not performed and merit investigation in the future.

Metformin improves angiogenesis

There is an imbalance towards an
antiangiogenic state in preeclampsia.16,19,20,23,82 We devised an assay to
examine the effects of metformin on
angiogenesis in maternal vessels. There
are a number of other angiogenesis
assays that could have been performed.
These include tube-forming assays
that use endothelial cells alone or are
cocultured with broblasts and mouse
or rat aortic ring assays.73 We believe
our assay more closely represents the
dysfunction that is present in preeclampsia because it examines human
vessels, examines angiogenesis in
heterogeneous tissues that includes
both endothelial and vascular smooth
muscle; we induced dysfunction with
sFlt-1, the antiangiogenic molecule
that is of direct relevance to preeclampsia. We demonstrated that sFlt-1
reduced human omental vessel
outgrowth and that metformin rescued
this effect. These data suggest that
metformin has angiogenic effects on
maternal vessels.

Metformin has potential to prevent

and treat preeclampsia
Metformin is safe in pregnancy and
currently is used to treat gestational
diabetes mellitus. Interestingly, a randomized trial that compared metformin
and insulin to treat gestational diabetes
mellitus49 showed a nonsignicant
reduction in the incidence of gestational
hypertension (3.9% metformin arm,
compared with 6.2% insulin treatment)
and preeclampsia (5.5% metformin arm,
7% insulin treatment) among those
treated with metformin. A randomized
trial86 that assessed the effect of metformin on reducing perinatal morbidity in
obese women did not identify a difference in preeclampsia risk. However, it is
possible that under-dosing and poor
compliance may be an explanation for
the reason that there was no effect on
the incidence of preeclampsia. Furthermore, a metaanalysis of 2 randomized
control trials that assessed the effect of
metformin on pregnancy outcome in

patients with polycystic ovarian syndrome also did not show a reduction in
preeclampsia.87
It is important to note that incidence
of hypertensive disorders of pregnancy
and preeclampsia were not the primary
outcome of these previous trials.49,86 In
light of our work, we propose that a
clinical trial that will examine whether
metformin can prevent or treat preeclampsia is justied. Given our ndings
of reduced sFlt-1 and sENG placental
secretion and improved endothelial
dysfunction, we believe metformin
may be able to reduce both placental
and maternal vascular aspects of
preeclampsia

Conclusion
We have performed preclinical studies
using primary human tissues to show
that mitochondrial electron transport
chain activity is up-regulated in preterm
preeclamptic placenta and that the
mitochondrial electron transport chain
regulate villous cytotrophoblast cells and
endothelial cell sFlt-1 and sENG secretion. Metformin reduces sFlt-1 and
sENG secretion by inhibiting complex 1
of the mitochondria. Furthermore,
metformin reduces key features of
endothelial dysfunction that are specic
to preeclampsia and enhances angiogenesis. Metformin may be a novel
preventative or be therapeutic for preeclampsia and has the potential to reduce
the burden of this major pregnancy
complication.
n
Acknowledgments
We thank the research midwives, Gabrielle Pell,
Genevieve Christophers, Rachel Murdoch, and
Debra Jinks, and the patients at Mercy Hospital
for Women for participating in this research.

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Author and article information

From the Translational Obstetrics Group, Department of
Obstetrics and Gynaecology, University of Melbourne,
Mercy Hospital for Women (Drs Brownfoot, Hannan,
Kaituu-Lino, and Tong and Ms Hastie, Cannon, and
Tuohey) Heidelberg, Victoria, Australia; Biosciences,
University of Melbourne, (Drs Parry and Senadheera),
Parkville, Victoria, Australia; and Department of Obstetrics and Gynecology, Laboratory of Reproductive Biology,
Faculty of Medicine, Universidad de Los Andes, Santiago,
Chile (Dr Illanes).
1
These authors have contributed equally to this article.
Received Nov. 30, 2015; revised Dec. 15, 2015;
accepted Dec. 15, 2015.
Supported by The National Health and Medical
Research Council of Australia (NHMRC; #1048707,
#1046484, #1101871) and an Arthur Wilson RANZCOG
scholarship; by an Australian Postgraduate Award and an
AVANT scholarship (F.B); by a CR Roper Research
Fellowship (N.J.R.); the NHMRC provided salary support
(#1050765 [S.T.]; #1062418 [T.K.L.]; #628549 [S.S.]).
The funders had no role in study design, data collection,
analysis, decision to publish or the preparation of the
manuscript.
The authors report no conflict of interest.
Corresponding author: Stephen Tong, MBBS, PhD.
s.tong@unimelb.edu.au

ajog.org
Supplementary:
Methods
Cell culture media
Placental tissues, endothelial cells,
and omental vessels were cultured in a
variety of media. Human umbilical
vein endothelial cells (HUVECs) were
cultured in M199 media (Life Technologies, Mulgrave, Australia) that contained
10% fetal calf serum, 1% antibioticantimicotic (Life Technologies), 1%
endothelial cell growth factor (Sigma
Chemical Company, St. Louis, MO), and
1% heparin; primary trophoblasts and
placental explants were cultured in
DMEM high Glutamax (Life Technologies) that contained 10% fetal calf serum
(Sigma Chemical Company) and 1%
antibiotic-antimicotic (Life Technologies).

Isolation of HUVECs and

trophoblasts from human placenta
Placentas were obtained from women who
had undergone elective cesarean delivery
at term. HUVECs were isolated by obtaining the umbilical cord, infusing it with
10 mL (1 mg/mL) of collagenase (Worthington, Lakewood, NJ), and ushing the
cells through. as previously described.1
Primary trophoblasts were isolated by
scraping 50 g of cotyledon tissue from the
placenta, subjecting it to enzyme digestion
buffer, and separating cells with the use of
a discontinuous Percoll gradient then
subjecting them to negative selection with
CD9 to remove contaminating cells, as
previously described.2

Cell viability assays (MTS assay

and calcein stain)
Cell viability assays were performed for
all HUVEC and primary trophoblast
functional experiments with the use of
either CellTiter 96-Aquesous One solution (Promega, Madison, WI) or calcein
stain (Merck Millipore, Darmstadt,
Germany) and were quantitated with a
Fluostar omega uorescent plate reader
(BMG Labtech, Victoria, Australia).

Whole omental vessel collection,

pressure myography, and omental
vessel explant outgrowth
Omental vessel tissue collection

Omental tissue was obtained from

women with normal pregnancies

OBSTETRICS
who had undergone elective cesarean
delivery at term. The omental biopsy
specimens were transferred to cold
Krebs physiologic solution (PSS) that
contained 112 mmol/L NaCl, 25 mmol/
L NaHCO3, 4.7 mmol/L KCl, 1.2 mmol/
L MgSO4.7H2O, 0.7 mmol/L KH2PO4,
10 mmol/L HEPES, 11.6 mmol/L Dglucose, and 2.5 mmol/L CaCl2.2H2O;
pH 7.4; Sigma Chemical Company) and
washed 3 times to remove the anesthetic.
The average inner lumen diameters of
arteries that were used were 280-320 mm
at 60 mm Hg in zero calcium. All experiments were performed at <20 hours
after surgery.
Whole vessel pressure
myography

Whole omental arteries were cleaned

carefully of connective and adipose tissue. Arteries were cultured for 3 hours at
4 C in treatment groups (control group,
M199 media [Sigma Chemical Company] or treatment groups, M199 with
25% conditioned media that had been
exposed to placental explants for 24
hours and M199 with 25% conditioned
media with 5 mmol/L metformin
[Sigma Chemical Company]) for incubation studies. All arteries were mounted in a pressure myograph organ bath
(Living Systems Instrumentation, Burlington, VT) and continuously superfused with PSS (37 C) at a rate of 4 mL/
min. In the absence of intraluminal ow,
arteries were pressurized gradually to
60 mm Hg, warmed to 37 C, and
checked for pressure leaks over a
50-minute equilibration period. The
outer diameters were measured through
video microscopy (Diamtrak Software,
Adelaide, SA, Australia). Before the start
of each experiment, the viability of the
smooth muscle was tested with potassium physiologic saline solution
(KPSS, 100 mmol/L) or thromboxane
agonist
9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2a (U46619, 1
mmol/L; Sigma Chemical Company)
washed with PSS to regain basal diameter. Arteries were preconstricted with
thromboxane agonist U46619; endothelial function was tested by applying
bradykinin (10 mmol/L; Auspep, West
Melbourne, Australia), and washed to

Original Research

regain basal diameter. Arteries were then

submaximally preconstricted (approximately 70% of maximal response to
KPSS or U46619) with U46619 (Sigma
Chemical Company); vascular reactivity
to drugs were assessed in the following
manner. In incubation studies, the effect
of treatments on vascular reactivity was
assessed with bradykinin (0.01-1000
nmol/L). At the end of the experiment,
smooth muscle integrity was tested
in response to the endotheliumindependent, nitric oxide donor, sodium nitroprusside (10 mmol/L). The
organ bath was then washed out with
PSS, and Ca2-free Krebs solution was
added for 20 minutes to determine the
maximum diameter. Ca2-free Krebs solution contained no added CaCl2.2H2O
but contained EGTA (2 mmol/L).
Human omental vessel explants

To assess changes in angiogenesis

potential, a human omental vessel
outgrowth assay was conceived and
performed. Omental samples were
collected from patients who had had an
elective cesarean delivery at term. An
arteriole was dissected carefully out of
the fat, and the vessel was ushed with
a 26G needle with OptiMEM (Life
Technologies). The vessel was cut into
0.5-mm rings and serum-starved in
OptiMEM at 20% O2, 5% CO2 at 37 C
overnight. The ring was embedded in a
collagen matrix (1 mg/mL in DMEM,
pH adjusted so slightly basic using
NaOH) in a 96-well plate with 1 omental
ring per well. Rings were treated in
media that contained OptiMEM with
2.5% fetal calf serum (Sigma Chemical
Company) and 1% antibiotic antimycotic (Life Technologies)  250 ng/
mL soluble fms-like tyrosine kinase 1
(sFlt-1) and 0 or 1 mmol/L metformin
(Sigma Chemical Company). For each
patient sample (n 4), we used 5 rings
(technical replicates) per treatment
group. Treatments were changed every
48 hours, and the experiment was
continued for 120 hours. Calcein AM
(Merck Millipore, Darmstadt, Germany) was added to the wells for 45
minutes at 37 C, and images were obtained at the same magnication (40)
with the EVOS FL microscope (Life

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356.e12

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Technologies). Vessel outgrowth at the

completion of the experiment was
determined by a calculation of the area
of growth with the computer program
Image J (http://imagej.nih.gov/ij/).

Assessment of mitochondrial
electron transport chain activity
in preterm preeclamptic placenta
and preterm gestationally
matched placenta
Placental collection for
mitochondrial electron transport
chain activity assay

Placental tissue was obtained from 23

women with severe preterm preeclampsia that was dened with the
American College of Obstetricians and
Gynecologists 2013 guidelines3 and
from 25 gestationally matched preterm
control subjects who delivered either
because of spontaneous preterm
labor without evidence of infection
(conrmed on histopathologic evaluation), hypertensive disease, or maternal
comorbidities. Baseline patient clinical
characteristics are detailed in the Supplementary Table.
Placental tissue was processed within
30 minutes of delivery. Placental tissue,
excluding fetal membranes, was washed
in sterile phosphate buffered saline,
frozen within 15 minutes, and stored
at e80 C.
Isolation of mitochondria from
preeclamptic and preterm
placental samples

Intact mitochondria were isolated, as

previously described.4 Briey, to isolate
intact mitochondria, the placental tissue
was homogenized in ice-cold Zheng
buffer (210 mmol/L mannitol, 70
mmol/L sucrose, 5 mmol/L HEPES, and
1 mmol/L EGTA, pH 7.2) and centrifuged at 600g for 10 minutes at 4 C; the
supernatant was transferred to an icecold microcentrifuge tube and freeze
thawed 3 times in a methanol bath
(1-minute freeze followed by 4-minute
thaw at room temperature).4
Electron transport chain activity

All mitochondrial respiratory complex

activity was performed by spectrophotometric assays, as previously

described.4 The Fluostar Omega Fluorescent Plate Reader (BMG Labtech,

Victoria, Australia) was used to measure
absorbance. First, the activity of the
mitochondrial matrix enzyme citrate
synthase was measured by the rate of free
sulfhydryl group production. This was
determined by the administration of thiol
reagent 5,5-dithio-bis-(2-nitrobenzoic
acid) to placenta, which reacts with free
sulfhydryl to produce thio nitrobenzoate
acid. Placental samples were sonicated on
ice for 1 minute to disrupt the mitochondrial membrane and 1 mmol/L 5,5dithio-bis-(2-nitrobenzoic acid) and 12.5
mmol/L acetyl CoA were added to 10 mL
of placental sample or blank, and the
absorbance was read every 15 seconds for
2 minutes at 412 nmol/L. To begin the
reaction, 5 mmol/L of oxaloacetic acid
was then added, and absorbance was read
every 15 seconds at 412 nmol/L for 4
minutes.
The activity of complex I was
measured by the determination of
the transfer of electrons from b-nicotinamide adenine dinucleotide to coenzyme
Q. This was achieved by the activation of
complex 1 by adding 5mmol/L b-nicotinamide adenine dinucleotide, 50
mmol/L potassium cyanide, 0.5 mmol/L
antimycin A, 10% bovine serum albumin, and 5.0 mmol/L oxidized coenzyme
Q to 10 mL of placental sample or blank
and the inhibiting of the complex by the
addition of rotenone 0.125 mmol/L and
reading the absorbance every 15 seconds
for 3 minutes at 340 nmol/L.
Complex II activity was next assessed
by the measurement of the reduction of
coenzyme Q on oxidation of succinate
to fumarate. Baseline activity was
assessed initially by the addition of 50
mmol/L potassium cyanide, 0.5 mmol/L
antimycin A, 100 mmol/L succinate,
and 0.125 mmol/L rotenone to 10 mL
of placental sample or blank; the
absorbance was read every 15 seconds
for 2 minutes at 280 nmol/L; then 5
mmol/L of coenzyme Q was added, and
the rate of reduction was assessed by the
reading of absorbance every 15 seconds
for 3 minutes at 280 nmol/L.
The activity of complex III was
assessed next by the measurement of the
reduced form of decyl benzoquinone,

356.e13 American Journal of Obstetrics & Gynecology MARCH 2016

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which is a short chain of analogue of
endogenous ubiquinone that donates
electrons to cytochrome c. Baseline activity was assessed by the addition of 50
mmol/L potassium cyanide, 0.125
mmol/L rotenone, 10% bovine serum
albumin, 25 mmol/L n-dodecyl-b-DMaltoside, 10 mmol/L reduced duroquinone, and 1 mmol/L oxidized cytochrome c to 10 mL of placental sample
or blank; absorbance read every 12 seconds for 4 minutes at 550 nmol/L; 25
mmol/L of L-ascorbate was added to
complete the reaction, and the absorbance was read every 15 seconds for 5
minutes at 550 nmol/L.
The activity of complex IV, the terminal enzyme in the electron transport
chain, was measured by the assessment
of the loss of reduced cytochrome c as it
is oxidized. Baseline activity was determined by the addition of reduced cytochrome c to 10 mL of placental sample or
blank; the absorbance was read every 15
seconds for 5 minutes at 550 nmol/L;
then 50 mmol/L of potassium ferricyanide was added to stop the oxidation of
cytochrome c, and the absorbance was
read every 15 seconds for 2 minutes at
550 nmol/L.
Electron transport chain activity was
calculated as rst-rate constants (rst
rate constant per minute per milligram)
that were determined by the subtraction
of the nal absorbance reading after the
addition of inhibiting substance from
the absorbance before and the data
plotted as a slope against time. The slope
is denoted the rst-order rate constant.
Activity was normalized to milligrams of
tissue or citrate synthase (maker of
mitochondrial content).
Enzyme-linked immunosorbent
assay (ELISA) analysis

Concentrations of sFlt-1 and soluble

endoglin were measured in conditioned
cell/tissue culture media with the use
of the DuoSet VEGF R1/Flt-1 kit
(R&D Systems by Bioscience, Waterloo,
Australia) and a DuoSet Human Endoglin CD/105 ELISA kit (R&D Systems)
according to the manufacturers instructions. The coefcient of variation
for our ELISA sFlt-1 was 3.4% and for
sENG was 2.4%.

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Real timeepolymerase chain
reaction (PCR)

RNA was extracted from placental explants and HUVECs using an RNeasy
mini kit (Qiagen, Valencia, CA) and
quantied using the Nanodrop ND 1000
spectrophotometer (NanoDrop technologies Inc, Wilmington, DE). 0.2 mg
of RNA was converted to complementary DNA with the use of Applied Biosystems high capacity complementary
DNA reverse transcriptase kit (Life
Technologies), as per manufacturer
guidelines. The coefcient of variation
for the PCR for vascular cell adhesion
molecule 1 was 5.6%; the coefcient of
variation for the PCR sFlt-1-i13 and
e15a was 3.24%.
A taqman gene expression assay was
performed for VCAM-1 (Life Technologies). Real timeePCR was performed
on the CFX 384 (Bio-Rad, Hercules, CA)
with the use of FAM-labeled Taqman
universal PCR mastermix (Life Technologies) with the following run conditions: 50 C for 2 minutes; 95 C for 10
minutes; 95 C for 15 seconds, and 60 C
for 1 minute (40 cycles). Sybr gene
expression assay for sFlt-1 e15a and sFlt1 i13 was used. Primers were designed as
previously described (Geneworks, South
Australia, Australia).5 RT-PCR was performed with the following run conditions: 95 C for 20 minutes; 95 C for
0.01 minutes, 60 C for 20 minutes, 95 C
for 1 minute (39 cycles), melt curve

OBSTETRICS
65 C to 95 C at 0.05 C increments at
0.05 seconds.
All data were normalized to GAPDH
as an internal control and calibrated
against the average Ct of the control
samples. Results were expressed as fold
change from control.

Statistical analysis
Technical triplicates were performed for
each experiment, with a minimum of 3
biologic replicates (different patient
samples) for each in vitro study. Data
were tested for normal distribution and
analyzed with the use of the appropriate
parametric or nonparametric test. The
statistical software we used was GraphPad Prism 6 (GraphPad Software, La
Jolla, CA). When 3 groups were
compared, a 1-way analysis of variance
(for parametric data) or Kruskal-Wallis
test (for nonparametric data) was used.
Post-hoc analysis was carried out with
either the Tukey (parametric) or Dunns
test (nonparametric). When 2 groups
were analyzed, either an unpaired t-test
(parametric) or a Mann-Whitney test
(nonparametric) was used. Concentration response curves from omental arteries were tted to a sigmoidal curve
with nonlinear regression to calculate
the sensitivity of each agonist (pEC50) or
maximum relaxation (Emax). All data
were expressed as mean  SEM; probability values of <.05 were considered
signicant.

Original Research

References
1. Brownfoot FC, Hannan N, Onda K, Tong S,
Kaituu-Lino T. Soluble endoglin production is
upregulated by oxysterols but not quenched by
pravastatin in primary placental and endothelial
cells. Placenta 2014;35:724-31.
2. Kaituu-Lino TJ, Tong S, Beard S, et al.
Characterization of protocols for primary
trophoblast purication, optimized for functional
investigation of sFlt-1 and soluble endoglin.
Pregnancy Hypertens 2014;4:287-95.
3. American College of Obstetricians and Gynecologists. Report of the American College of
Obstetricians and Gynecologists task force on
hypertension in pregnancy. Obstet Gynecol
2013;122:1122-31.
4. Frazier AE, Thorburn DR. Biochemical analyses of the electron transport chain complexes
by spectrophotometry. Methods Mol Biol
2012;837:49-62.
5. Whitehead CL, Palmer KR, Nilsson U, et al.
Placental expression of a novel primate-specic
splice variant of sFlt-1 is upregulated in pregnancies complicated by severe early onset preeclampsia. BJOG 2011;118:1268-71.

MARCH 2016 American Journal of Obstetrics & Gynecology

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SUPPLEMENTARY TABLE

Baseline characteristics of placenta collected that compares mitochondrial electron transport chain activity
in placentas that were complicated by preterm preeclampsia vs controls (normotensive, preterm delivery)
Characteristic

Preeclampsia (n 23)

Preterm (n 25)

P value

Maternal age, y

30.6  5.9

31.1  6.6

.78

Gestation, wka

29.6  2.3

29.4  2.4

.75

28.3  5.5

27.6  7.9

.58

169.9  15.4

119.8  14.7

< .0001

103.6  9.9

70.4  14.1

< .0001

1190  448.5

2a

Body mass index, kg/m

Systolic blood pressure, mm Hg

Diastolic blood pressure, mm Hg

1393  475.7

.14

Nulliparity, n (%)

15 (65)

9 (36)

.08

Intrauterine growth restriction (birthweight, <10%), n (%)

11 (48)

10 (40)

.77

Birthweight, g

Data are shown is mean  standard deviation.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016.

356.e15 American Journal of Obstetrics & Gynecology MARCH 2016