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RESEARCH ARTICLE
Melatonin enhances antioxidant molecules in
the placenta, reduces secretion of soluble
fms-like tyrosine kinase 1 (sFLT) from primary
trophoblast but does not rescue endothelial
dysfunction: An evaluation of its potential to
treat preeclampsia
Natalie J. Hannan*, Natalie K. Binder, Sally Beard, Tuong-Vi Nguyen, Tu’uhevaha
J. Kaitu’u-Lino, Stephen Tong
Translational Obstetrics Group, Mercy Perinatal, Department of Obstetrics and Gynaecology, University of
Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia
*nhannan@unimelb.edu.au
Abstract
Preeclampsia is one of the most serious complications of pregnancy. Currently there are no
medical treatments. Given placental oxidative stress may be an early trigger in the patho-
genesis of preeclampsia, therapies that enhance antioxidant pathways have been proposed
as treatments. Melatonin is a direct free-radical scavenger and indirect antioxidant. We per-
formed in vitro assays to assess whether melatonin 1) enhances the antioxidant response
element genes (heme-oxygenase 1, (HO-1), glutamate-cysteine ligase (GCLC), NAD(P)H:
quinone acceptor oxidoreductase 1 (NQO1), thioredoxin (TXN)) or 2) alters secretion of the
anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFLT) or soluble endoglin (sENG)
from human primary trophoblasts, placental explants and human umbilical vein endothelial
cells (HUVECs) and 3) can rescue TNF-αinduced endothelial dysfunction. In primary
trophoblast melatonin treatment increased expression of the antioxidant enzyme TXN.
Expression of TXN, GCLC and NQO1 was upregulated in placental tissue with melatonin
treatment. HUVECs treated with melatonin showed an increase in both TXN and GCLC.
Melatonin did not increase HO-1 expression in any of the tissues examined. Melatonin
reduced sFLT secretion from primary trophoblasts, but had no effect on sFLT or sENG
secretion from placental explants or HUVECs. Melatonin did not rescue TNF-αinduced
VCAM-1 and ET-1 expression in endothelial cells. Our findings suggest that melatonin
induces antioxidant pathways in placenta and endothelial cells. Furthermore, it may have
effects in reducing sFLT secretion from trophoblast, but does not reduce endothelial dys-
function. Given it is likely to be safe in pregnancy, it may have potential as a therapeutic
agent to treat or prevent preeclampsia.
PLOS ONE | https://doi.org/10.1371/journal.pone.0187082 April 11, 2018 1 / 13
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OPEN ACCESS
Citation: Hannan NJ, Binder NK, Beard S, Nguyen
T-V, Kaitu’u-Lino TJ, Tong S (2018) Melatonin
enhances antioxidant molecules in the placenta,
reduces secretion of soluble fms-like tyrosine
kinase 1 (sFLT) from primary trophoblast but does
not rescue endothelial dysfunction: An evaluation
of its potential to treat preeclampsia. PLoS ONE 13
(4): e0187082. https://doi.org/10.1371/journal.
pone.0187082
Editor: Sinuhe Hahn, University Hospital Basel,
SWITZERLAND
Received: March 22, 2017
Accepted: September 21, 2017
Published: April 11, 2018
Copyright: ©2018 Hannan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The National Health and Medical
Research Council of Australia (NHMRC) provided
salary support (#1050765 to ST; #1062418 to
TKL). NJH salary was supported by a University of
Melbourne CR Roper Fellowship. The funders had
Introduction
Complicating around 3% - 5% of all pregnancies, preeclampsia is a leading cause of maternal
mortality, especially in developing countries [1,2]. It is estimated to be responsible for over
60,000 maternal deaths annually [3] and far higher rates of perinatal loss [3]. Since there are
no treatments to arrest disease progression, the only known way to stop this disease is deliv-
ery of the placenta, removing the source of the pathogenic circulating factors that contrib-
utes to the maternal disease. As such, preeclampsia is a major cause of premature delivery,
especially when preeclampsia occurs at preterm gestation (<34 weeks). Thus, an effective
therapy to treat or prevent preeclampsia would be a major advance, saving many lives
globally.
Preeclampsia is a multi-system disorder affecting maternal vessels (causing high blood pres-
sure (hypertension) and vascular injury (endothelial dysfunction)), kidneys, liver, the haema-
tological system, brain (causing seizures or eclampsia) and the fetoplacental unit (resulting in
fetal growth restriction) [3]. In preeclampsia, hypertension is characterized by the mother’s
peripheral vasoconstriction and decreased arterial compliance [4] and the high levels of pro-
tein in the urine comes from acute inflammation of the kidneys endothelial cells (lining the
glomerular) [4], which leads to significant tissue damage and poor filtration.
Placental and systemic oxidative stress is thought to be a key aspect in the pathogenesis of
preeclampsia [5]. It is widely thought that the pathological process of preeclampsia can be
attributed to two main events: inadequate implantation and subsequent maternal endothelial
dysfunction [3]. In preeclamptic pregnancies, the placenta fails to adequately invade and
remodel the maternal uterine spiral arteries. This compromises placental perfusion, resulting
in high levels of oxidative stress in the placenta [5–7]. The dysfunctional stressed placenta
releases excess levels of anti-angiogenic factors, specifically soluble fms-like tyrosine kinase 1
(sFLT) [8,9] and soluble endoglin (sENG) [10,11] into the maternal circulation, where they
injure the maternal vasculature causing widespread endothelial dysfunction and multi-system
maternal organ injury [3,4,12]. A treatment that can reduce the oxidative stress in the pre-
eclamptic placenta could help to reduce tissue damage. Such a therapy could be a novel strat-
egy to prevent the disease, or decrease severity.
Antioxidants are molecules that inhibit the oxidation of other molecules, and thus reduce
or stop the chemical chain reaction of free radical production that results in oxidative stress
[13]. Melatonin (5-methoxy-N-acetyltryptamine), a potent endogenous antioxidant, functions
both directly as a free radical scavenger and indirectly by activating antioxidant enzymes [14–
16]. Melatonin is primarily synthesized in the pineal gland [17], however throughout gestation
the placenta is a significant extrapineal source, with maternal melatonin levels peaking at term
[18,19]. As well as synthesis, melatonin readily crosses the placental barrier and acts to pro-
mote trophoblast survival through its MT1 and MT2 receptors [19–21]. Aside from cellular
antioxidant defenses, melatonin also has vasodilatory properties [22–24]. Melatonin is largely
considered vital in pregnancy success [25].
Circulating melatonin levels are significantly decreased in cases of severe preeclampsia
[26] concurrent with decreased placental melatonin synthesis and receptor abundance [27].
Serotonin, melatonin’s immediate precursor, is increased in preeclamptic placentas due to an
inhibition of aralkylamine N-acetyltransferase activity, the rate limiting enzyme in melatonin
synthesis [27]. Use of melatonin as a therapeutic in animal models of placental ischemia has
shown reduced tissue and DNA damage from oxidative stress [28,29], with no reported nega-
tive side-effects [30]. Given this, and the likely safety of melatonin supplementation during
pregnancy for mother and fetus, we set out to test the possibility that melatonin may be a treat-
ment for preeclampsia in models of preeclampsia in vitro.
Melatonin to treat preeclampsia
PLOS ONE | https://doi.org/10.1371/journal.pone.0187082 April 11, 2018 2 / 13
no role in study design, data collection, analysis or
decision to publish.
Competing interests: The authors have declared
that no competing interests exist.
Materials and methods
Tissue collection
Ethical approval was obtained for this study from the Mercy Health Human Research Ethics
Committee. Women presenting to the Mercy Hospital for Women, Melbourne gave informed
written consent for tissue collection.
Placentas and umbilical cords were obtained from normal term pregnancies (>38 weeks
gestation) at elective cesarean section for functional studies. Placentas and umbilical cords
were collected within 30min of delivery and washed in sterile phosphate buffered saline
(PBS).
Primary cytotrophoblast isolation
As described previously [31,32] approximately 150g of placental tissue was washed with sterile
PBS and maternal and fetal surfaces were removed. Placental cotyledons were scraped with a
scalpel to dissociate placental villi from vessels. Placental tissue was washed with 0.9% NaCl to
remove blood cells then subjected to three 20 minute digestion cycles with 0.25% trypsin and
0.2mg/ml DNAse in Enzyme Digestion Buffer containing 10 x Hanks Buffered Salt Solution,
sodium bicarbonate, HEPES and deionised H
2
O. Cell suspensions were filtered and then
separated using a discontinuous Percoll gradient centrifugation. The layer containing cyto-
trophoblasts were then collected and subjected to a CD9 negative selection step, to remove
contaminating non-trophoblast cells (resulting in >98% pure trophoblast population[33]).
Primary cytotrophoblasts were plated at 5x10
5
/cm
2
and cultured in DMEM high Glutamax
(Thermofisher; Scoresby VIC) containing 10% Fetal Calf Serum (Sigma, St Louis, United
States) and 1% anti-anti (Life Technologies) on fibronectin (10ug/mL; BD Bioscience, USA)
coated plates, cells were cultured at 37˚C under 8% O2. Viable cells attached overnight and
were then washed twice with sterile PBS to remove non-viable cells and cell debris. Isolated
primary cytotrophoblasts were treated with increasing doses of melatonin (1–1000μM) for 48
h. This culture period and cell density would include a mixed population of cytotrophoblast
and syncytiotrophoblast [33].
Isolation and culture of placental explants
Small pieces of villous tissue were cut from the mid-portion of the placenta to avoid the mater-
nal and fetal surfaces. These were thoroughly washed with PBS and allowed to equilibrate at
37˚C for 1 hour in DMEM (Thermofisher) containing 1% anti-anti and 10% fetal calf serum
(Thermofisher). Tissue explants were then dissected into small fragments of 1-2mm size and
three pieces put into each well of a 24 well plate and were cultured at 37˚C under 8% O2. Pla-
cental explants were treated with melatonin (100μM and 1000μM) for 48h.
Primary human umbilical vein endothelial cell (HUVEC) isolation
The cord vein of umbilical cords of normal term placentas was cannulated and infused with
PBS to wash out fetal blood. Next, approximately 10ml (1mg/ml) of collagenase (Worthington,
Lakewood, New Jersey) was infused into the cord followed by incubation at 37˚C for 10 min-
utes. The dissociated HUVEC cells were recovered by pelleting and resuspension followed by
culture in M199 media (Life Technologies) containing 20% fetal calf serum, 1% anti-anti and
1% endothelial cell growth factor (Sigma) and 1% heparin. HUVECs were cultured at 37˚C
under 20% O2. Isolated primary HUVECs (between passage 2 and 4) were treated with
increasing doses of melatonin (Sigma (M5250); 1–1000μM) for 48 h.
Melatonin to treat preeclampsia
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Endothelial dysfunction rescue studies
Endothelial cells (HUVECs) were treated with TNF-α(Thermofisher; 10ng/mL) to induce
dysfunction. Cells were then cultured in the presence of either TNF-α(10ng/ml) alone; with
both TNF-α(10ng/ml) and melatonin (50μM and 100μM); or control media for 8–12 h.
Quantitative RT-PCR
Total RNA was extracted from isolated cytotrophoblast, endothelial cells (HUVEC) and pla-
cental explant tissue using the RNeasy mini kit (Qiagen, Valencia, CA) and quantified using a
Nanodrop ND 1000 spectrophotometer (NanoDrop technologies Inc, Wilmington, DE) and
converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit
(Thermofisher) as per manufacturer guidelines. Quantitative PCR was performed using Taq-
man gene expression assays for: HO-1, GCLC, TXN, NQO1, VCAM, ET-1. PCR was per-
formed on the CFX 384 (Biorad, Hercules, CA) using FAM-labeled Taqman universal PCR
mastermix (Applied Biosystems) with the following run conditions: 50
o
C for 2 minutes; 95
o
C
for 10 minutes, 95
o
C for for 15 seconds, 60
o
C for 1 minute (40 cycles). All data were nor-
malized to an appropriate house-keeping gene (isolated cells normalized to: GAPDH and
YWHAZ; placental tissue to: TOP1 and CYC1) as an internal control and calibrated against
the average C
t
of the control samples. The results were expressed as fold change relative to con-
trols. All samples were run in triplicate.
sFLT and sENG ELISAs. Conditioned media from primary cytotrophoblast, placental
explants and endothelial cells (HUVECs) was assessed using ELISA for the presence of the fol-
lowing soluble factors: soluble Flt-1 (sFLT) DuoSet VEGF R1/Flt-1 kit (R&D systems by Bio-
science, Waterloo, Australia), soluble endoglin (sENG) DuoSet Human Endoglin CD/105
(R&D systems). Optical density for all ELISAs was determined using a BioRad X-Mark micro-
plate spectrophotometer (BioRad), Protein levels determined using BioRad Microplate man-
ager 6 software. The assay lower detection limit was 125pg/mL and the upper detection limit
was 8000pg/mL. The coefficients of variants were: intra-assay <10% and inter-assay <15%.
Effects of melatonin on cell viability. Primary cytotrophoblasts and human umbilical
vein endothelial cells (HUVECs) treated with increasing doses of melatonin (1–1000μM)
showed no significant difference in cell viability (see S1 File). Cell viability assays were per-
formed using CellTiter 96-Aqueous One solution (Promega, Madison WI) according to the
manufacturer’s instructions.
Statistical analysis
All in vitro and ex vivo experiments were performed with technical triplicates and all experi-
ments were repeated a minimum of three times. Data was tested for normal distribution and
statistically tested as appropriate. Data was tested non-parametrically using Kruskal-Wallis
test. Data is expressed as mean ±SEM. P-values <0.05 were considered significant. Statistical
analysis was performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA).
Results
Effects of melatonin on anti-oxidant gene expression in primary human
placental tissues and endothelial cells
Melatonin was added to primary placental explants, and expression of anti-oxidant genes was
measured. Melatonin had no effect on HO-1 mRNA expression (Fig 1A; see S2 File), but sig-
nificantly increased expression of GCLC, NQO1 and TXN (Fig 1B–1D; see S2 File) at the top
Melatonin to treat preeclampsia
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dose. However melatonin treatment did not increase NQO1 protein production by placental
explants (Fig 1E; see S2 File).
When added to isolated primary trophoblast, melatonin treatment did not affect heme-oxy-
genase 1 (HO-1) expression (Fig 1F; see S2 File). Increasing doses of melatonin induced a
non-significant trend toward an increase in mRNA expression of both glutamate-cysteine
ligase (GCLC) and NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) (Fig 1G–1I). Mela-
tonin significantly increased the mRNA expression of the anti-oxidant enzyme thioredoxin
(TXN) (Fig 1I; see S2 File). When added to primary human umbilical vein endothelial cells
(HUVECs), melatonin did not alter mRNA expression of HO-1 (Fig 1J; see S2 File), or NQO1
(Fig 1L; see S2 File), but significantly increased GCLC mRNA expression in HUVECs at the
top dose (Fig 1K; see S2 File). Melatonin increased TXN mRNA expression at the 10μM con-
centration (Fig 1M; see S2 File). Consistently NQO1 protein was not increased in HUVECs
with melatonin treatment, however while TXN mRNA was increased in HUVECs treated with
melatonin (Fig 1M; see S2 File) there was no change in TXN protein in HUVECs treated with
melatonin (Fig 1O; see S2 File).
In summary, addition of melatonin to primary placental tissues and cells caused an up-reg-
ulation in the mRNA expression of anti-oxidant genes. This possibly also occurs when admin-
istered to endothelial cells although the effects appear more modest.
Effects of melatonin on sFLT and sENG secretion
The excess release of anti-angiogenic factors by the preeclamptic placenta into the maternal
circulation is thought to lead to widespread maternal endothelial dysfunction and multi-organ
injury seen in clinical disease. Drugs that can decrease the release of these factors may have
potential as a treatment [31,34,35]. Therefore, we examined whether melatonin can decrease
sFLT and sENG secretion from placental tissues and cells.
Melatonin did not affect placental secretion of sFLT or sENG when administered to placen-
tal explants (Fig 2A and 2B; see S2 File). Melatonin significantly reduced sFLT secretion from
primary human trophoblast at the 1000μM dose (Fig 2C; see S2 File). However, similarly to
placental explant tissue, the addition of melatonin to HUVECs did not reduce either sFLT or
sENG secretion (Fig 2D and 2E; see S2 File).
Effects of melatonin on TNF-αinduced endothelial dysfunction
Given endothelial dysfunction is an important aspect of the pathophysiology of preeclampsia,
we examined whether melatonin might have actions to rescue endothelial dysfunction. As
expected, administering tumor necrosis factor α(TNF-α) caused a potent increase in vascular
cell adhesion molecule 1 (VCAM-1, Fig 3A; see S2 File) and Endothelin-1 (ET-1, Fig 3B; see S2
File), markers of endothelial dysfunction. The administration of melatonin at 50 and 100μM
did not rescue TNF-αinduced VCAM-1 or ET-1 mRNA up-regulation (Fig 3A and 3B; see
S2 File).
Fig 1. Effects of melatonin on anti-oxidant gene expression in primary human placenta.Normal term placental explant tissue, isolated
primary cytotrophoblasts and human umbilical vein endothelial cells (HUVECs) were treated with melatonin (100–1000μM placental explant
tissue and 1–1000μM for isolated cells) for 48 hours. Placental explant tissue mRNA expression for heme-oxygenase 1 (HO-1) (A), glutamate-
cysteine ligase (GCLC) (B), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) (C) and thioredoxin (TXN) (D) follwoing melatonin
treatment. Densitometric analysis of placental explant NQO1 protein levels with melatonin treatment (E). Primary trophoblast mRNA expression
of HO-1 (F), GCLC (G), NQO1 (H) and TXN (I) with melatonin treatment. Primary HUVEC mRNA expression of HO-1 (J), GCLC (K), NQO1
(L) and TXN (M). Experiments were performed with technical triplicates and all experiments were repeated a minimum of three times. Data is
expressed as the mean % change from control ±SEM. All data were analyzed by Kruskal-Wallis followed by Dunn’s multiple comparisons test
(p0.05; p0.01).
https://doi.org/10.1371/journal.pone.0187082.g001
Melatonin to treat preeclampsia
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In accordance with the PLOS ONE journal’s Data Availability requirements the full data
sets are provided in a supporting information file S2 File.
Discussion
Melatonin is a potent endogenous antioxidant hormone, with extrapineal synthesis occurring
in the placenta throughout pregnancy. Both circulating melatonin and placental melatonin
synthesis are decreased with preeclampsia, suggesting melatonin supplementation may be a
potential therapeutic to treat the disease. Here we demonstrate for the first time that melatonin
has increases antioxidant enzyme expression in placental tissue, with more modest effects on
isolated trophoblast and endothelial cells (HUVECs). In addition, we show that melatonin has
little to no effect on sFLT and sENG secretion, and does not mitigate TNF-αinduced endothe-
lial dysfunction in vitro.
In many disease models, melatonin has been shown to protect tissue from damage caused
by inflammation and oxidative stress. Melatonin is a powerful antioxidant, acting as a direct
scavenger of oxygen free radicals, especially the highly damaging hydroxyl radical, and indi-
rectly through upregulation of antioxidant enzymes[36]. In animal models of fetal growth
restriction (FGR), the administration of melatonin had positive effects on the newborn lamb
with overall reduced hypoxia, improved neurodevelopment and decreased brain injury[37].
Furthermore in rodent models melatonin has been observed to have protective benefits in
Fig 2. Effects of melatonin on sFLT and sENG secretion. Placental explant tissue, primary cytotrophoblasts and human umbilical vein endothelial cells
(HUVECs) were treated with melatonin (100–1000μM placental explant tissue and 1–1000μM for isolated cells) for 48 h. Placental explant secretion of
sFLT (A) and sENG (B) was assessed following melatonin treatment. Cytotrophoblast secretion of sFLT (C) following melatonin. HUVEC secretion of
sFLT (D) and sENG (E) with increasing doses of melatonin. Experiments were performed with technical triplicates and all experiments were repeated a
minimum of three times. Data is expressed as mean % change from control ±SEM. All data were analyzed by Kruskal-Wallis followed by Dunn’s multiple
comparisons test (p0.001).
https://doi.org/10.1371/journal.pone.0187082.g002
Fig 3. Effects of melatonin on TNF-α-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were treated with tumor necrosis factor α
(TNF-α; 10ng/mL) for 2 hours, followed by addition of melatonin (50 and 100μM) in the presence of TNF-α(10 ng/mL) for an additional 24 hours. The mRNA
expression of endothelial dysfunction markers was assessed VCAM (A) and ET-1 (B). Experiments were performed with technical triplicates and all experiments were
repeated a minimum of three times. Data is expressed as relative mRNA expression ±SEM. All data were analyzed by Kruskal-Wallis followed by Dunn’s multiple
comparisons test (p0.01; p0.001).
https://doi.org/10.1371/journal.pone.0187082.g003
Melatonin to treat preeclampsia
PLOS ONE | https://doi.org/10.1371/journal.pone.0187082 April 11, 2018 8 / 13
ischemic-reperfusion studies, reducing the overall induced oxidative damage to placental
mitochondria[29], and melatonin administration was shown to improve the fetal to placental
weight ratio, birth weight and enhance antioxidant enzyme production in a model of maternal
undernourishment (which leads to oxidative damage) [38]. Given such promising data in ani-
mal models the PAMPR trial, an early Phase I (single arm, open label clinical trial) was estab-
lished, to assess the potential clinical and biochemical effects of melatonin (daily oral
administration (10 mg; 3 times)) in pregnancies complicated with pre-term preeclampsia [39].
Our group has a long standing interest in assessing drugs safe in pregnancy, to determine
whether they may be able to mitigate the pathogenesis of preeclampsia. Our approach is to
assess the efficacy of the agent to 1) enhance antioxidant pathways, 2) reduce the anti-angio-
genic imbalance and 3) reduce maternal systemic oxidative stress and endothelial dysfunction.
We have now shown that both metformin and the proton pump inhibitors are able to potently
induce Nrf2 regulated anti-oxidant enzymes, reduce secretion of sFLT and sENG and mitigate
endothelial dysfunction (in vitro and ex vivo)[40,41] as well as hypertension in a mouse
model of preeclampsia[41]. Thus using a dose range 1–1000μM of melatonin (previously
shown to have actions on cytotrophoblast [42] and relevant to that being investigated in the
PAMPR trial [39]) we aimed to assess whether melatonin was able to enhance antioxidant
defences, reduce sFLT and sENG secretion and quench endothelial dysfunction. However we
found that HO-1 production was not stimulated by melatonin in primary trophoblasts, placen-
tal explant tissue, or primary HUVECs.
In the current study, melatonin treatment upregulated the mRNA expression of ARE genes;
TXN in primary trophoblasts, placental explant tissue and primary HUVECs; GCLC in placen-
tal explant tissue and primary HUVECs, but not primary trophoblasts; NQO1 dose-depen-
dently in placental explant tissue, but not primary trophoblasts or primary HUVECs. However
at these doses, melatonin was unable to increase the protein levels of these antioxidant mole-
cules. Melatonin may act as an indirect antioxidant in trophoblast, placental explant tissue and
primary HUVECs and may be useful to combat the rise in oxidative stress in the placenta dur-
ing preeclampsia [43–45].
Anti-angiogenic factors (sFLT and sENG) are released in excess into the maternal circula-
tion where they circulate causing widespread endothelial dysfunction [8,9]. Quenching sFLT
and sENG secretion would likely halt the damage to the maternal vasculature and end organ
injury associated with preeclampsia. Melatonin treatment did not decrease the production of
either sFLT or sENG by placental explant tissue or isolated primary HUVECs. However, the
secretion of sFLT by primary trophoblast was significantly reduced following treatment with
1000 μM melatonin. It is however important to note, although not statistically significant, at
this dose (1000 μM) melatonin had a slight negative effect on primary trophoblast cell viability,
and as such this decrease in sFLT secretion may in fact reflect altered viability rather than a
true protective effect of melatonin. In addition the placentae obtained and used in these exper-
iments were collected from normal pregnancies. While the isolation of trophoblast and cell
culture effects have been shown to enhance sFLT secretion[33] and thus provide a good model
of preeclampsia in vitro, examination of Melatonin in placentae from preeclamptic pregnan-
cies may provide further insight.
Our group [40,41,46–48] and others[49,50] have used the pro-inflammatory cytokine
TNF-αto model endothelial dysfunction in vitro with respect to preeclampsia. Importantly,
endothelial sensitivity to TNF-αis enhanced by exposure to recombinant sFLT (as well as
other agents that block VEGF signaling) [50]. We used TNF-αto induce endothelial dysfunc-
tion in vitro, consistent with our previous studies we found that TNF-αsignificantly increased
expression of VCAM and ET-1. However melatonin was unable to mitigate the effects of TNF-
αinduced endothelial dysfunction in primary HUVECs. Interestingly, several animal models
Melatonin to treat preeclampsia
PLOS ONE | https://doi.org/10.1371/journal.pone.0187082 April 11, 2018 9 / 13
of vascular injury (smoke-induced and chronic intermittent hypoxia) have shown a recovery
from endothelial dysfunction with melatonin treatment, reducing expression of both VCAM
and ET-1 [51,52]. Melatonin appears to have little ability to alleviate the effects of endothelial
dysfunction in primary HUVECs isolated from the human placenta.
We have demonstrated that in vitro, melatonin (1–100μM) does not significantly quench
sFLT and sENG production by placental explant tissue or primary HUVECs despite a decrease
in sFLT production by primary trophoblasts (at top dose), nor does it mitigate the effects of
endothelial dysfunction in primary HUVECs. Melatonin did however successfully act as an
indirect antioxidant in these cells and tissues, differentially upregulating the expression of sev-
eral ARE genes. It is still possible that melatonin’s antioxidant properties and safety profile
may prove to be beneficial in maintaining an ongoing healthy pregnancy. We eagerly await the
results of a phase 1 pilot clinical trial currently underway to test the effect of melatonin in pre-
eclamptic pregnancies [39].
Supporting information
S1 File. Effects of melatonin on trophoblast and endothelial (HUVEC) viability. Primary
cytotrophoblasts (A) and human umbilical vein endothelial cells (B) were isolated from term
placentas and treated with increasing doses of melatonin (1–1000μM) for 48 h cell viability
was assessed using a MTS assay. There was no significant effect on cell viability with melatonin
treatment. Data is expressed as relative mRNA expression ±SEM. Data were analyzed by Krus-
kal-Wallis followed by Dunn’s multiple comparisons test.
(TIFF)
S2 File. Raw data files and statistical analysis used for generation of Figs 1–3.
(PDF)
Acknowledgments
The authors acknowledge Clinical Research midwives Gabrielle Pell, Rachel Murdoch, Gene-
vieve Christophers and Debra Jinks and the Obstetrics midwifery staff and patients at the
Mercy Hospital for Women (Heidelberg) for provision of placental tissue.
Author Contributions
Conceptualization: Natalie J. Hannan, Stephen Tong.
Data curation: Natalie J. Hannan, Natalie K. Binder, Sally Beard, Tuong-Vi Nguyen.
Formal analysis: Natalie J. Hannan, Sally Beard.
Funding acquisition: Natalie J. Hannan, Tu’uhevaha J. Kaitu’u-Lino, Stephen Tong.
Investigation: Natalie J. Hannan, Natalie K. Binder, Sally Beard, Tu’uhevaha J. Kaitu’u-Lino,
Stephen Tong.
Methodology: Natalie J. Hannan, Natalie K. Binder, Sally Beard, Tuong-Vi Nguyen.
Project administration: Natalie J. Hannan, Tu’uhevaha J. Kaitu’u-Lino, Stephen Tong.
Resources: Natalie J. Hannan, Stephen Tong.
Supervision: Natalie J. Hannan, Tu’uhevaha J. Kaitu’u-Lino.
Validation: Natalie K. Binder, Sally Beard, Tuong-Vi Nguyen.
Visualization: Sally Beard.
Melatonin to treat preeclampsia
PLOS ONE | https://doi.org/10.1371/journal.pone.0187082 April 11, 2018 10 / 13
Writing – original draft: Natalie J. Hannan, Natalie K. Binder.
Writing – review & editing: Natalie J. Hannan, Sally Beard, Tuong-Vi Nguyen, Tu’uhevaha J.
Kaitu’u-Lino, Stephen Tong.
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