Review

Special Issue: Nurturing the Next Generation

Molecular mechanisms of maternal

vascular dysfunction in preeclampsia
Styliani Goulopoulou1,2 and Sandra T. Davidge3,4,5
1

Department of Integrative Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, USA
Department of Obstetrics and Gynecology, University of North Texas Health Science Center, Fort Worth, TX, USA
3
Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Canada
4
Department of Physiology, University of Alberta, Edmonton, Canada
5
Women and Childrens Health Research Institute, Edmonton, Canada
2

In preeclampsia, as a heterogeneous syndrome, multiple

pathways have been proposed for both the causal as
well as the perpetuating factors leading to maternal
vascular dysfunction. Postulated mechanisms include
imbalance in the bioavailability and activity of endothelium-derived contracting and relaxing factors and oxidative stress. Studies have shown that placenta-derived
factors [antiangiogenic factors, microparticles (MPs),
cell-free nucleic acids] are released into the maternal
circulation and act on the vascular wall to modify the
secretory capacity of endothelial cells and alter the responsiveness of vascular smooth muscle cells to constricting and relaxing stimuli. These molecules signal
their deleterious effects on the maternal vascular wall
via pathways that provide the molecular basis for novel
and effective therapeutic interventions.
Preeclampsia: a pregnancy-specific syndrome with
gestational and postpartum vascular implications
Preeclampsia is a pregnancy-specific syndrome and one of
the leading causes of maternal and fetal mortality and
morbidity. This syndrome is characterized by newly developed hypertension and proteinuria diagnosed after
20 weeks of gestation [1]. Common clinical manifestations
include maternal vascular dysfunction, chronic immune
system activation, renal dysfunction, and intrauterine
growth restriction (IUGR). If left unmanaged, preeclampsia can lead to maternal seizures, stroke, multiorgan failure, and death [2]. While approximately 63 000 maternal
and 500 000 infant deaths are attributed to preeclampsia
annually [3], no early diagnosis criteria are established
and there is no cure for this pregnancy syndrome.
The exact cause of preeclampsia is currently unknown
but there is a consensus that the placenta plays a cardinal
role in the pathogenesis of preeclampsia because delivery
of the placenta resolves the clinical symptoms. Changes in
the oxygenation levels of the placenta due to failure of
spiral artery transformation are thought to be responsible
Corresponding author: Davidge, S.T. (sandra.davidge@ualberta.ca).
Keywords: endothelial function; pregnancy; hypertension; preeclampsia.
1471-4914/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.11.009

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Trends in Molecular Medicine, February 2015, Vol. 21, No. 2

for the underlying pathology of preeclampsia [4]. Recent

discoveries of an imbalance in antiangiogenic factors and
loss of cytoprotective mechanisms in preeclamptic placentas have provided new insights into the pathophysiology of this pregnancy syndrome and are promising
prognostic and therapeutic targets [59].
Systemic maternal vascular dysfunction is a major phenotype of pregnancies with preeclampsia, contributing to

Glossary
Endothelium-derived contracting factors (EDCFs): molecules produced in the
vascular endothelium that signal smooth muscle cells in blood vessel walls to
contract, constricting the blood vessel. EDCFs are released in response to
various stimuli, such as Ang II, arachidonic acid, hypoxia, stretch, and the
superoxide anion. Release of EDCFs is associated with pathological conditions.
Endothelium-derived relaxing factors (EDRFs): molecules produced in the
vascular endothelium that signal smooth muscle cells in blood vessel walls to
relax, dilating the blood vessel.
Hypertension: a chronic medical condition in which the blood pressure in the
arteries is elevated.
Intrauterine growth restriction (IUGR): attenuated growth of a fetus; the fetus
does not reach its growth potential while in the mothers womb. In cases of
IUGR, the growing fetus weighs less than 90% of other babies at the same
gestational age.
Lectin-like oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1): a
lectin-like 52-kD receptor that mediates the recognition, internalization, and
degradation of oxLDL by vascular endothelial cells.
Microparticles (MPs): vesicles >100 nm in diameter that are released into the
circulation via shedding from activated or apoptotic cells.
Mitochondrial DNA (mtDNA): contains 37 genes and approximately 16 600 bp;
organized as a circular, covalently closed, double-stranded DNA in most
multicellular organisms.
Nitric oxide (NO): free radical with cell signaling properties.
Oxidative stress: a physiological state that describes an imbalance between the
production of free radicals and antioxidant defense mechanisms.
Postpartum: relating to or occurring in the period of time following the birth of
a child.
Prostacyclin (PGI2): a lipid molecule and member of the eicosanoid family that
is released by healthy endothelium and induces smooth muscle relaxation.
Proteinuria: the presence of an excess of serum proteins in the urine.
Reactive oxygen species (ROS): chemically reactive molecules containing
oxygen that play a role in cell signaling and homeostasis.
Resistance vessels: small blood vessels that constitute the main part of total
vascular resistance to blood flow.
Spiral arteries: corkscrew-like arteries of the endometrium that are sensitive to
hormonal and growth factor influences in the non-pregnant endometrium.
Undergo remodeling and vascular smooth muscle disorganization during
pregnancy due to the phenomenon of trophoblast invasion.
Toll-like receptors (TLRs): single, membrane-spanning, noncatalytic receptors
that recognize structurally conserved molecules derived from microbes
(pathogen-associated molecular patterns) or dying cells (damage-associated
molecular patterns).
Trophoblast: specialized cells of the placenta.

Review
increased peripheral vascular resistance, maternal hypertension, and proteinuria [10]. The mechanisms of systemic
vascular dysfunction in preeclamptic pregnancies involve
an imbalance in the production of constrictors and dilators
in vascular cells, hyper-responsiveness to constrictor stimuli, reduced endothelium-dependent dilation, and oxidative stress [1,10,11]. Many studies have shown that the
interaction between circulating factors and the maternal
endothelium significantly contribute to generalized vascular dysfunction [1214]. These circulating factors are partly of placental origin, providing a link between placental
and maternal vascular dysfunction [15]. The maternal
response to these factors depends on the womans vascular
health, which may be compromised due to pre-existing
conditions such as obesity, diabetes, or poor nutrition,
all of which are risk factors for developing preeclampsia [1].
The implications of preeclampsia extend beyond the
course of gestation. Delivery of the placenta resolves the
clinical symptoms; however, women with a history of preeclampsia have increased risk of developing cardiovascular
disease later in life [16,17]. Women affected by preeclampsia have a three- or fourfold greater risk of developing
hypertension [18], stroke, or heart disease 515 years after
pregnancy [17] and a twofold greater risk of dying from
cardiovascular or cerebrovascular disease compared with
women who had a normal pregnancy [10]. The molecular
mechanisms that link gestational complications with risk
for cardiovascular disease after pregnancy are not well
understood. Vascular dysfunction is an early marker of
atherosclerosis and cardiovascular disease and a common
feature of pregnancies with preeclampsia that persists
many years after a pregnancy affected by this syndrome
[1921]. Thus, vascular dysfunction during gestation may
account for an elevated risk of cardiovascular disease in
women with a history of preeclampsia [10].
Preeclampsia is a heterogeneous syndrome and multiple pathways have been proposed for both the causal as
well as the perpetuating factors leading to maternal vascular dysfunction. This review synthesizes knowledge from
recent discoveries on the mechanisms of maternal vascular
dysfunction in preeclampsia, elaborating specifically on
the molecular changes in vascular endothelial and smooth
muscle layers and on the mechanisms through which
circulating factors affect vascular reactivity thus contributing to maternal vascular dysfunction. We propose that
understanding the molecular mechanisms underlying preeclampsia-associated vascular dysfunction will provide
insight into the clinical manifestations of this syndrome
and also explain how pregnancy complications predispose
the mother to future cardiovascular events.
Endothelium-derived regulators of maternal vascular
reactivity in preeclampsia
Reduction of peripheral vascular resistance in early pregnancy followed by modest attenuation of mean arterial
pressure in mid-gestation is one of the major hemodynamic
adaptations to normal pregnancy [22]. In preeclampsia,
however, the maternal vascular adaptations are aberrant
and pregnant women with preeclampsia exhibit increased
total vascular resistance and arterial blood pressure that
persist until delivery [22]. Systemic endothelial dysfunction

Trends in Molecular Medicine February 2015, Vol. 21, No. 2

is central to the pathophysiology of preeclampsia and an

underlying mechanism of increased vascular resistance and
hypertension [1]. In response to mechanical and chemical
stimuli, the endothelial cells release vasoactive molecules,
which induce vascular smooth muscle relaxation [also
known as endothelium-derived relaxing factors (EDRFs)]
or contraction [also known as endothelium-derived contracting factors (EDCFs)], modulating vascular tone [23]. An
imbalance between EDRF and EDCF bioavailability, and
alterations in vascular smooth muscle responsiveness to
these factors, may account for the hypertensive phenotype
of preeclampsia, which is characterized by increased vasoconstriction and reduced vasodilation in the systemic maternal circulation.
EDRFs
Isolated arteries from women with preeclampsia exhibit
reduced endothelium-dependent relaxation [24,25]. Experimental animal models of preeclampsia show signs of
endothelial dysfunction in reproductive [26] and nonreproductive vessels [27,28]. The reduced endothelium-dependent
relaxation observed in pregnancies with preeclampsia is
attributed partly to reduced bioavailability of nitric oxide
(NO) [29] and prostacyclin (PGI2) [30]. NO induces vascular
smooth muscle relaxation via soluble guanylyl cyclase/cyclic
guanosine monophosphate (sGC/cGMP)-dependent and independent mechanisms [31]. PGI2 is a product of arachidonic acid metabolism that induces vascular smooth muscle
relaxation via receptor-mediated cyclic adenosine monophosphate (cAMP)-dependent mechanisms. Previous studies
have shown increased endothelial NO synthase (eNOS)
expression [32] in preeclampsia; however, increased arginase expression (a reciprocal regulator of NOS) [33],
elevated levels of asymmetric dimethylarginine (ADMA)
(a natural NOS inhibitor) [34], and increased peroxynitrite
formation (formed through scavenging of NO by superoxide
anions) [35] suggest that NO bioavailability is reduced
(Figure 1). Urinary and plasma concentrations of PGI2
are also reduced in women with preeclampsia [36], favoring
an imbalance between vasodilatory and vasoconstrictor
prostanoids.
Relaxation responses in some arteries cannot be fully
explained by the contributions of NO and PGI2. Vascular
smooth muscle hyperpolarization by endothelium-derived
factors other than prostanoids and NO has been previously
described [23]. The exact nature of these factors and mechanisms has not been defined, but myoendothelial gap
junctions and hydrogen sulfate (H2S) are potential candidates [9,23]. Small myometrial arteries from women with
preeclampsia showed reduced vasodilatory responses,
which were attributed to attenuation in the contribution
of endothelium-derived hyperpolarization (EDH) because
of physical disruption of myoendothelial junctions [37]
(Figure 1). Reduction in plasma H2S in pregnancies with
preeclampsia has also been reported [9]. In addition,
cystathionine-g-lyase (CSE), the primary H2S-synthesizing enzyme in the vasculature, is reduced in preeclampsia
[9] (Figure 1). The EDH-mediated response involves the
actions of small- (SKCa) and intermediate- (IKCa) but not
large- (BKCa) conductance calcium-activated potassium
channels. Chronic hypoxia inhibits pregnancy-induced
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Trends in Molecular Medicine February 2015, Vol. 21, No. 2

oxLDL

(6)

(7)

Endothelial cell

sFIt-1

sEng

cfDNA

STBMs

Placenta-derived factors

LOX-1

Arginase

AA

O2

ONOO

VEGF/TGF signaling

ROS

Cytokines

L-citrulline

L-arginine

O2

CSE

NO

ADMA
(1)

bET-1

L-cysteine

eNOS
uncoupling

NO

IKCa

H2S

SKCa

MMPs

ET-1

K+

MEGJ

PGI2

ETB

(3) Hyperpolarizaon

ETA

GTP

Vascular smooth
muscle cell

TxA2

ANGII
K+

NO

PDE

(4)

ONOO
NO

(2) sGC

PGHS

TP

AT1R

(5)

Fe
cGMP
Relaxaon

Vasodilaon
Vasoconstricon

Constricon
AT1-AA

TRENDS in Molecular Medicine

Figure 1. Simplified schematic representation of vascular processes affected by circulating and endothelium-derived factors in preeclampsia. (1) Increased arginase
expression [reciprocally regulates nitric oxide (NO) synthase (NOS)], elevated levels of asymmetric dimethylarginine (ADMA), a natural NOS inhibitor, and increased
peroxynitrite formation (formed through scavenging of NO by superoxide anions) contribute to a reduction in NO bioavailability, increased oxidative stress, and reduced
endothelium-dependent relaxation. (2) Reduced levels of cyclic guanosine monophosphate (cGMP) due to enhanced phosphodiesterase (PDE) and soluble guanylyl cyclase
(sGC) activity attenuate vascular smooth muscle relaxation. (3) Physical disruption of myoendothelial gap junctions (MEGJs), reduced production of hydrogen sulfate (H2S)
due to reduced cystathionine-g-lyase (CSE) activity, and downregulation of small- (SKCa) and intermediate- (IKCa) conductance calcium-activated potassium channels lead to
reduced endothelium-derived hyperpolarization (EDH), contributing to reduced vasodilation. (4) Increased production of endothelin (ET)-1 due to increased activity of
matrix metalloproteinases (MMPs) and thromboxane A2 (TxA2) results in augmented vascular constriction via activation of ET receptor A (ETA) and the TxA2 receptor (TP),
respectively. (5) Vasoconstrictor responses are potentiated by increased angiotensin (Ang) I receptor (AT1R) signaling. Circulating levels of AT1R autoantibodies (AT1-AAs)
also enhance AT1R signaling, augmenting vasoconstriction. (6) Increased circulating oxidized low-density lipoprotein (oxLDL) binds and activates low-density lipoprotein
receptor-1 (LOX-1) in endothelial cells causing the production of superoxide and peroxynitrite. (7) Overproduction of the antiangiogenic factors fms-like tyrosine kinase-1
(sFlt-1) and soluble endoglin (sEng) antagonize the vasodilatory properties of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and transforming
growth factor beta (TGF-b), respectively. Placenta-derived cell-free DNA (cfDNA), including mitochondrial and fetal DNA, and syncytiotrophoblast-derived microparticles
(STBMs), interacts with endothelial cells, monocytes, and neutrophils to promote oxidative stress and inflammation. Abbreviations: AA, arachidonic acid; PGI2,
prostacyclin; PGHS, prostaglandin endoperoxide synthase; bET-1, big ET-1; eNOS, endothelial NOS; ETB, ET receptor B.

upregulation of SKCa channel function in sheep uterine

arteries [38]. The function of endothelial SKCa channels
may be relevant to the vascular pathophysiology of preeclampsia since placental ischemia/hypoxia is considered a
feature of this syndrome.
The vascular smooth muscle responsiveness to EDRFs in
pregnancies with preeclampsia has been understudied, but
evidence suggests that it may contribute to maternal vascular dysfunction. cGMP is a second messenger downstream
of NO/sGC signaling that facilitates vascular smooth muscle
relaxation via activation of protein kinases or direct influences on potassium channels [39]. cGMP activity is regulated by the phosphodiesterase (PDE) enzyme family
[31]. cGMP levels in the placental circulation are reduced
and serum PDE activity is increased in pregnancies with
90

preeclampsia [31]. Reductions in endothelium-independent

relaxation of the thoracic aorta with concomitant decreases
in cGMP levels have been also found in animals with preeclampsia-like signs [40]. These findings were attributed to
a reduction in sGC activity [40] (Figure 1).
EDCFs
In addition to EDRFs, the endothelium produces endothelin (ET)-1, prostanoids [e.g., thromboxane A2 (TxA2)], and
angiotensin II (Ang II), which are all vasoconstrictors and
have been implicated in the vascular dysfunction of preeclampsia and other hypertensive disorders. Elevated
circulating levels of ET-1 have been reported in women
with preeclampsia [36]. There is no evidence to suggest
that increased ET-1 levels precede the manifestation of

Review
preeclampsia, suggesting that ET-1 may not play role in
the genesis of the syndrome. This claim, however, warrants further investigation because circulating levels do
not reflect tissue levels of the peptide. Nevertheless, many
studies have confirmed the implication of ET-1 signaling
in the excessive vasoconstriction seen in pregnancies with
preeclampsia [4144]. ET-1 transduces its effects via activation of ET receptor A (ETA) and ET receptor B (ETB),
which are expressed in vascular smooth muscle and endothelial cells [44]. Activation of ETA in vascular smooth
muscle cells induces vasoconstriction, whereas ETB activation in endothelial cells induces the release of NO and
PGI2, promoting vasodilation, and facilitates ET-1 clearance [44]. In preeclampsia there is an imbalance between
ET-1 production and receptor actions, promoting a vasoconstrictive state [43,44]. Enhanced ET-1-induced constriction was found in microvessels from the reduced
uteroplacental perfusion (RUPP) rat model of preeclampsia [44] and this was associated with an increased intracellular calcium initial peak. Thus, it is possible that
enhanced ET-1-induced vasoconstriction is due to a defect
in ET-1-induced calcium influx involving voltage-gated
and/or store- and receptor-operated channels. ETA antagonism ameliorated the hypertensive phenotype in various
animal models of preeclampsia, suggesting that enhanced
vasoconstrictor responses to ET-1 in preeclampsia may be
ETA dependent [45]. Recently, the involvement of ETB in
augmented ET-1-induced vasoconstrictor responses was
also reported [44]. Expression of the ETB receptor and its
ability to induce relaxation were attenuated in RUPP rats,
suggesting that downregulation of the ETB receptor and
its activity further contributes to enhanced ET-1 vasoconstrictive effects [44]. Abdalvand et al. [41] showed increased mesenteric artery responses to big ET-1 (bET-1)
in the RUPP model of preeclampsia. These enhanced
vasoconstrictor responses were attributed to alterations
in the conversion of bET-1 to ET-1 within the endothelium
due to increased activity of matrix metalloproteinases
(MMPs), particularly MMP-2 and MMP-9 [41]
(Figure 1). Thus, derangements in the functionality of
ET receptors, as well as alterations in ET-1 production,
may occur in preeclampsia.
TxA2 is an arachidonic acid metabolite derived from
prostaglandin H synthase (PGHS) that is produced in platelets and endothelial cells. The constrictor effects of TxA2 on
vascular smooth muscle are mediated by the TxA2 (TP)
receptor, a member of the prostanoid family of heterotrimeric G protein-coupled receptors [46]. TxA2 is considered to
play a role in preeclampsia-associated enhanced vasoconstriction [46] (Figure 1) and circulating levels of TxB2 (a
TxA2 metabolite) are significantly increased in pregnancies
with preeclampsia [30]. The vasoconstrictor effects of TxA2
in preeclampsia are amplified by its ability to potentiate the
vasoconstrictor effects of Ang II [47] and ET-1 [48]. NO and
PGI2 inhibit the actions of TxA2 via TP receptor desensitization [49]; however, this is not the case in pregnancies with
preeclampsia, as NO and PGI2 production is impaired.
Investigation of the global DNA methylation profiles of
omental arteries from women with preeclampsia revealed
that the thromboxane synthase gene (TBXAS1; encodes an
enzyme that catalyzes the isomerization of prostaglandin

Trends in Molecular Medicine February 2015, Vol. 21, No. 2

H2 into thromboxane) was the most significantly hypomethylated gene in vessels from women with preeclampsia
[50]. Reduced DNA methylation of the TBXAS1 promoter
was associated with increased expression of thromboxane
synthase in omental arteries of preeclamptic women
[51]. Taken together, these data suggest that in preeclampsia, there is an imbalance in the production of vasoconstrictor (TxA2) and vasodilatory prostanoids (PGI2), which is
modulated by epigenetic modifications.
Women with preeclampsia also exhibit an augmented
pressor response to Ang II and this response can be observed even before the manifestation of this syndrome
[52]. Interestingly, circulating Ang II levels are not greater
in women with preeclampsia compared with normal pregnant women [53]; however, Ang II signaling is augmented
in preeclampsia, suggesting effects on Ang II receptor
(AT1R) expression and/or cell signaling. Expression of
AT1R is indeed increased in women with preeclampsia
[54] (Figure 1). Furthermore, agonistic AT1R autoantibodies (AT1-AAs) have been found in the circulation of women with preeclampsia and these autoantibodies may be
responsible for enhanced Ang II signaling [55,56]
(Figure 1). AT1-AAs are able to activate the AT1R in a
similar way to Ang II [56]. Signal cascades associated with
binding of AT1R by AT1-AAs (or Ang II) involve phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/
2), induction of NADPH oxidase, phosphorylation of nuclear factor kappa light chain enhancer of activated B cells
(NF-kB), and promoter activation in the nucleus [55]. These
intracellular cascades lead to upregulation of tissue factor,
soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin
(sEng), and ET-1 [57], all of which are notably upregulated
in pregnancies with preeclampsia.
Placenta-derived circulating factors as links between
placental ischemia and maternal vascular dysfunction in
preeclampsia
Shallow trophoblast invasion of the spiral arteries during
the early placentation process is a common feature of
preeclampsia and contributes to inappropriate fluctuations of oxygen tension in the uteroplacental unit, leading
to various cellular events that induce the release of antiangiogenic and proapoptotic factors [4,58]. Consequently,
the placenta acts as a vector of these factors in the maternal systemic circulation. In support of the notion that
placenta-derived factors contribute to maternal vascular
dysfunction, addition of plasma or serum from pregnancies
with preeclampsia to cultured endothelial cells increased
endothelial cell platelet-derived growth factor (PDGF)
mRNA and protein expression and the release of fibronectin, NO, and PGI2 [12,14]. In addition, incubation of isolated myometrial resistance vessels with plasma from
women with preeclampsia reduced vascular relaxation
responses [13]. The exact molecular pathways by which
placenta-derived factors induce dysfunction of the maternal peripheral arteries are not fully understood. Evidence
suggests that placenta-derived factors act on endothelial
cells to induce secretion of proinflammatory cytokines and
modify the production of vasoactive substances and reactive oxygen species (ROS), promoting vascular inflammation and vasoconstriction [5961].
91

Review
Angiogenic factors
Vascular endothelial growth factor (VEGF), placental
growth factor (PlGF), and sEng have angiogenic and vasoactive properties [10]. They play an important role in
placental development and modulate the function of the
placental and maternal systemic vasculatures [10]. In preeclampsia, however, changes in the production and signaling of these angiogenic factors account for the development
of placental and maternal vascular dysfunction and the
hypertensive phenotype [10]. VEGF transduces its biological effects through two high-affinity receptor tyrosine
kinases, VEGF receptor-1 [also known as fetal liver tyrosine-like (Flt-1)] and VEGF receptor-2 [also known as
kinase domain-related receptor (KDR) or Flk-1] [10]. Activation of KDR in endothelial cells induces NO and PGI2
release, promoting endothelium-dependent relaxation
[62]. PlGF has 54% homology with VEGF, binds to Flt-1
but not to KDR, and potentiates the actions of VEGF
[10]. The antiangiogenic protein soluble Flt-1 (sFlt-1) is
the soluble form of the VEGF/PIGF receptor Flt-1 and is
produced by alternative splicing of Flt-1 mRNA [10]. sFlt-1
antagonizes the binding of VEGF and PIGF with their
receptors [63], inhibiting their vasodilatory effects
(Figure 1). Circulating levels of VEGF and PIGF are
reduced in women with preeclampsia [5] and experimental
animal models with preeclampsia-like signs [64] and this
may be attributed to increased levels of circulating sFlt-1
[5]. Infusion of sFlt-1 in pregnant rodents induces preeclampsia-like signs, increased expression of VEGF receptors, and reduced circulating levels of VEGF [65],
suggesting a causal role of sFlt-1 in the pathogenesis of
preeclampsia. In addition to attenuated endothelium-dependent relaxation, rodents infused with sFlt-1 exhibit a
reduction in endothelium-independent relaxation [65]. The
latter effect was abolished following incubation with a
superoxide scavenger, suggesting that the detrimental
effects of sFlt-1 on maternal vascular function are oxidative stress dependent.
Circulating sFlt-1 in preeclampsia was considered to be
of placental origin but additional sources of sFlt-1 have
also been reported. Plateletmonocyte aggregates (PMAs)
are able to produce sFlt-1 and PMA-specific sFlt-1 production is greater in women with preeclampsia [66]. Production of sFlt-1 in PMAs is regulated at the transcriptional
level, through NF-kB-related mechanisms [66] that also
contribute to the production of proinflammatory cytokines
commonly found in the circulation of women with preeclampsia.
Gene-expression profiling of placentas from women with
preeclampsia revealed the involvement of sEng as another
antiangiogenic factor in preeclampsia [67]. sEng is the
soluble form of endoglin, a type I membrane glycoprotein
located on cell surfaces and coreceptor for transforming
growth factor beta (TGF-b) receptor complex. TGF-b1
signaling in vascular tissues induces endothelium-dependent relaxation via an eNOS-dependent mechanism. sEng
inhibits binding of TGF-b1 to its receptor and consequently
downregulates eNOS, leading to reduced NO formation
and attenuated vasodilatory responses [8] (Figure 1). Circulating levels of sEng are elevated in women with preeclampsia [68]. It has been suggested that sFlt-1 and sEng
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act in concert to induce endothelial dysfunction, as mice

injected with both molecules exhibited decreased cerebral
perfusion, vascular thrombi, and endothelial swelling, but
these effects were not seen in mice injected with either
molecule alone [69].
MPs
MPs are membrane vesicles (>100 nm) shed into the extracellular space under conditions of cell stress or injury
[70]. MPs promote the production of oxidative stress in the
vascular endothelium through processes that involve
NADPH oxidase, PGHS, xanthine oxidase, and eNOS
[70] (Figure 1). Further, MPs have vasoconstrictor effects
by acting as a source of TxA2 for the vascular wall [71] and
by increasing the expression of inducible NOS (iNOS) and
PGHS-2 (inducible isoform) in smooth muscle cells [71].
Increased circulating levels of MPs have been found in
several inflammatory and thrombotic diseases. Placentaderived syncytiotrophoblast MPs (STBMs) are found in the
circulation by the third trimester of pregnancy and are
increased in pregnancies with preeclampsia [72]. In vitro
studies showed that artificially prepared STMBs reduced
the viability of endothelial cells and affected their function
[73,74]. Maternal omental arteries perfused with STBMs
exhibited reduced endothelium-dependent relaxation
[75]. By contrast, Van Wijk et al. [76] reported no direct
vascular effect of STBMs on isolated mesometrial arteries
from pregnant women. This dichotomy may be due to
vascular bed differences.
STBMs may also indirectly affect endothelial function
via their interaction with other factors. Circulating STBMs
come into contact not only with endothelial cells but also
with maternal blood cells. Supernatants from cocultured
human umbilical vein endothelial cells with STBMs activated isolated peripheral blood leukocytes and potentiated
peripheral monocyte responses in vitro [60]. Messerli et al.
[77] reported that STBMs shed by placental explants
upregulated monocyte cell-surface expression of the adhesion molecule CD54 and induced the production of the
proinflammatory cytokines IL-8, IL-6, and IL-1b. Given
that STBM levels and activation of monocytes are elevated
in preeclampsia, it is possible that STBM-induced monocytes produce soluble and cell-surface mediators of inflammation that transduce a proinflammatory signal in
endothelial cells. Others have shown that STBMs activate
neutrophils and induce the production of superoxide radicals, which may be the mediator of endothelial dysfunction
in preeclampsia [78].
STBMs are potential contributors to preeclampsia-associated vascular dysfunction, but MPs of platelet and
endothelial cell origin have also been found to be elevated
in preeclampsia [79]. To date, there are no reports on the
role of leukocyte- and vascular smooth muscle cell-derived
MPs in pregnancies with preeclampsia. Given the diverse
biological effects of MPs based on the vascular bed, the cell
type of origin, and the nature of their interaction with the
target cells, efforts should be made to identify the major
cell contributor to preeclampsia-associated vascular dysfunction and the molecular mechanism by which MPs
transduce their detrimental effects on the maternal vascular wall.

Review
Cell-free nucleic acids
Cell-free fetal DNA is increased in pregnancies with preeclampsia and, most importantly, was increased in the
circulation of women who were at high risk of developing
preeclampsia before the onset of the symptoms [80]. Cellfree fetal DNA is almost exclusively of trophoblast origin
and is not derived from the demise of circulating fetal cells
[80,81]. Scharfe-Nugent et al. [82] demonstrated that fetal
DNA is highly inflammatory, because fetal, but not adult,
DNA activated human peripheral blood mononucleated
cells (PBMCs), thereby inducing the release of the inflammatory cytokine IL-6. Treatment of mice with fetal DNA
resulted in fetal resorption and systemic maternal inflammation and these effects were facilitated by activation of
the immune receptor Toll-like receptor (TLR) 9 [82]. Circulating cell-free nucleic acids in the circulation of women
with preeclampsia also include mitochondrial DNA
(mtDNA), which is also increased in women with IUGR
[83]. In a seminal paper, Zhang et al. [84] showed that
mitochondrial fragments, including mtDNA, that are released into the extracellular space due to cell injury and
death have proinflammatory and immunogenic properties
via activation of pattern recognition receptors. mtDNA was
able to specifically bind and activate TLR9 [84]. TLR9 is
activated by hypomethylated CpG DNA, a common feature
of fetal DNA and mtDNA sequences as well as bacterial
DNA but not common in adult vertebrate DNA [81]. Therefore, bacterial infections, trophoblast shedding, and placental cell death (all characteristics of preeclampsia) may
give rise to an immune response via a converging pathway
related to TLR9 signaling (Figure 2). Interestingly, activation of other TLRs, such as TLR4 and the endolysosomic
TLR3, TLR7, and TLR8, has been previously implicated in
the development of preeclampsia [27,85,86]. Pregnant rats
treated with synthetic ligands of TLR developed endothelial dysfunction, hypertension, systemic inflammation, and
proteinuria [86], all characteristics of pregnancies with
preeclampsia.
Neutrophils have been suggested as a potential contributor to the increases in total cell-free DNA seen in pregnancies with preeclampsia [81]. Neutrophils are able to
expel their genomic DNA into the extracellular environment in the form of neutrophil extracellular traps (NETs)
[81]. NETs have been found in the intervillous space of
placentas from women with preeclampsia and it is of
particular interest that STBMs are able to induce NETosis
and become trapped in the extruded NET structures
[87]. These findings raise the possibility that neutrophils
and the process of NETosis are links between placental
shedding and injury with the maternal syndrome of preeclampsia [81].
Hypoxia-inducible factor 1 (HIF-1)
HIF-1a is a subunit of the heterodimeric transcription
factor HIF-1 that regulates the cellular response to low
oxygen tension [88]. HIF-1a expression is increased in
placentas from women with preeclampsia and overexpression of HIF-1a in pregnant mice upregulates sFLt-1, sEng,
and ET-1, inducing a preeclamptic phenotype [89]. Taken
together, these data suggest that HIF-1a is a possible
mediator between the placenta and vasoactive molecules

Trends in Molecular Medicine February 2015, Vol. 21, No. 2

Placental
apoptosis/necrosis

mtDNA

Maternal
circulaon

Fetal DNA

Extracellular
Intracellular
PI3K

PI3K

Endolysosme

TLR9

MyD88
IRAK4
TRAF6

IRF7

NF-B

MAPKs

NF-B

AP-1

Nucleus
IRF7

Transcripon of proinammatory genes and interferon-inducible genes

Inammaon and type I interferon response
TRENDS in Molecular Medicine

Figure 2. Abbreviated schematic of Toll-like receptor (TLR) 9 signaling. Fetal and

mitochondrial DNA (mtDNA) are increased in the circulation of women with
preeclampsia [80,83]. Fetal DNA is released during placenta trophoblast shedding
and mtDNA may be released from dying trophoblast cells. Fetal DNA and mtDNA
contain unmethylated CpG DNA, which enters the intracellular space via class III
phosphatidylinositol 3-kinase (PI3K)-mediated endocytosis to activate the pattern
recognition receptor TLR9 located in endolysosomes. CpG DNA binding leads to
recruitment of the adapter protein myeloid differentiation factor 88 (MyD88). The
interaction of TLR9 with MyD88 activates signal transduction proteins such as
members of the IL-1 receptor-associated kinase family (IRAK). Subsequently, IRAK
proteins interact with tumor necrosis factor (TNF) receptor-associated factor 6
(TRAF-6) leading to its ubiquitination. These events result in nuclear localization of
nuclear factor kappa light chain enhancer of activated B cells (NF-kB) and mitogenactivated protein kinase (MAPK) stimulation of the transcription factor activator
protein 1 (AP-1), which trigger the production of proinflammatory cytokines. TLR9
also stimulates type I interferon production via activation of interferon regulatory
factor 7 (IRF7).

that have deleterious effects on the maternal vascular wall

in preeclampsia.
Oxidative stress: a mediator of maternal vascular
dysfunction in preeclampsia
An imbalance between production of pro-oxidants and
antioxidant scavenging mechanisms, namely oxidative
stress, is considered a convergence point of various molecular pathways, proximal to endothelial dysfunction in
preeclampsia [1,11]. The preeclamptic placenta shows
signs of increased production of oxidants, lipid peroxides,
and isoprostanes and reduced levels of antioxidant mechanisms [11]. Further, vessels from women with preeclampsia show increased eNOS in the presence of increased
markers of the potent oxidant peroxynitrite and a decrease
in the antioxidant superoxide dismutase [90]. Activation of
the NADPH oxidase enzyme system plays a critical role in
the production of superoxide [91] and has been proposed as
a central mechanism in the induction of a pro-oxidant
93

Concluding remarks and future perspectives

Significant progress has been achieved in the past few
years regarding the molecular mechanisms associated
with the pathogenesis of preeclampsia. Specifically, the
characterization of antiangiogenic factors (i.e., sFlt-1) [103]
and endogenous cytoprotective pathways (i.e., HO-1/CO,
CSE/H2S) [9,96,97] have provided scientific justification for
recent therapeutic efforts such as sFlt-1 apheresis to reduce sFlt-1 in the maternal circulation [7] and statins to
improve maternal vascular health [104]. In addition, work
on the vasodilatory properties of relaxin [22], a hormone of
placental origin, has led to the identification of relaxin as a
potential therapy to improve maternal vascular reactivity
in pregnancies with preeclampsia.

Environmental factors

Pre-exisng condions

Placental factors

Genec factors

Circulang factors

Nrf2
HO-1

Va PGHS-2
so
co

ess
str
ve

on
tric
ns

MMPs

es
a pons
on e

Maternal vascular
dysfuncon

LOX-1

as
od

94

oxLDL/LOX-1, Nrf2, and HO-1 and multiple targets for

novel therapies in preeclampsia.

environment in the vascular endothelium of women with

preeclampsia [1]. Superoxide induces eNOS uncoupling
and reduces bioavailable NO, which also leads to increased
peroxynitrite production. Subsequently, peroxynitrite promotes the production of EDCFs such as ET-1 while inhibiting the production of EDRFs such as PGI2 [1].
Formation of peroxynitrite and superoxide in the maternal vasculature in preeclampsia can be also induced by
activation of lectin-like oxidized low-density lipoprotein
receptor-1 (LOX-1). Previous studies showed that LOX-1
activation in endothelial cells by plasma from women with
preeclampsia increased the activity of NADPH oxidase,
superoxide, and peroxynitrite production [35] (Figure 1).
Further, peroxynitrite upregulated and maintained higher
LOX-1 expression, suggesting a positive feedback loop
where LOX-1 induces oxidative stress and, in turn, oxidative
stress upregulates LOX-1 [35]. Recent evidence suggests a
new regulatory mechanism for LOX-1, as activation of TLR4
signaling induced LOX-1 expression via a p38 mitogenactivated protein kinase (MAPK)/NF-kB pathway in mouse
aorta [92]. Further, activation of LOX-1 downregulated
endothelial IKCa3.1 channels [93], which are important electrical triggers in vasorelaxation that contribute significantly
to endothelium-dependent dilatation. Oxidized low-density
lipoprotein (oxLDL) is a ligand for LOX-1 and is increased in
the circulation of women with preeclampsia [94]. In addition
to oxLDL, other circulating factors that are increased in
preeclampsia are able to activate LOX-1. These include
anionic phospholipids, apoptotic cells, activated platelets,
and bacteria [35]. The LOX-1 pathway may be one of the
molecular links between circulating factors, oxidative
stress, and maternal vascular dysfunction in preeclampsia.
LOX-1 is expressed in endothelial cells and also in vascular
smooth muscle cells [95]; however, the role of LOX-1 in
vascular smooth muscle cells from pregnancies with preeclampsia has not been investigated.
Heme oxygenase (HO)-1, the inducible form of HO, has
been identified as a major placental protector from cellular
damage during pregnancy [96]. HO-1 has antioxidant, antiapoptotic, and vasodilatory properties via the actions of its
metabolites biliverdin, bilirubin, and carbon monoxide (CO)
[97,98]. Expression of HO-1 was reduced in preeclamptic
placentas and levels of CO were attenuated in the exhaled
breath of women with preeclampsia, suggesting decreased
HO-1 activity. HO-1 inhibition potentiated sFlt-1 and sEng
production from placental villous explants [99] and in vivo
induction of HO-1 reduced maternal hypertension, decreased placental sFlt-1 and oxidative stress, and increased
circulating VEGF in an animal model of preeclampsia
[100]. Thus, loss of HO-1 cytoprotective actions may be a
significant contributor to the pathogenesis of preeclampsia
[97]. The human HO-1 genes contain binding sites for
multiple transcription factors, among which nuclear factor
erythroid 2-related factor 2 (Nrf2) is considered the most
important [101]. Interestingly, oxLDL is a regulator of Nrf2
but Nrf2 was less activated in placentas from preeclamptic
pregnancies despite a high serum concentration of oxLDL
[102]. Chigusa et al. [102] suggested that in preeclampsia
internalization of oxLDL is reduced due to lower placental
expression of LOX-1, leading to reduced Nrf2 and HO-1.
These data suggest a common pathway for oxidative stress,

Trends in Molecular Medicine February 2015, Vol. 21, No. 2

Oxi
da


Review

il a

MPs

o

ETB

er
un m
I m m a m
n
i
TLR
AT1-AA

Gestaonal maternal
syndrome

Postpartum maternal
cardiovascular risk
TRENDS in Molecular Medicine

Figure 3. Phenotypic characteristics of maternal vascular dysfunction and potential

molecular targets. Oxidative stress, inflammation, and imbalance between
constrictor and dilatory mechanisms are the main features of maternal vascular
dysfunction during gestation and may predispose the mother to a high risk of
cardiovascular disease later in life. This vascular phenotype is the result of the
interaction between the actions of placenta-derived factors, other circulating factors,
and the environment, as well as pre-existing conditions and genetic factors.
Pharmacological manipulation of nuclear factor erythroid 2-related factor 2 (Nrf2),
heme oxygenase-1 (HO-1), lectin-like oxidized low-density lipoprotein receptor-1
(LOX-1), and Toll-like receptor (TLR) signaling may provide novel strategies to target
maternal vascular dysfunction due to the role of these molecules/pathways in central
inflammatory and vasoactive mechanisms. Furthermore, development of selective
pharmacological agents that modulate the production and/or signaling of
angiotensin receptor autoantibodies (AT1-AAs), microparticles (MPs), and cell-free
nucleic acids (CpG DNA) will allow the investigation of their cellular mechanisms and
will further our understanding regarding the vascular pathophysiology of
preeclampsia. Finally, studies are needed to investigate the molecular mechanisms
by which prostaglandin endoperoxide synthase 2 (PGHS-2), endothelin receptor B
(ETB), and matrix metalloproteinases (MMPs) facilitate derangements of maternal
vascular biology during preeclampsia.

Review
Box 1. Outstanding questions
 What are the converging pathways in the occurrence of endothelial and vascular smooth muscle dysfunction in preeclampsia?
 What are the contributions of placental debris (i.e., fetal and
mtDNA) overproduction and deficiencies in clearance mechanisms (i.e., autophagy and mitophagy) to increased circulating cellfree DNA in preeclampsia?
 What are the molecular links between maternal vascular dysfunction during a preeclamptic pregnancy and maternal cardiovascular risk following pregnancy?

Currently, the molecular links between preeclampsiaassociated maternal vascular dysfunction and maternal
risk of cardiovascular disease later in life are unknown.
The vascular pathophysiology of preeclampsia involves
various circulating factors that act on the maternal vascular wall to disturb the balance between vasodilatory and
vasoconstrictor mechanisms (Figure 1). It is unlikely that
poor maternal vascular responses in preeclampsia are the
result of the actions of a single factor/molecular pathway.
The diversity in the nature of the potential instigators and
their source, as well as the complex interactions between
the proposed pathways, underscores the need for the discovery of common converging pathways that link placental
dysfunction with systemic maternal vascular pathology. It
is possible that these converging pathways are also links to
maternal risk of cardiovascular disease following a pregnancy with preeclampsia. The pursuit of the identity of the
molecular links between gestational vascular dysfunction
and future cardiovascular risk should include investigation of the role of preeclampsia-related damage of the
maternal vasculature versus the presence of pre-existing
factors leading to cardiovascular disease.
The studies summarized here describe the multiple and
complex mechanisms associated with the development and
perpetuating mechanisms of maternal vascular dysfunction. To the best of our knowledge, there are no data to
support the importance of one pathway over the others in
the end result of maternal vascular dysfunction in the
heterogeneous syndrome of preeclampsia. We propose certain molecules (HO-1, Nrf2), biological processes (epigenetic modifications), and pathways (TLR signaling) as targets
with potential therapeutic implications (Figure 3). Nevertheless, important basic questions remain to be answered
(Box 1) before we can develop a comprehensive strategy
targeting preeclampsia and maternal vascular pathophysiology. Although we have animal models to test hypotheses, we lack a robust animal model to address the
complexity of preeclampsia. Thus, many of the molecular
pathways reviewed here have been described and investigated in studies of other cardiovascular or chronic disease
complications (i.e., hypertension, kidney disease, obesity,
and diabetes). It is imperative, however, that preeclampsia
be recognized as a unique syndrome, with the placenta
serving as a source of activating factors and the maternal
response being modulated by pregnancy.
Acknowledgments
S.T.D. is a Canada Research Chair in Maternal and Perinatal
Cardiovascular Health. The Davidge laboratory receives funding from
the Canadian Institutes of Health Research, the Heart and Stroke
Foundation of Canada, and the Women and Childrens Health Research

Trends in Molecular Medicine February 2015, Vol. 21, No. 2

Institute through the generous support of the Stollery Childrens Hospital

Foundation and the Royal Alexandra Hospital Foundation. Research in
the laboratory of S.G. is supported by the American Heart Association
(13SDG17050056).

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