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Review Article
Recent Res. Devel. Physiol., 6(2012): 105-158 ISBN: 978-81-308-0489-7
7. Fetoplacental vascular pathophysiology

in preeclampsia
Sobrevia L1, Guzmn-Gutirrez E1, Westermeier F1, Salomn C1, Arroyo P1,
Palacios E1,2, Bugueo K1,3, Santos M1, Diez de Medina C1, Gonzlez M4,
Escudero C5, Salsoso R6, Mate A6, Vsquez CM6, Pardo F1 and Leiva A1
1
Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology
School of Medicine, Faculty of Medicine, Pontificia Universidad Catlica de Chile, P.O. Box 114-D
Santiago, Chile; 2Pontificia Universidad Catlica de Valparaiso, Valparaso, Chile; 3Faculty of Health
Sciences, Universidad de Antofagasta, Antofagasta, Chile; 4Vascular Physiology Laboratory
Department of Physiology, Faculty of Biological Sciences, Universidad de Concepcin, Concepcin
Chile; 5Vascular Physiology Laboratory, Department of Basic Sciences, Faculty of Sciences,
Universidad del Bo-Bo, Chilln, Chile; 6Department of Physiology, Faculty of Pharmacy. Universidad
de Sevilla Sevilla, Spain
Abstract. Preeclampsia is a syndrome that affects a significant

proportion of pregnant women worldwide. The several hypotheses
proposed regarding the aetiology of preeclampsia highlight the
possibility of a critical role played by the placenta and the multiple
molecules released by this organ in the onset of the syndrome.
Recent reports suggest that not only the placenta itself but also
fetal circulating factors such as lipids, including cholesterol, and
the endogenous purine nucleoside adenosine, via activation A2A
adenosine receptors, are determinant in this syndrome.
Alternatively, maternal pregestational obesity and obesity in
pregnancy determines the appearance of preeclampsia, with leptin
as a key factor in this phenomenon. In addition, maternal and fetal
insulin resistance associated with maternal obesity in pregnancy
Correspondence/Reprint request: Prof. Luis Sobrevia, Cellular and Molecular Physiology Laboratory (CMPL)
Division of Obstetrics and Gynaecology, School of Medicine, Pontificia Universidad Catlica de Chile, P.O.
Box 114-D, Santiago, Chile. E-mail: sobrevia@med.puc.cl
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Luis Sobrevia et al.
could result from differential expression of insulin receptor isoforms A and B.

Fetoplacental endothelium is a primary target for the action of these factors resulting
in endothelial dysfunction. Thus, reduced synthesis and release of nitric oxide and
increased plasma levels of adenosine are mechanisms involved in the genesis of
preeclampsia. We here summarize these observations and propose potential links
between these factors in the aetiology of this syndrome. We highlight the possibility
that the placenta and the developing fetus are key elements leading to the onset of
preeclampsia. Therapies could include modulation of the serum level of adenosine,
cholesterol, insulin, and expression of adenosine and insulin receptors, as well as
membrane transporters of nucleosides and cholesterol.
1. Introduction
A large number (~15%) of the women who become pregnant will
develop complications leading to maternal or fetal vital risk. One of the
perinatal diseases with significant impact is preeclampsia (PE) whose
incidence varies between 2-15% in people from developed and undeveloped
countries. The aetiology of PE is multiple, and, unfortunately, a significant
number of cases are idiopathic, with no clear cause. Placental dysfunction in
PE is a common condition also for several other diseases of pregnancy,
where altered placental function associates with vascular and endothelial
dysfunction, and/or a local environment limiting oxygenation to the
developing fetus. However, there is no information addressing the
mechanisms, and whether those leading to endothelial dysfunction are
transversal for placental vascular dysfunction in terms of its macro and
microcirculation. The normal development of the placenta is essential to
maintain an adequate fetal development and growth. The human
fetoplacental circulation under physiological conditions exhibits a high
blood flow and reduced vascular resistance. Since the placenta lacks
autonomic innervation, circulating, and locally released vasoactive
molecules, such as nitric oxide (NO) or adenosine, are essential to maintain
control of the fetoplacental haemodynamic. An alteration in this process,
where the placental function is abnormal, referred as placental dysfunction,
leads to different clinical manifestations, leading to a vast amount of clinical
possibilities, maternal and/or fetal symptoms, as well as different short- and
long-term prognosis.
2. Preeclampsia
Preeclampsia is a syndrome presented in human pregnancies since the
20th week of gestation, clinically characterized by hypertension and
proteinuria (Brown et al., 2001; WHO, 2011a). Thus, PE could be mild
Fetoplacental vasculature in preeclampsia
107
(proteinuria 3 g/24 hours, blood pressure 140/90 mmHg) or severe

(proteinuria 5 g/24 hours, blood pressure 160/110 mmHg) (Geller et al.,
2004). A recent classification includes the gestational age at which this
syndrome appears to be PE of early-onset (<34 weeks of gestation (wg)) or
late-onset (>34 wg) (Fugelseth et al., 2011). PE is a leading cause of
maternal and infant morbidity and mortality related with a high risk (2 to 4
fold) for stillbirth, growth restriction and preterm births (Xiong et al., 2002a;
Villar et al., 2006; Duley, 2009). The risk of maternal death due to severe PE
is four times greater if the diagnose is done before the 32 wg, and most of
these deaths result from complications such as brain haemorrhage, kidney or
liver failure (Xiong et al., 2002b; Duley et al., 2006; Duley, 2009; Young et
al., 2010). A commonly accepted treatment of this syndrome is the
interruption of pregnancy if it develops after 34 wg. However, the benefits of
this action must be considered together with the likelihood of fetal
immaturity.
There are certain risk factors that predispose to the occurrence of PE.
These factors relate mainly to the pre-existence of endothelial injury or
chronic inflammation, especially in essential hypertension, diabetes mellitus
and chronic kidney damage. Also, a history of severe PE or eclampsia in
previous pregnancies confers an increased risk for future pregnancy (Sibai et al.,
2005; Backes et al, 2011). It is generally agreed that PE results from the
existence of trophoblasts, which form the syncytiotrophoblast in direct
contact with maternal blood (OSullivan et al., 1982; Myatt, 2010). From a
clinical point of view, it is widely accepted that the development of PE
during pregnancy requires the presence of the placenta, as the clinical
pictures of the syndrome will not appear if this organ is not present and
disappears after childbirth. Moreover, the presence of the fetus for the
development of this condition is not necessary because it can happen in a
hydatidiform mole (Page, 1939). The pathophysiological mechanism
associated with this phenomenon involves a failure in the placentation that
precedes the characteristic clinical manifestation of the syndrome (Burton et al.,
2009a; Roberts & Rajakumar, 2009; Roberts & Escudero, 2012).
The physiological placentation process involves at least two stages: the
first, where predominates cytotrophoblast exhibiting a proliferative
phenotype until the 12th wg, characterized by a relative hypoxia; and the
second beginning at 12th wg and that will change the proliferative
cytotrophoblast of the villi core towards an invasive phenotype (extravillious
trophoblasts) which is mediated by changes in the concentration of oxygen
(O2) in the intervillious space (Casanello & Sobrevia, 2010). The hypothesis
widely accepted for the pathophysiology of PE indicates the following
events: an impaired cytothophoblasts transformation toward extravillous
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trophoblasts resulting in a narrow invasion into the maternal vascular bed

(Genbacev et al., 1997; Red-Horse et al., 2004). This phenomenon leads to a
reduced trophoblasts invasion into maternal spiral vessels preventing its
transformation into capacitance vessels. This will compromise the maternal
blood flow towards the placenta, which in turn would trigger an ischemiareperfusion phenomenon associated to high perfusion pressure generating a
continues shear stress against the placenta and on syncytiotrophoblasts (the
cell layer in direct contact with maternal blood) (Burton et al., 2009a). These
phenomena trigger cell damage (i.e., necrosis) leading to detachment and
release of cell fragments (microparticles or nanoparticles) towards maternal
circulation (Burton & Jones 2009). Within these microparticles harmful
elements (such as phospholipids with an altered biochemistry, free radicals,
purines) will be transported affecting the maternal endothelium. These
elements will be responsible for triggering an exacerbated inflammatory
response, endothelial dysfunction, vasoconstriction (hypertension) and
glomerular endotheliosis (proteinuria). At the same time, these changes
would generate a vicious cycle that will chronically affect the placental
blood flow and maternal endothelial function (Roberts & Rajakumar, 2009;
Roberts & Escudero, 2012). In addition, even when the mechanisms
involved in these alterations are still unknown it has been proposed that an
impaired maternal immune recognition associates with increased expression
of the hypoxia inducible factor 1 (HIF-1), transforming growth factor 3
(TGF-3) and the vascular endothelial growth factor 1 (sFlt1, soluble fmslike tyrosine kinase 1), but decreased PlGF and NO (Casanello & Sobrevia,
2010; Escudero et al., 2008).
Nevertheless the actual evidences suggest that in severe PE a reduction in
placental angiogenesis could also be a mechanism responsible for the observed
reduced placental blood flow, while in mild PE the observed normal blood
flow could result from either not alterations or increased placental
angiogenesis, and/or reduced vessel resistance. However, despite all this
information, cellular mechanisms involved in the syndrome are not well
understood (Ahmad & Ahmed, 2004; Escudero et al., 2008; Roberts &
Escudero, 2012). An altered NO synthesis and plasma membrane transport
mechanisms (amino acids and nucleosides transport) in human placental cells
including the endothelium have been reported in PE (Weiner et al., 1994); as
well as with an increased level of the soluble receptor for vascular endothelial
growth factor 1 (sFlt1, soluble fms-like tyrosine kinase 1) (Levine et al., 2004).
The proposed mechanism is that sFlt1 will binds vascular endothelial growth
factor (VEGF) and placental growth factor (PlGF) depriving the endothelium
of these essential survival proteins (Escudero & Sobrevia, 2008) and reducing
NO synthesis (Ahmad et al., 2011).
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In parallel with the characterization of the intrinsic cellular mechanisms

explaining the aetiology of PE, numerous methods have been used to predict
the onset of PE with varying degrees of efficiency. Among them altered
blood pressure, uric acid level and Doppler of the uterine arteries are
considered risk factors (Monte, 2011). It has been possible to separate
makers at the plasma or serum in early, intermediate and late stages of the
syndrome (Sidani & Siddik-Sayyid, 2011; Monte, 2011). Early factors
(assessed in the second trimester of pregnancy) are responsible for
migration/inadequate trophoblastic invasion, including placental hypoxia and
oxidative stress (i.e., leptin, uric acid, ascorbic acid, placental growth factor
(PlGF)) and alteration of placentation (plasminogen activator inhibitor type 2
(PAI-2) antigen, PAI-1/PAI-2, vascular cell adhesion molecule (VCAM),
intercellular adhesion molecule (ICAM) and inhibin. As described, the
intermediate factors appear in the maternal circulation as a result of
decreased placental perfusion and endothelial activation. These factors are
present in the maternal plasma or serum before the onset of clinical signs of
the disease and are associated with placental deficit (transforming growth
factor 1 (TGF-1) and SS3 (TGF-SS3), PAI-1, PAI-2) and in the response
of the placenta to hypoxia or oxidative stress (leptin, endothelin-1,
asymmetric dimethyl arginine (ADMA), malonyldialdehyde (MDA), F2isoprostano, nitrotyrosine, homocisteinemia, vitamin B12, folic acid, VEGF,
PlGF, uric acid). The late factors appear as a result of the activation of the
endothelium or in a state of endothelial dysfunction including those
associated with vasoconstriction (neurokinin-B, neuropeptide Y, endothelin
1 (ET-1), ADMA), compensation by vasoconstriction (NO release), impaired
endothelial function (VEGF, IL-12, IL-6, tumour necrosis factor (TNF),
VCAM-1) and impaired renal function (uric acid). While these markers in
general have not increased the predictor capacity of the Doppler of the
uterine artery in the second trimester of pregnancy, some of these, especially
those related to antiangiogenesis such as sFlt1, PlGF and Seng, precede the
onset of the signs of PE in at 5 to 8 weeks. However, despite that, it would
be extremely useful to have predictive markers of PE within the first
trimester of pregnancy in order to design appropriate prevention strategies
(Monte, 2011).
2.1. The fetus in preeclampsia

It is well known that PE has a substantial impact on the intrauterine
environment leading to fetal and neonatal morbidity and mortality. Thus, in a
study conducted by the World Health Organization (Villar et al., 2001), it
has been shown that the rate of preterm delivery was higher in newborns
110
from PE compared with newborns from normal pregnancies (Villar et al.,

2006). Moreover, diagnosis of intrauterine growth restriction (IUGR) is
more often associated to PE compared to idiopathic IUGR (Sibai et al.,
2005; Villar et al., 2006; Bertoglia, 2010). Thus, offspring from PE,
principally those exposed to severe PE, usually exhibit prematurity (below
37 wg) or severe prematurity (below 27 wg), low birth weight and
consequently high risk for stillbirth or perinatal death (Sibai et al., 2005). In
fact, the fetal and neonatal mortality in PE resulted to be ~2.2 and 2.4%,
respectively (Villar et al., 2006).
High infant morbidity and mortality associated with PE is associated to
prematurity or IUGR. However, cerebral palsy risk is markedly increased in
infants born preterm or in those children born with low weight
(Argyropoulou, 2010) whereas babies from PE (mainly severe PE) have a
lower risk for cerebral palsy (Xiong et al., 2002b). Additionally in preterm
newborns from severe PE exhibit elevated systemic blood pressure
compared to preterm newborns from normal pregnancies (Swarup et al.,
2005). PE has been also associated with the presence of febrile seizures
(Vestergaard et al., 2003) or transient hearing defects (Bakhshaee et al.,
2008). In a prospective cohort study of 324 preterm neonates with birth
weight 1500 g and gestational age 32 weeks, PE was associated with by
60% reduction in the risk of retinopathy associated to prematurity and by
60% in the severe retinopathy cases (Fortes et al., 2011). Thus, it is
intriguing that PE could be associated with fetal morbidity and in some cases
with fetal protection against defect observed in premature babies. Therefore,
fetal adaptation to PE might generate a particular phenotype different from
premature or growth restricted babies.
2.2. The placenta in preeclampsia

The placenta has long been recognized as the necessary component for
the genesis of PE. Initial evidences show that PE is present in cases of
hydatidiform moles with abundant trophoblasts but no the fetus (Page,
1939), or the development of this disease in cases of abdominal pregnancies
where placenta is implanted outside the uterus and the resolution of PE is
delayed as long as the placenta is reabsorbed (Shembrey & Noble, 1995) or
the disappearance of the disease after the placenta extraction in normal
deliveries of caesarean sections (Sibai et al., 2005). Nowadays, there are
several evidences linking reduced placental perfusion associated with PE
development (Roberts & Gammill 2005; Escudero et al., 2009a; Roberts &
Escudero 2012). The causes for the reduced placental perfusion in PE could
be due to preplacental factors, where the reduced blood flow towards the
111
placenta results from preservation in the capacity of contraction of the spiral

arteries offering high resistance and intense pressure against the placenta
(Burton et al., 2009b). Another cause could be placental factors,
characterized by a failure in the process where endothelium and smooth
muscle cells in the maternal spiral arteries must be replaced by invasive
syncytiotrophoblast, allowing the transformation of the maternal vessels into
a capacitance phenotype, reduced placental perfusion due to reduced
placental angiogenesis especially in severe PE, or the presence of large
placentas with a relative hypoperfusion (i.e., hydatidiform moles, multiple
gestations). Furthermore, combined preplacental and placental factors could
also be the cause, a condition that is, in fact, the most common in PE.
3. Vascular dysfunction in preeclampsia

The greatest risk of fetal morbidity and mortality in PE might be
associated to a reduction in placental blood flow (Di Paolo et al., 2003;
Kofinas et al., 2007), leading to limited fetal access to nutrients that
eventually will affect its development. The causes for the decrease in blood
flow to the placenta in pregnancies with PE are not entirely clear, but might
be associated with maternal and placental vascular dysfunction (Escudero &
Sobrevia, 2008, Escudero et al., 2009a; Roberts & Escudero, 2012).
3.1. Regulation of placental vascular tone in preeclampsia

The regulation of vascular tone in the placenta depends on its capacity of
synthesis and reactivity to local and circulating vasoconstrictors and
vasodilators (King et al., 1995, Webster et al., 2008; Casanello et al., 2007;
Escudero & Sobrevia, 2008; Sobrevia & Casanello, 2010; Roberts &
Escudero, 2012). The analysis of the information derived from echography
show an increased index of uterine artery pulsatility (Di Paolo et al., 2003)
and higher hypoechoic areas (Kofinas et al., 2007) in the placenta from PE
suggesting reduced in the uterine artery and placenta blood flow,
respectively. Moreover, there is no significant difference in the index of
umbilical artery pulsatility in PE compared with normal pregnancies (Luzi et al.,
1999). Thus, it is suggested that PE was associated with alterations in
placental and fetal blood flow probably due to its redistribution in the
placenta.
A reduction in the placental blood flow associates with increased
sensitivity of placental vasculature to vasoconstrictors such as angiotensin II,
ET-1 and ET-3, prostaglandin F2 (PGF2), PGE2 and PGD2, as well as
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thromboxane A2 is reported in PE (Walters et al., 1991; Ajne et al., 2005).

Similarly, even when the fetoplacental vasculature is highly sensitive to
vasodilators such as prostacyclin (PGI2) and NO (Walters et al., 1991, King
et al., 1995), several studies report that in PE the feto-placental tissue show
reduced synthesis, less biological effect and/or diminished bioavailability of
NO (Escudero & Sobrevia, 2008; Escudero et al., 2009a,b; Myatt 2002,
2010; Veas et al., 2011). It now clearer that synthesis and activity of NO
depends on the placenta cell type where it is investigated. For instance, in a
cross sectional study it is shown that human umbilical vein endothelial cells
(HUVEC) isolated from mild and severe PE exhibit low capacity of
histamine-stimulated NO synthesis compared with cells from normal
pregnancies (Veas et al., 2011). This phenomenon was negatively correlated
with maternal serum levels of VCAM-1 and sFlt1. Therefore, severe PE is
characterized by high maternal level of soluble VCAM-1 and Flt1 associated
with decreased production of NO in the fetal endothelium.
Adenosine is a modulator of placental vascular tone with a differential
effect on chorionic and umbilical vessels (Donoso et al., 2005; Westermeier
et al., 2011; Leiva et al., 2011; Pardo et al., 2012). A higher expression of
adenosine receptors (ARs) in placental homogenate have been reported in
PE (von Versen-Hynck et al., 2009), but reduced expression of A2A
adenosine receptor (A2AAR) without changes in A2BAR, in primary culture
of human placental microvascular endothelial cells (hPMEC) was shown
(Escudero et al., 2008). In addition, it has been suggested that adenosine
increases NO synthesis in HUVEC (Vsquez G, 2003; Vsquez et al., 2004)
and hPMEC (Escudero et al., 2008) by activation of adenosine A2A
adenosine receptors (A2AAR), suggesting a role for adenosine as a
vasodilator in the human fetoplacental vasculature (Sobrevia et al., 1996;
Sobrevia & Mann, 1997; Wyatt et al., 2002; Sobrevia & San Martn, 2006;
Escudero et al., 2008; Sobrevia et al., 2011; Westermeier et al., 2009, 2011;
Guzmn-Gutirrez et al., 2011; Leiva et al., 2011; Pardo et al., 2012).
However, the activation of another group of adenosine receptors, i.e.,
A2BAR, increases the synthesis of thromboxane and causes vasoconstriction
in chorionic vessels (Donoso et al., 2005). The physiological significance of
these findings in umbilical and chorionic vessels is not well understood, but
might be related to the mechanisms of redistribution of blood flow to areas
with low partial O2 pressure (PO2) as found in other tissues such as the lung
(Gottlieb et al., 1984). These differential events (i.e., vasodilation or
vasoconstriction depending on the vascular bed) are of great importance in
pathological conditions associated with decreased blood supply as in the
placenta of pregnancies with PE (Di Paolo et al., 2003; Kofinas et al., 2007).
113
3.2. Preeclampsia and intrauterine growth restriction

The current evidence suggests that PE is a maternal condition that also
affects the developing fetus in a defined group of patients (~30% of pregnant
women with PE have children with IUGR). However, other pregnancies with
PE (~70%) undergo a normal fetal development. Since placentation a
continuous and growing release of trophoblastic material (including the fetal
DNA) to the maternal circulation occurs. The first pathophysiological stage
of the disease is hard to diagnose, but the second one is the clinical
syndrome itself. Similarly, uterine artery Doppler allows the study of PE and
IUGR in unselected populations with high variability. This is due to the
variability in gestational age at the time of the patient examination as well as
methodology and definitions of poor perinatal outcomes. Harrington and
colleagues (1997) found that 4% of pregnant women showed increased
resistance of uterine arteries at 24 wg with 55 and 30% of pregnant
women developing PE and IUGR, respectively. However, when adverse
perinatal outcome was defined as PE or IUGR before the 34 wg an abnormal
Doppler increased the sensibility to 90 and 60%, respectively. In a metaanalysis including 27 studies and 12.994 pregnant women, where uterine
artery Doppler was used as predictor of IUGR and PE, it was concluded that
Doppler has a moderate skill in predicting these pathologies. However an
abnormal Doppler in the second trimester of pregnancy increases the risk of
PE by 6 fold, making it clinically relevant to the mother. The usefulness of
uterine artery Doppler in the first trimester of pregnancy is of less impact
than in the second trimester. It has been shown that a pulsatility index on the
95 percentile at 12 wg predicts 60% of severe PE and 30% of severe
IUGR (defined as those pregnancies that were interrupted prior the 32 wg).
On the other hand, patients with PE have uterine arteries pulsatility index
that increased significantly at 12 wg compared with controls (Parra et al.,
2001). In addition, its ability to predict severe PE (defined as those
pregnancies interrupted before the 35 wg) was higher in 40%.
Despite the development of PE in the mother, in the fetus and possibly
also in the placenta, several mechanisms helping to ensure the normal
growth of the developing fetus are likely triggered. Although the specific
mechanisms are still unknown, it has been proposed that in PE and IUGR
there is a reduction in the formation of placental blood vessels or an increase
in placental vasoconstriction, given for example by a reduction in the
synthesis of vasodilators such as NO (Table 1) (Webster et al., 2008;
Bernardi et al., 2008, Roberts & Hubel, 2009). Thus, the relative reduction in
utero-placental blood flow, secondary to alterations in placentation,
could be, in fact, what ultimately leads to the development of these pathologies.
Table 1. Alterations in preeclampsia coursing with or without intrauterine growth restriction.
114
115
This is evidenced by histopathological changes in the placental implantation

site where 80-100% of patients with PE and ~75% of patients with IUGR
have a deficit in the extravillous trophoblast invasion into maternal spiral
arteries. It is fairly agreed that the sequence of events leading to the
development of PE and/or IUGR may be explained by a first stage of
defective trophoblastic invasion, which occurs early in pregnancy and leads
a state of high resistance of the utero-placental circulation, detected by an
increase in resistance of the uterine arteries. In addition, the persistence of a
state of reduced infusion leads to placental hypoxia, which will increase the
local oxidative stress. This phenomenon will affect the growth of the fetus,
but if these alterations happen in almost all cases of IUGR and PE, a
question that remains unanswered is how the same pathophysiological
processes trigger different clinical maternal conditions? One of the
possibilities is that PE is mainly associated with the release of
syncytiotrophoblast microparticles (STBM) (Goswami et al., 2006). This
study showed that women with PE exhibit higher level (~2 fold) of STBM
than normal pregnancies (matched by gestational age). However, the STBM
level was similar between IUGR pregnancies compared with normal
pregnancies. These results were further confirmed by others groups
(Germain et al., 2007; Than et al., 2008) suggesting that depending on the
quantity of STBM released into the maternal circulation a different
pathophysiological process leading to PE or IUGR could account.
4. Adenosine in the placenta from preeclampsia

Adenosine is an endogenous purine nucleoside that maintains the
homeostatic equilibrium via activation of adenosine receptors (ARs)
(Schiemann et al., 1990). Adenosine biological effects include modulation of
energy homeostasis (ATP metabolism), regulation of vascular tone (Vazquez
et al., 2004; Westermeier et al., 2011), angiogenesis (Feoktistov et al., 2002,
2004, 2011), proliferation (Grant et al., 2001), endothelial permeability
(Richard et al., 1998), inflammation (Olah et al., 2000), and protection
against oxidative stress (Yang et al., 2005). Preeclampsia also associates
with increased plasma adenosine concentration with the subsequent
alterations of specific endothelial cell functions such as angiogenesis and
endothelial cell proliferation (Escudero et al., 2009a) (Figure 1). Several
reports show that adenosine plasma level in the fetal and maternal blood in
PE is higher compared with normal pregnancies (Yoneyama et al., 1996,
2002). Adenosine plasma level in umbilical blood samples from PE without
116
Figure 1. Adenosine effect in human placenta endothelial cell function in

preeclampsia. Adenosine fetal plasma concentration is increased in preeclampsia
thus activating adenosine receptors subtypes A1, A2A, A2B and A3 in the human
placental tissue. Changes in adenosine receptor expression caused by or associated
with preeclampsia could lead to a defective signalling resulting in altered function of
the endothelial cells causing for example abnormal angiogenesis and cell
proliferation in the human placenta (Escudero et al., 2008; von Versen-Hynck et al.,
2009.
fetal hypoxia was reported as ~600 nM (Yoneyama et al., 1996), a

concentration similar to that found in human umbilical blood in normal
pregnancies (Maguire et al., 1998), whereas this level of adenosine increased
by ~3 fold (i.e., ~1800 nM) in PE coursing with fetal hypoxia. Similarly,
high plasma levels of adenosine have been reported in the maternal blood in
PE (~680 nM) compared with normal pregnancies (~390 nM) (Yoneyama et al.,
2002). A significant positive correlation between increased adenosine
plasma level and PE severity has been proposed (Yoneyama et al., 2002).
Only few studies show that adenosine deaminase (ADA) activity is, in fact,
increased in maternal and umbilical vein whole blood and in placental tissue
from women with mild or severe PE (Kafkafsli et al., 2006). This study
established that increased ADA activity was related to the presence of the
117
disease but not to the severity of patients clinical symptoms. In addition,

ADA activity, and possibly the isotype 2 of ADA (ADA2) (Yoneyama et al.,
2002), was also found increased in maternal and umbilical cord whole blood.
This finding was proposed as a marker of immunological disorders that
could potentially be related to the pathogenesis of PE. In addition, increased
ADA activity could be an adaptive response of the fetoplacental vasculature
to PE intending to reduce the elevated adenosine plasma levels detected in
the maternal and fetal blood in this syndrome.
4.1. Adenosine receptors and preeclampsia

Four subtypes of ARs have been identified, i.e., A1AR, A2AAR, A2BAR
and A3AR. These subtypes exhibit different pharmacological properties, and
are expressed in different tissues with different order of potency of ligand
binding and cell signalling associated mechanisms (Ralevic & Burnstock,
1998; Fredholm et al., 2011). These characteristics show that differential
ARs expression is key in adenosine biological effects (Schulte & Fredholm,
2003; Eltzschig, 2009; Fredholm et al., 2011). Activation of A1AR and
A3AR leads to inhibition of adenylyl cyclase and activation of
phosphatidylinositol 3-kinase (PI3k)/Akt pathway, while A2AAR and A2BAR
activates adenylyl cyclase (Eltzschig, 2009). In the placenta from PE higher
ARs expression in syncytiotrophoblast, endothelium and myofibroblasts
compared with normal pregnancies is found (von Versen-Hynck et al.,
2009). However, in hPMEC, a cell type representative of fetoplacental
microvascular endothelium (Sobrevia et al., 2011), it has been seen that only
A2AAR expression is decreased when compared with hPMEC from normal
pregnancies (Escudero et al., 2008). In HUVEC, a cell type representative of
fetoplacental macrovascular endothelium (Sobrevia et al., 2011), the
expression of A2AAR, A2BAR and A3AR, and a reduced but still detectable
expression of A1AR are reported (Liu et al., 2002; Wyatt et al., 2002;
Feoktistov et al., 2002). However, ARs expression in HUVEC from PE has
not been studied (Escudero et al., 2008; Roberts & Escudero, 2012).
Interestingly, it has been shown that expression and angiogenesis-associated
to activation of A2BAR are upregulated in this cell type in response to
hypoxia (Feoktistov et al., 2002; Eltzschig et al., 2003). A potential crosstalk
between adenosine and other molecules (i.e., hormones, cytokines) have not
been described in placental tissue from PE, suggesting that differential
expression of ARs subtypes could contribute to understand functional
heterogeneity of human placental vascular endothelial cells in PE
(Sobrevia et al., 2011).
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4.2. Adenosine transporters and preeclampsia

Endothelial cells from placental tissue (i.e., HUVEC and hPMEC)
maintain normal adenosine extracellular levels by an efficient uptake of this
nucleoside (Escudero et al., 2008; Westermeier et al., 2011; Salomn et al.,
2011), thus modulating its broad biological effects (Eltzschig, 2009). At
present, nucleoside transport in human placental endothelial cells is
mediated via the Na+-independent, human equilibrative nucleoside
transporters (hENTs). Four members of the ENTs family of solute carriers
(SLC29A genes) have been cloned from human tissues, i.e., hENT1, hENT2,
hENT3 and hENT4 (Baldwin et al., 2004; Westermeier et al., 2009; Leiva et al.,
2011). In HUVEC, adenosine transport is mainly (~80%) mediated by
hENT1 (inhibited by <1 M nitrobenzylthioinosine, NBTI) with a remaining
transport mediated by hENT2 (inhibited by >1 M NBTI) (Molina-Arcas et al.,
2009; Westermeier et al., 2009). On the contrary hENT3 and hENT4 seems
not to play a physiological role in endothelium (Westermeier et al., 2009;
Leiva et al., 2011).
hENT1 and hENT2 are also expressed in hPMEC; however, in this cell
type these membrane transporters have a comparable relative contribution to
the total adenosine transport (Escudero et al., 2008). We have shown that
hPMEC isolated from pregnancies with PE exhibit reduced maximal velocity
(Vmax) of hENT1-mediated adenosine transport, but increased Vmax for
hENT2-mediated transport (Escudero et al., 2008). Since the apparent Km
values were unaltered in PE, the relative effect of this pathological condition
on the maximal transport capacity (Vmax/Km) mediated by hENT2 compared
with cells from normal pregnancies was higher (~2 fold), but for hENT1 it
was reduced (~75%). In addition, the relative contribution of hENT1
compared with hENT2 to total adenosine transport (i.e., hENT1 + hENT2) in
hPMEC from normal pregnancies was higher (~7.9 fold) compared with
cells from PE. Thus, even when adenosine transport in hPMEC from PE
seems preferentially mediated by hENT2, this phenomenon might be
insufficient to reverse the reduced hENT1-mediated transport seen in this
syndrome. Moreover, high extracellular adenosine levels detected in
HUVEC from pathological pregnancies (such as in gestational diabetes)
(Westermeier et al., 2011) compared with cells normal pregnancies, leads to
activation of adenosine receptors increasing the NO synthesis and membrane
transport of the cationic amino acid L-arginine (Vsquez G, 2003;
Vsquez et al., 2004), the substrate for NO synthesis via NO synthases
(Moncada et al., 2006).
The functional relationship between adenosine and L-arginine/NO
pathway was first characterized and more recently referred as ALANO
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(Adenosine/L-Arginine/Nitric Oxide) signalling pathway in HUVEC (San

Martn & Sobrevia, 2006; Leiva et al., 2011; Pardo et al., 2012). It was
initially proposed that adenosine opens K+ channels, likely ATP-activated K+
channels (K+ATP) via activation of A2AAR in HUVEC, a possibility
confirmed by observations showing an increase in the outward K+ current in
response to adenosine in this cell type (Wyatt et al., 2002). Interestingly, this
phenomenon was blocked by the NOS inhibitor NG-nitro-L-arginine methyl
ester (L-NAME) and mimicked by the NO donors sodium nitroprusside
(SNP) and S-nitroso-L-acetylpenicilamine (SNAP), suggesting that a
NO-mediated membrane hyperpolarization may account for the stimulation
of L-arginine transport in HUVEC challenged with adenosine. This pathway
could be essential in diseases of pregnancies associated with fetal endothelial
dysfunction such as PE where L-arginine transport via hCATs is reduced
(Escudero, 2009; C Escudero, L Sobrevia, unpublished results).
5. Preeclampsia and obesity in pregnancy

5.1. Obesity
One of the major public health problems is obesity. This syndrome
occurs with a misbalance between the energy intake and energy used
accompanied with an over-storage of lipids in adipose tissue (Shoelson et al.,
2007). Obesity contributes to the generation of the metabolic syndrome
leading to alterations in several tissues triggering multiple systemic
complications such as hypertension, dyslipidaemia and insulin resistance
(Nishimura et al., 2009). According with the last World Health Organization
report ~14% of the women suffer obesity (body mass index (BMI) >30) with
~20% with some level of overweight (BMI >25) (Heslehurst et al., 2007,
2010; Flegal et al., 2011; WHO, 2011b). The major effect of obesity is the
manifestation of several metabolic alterations including endothelial
dysfunction, which leads to hypertensive disorders (Tesauro & Cardillo, 2011).
Worryingly, it is expected that obesity during pregnancy could becomes a
condition altering fetal development and growth due to the abovementioned
maternal obesity-associated metabolic changes. It is well documented that
PE incidence associates with a higher BMI (Aliyu et al., 2010; Seabra et al.,
2011; Chung et al., 2012; Yazdani et al., 2012; Mandal et al., 2011).
Nevertheless, there is not a clear correlation between the increase in BMI
with the severity of PE (Roberts et al., 2011; Sohlberg et al., 2012). These
findings correspond well with the hypothesis of two subsets of PE, the earlyonset disease with a predominantly placental and genetic origin and the lateonset disease with a stronger metabolic origin (Sohlberg et al., 2012).
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Figure 2. Obesity as a factor to develop preeclampsia. Obesity in pregnancy is

primarily related with mild preeclampsia most likely due to increased () in
cytokines such as interleukin 6 (IL-6) and tumour necrosis factor (TNF) and free
fatty acids. This phenomenon leads to oxidative stress increasing the incidence of this
syndrome. Insulin resistance and increased leptin level could also promote placental
dysfunction in preeclampsia. The severe preeclampsia results from lean pregnant
women (lean pregnancy) and is more related to a placental origin with a large genetic
background (Roberts et al., 2011; Tosun et al., 2011).
Some groups report that obesity correlates with PE through a state of

dyslipidaemia and increased level of inflammatory cytokines; however, these
alterations are also seen in lean pregnant women coursing with PE (ZalvalzaGmez et al., 2011). The type of fat accumulated during pregnancy and in
the pregestational period seems crucial and correlates with a higher risk of
developing insulin resistance, diabetes mellitus type 2 (DMT2) and
cardiovascular disease (CVD) (Strasser et al., 2012). Roberts and colleagues
(2011) proposed that lean women showing an increase in the weight gain
during pregnancy could exhibit higher incidence of PE likely resulting from
increased liquid retention in response to cytokines release in the placenta. In
addition, an increase in the level of free fatty acids in obesity could be a
factor leading to oxidative stress associated with PE (Dandona et al., 2005;
Roberts et al., 2011).
5.2. Cytokines/interleukins and obesity in pregnancy

Several studies associate obesity with chronic inflammation since blood
markers such us IL-6, TNF, adipokins (including leptin) and others are
increased in patients suffering of this syndrome (Table 2) (Dandona et al.,
121
2004; Dandona et al., 1998; Kern et al., 2001; Pradhan et al., 2001;
Vozarova et al., 2001). The endothelium is the first cell line exposed to these
cytokines (Fantuzzi, 2005; Meijer et al., 2011; Tilg & Moschen, 2006;
Weisberg et al., 2003) leading to altered eNOS expression and activity, and
reduced bioavailability of the vasodilator NO (Kim et al., 2008; Suwaidi et al.,
2000; Yamamoto et al., 2010). IL-6 stimulates B-lymphocytes and regulates
T-cell signalling processes in PE (Lamarca et al., 2011). In the placenta, IL-6
is also expressed in the trophoblasts (Kk et al., 2012), thus, since IL-6
level is elevated in PE both in the maternal (Amash et al., 2010, Tosun et al.,
2010, Xie et al., 2011, Kalinderis et al., 2011) and the umbilical sera, and
correlates with the severity of PE (Tosun et al., 2010; Kk et al., 2012;
degrd et al., 2001), this cell type could play key roles in this syndrome. In
addition, elevated IL-6 level in PE could be a key phenomenon promoting
endothelial cell dysfunction, not only in the mother but also in the fetus
(Loockwood et al., 2008) thus preconceiving an inflammatory state in the
newborn from this syndrome. Increased IL-6 levels associates with an altered
biological action of ET-1, characteristic of hypertension (Epstein et al., 2009).
Since both IL-6and ET-1 maternal serum level are increased in PE (Taylor et al.,
1990) this could be a phenomenon that contributes to the abnormal
trophoblast invasion in this syndrome by increasing TNF- levels (Zhou et al.,
2011). Even when IL-6 is increased in the maternal serum from
obese pregnant women (Lager et al., 2011), there is no information regarding
Table 2. Maternal and umbilical cord level of cytokines in preeclampsia.
122
whether obesity is a factor leading to an increase of IL-6 higher than what is

seen in non-obese pregnant women coursing with PE. This phenomenon
could result in an increased incidence rather than the severity of the
syndrome. Elevated TNF maternal serum level has been correlated with
severe PE (Tosun et al., 2010; Xie et al., 2011) and in a minor level with
mild PE (Yoneyama et al., 2002; Canakci et al., 2007; Guven et al., 2009;
Tosun et al., 2010; Alanbay et al., 2011; Vitoratos et al., 2010; Sharma et al.,
2011). Nevertheless, there are not reports describing increased TNF level in
the human umbilical serum from obese women with PE (Tosun et al., 2010).
Other studies show that placental NFkB activation in PE may be due to
elevated fetal serum level of TNF (Vaughan et al., 2012). This phenomenon
could be the result of an increased release of this cytokine from the
trophoblasts (Ma et al., 2011) rather than the syncytiotrophoblasts (Goksu et al.,
2011), thus inhibiting trophoblasts integration with the endothelium (Xu et al.,
2011; Chen et al., 2010). Even when obese pregnant women do not exhibit
increased TNF serum level it is unclear whether a potential increase in the
level of this cytokine in obesity in pregnancy is a cause or is a consequence
of PE; however, obesity could accelerates the pathogenesis of PE.
5.3. Leptin and obesity in pregnancy

Leptin is primarily secreted by the white adipose tissue (Masuzaki et al.,
1995), and is also secreted by the human placenta (Masuzaki et al., 1997). In
obese patients leptin serum level is increased and patients develop leptin
resistance exhibiting a lost of its biological functions in target cells. It is
known that leptin level in the serum (Mise et al., 1998; Adali et al., 2009;
Molvarec et al., 2011) and leptin placental content (Lepercq et al., 2003,
Hoegh et al., 2010) in women with PE are increased compared with women
coursing with normal pregnancies, but its biological role is not fully
understood (Table 3). Controversially, only one study has reported leptin
levels in the human umbilical serum in PE showing that there are not
differences compared with normal pregnancies; however, a reduced level of
this molecule in the maternal serum in PE compared with normal pregnancy
was detected (Lam et al., 2001). On the contrary, in another study an
increase of leptin level was detected in umbilical vein blood in PE (Vitoratos
et al., 2001). These apparent discrepancies could result from recruitment of
patients that exhibit different weights in pregnancy and by the fact that some
of the studies were performed in patients with term pregnancies, an unusual
characteristic for pregnancies with severe PE. In obese pregnant women
maternal leptin level is increased (Fattah et al., 2011); however, nothing is
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Table 3. Maternal and umbilical cord leptin levels in preeclampsia.
reported regarding leptin level in the fetal serum in these pregnancies. Thus,
we highlight the fact that is crucial to determine the consequences of leptin
increase in the fetus from PE since this phenomenon could lead to
endothelial dysfunction (Korda et al., 2008).
6. Insulin resistance and vascular dysfunction in preeclampsia

Insulin resistance is defined as the inability of a known quantity of
exogenous or endogenous insulin to increase glucose uptake and its
metabolism in an individual as much as it does in a normal population
(Lebovitz, 2001). Interestingly, insulin resistance has also been reported in
124
patients with PE (Scioscia et al., 2009); however, the underlying

mechanisms by which insulin resistance is associated with PE and the
subsequent vascular dysfunction remains unclear. Endothelial dysfunction
has been demonstrated in vessels from women with PE (Chambers et al.,
2001), an effect that has been demonstrated in studies in vivo that impaired
endothelial function persists into the immediate postpartum period (Ramsay
et al., 2003) or impaired insulin sensitivity persists at least for six months
after delivery following a pregnancy coursing with PE compared with
normal pregnancies (Kaaja et al., 1999; Sattar & Greer, 2002; D'Anna et al.,
2006). Thus, insulin resistance and vascular dysfunction syndrome seems to
be crucial as an adverse intrauterine environment that could leads to diseases
in the adulthood (Barker, 2004).
6.1. Impaired insulin signalling

Insulin is involved in fetal growth (Hattersley & Tooke, 1999) and their
metabolic and mitogenic effects (Belfiore et al., 2009) are mediated via two
cell surface tyrosine kinase receptors (Ebina et al., 1985; Ullrich et al.,
1985). The human insulin receptor (IR) is encoded by a single INSR gene
(insulin receptor gene) located on chromosome 19 and composed of 22
exons. INSR codes for two protein isoforms differing by a 12-amino acids
insertion in the hormone-binding domain of the receptor resulting from exon
11 alternative splicing (Ebina et al., 1985; Ullrich et al., 1985; Sen et al.,
2009, 2010; Talukdar et al., 2011). The IR isoform lacking exon 11 (11)
(called IR-A) binds both insulin and insulin-like growth factor-II (IGFII),
whereas the isoform expressing the exon 11 (11+) (called IR-B) binds only
insulin (Frasca et al., 1999). A lack of regulation of the alternative splicing
of INSR may therefore have important consequences for insulin and IGFII
sensitivity and responsiveness. In fact, the mechanism involved in the
control of the IR-B/IR-A ratio is of critical importance for the understanding
of the role of IR in different disease states, including cancer (Belfiore et al.,
2011), myotonic dystrophy (Savkur et al., 2001; 2004), type 2 diabetes
mellitus (Sesti et al., 2001) and gestational diabetes (Westermeier et al.,
2011). Interestingly, the potentially role of insulin receptor isoforms in PE is
not yet reported.
Daz and colleagues (2005) have shown a lower affinity of placental IR
in PE compared with normotensive pregnant women. Moreover, expression
of IR and its signalling related proteins in trophoblast microvillous plasma
membranes from PE and normal pregnancies was similar, although impaired
signalling cascade activation was observed following incubation with insulin
in preparations from PE. Insulin increased the tyrosine phosphorylation of IR
125
in preparations from PE and normal pregnancies, but failed to induce

tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), IR-2 and the
p85 regulatory subunit of PI3K in PE (Kunjara et al., 2000). Other studies
show that there was no difference in the phosphorylation state of Akt/protein
kinase B (PKB) in the absence (Orcy et al., 2008) or in presence (Ferreira et
al., 2011) of insulin in placentas from PE or normal pregnancies. However, a
role for Akt/PKB signalling pathway in the pathophysiology of PE
associated with insulin resistance cannot be ruled out. In other hands, P-type
inositol phosphoglycan (P-IPG), a second messenger of insulin, enhances the
metabolic effects of insulin and is associated with insulin resistance
(Scioscia et al., 2009). In addition, the expression of P-IPG is higher in the
placenta (Kunjara et al., 2000), the fetus (detected in umbilical cord blood
and amniotic fluid) (Paine et al., 2006; Scioscia et al., 2006) and in the
mother (detected in the urine) (Paine et al., 2006; Williams et al., 2007) in
pregnancies coursing with PE, effects that were not extended to newborns
from PE (Scioscia et al., 2012). The latter results may help to elucidate how
these abnormal metabolic conditions influence fetal growth in pregnancies
with early or late onset of PE. In a recent study, 46 normotensive pregnant
women and 43 pregnant women coursing with PE were included in a search
for polymorphisms (Machorro-Lazo et al., 2009) in INS (insulin gene), INSR
and the mutations Ala513Pro and Gly972Arg of IRS-1. The protein products
of these genes form a functional complex in response to insulin (SnchezCorona et al., 2004). In this study polymorphisms in INS or INSR were not
detected in PE, suggesting that these genes would not be related with insulin
resistance in this syndrome (Machorro-Lazo et al., 2009). However, further
studies are needed to elucidate the complex relationship between functional
polymorphisms and PE.
6.2. Insulin and programming

Preeclampsia is a pathological condition with heritability of ~55%
(Williams et al., 2011), which is contributed to by both maternal and fetal
genes. It is feasible that some of the alterations in the risk of developing PE
seen in the offspring from pregnancies coursing with this syndrome relate to
share genetic factors between the mother and child. The modification of
these pathways also appear to be induced specifically by in utero exposure to
the syndrome. The underlying programming of vascular function may relate
to structural, hypoxic or epigenetic programming of endothelial development
and pathophysiology in PE (Davis et al., 2012a). By the other hand PE
correlates with changes in the metabolism and hormones that may lead to
alterations in tissue and organ development later in life (Gluckman &
126
Hanson, 2004). Epigenetic mechanisms may cause long-term functional and

structural changes (Gluckman et al., 2005; Goldberg et al., 2007). To date,
women who have had a pregnancy complicated by PE are more likely to
have heart disease in later life, and studies from the Helsinki Birth Cohort
show that the offspring from mothers with PE have an elevated risk for
stroke (Kajantie et al., 2009). Meta-analysis of available blood pressure data
indicates that children exposed to PE in utero exhibit a systolic blood
pressure that varies between 2-3 mmHg higher during childhood and young
adult life (Davis et al., 2012b). Fugelseth and colleagues (2011) studied
whether an altered myocardial function assessed by tissue Doppler
echocardiography could be detected at the age of 5-8 years in offspring from
pregnancies coursing by PE. The results show that offspring from PE have
smaller hearts, increased heart rate and late diastolic velocity at mitral valve
compared with offspring from normal pregnancies. Thus, pregnancies
complicated by PE could have a negative impact on the heart already at the
age of 5-8 years. Therefore, a better understanding of the mechanisms
behind these phenomena this will provide the basis for future novel
strategies to prevent CVD associated with PE pregnancy.
7. Dyslipidaemia in preeclampsia
Increased triglycerides and low-density lipoprotein (LDL) levels, with a
reduction in high-density lipoprotein (HDL) levels are typically described in
PE. This abnormal lipid metabolism may not only be a manifestation of PE,
but dyslipidaemia could also be involved in its pathogenesis and may play an
essential role in PE development (Thadhani et al., 1999; Demirsi et al., 2011;
Sep et al., 2011). Additionally, different risk factors to develop PE have been
assayed. Between those, dyslipidaemia, including hypercholesterolemia and
hypertriglyceridemia, have been characterized as risk factors (Lorentzen et al.,
1995; Grtacos et al., 1996; Enquobahrie et al., 2004; Magnussen et al., 2007;
Demirci et al., 2012). Longitudinal studies in have shown that increased
levels of cholesterol and triglycerides before pregnancy or in early pregnancy
associate with increased risk to develop PE (Table 4) (Thadhani et al., 1999;
Demirsi et al., 2011; Sep et al., 2011). Even when normal pregnancy is
associated with a physiological increase in the plasma level of triglycerides
and cholesterol (Potter & Nestel, 1979; Montes et al., 1984; Leiva et al.,
2011), PE is associated with an increase in the plasma level of these
type of molecules that is higher than the physiological process, a
phenomenon that perhaps intends to assure fetal growth (Potter & Nestel, 1979;
Belo et al., 2002; Demirsi et al., 2011; Sep et al., 2011). This phenomenon
127
Table 4. Triglyceride and cholesterol level in early pregnancy: relative risk to

develop preeclampsia.
has been associated with the occurrence of endothelial dysfunction (Van

Wijk et al., 2000; Escudero et al., 2008) and acute atherosis in the placenta
(Harsem et al., 2007; Staff et al., 2010) from PE such as in other vascular
beds exposed to this lipidic profile (Demirsi et al., 2011; Leiva et al., 2011).
7.1. Endothelial dysfunction and dyslipidaemia in preeclampsia

Several studies suggest that endothelial function and vascular reactivity in
both maternal and fetal-placental vasculature are altered in PE. In this regard,
expression of VCAM-1 (marker of vascular dysfunction) correlates directly
with the plasma concentration of LDL in PE, but show no correlation with
other lipids, suggesting that LDL cholesterol could be involved in the
endothelial dysfunction exhibited in patients with PE (Hubel et al., 1998).
Endothelium dependent relaxation of umbilical and placental vessels has been
reported in PE (Wareing & Baker, 2004; Dong et al., 2005; Karadas et al.,
2007). The response to calcitonin gene related peptide (CGRP), an
endothelium dependent vasodilator, is reduced in umbilical vessels from PE
and is associated with a reduced expression of its receptor (Dong et al., 2005).
Interestingly, the response to CGRP is also reduced in umbilical vessels from
pregnancies coursing with maternal supraphysiological hypercholesterolemia
(MSPH) without PE or another recognized pregnancy disease such as GDM or
IUGR (Leiva et al., 2011). This phenomenon has been associated with the
increased development of fetal atherosclerosis (Napoli et al., 1997, 1999).
Since MSPH leads to reduced endothelial-dependent vascular umbilical
128
response (Leiva et al., 2011) and fetal complications in PE have been related
with reduced fetoplacental blood flow (Wareing & Baker, 2004) it is
hypothesized that high levels of cholesterol in pregnancies with PE could
contribute, at least in part, to the observed placental endothelial dysfunction in
patients coursing with this syndrome.
Several in vitro studies suggest that LDL leads to endothelial
dysfunction by several pathways in different vascular beds. LDL and
oxidized LDL (oxLDL) leads to reduced NO synthesis by several
mechanisms including reduced L-arginine uptake (Kikuta et al., 1998;
Schwartz et al., 2007), eNOS expression and function (Liao et al., 1995,
Jimenez et al., 2001), tetrahydrobiopterin (BH4) synthesis (Ozaki et al.,
2002; Tang et al., 2005), but increased arginases expression and activity
(Ryoo et al., 2006, 2008). The levels of cholesterol and particularly LDL
and oxLDL are higher in PE, and even when the expression of the receptor
for oxLDL (lectin-like oxidized low-density lipoprotein receptor 1, LOX-1)
is induced in maternal and placental cells from PE (Sankaralingam et al.,
2009; Zhang et al., 2009) the effect of LDL from women with PE on the
L-arginine transport and NO synthesis (i.e., the L-arginine/NO signalling
pathway) in placental endothelium have been not yet reported. In PE the
expression of eNOS in unchanged or even increased (Barden et al., 1999;
Wang et al., 2004; Kanter et al., 2010), an effect that could corresponds
almost partially to a lipidic-dependent effect. This is supported by the
findings showing that HUVEC from pregnancies coursing with MSPH,
whose comparable cholesterol level (>280 mg/dl at birth) to those in PE
(~290 mg/dl at birth) (Solomon et al., 1999; Catarino et al., 2008), exhibits
reduced NO synthesis without changes in eNOS expression (Leiva et al.,
2012a). Additionally, other studies have shown increased expression and
activity of arginases (a group of enzymes that compete by the common
substrate L-arginine with eNOS in HUVEC) (Prieto et al., 2011) in the
maternal blood (Bernardi et al., 2008) and in HUVEC from normal
pregnancies in response to serum from women with PE (Sankaralingam et al.,
2009, 2010a,b). Thus, arginase would be upregulated within the fetal
endothelium and could contribute to the PE-associated endothelial
dysfunction. This increased endothelial arginase activity has been also
reported in hypercholesterolemic mice (Ryoo et al., 2006, 2008) and in
HUVEC from pregnancies coursing with MSPH (Leiva et al., 2012b).
7.2. Atherosclerosis and dyslipidaemia in preeclampsia

The link between endothelial dysfunction and the latter development of
atherosclerosis is widely recognized, where high levels of cholesterol are the
main factor involved in both phenomena. In PE the oxidation of LDL
129
lipoproteins is increased along with a reduction in the antioxidant effect of

HDL (Sarandol et al., 2004; Demir et al., 2011), thus promoting endothelial
dysfunction and foam cell formation which contributes to the formation of
atheroma in human placental vasculature (Staff et al., 2010). In this regards,
PE is associated with the appearance of atheroma in the placental
vasculature, especially in the maternal uterine spiral arteries. The
histological evidence in these vessels demonstrates visible lipid deposition in
the wall of the spiral arteries, a phenomenon known as acute atherosis, i.e., a
phenomenon requiring shorten times than for the development of
atherosclerosis (Lorentzen & Henriksen, 1998; Staff et al., 1999; Harsem et al.,
2007; Staff et al., 2010). Lipids deposition and foam cells in the acute
atherosis in placental vasculature of women with PE are similar to
atherosclerotic lesions in adults, both displaying vessel wall necrosis and
accumulation of subendothelial lipids-laden foam cells (a hallmark of
oxLDL), vascular fibrosis and perivascular lymphocytic infiltration. Even
when the precise mechanisms involved in placental atherosis or the direct
link between high levels of cholesterol and placental atheroma in PE are
unknown (Satff et al., 2010), it is feasible to hypothesize that
hypercholesterolemia leading to endothelial dysfunction and altered
L-arginine/NO signalling pathway may be involved in PE.
7.3. Dyslipidaemia in preeclampsia and fetal programming

Additionally to the effect reported in maternal and the fetoplacental
vasculature, the effect of dyslipidaemia on PE affect directly the offspring.
Newborns of mothers with PE have increased triglycerides and reduced
blood level of HDL (Rodie et al., 2004; Catarino et al., 2008; Akcakus et al.,
2010). The latter is a phenomenon related with a pro-atherogenic lipidic
profile that could be related with the future development of cardiovascular
disease in these children (Kajantie et al., 2009; Davis et al., 2012a,b).
Indeed, neonates from mothers with PE present an increased thickness of the
aortic intima-media thickness, a parameter useful to determine
atherosclerosis risk (Akcakus et al., 2010). In this regard, experimental
evidence demonstrate that in placental tissue from PE, the expression of the
transporter of cholesterol ABCA1 is increased in the first 25-33 wg (Plosh et al.,
2010) coinciding with the fetal period where a positive correlation between
the maternal and fetal levels of cholesterol has been documented (Napoli et al.,
1997). Thus, the higher expression of ABCA1 in the placenta could result in
increased cholesterol efflux from the maternal circulation to the placental
tissue and eventually reaching the growing fetus, a phenomenon which may
have physiological consequences such as the above mentioned increased
130
thickness of the aorta intima media in neonates from PE (Akcakus et al.,

2010; Plosh et al., 2010).
7.4. Preeclampsia, hypercholesterolemia and insulin signalling

Even when in PE there is no evidence regarding expression and function
of IR-A and IR-B, the dyslipidaemia coursing with this syndrome could be
involved in the alterations of the activity of both type of receptors as
reported in other pathologies of pregnancy such as GDM, where the
signalling of insulin mediated by IR-A and IR-B is altered and related with
endothelial dysfunction due, most likely, to altered adenosine transport
(Westermeier et al., 2009, 2011). Interestingly, in different endothelial cell
types, including bovine and mice aortic endothelial cells (Wang et al., 2006,
2011), insulin receptors are located in caveolae, a microdomain of the
plasma membrane enriched in caveolin (Parpal et al., 2000; Saltiel & Pessin,
2003; Ishikawa et al., 2005). Additionally, it has been determined that
deficiency of caveolin disrupts the insulin signalling in mice aortic
endothelial cells by reduction of IR protein expression, IR translocation to
membrane and insulin-stimulated Akt phosphorylation (Wang et al., 2011).
Because high levels of cholesterol increase the expression of caveolin and
alter the caveolae structure modulating the activity of proteins located in
caveolae (Feron et al., 1999), such as eNOS (Feron et al., 1999, 2001) and
hCAT-1 (McDonald et al., 1997), it is feasible that alterations in insulin
signalling in pregnancies coursing with increased levels of cholesterol
including PE, a phenomenon directly related with endothelial dysfunction
(Gonzlez et al., 2004; Westermeier et al., 2011), could be found.
8. Free radicals in preeclampsia

Oxidative stress related with abnormal vascular reactivity has been
considered a key stage in the aetiology of PE (Myatt & Webster, 2009). This
state is described as in imbalance in the production of reactive oxygen
species (ROS) and the scavenger ability of antioxidant defences of
fetoplacental tissues (Myatt et al., 2000; Myatt & Cui, 2004). The origin of
PE has not well established, but there is increased evidence demonstrating
that there is a higher accumulation of ROS and reactive nitrogen species
(RNS) in the fetoplacental vasculature and the trophoblast (Webster et al.,
2006, 2008; Myatt & Webster, 2009). The sources of ROS in the placental
tissues are NADPH oxidase, mitochondria, cytochrome P450, endoplasmic
reticulum, uncoupled eNOS, xanthine oxidase (XO) (Burton & Janiaux,
131
2011). The main ROS involved in PE are superoxide (O2-), hydroxyl ion
(OH-), hydrogen peroxide (H2O2), nitric oxide (NO) and peroxinitrite
(ONOO-) (Burton & Janiaux, 2011; Gonzlez et al., 2011). In vascular cells
of umbilical and placental tissues, the concomitant increase in the synthesis
of NO and O2- results in higher levels of ONOO-, a very deleterious ROS
that causes protein nitration and alters protein activity. The reaction leading
to formation of ONOO- is kinetically favoured because it is higher than the
reaction of NO with the soluble guanilyl cyclase (sGC) or O2- with
superoxide dismutase (SOD) (Webster & Myatt, 2008). For these reasons
stimuli for example, high extracellular D-glucose) causing increase of NO
and ROS synthesis in endothelium do not result in relaxation of smooth
muscle cells, but in endothelial dysfunction, thus triggering of vascular
diseases (Sobrevia & Gonzlez, 2009).
In animal models the reduction of uteroplacental blood flow causes
hypertension, characteristic of PE (Myatt & Webster, 2009). The reduction
of uteroplacental blood flow could be focused in alterations of vascular
reactive of placental and umbilical vessels, alterations with an origin in early
stages of placentation resulting in reduced blood flow to the fetus and
increased placental sensitivity to vasoconstrictors. In placental tissue from
normal pregnancies it has been demonstrate that acute exposure of
xanthine/xanthine oxidase or hydrogen peroxide (H2O2) increases the
contractility of human chorionic arteries, an effect abolished by catalase, an
enzyme that catalyses the conversion of H2O2 into water and O2- (Mills et al.,
2009). Administration of ONOO- caused relaxation in chorionic arteries
preconstricted with analogues of thromboxane A2. Thus, it is likely that the
main ROS involved in vasoconstriction in the chorionic plate could be H2O2
and/or O2-. Similar effects have been described in human chorionic vein and
isolated perfused cotyledon. In addition, cardiovascular active hormones,
such as the insulin, abolished the H2O2-induced contraction (Gonzlez et al.,
2011; Rojas et al., 2011).
The mechanisms involved in placental oxidative stress involve activity
of the catalytic subunits of nicotinamide adenine dinucleotide phosphateoxidase (NADPH oxidase) NOX2 and/or NOX4; both of these expressed in
human endothelial cells (Drummond et al., 2011). Recently it was
demonstrated that NOX4 could synthesize H2O2 (Takac et al., 2011), thus
supporting the possibility that the H2O2 is a relevant ROS in oxidative stress
mediated by NOX4. However, the main ROS or RNS involved in the
alterations in fetoplacental blood flow seen in PE is still unclear. In the other
hand, L-arginine, precursor of NO, is implicated in the fetoplacental
regulation of vascular tone. In fetal plasma from PE pregnancies there is a
132
decrease in L-arginine concentration correlated with an increase of the

expression of human cationic amino acid transporters 1 (hCAT-1) and
arginase II (ARGII) in the placental villi from pregnant women with PE
(Noris et al., 2004). Arginases are enzymes constitutively expressed in the
vascular endothelium with ARGII as the predominant isoform. ARGII
catalyses the hydrolysis of L-arginine into urea and ornithine, and evidences
show that the catalytic activity of arginases in endothelial cells limits the NO
synthesis and the NO-dependent vasodilatation (Morris, 2009). In HUVEC
exposed to hypoxia there is an upregulation of ARGII associated with
reduced eNOS activity by a mechanism most likely involving colocalization
of ARGII with eNOS. This potential colocalization results in reduced
substrate availability for eNOS inducing oxidative stress derived from eNOS
uncoupling (Prieto et al., 2011).
Hypoxia is a physiological condition in the placenta from normal
pregnancies. In fact, foetuses whose mothers have normal placental
perfusion are exposed to intervillous O2 levels of 8-11%. Abnormal
placentation in PE results in lower O2 delivery to the fetus, but the hypoxia
per se is not enough to explain the alterations in the newborn associated with
PE (Davis et al., 2012a). Thus, a link between hypoxia and oxidative stress
in early stages of PE is proposed. In placental explants, hypoxia increases
the synthesis of O2-, an effect blocked by activation of hemeoxygenase 1
(HO-1) (George et al., 2012), an enzyme involved in antioxidant responses,
angiogenesis, vasculogenesis and early placentation (Grochot-Przeczek et
al., 2012; Marcantoni et al., 2012). In addition, activation of HO-1 is related
with attenuation of O2- levels, NADPH oxidase activity and increase in the
arterial pressure in animal models of placental ischemia-induced
hypertension (George et al., 2011). Thus, oxidative stress and hypoxia are in
a close interaction and involve the regulation of signalling pathways related
with synthesis of ROS, decrease activity of antioxidant enzymes, reduced
vasodilation and lower supply of O2 to the fetus. Unfortunately, it is still
unclear whether the oxidative stress is an early state or a consequence of the
alterations in trophoblast invasion and placentation, but a correlation
between the hypoxia caused by poor placentation and increased levels of O2and/or ONOO- in placenta or umbilical cells seems clear.
9. Proteomics in preeclampsia
Preeclampsia-associated complications are responsible for the increased
obstetric and paediatric risk (Firoz et al., 2011), and have a permanent
impact in the health in adult life (Tuovinen et al., 2012). When the PE is
133
diagnosed (usually after 20 weeks) the syndrome is probably established and

potential adverse perinatal outcome is likely (Grunewald, 2011). Thus,
proteomics is a valuable tool for the identification of proteins or peptides
that are informative about the risk in presymptomatic pregnant women who
later develop pregnancy complications. One of the most common methods is
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Mass
Spectrometry (MS). These methods are conceptually simple, i.e., proteins
are separated by isoelectric point (pI) in a first dimension followed by
separation by its molecular weight (Choolani et al., 2009). Several studies
show different expression protein patterns in trophoblasts and plasma
proteins at the blood-tissue interface from placenta in PE, which are
participating in multiple processes including the hormone biosynthesis and
metabolism (Cox et al., 2011). Studies performed in trophoblasts from
placentas obtained from PE revealed that expression of 11 proteins was
reduced in PE, including actin, glutathione S-transferase, peroxiredoxin 6,
aldose reductase, heat shock protein 60, two molecular forms of heat shock
protein 70, -tubulin, subunit proteasome, ezrin, protein disulphide
isomerase and phosphoglycerate mutase 1; however, only -2-HSglycoprotein (fetuin) was found increased in samples from PE (Johnstone et al.,
2011). Altogether these findings are of interest since many of the proteins
have been associated with a reduced cytotrophoblast response to oxidative
stress.
Studies using placental tissue from full-term pregnancies show that
apolipoprotein A1 was accumulated in the placenta from PE (Centlow et al.,
2010; Zhang et al., 2011a). Other studies using two-dimensional
electrophoresis (2-DE) and matrix-assisted laser desorption ionization time
of flight mass spectrometry (MALDI-TOF-MS) found overexpression of
Hsp27 and Hsp90 in the placenta from PE (Gharesi-Fard et al., 2010; Shin et
al., 2011). These results suggest that oxidative stress exists in the placenta
from PE perhaps leading to a reduced cytotrophoblast defence and response
to oxidative stress. Also there is down-regulation of proteins with
antioxidant activities (peroxiredoxin 2 and peroxiredoxin 3), supporting the
latter feasibility in PE. In addition, altered expression of the stressresponsive proteins Hsc70, Hsp gp96 and protein disulphide isomerase,
might play a role in the pathogenesis of PE.
Other studies performed in homogenized chorionic villus explants by
MS/MS spectra show that the protein calcyclin (involved in promoting the
growth and proliferation of diverse cancer types) (Zhang et al., 2011b) is
overexpressed in PE (de Groot et al., 2007; Gzel et al., 2011). These data
complement other assays performed in HUVEC exposed to plasma from
women with PE where a reduced expression of structural proteins such as
134
cytokeratin, actin--1 and moesin, and the calcium-binding protein

calumenin (Sankaralingam et al., 2010b). Similar results were found in
HUVEC incubated with peroxynitrite scavenger FeTPPS ((5,10,15,20tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride) and subjected to
2D gel electrophoresis and mass spectrometry (Sankaralingam et al., 2010a).
These results suggest that the prooxidant and nitrating agent ONOO- might
damage a wide range of molecules in the cells including DNA and proteins
that are altered in PE.
Despite the great advances in proteomic techniques there are still several
problems such as low reproducibility and detection. In this context, the gelfree strategies represent a breakthrough in mass spectrometry. In this
technique there is a fractionation of the peptide, which is produced by liquid
chromatographic (LC), as long as proteins remain in solution. Data obtained
with this technique identifies 499 proteins in term placental. A fraction of
these proteins (45 proteins) were differentially expressed including IL-8
(Blankley et al., 2011). Additionally, serum from patients with diagnoses of
PE as well as in asymptomatic women that later on were diagnosed with PE
has also been analysed. The results show that antiangiogenic proteins are
higher in women with clinical PE compared with the proteomic profile
obtained in women with preclinical PE (Rasanen et al., 2010). In addition,
using one peptide ligand library combining with 1D gel-LC-MS/MS analysis
it was shown that the chorionic human chorionic somatomammotropin
hormone (CSH1) was overexpressed in serum from patients with PE (Liu et al.,
2011). These findings suggest that the metabolism of the mother is altered in
the sense of ensuring delivery of D-glucose to the fetus to comply with
energy demand and delivery of proteins for fetal growth. Other experiments
show significant reduction in the H-ficolin (1.3 fold) and L-ficolin (3.2 fold)
expression in plasma from women with PE (Wang et al., 2007). Ficolins are
pattern-recognition lectins involved in the lectin-complement pathway, and
play an important role in innate immunity, which might be responsible for
the systemic inflammatory response seen in PE.
In addition, analysis of urine samples from patients with severe or mild PE
and normotensive controls by using surface-enhanced laser desorption
ionization time-of-flight mass spectrometry (SELDI-TOF-MS) a different
Preeclampsia Proteomic Score of Urine (PPSU) was found compared with
subject with normal pregnancies (Lee et al., 2011). In another assay, using
isobaric tagging for relative and absolute quantification (iTRAQ) coupled with
Two-Dimensional Multidimensional Liquid Chromatography Tandem Mass
Spectrometry, a total of 113 proteins were found differentially expressed when
compared urine samples from women with PE compared with women
coursing with a normal pregnancy (Chen et al., 2011; Zhang et al., 2011a).
135
These proteins were associated with several biological processes including

blood coagulation, cell adhesion and differentiation, immune response and
cytoskeleton development. In the latter study the urinary angiotensinogen
(AGT), which is higher in PE by conventional ELISA determination, show the
same result when compared with a proteomic approach. Mass spectrometry
has exponentially increased in the recent years, and its development responds
to the need of finding specific biomarkers that could lead to a better clinical
diagnose and management and that will determine the susceptibility of
pregnant women to develop PE before that the symptoms appear.
10. Concluding remarks

Preeclampsia is a syndrome with high incidence worldwide that courses
with alterations in the fetoplacental vascular function, with clear alterations
in the macro and microvascular endothelium of the placenta. Among the
several hypotheses it is proposed that abnormal L-arginine/NO signalling
pathway is key in the aetiology of the syndrome. It has been proposed that a
key role is played by the endogenous nucleoside adenosine, which acts via
adenosine receptors, most likely A2AAR, in the endothelium. This
phenomenon is a likely a potential defence mechanism for PE-associated
altered endothelial function. Along with this specific mechanism, several
other metabolic pathways seem involved in the aetiology or at least are
making more susceptible the patients to develop this syndrome. Thus,
pregestational as well as obesity in pregnancy and dyslipidaemia, along with
altered protein expression profiles could be determinant in the onset of PE.
In addition to this, insulin and the involvement of the two isoforms of insulin
receptors, IR-A and IR-B, and its differential expression in placenta tissue
from PE could be determinant in understanding the role of insulin in this
syndrome. It is feasible that a differential action of insulin on these isoforms
of insulin receptors could be the bases of a future selective therapy to
improve endothelial cell function. Adenosine could, in fact, play a role in
this phenomenon since adenosine activation of A2AAR seems to be required
to allow the biological effects of insulin in human umbilical macro and
microvascular endothelium (Guzmn-Gutirrez et al., 2011; Leiva et al.,
2011; Pardo et al., 2012).
Acknowledgements
Supported by Fondo Nacional de Desarrollo Cientfico y Tecnolgico
(FONDECYT 1110977, 1120928, 11110059, 1100684), Comisin Nacional
de Investigacin en Ciencia y Tecnologa (CONICYT PIA Anillos ACT-73,
136
AT-24100210), Chile. Instituto de Salud Carlos III-Subdireccin General de

Evaluacin y Fomento de la Investigacin, PN de I+D+I 2008-2011
(PS09/01395), Junta de Andaluca, Consejera de Salud (PI-0034), Agencia
Espaola de Cooperacin Internacional para el Desarrollo (AECID)
(D/031187/10, A1/036123/11), Spain. FP holds a postdoctoral position
(CONICYT 3130583 and previously a CONICYT PIA ANillos ACT-73
postdoctoral research associate at CMPL, Pontificia Universidad Catlica de
Chile (PUC)). PA, CS, FW and EG-G hold CONICYT-PhD (Chile)
fellowships. PA and CS hold a Faculty of Medicine, PUC-PhD fellowship.
The authors thank the personnel of the labor ward at the Division of
Obstetrics and Gynaecology, PUC.
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Recent Res. Devel. Physiol., 6(2012): 105-158 ISBN: 978-81-308-0489-7

7. Fetoplacental vascular pathophysiology

Abstract. Preeclampsia is a syndrome that affects a significant

Luis Sobrevia et al.

could result from differential expression of insulin receptor isoforms A and B.

Fetoplacental vasculature in preeclampsia

(proteinuria 3 g/24 hours, blood pressure 140/90 mmHg) or severe

Luis Sobrevia et al.

trophoblasts resulting in a narrow invasion into the maternal vascular bed

Fetoplacental vasculature in preeclampsia

In parallel with the characterization of the intrinsic cellular mechanisms

2.1. The fetus in preeclampsia

Luis Sobrevia et al.

from PE compared with newborns from normal pregnancies (Villar et al.,

2.2. The placenta in preeclampsia

Fetoplacental vasculature in preeclampsia

placenta results from preservation in the capacity of contraction of the spiral

3. Vascular dysfunction in preeclampsia

3.1. Regulation of placental vascular tone in preeclampsia

Luis Sobrevia et al.

thromboxane A2 is reported in PE (Walters et al., 1991; Ajne et al., 2005).

Fetoplacental vasculature in preeclampsia

3.2. Preeclampsia and intrauterine growth restriction

Table 1. Alterations in preeclampsia coursing with or without intrauterine growth restriction.

Fetoplacental vasculature in preeclampsia

This is evidenced by histopathological changes in the placental implantation

4. Adenosine in the placenta from preeclampsia

Luis Sobrevia et al.

Figure 1. Adenosine effect in human placenta endothelial cell function in

fetal hypoxia was reported as ~600 nM (Yoneyama et al., 1996), a

Fetoplacental vasculature in preeclampsia

disease but not to the severity of patients clinical symptoms. In addition,

4.1. Adenosine receptors and preeclampsia

Luis Sobrevia et al.

4.2. Adenosine transporters and preeclampsia

Fetoplacental vasculature in preeclampsia

(Adenosine/L-Arginine/Nitric Oxide) signalling pathway in HUVEC (San

5. Preeclampsia and obesity in pregnancy

Luis Sobrevia et al.

Figure 2. Obesity as a factor to develop preeclampsia. Obesity in pregnancy is

Some groups report that obesity correlates with PE through a state of

5.2. Cytokines/interleukins and obesity in pregnancy

Fetoplacental vasculature in preeclampsia

Luis Sobrevia et al.

whether obesity is a factor leading to an increase of IL-6 higher than what is

5.3. Leptin and obesity in pregnancy

Fetoplacental vasculature in preeclampsia

Table 3. Maternal and umbilical cord leptin levels in preeclampsia.

6. Insulin resistance and vascular dysfunction in preeclampsia

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patients with PE (Scioscia et al., 2009); however, the underlying

6.1. Impaired insulin signalling

Fetoplacental vasculature in preeclampsia

in preparations from PE and normal pregnancies, but failed to induce

6.2. Insulin and programming

Luis Sobrevia et al.

Hanson, 2004). Epigenetic mechanisms may cause long-term functional and

Fetoplacental vasculature in preeclampsia

Table 4. Triglyceride and cholesterol level in early pregnancy: relative risk to

has been associated with the occurrence of endothelial dysfunction (Van

7.1. Endothelial dysfunction and dyslipidaemia in preeclampsia

Luis Sobrevia et al.