Life Sciences 91 (2012) 710–715

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Life Sciences
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Complex expression changes of the placental endothelin system in

early and late onset preeclampsia, fetal growth restriction and
gestational diabetes
Martina Dieber-Rotheneder ⁎, Sanja Beganovic, Gernot Desoye, Uwe Lang, Mila Cervar-Zivkovic
Department of Obstetrics and Gynaecology, Medical University of Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Preeclampsia (PE), fetal growth restriction (FGR) and gestational diabetes mellitus (GDM) are major
Received 2 November 2011 pregnancy complications affecting maternal and fetal health. The placenta and the vasoconstrictor endothelin-1
Accepted 24 April 2012 (ET-1) have a controlling and mediating role in these conditions. This study tested the hypothesis that the expres-
sion of ET-1 and its receptors (ETA and ETB) is altered in these pathologies and differs between early (gestational
Keywords:
week [GW] ≤34) and late (GW> 34) third trimester pregnancies.
Human placenta
Main methods: The study included 88 women (GW 28-41) with PE (blood pressure > 140/90 mmHg,
endothelin
preeclampsia
protein > 300 mg/24 hrs; n = 14), FGR (b 10th birthweight centile and pathological umbilical blood
fetal growth restriction flow; n = 13), PE + FGR (n = 5) and GDM (n = 21), and gestational age-matched controls (n= 35). ET-1,
gestational diabetes ETA and ETB mRNA and ETA and ETB protein were quantified in placental tissues by real-time PCR and immuno-
blotting.
Key findings: The ET/ETR mRNA system is altered in PE and PE+ FGR and GDM. Expression of ET-1, ETA and ETB
is upregulated in early onset PE and PE + FGR with stronger effect in PE + FGR. GDM down regulated ET/ETR
mRNA in the placentas in late third trimester of pregnancy. ET/ETR protein is virtually unchanged.
Significance: Early onset PE (≤GW34) with or without FGR is associated with increased mRNA expression of the
ET/ETR system, while in late onset PE and GDM the opposite effect was observed. This study supports the
emerging concept that early and late onset PE are different diseases.
© 2012 Elsevier Inc. Open access under CC BY-NC-ND license.

Introduction
Preeclampsia (PE) is one of the most common complications in preg-
nancy occurring in approximately 8% of all pregnancies. It is a leading
cause of maternal morbidity and mortality characterised by new develop-
ment of maternal hypertension and significant proteinuria preceded by
endothelial cell activation and an inappropriate inflammatory response
(Redman and Sargent, 2005; Roberts and Cooper, 2001). The cause of
PE and the underlying mechanism are poorly understood. It is clear, how-
ever, that the central organ of dysfunction is the placenta as the only truly
definite intervention is its delivery (George and Granger, 2011b;
Huppertz, 2008). Placental ischemia and hypoxia are proposed to act as
central causative factors in the etiology of this disorder (Burton and
Jauniaux, 2004; George and Granger, 2010).
For several years a two-stage model of PE has been proposed (Roberts
and Gammill, 2005). The first stage is characterised by a reduced placental
perfusion, resulting in fetal growth restriction (FGR) and/or preterm birth,
the second stage is a widespread maternal multisystem disorder, which
⁎ Corresponding author at: Department of Obstetrics and Gynaecology, Medical Univer-
sity Graz, Auenbruggerplatz 14, A-8036 Graz, Austria. Tel.: +43 316 385 12476; fax: +43
316 385 12477.
E-mail address: martina.dieber@medunigraz.at (M. Dieber-Rotheneder).
constitutes the symptomatic phase of the disease (George and Granger,
2010). PE occurs only, if “constitutional factors” (genetics, behaviour, en-
vironment, obesity, diabetes, etc.) render the mother sensitive to the ef-
fects of the reduced placental perfusion (Roberts and Hubel, 2009). This
model, however, had to be modified since changes relevant to PE can be
detected already in the first trimester, long before vascular remodelling
occurs. The concentrations of certain proteins in maternal blood such as
placental growth factor, soluble vascular endothelial growth factor
receptor-1 (sFlt1), soluble endoglin and others are altered weeks
before the onset of clinical symptoms (Than et al., 2008).
An important factor implicated in the maternal endothelial dysfunc-
tion associated with PE is elevated endothelin-1 (ET-1) (George and
Granger, 2011a). This peptide was first identified in 1988 by Yanagisawa
et al. as the most potent known vasoconstrictor. ET-1 is produced by a
two stage proteolytic degradation of the protein prepro-ET into the ac-
tive 21-amino acid peptide (Yanagisawa et al., 1988) and acts via two
G-protein coupled receptors, endothelin receptor-A and -B (ETA, ETB).
Circulating ET-1 concentration is increased in preeclamptic women com-
pared with non pregnant controls (Taylor et al., 1990) and returns to
normal levels after delivery.
In several animal models ETA blockade resulted in an attenuation
of the pregnancy-induced hypertension. This argues for an important
role of ET-1 in PE pathology (George and Granger, 2011b). ET-1 may

0024-3205 © 2012 Elsevier Inc. Open access under CC BY-NC-ND license.

doi:10.1016/j.lfs.2012.04.040
 M. Dieber-Rotheneder et al. / Life Sciences 91 (2012) 710–715
be the key link between the poorly perfused placenta and the im-
paired maternal vascular function associated with PE by triggering
oxidative stress in the human placenta (Fiore et al., 2005). ET-1 has
also been implicated in the pathophysiology of FGR in several animal
models as well as in humans (Neerhof et al., 2011; Nezar et al., 2009).
Placental vascular dysfunction is also associated with gestational di-
abetes mellitus (GDM), a further pregnancy complication which occurs
in approximately 2-10% of all pregnancies. GDM women demonstrate a
reduced vascular sensitivity to ET-1 (Ang et al., 2001). Women with
GDM are at increased risk of unfavorable obstetrics outcome and for
the development of Type 2 diabetes after pregnancy.
Because the human placenta expresses ET-1 and its circulating levels
are elevated in pregnancies complicated by PE and/or FGR, we tested the
hypothesis that the expression of ET-1 and ETRs in the human placenta is
upregulated in PE, FGR, PE+ FGR and GDM depending on severity and
onset of the disease. We further postulated that this upregulation is
more pronounced in more severe forms of the underlying placental dis-
ease ie., in FGR in combination with PE.
Subjects and methods
The study included 88 women (gestational week [GW] 28-41) with
pregnancies complicated with PE, FGR, PE plus FGR and GDM, as well as
gestational age-matched controls. Both, the PE and FGR group were divid-
ed into an early (≤GW34) and late onset group (>GW34). Only singleton
pregnancies were investigated. Patients had no other complications of
pregnancy and no reported co-morbidities such as smoking or drug use.
PE was defined as hypertension (>140/90 mmHg on at least 2 occa-
sions) and proteinuria of >300 mg protein /24 hrs urine collection. Se-
vere PE was defined as hypertension (>160/110 mmHg) plus mild
proteinuria or mild hypertension plus severe proteinuria (≥5 g protein/
24 hrs urine collection) or elevated blood pressure and proteinuria to-
gether with the occurrence of any maternal or fetal complication. FGR
was defined as infant birth weight >10th percentile and pathological um-
bilical blood flow, and PE plus FGR defined as the combination of both dis-
eases. GDM was diagnosed on the basis of a pathologic glucose tolerance
test between GW24 and 28 using a 75 g glucose load if one or more plas-
ma glucose concentrations met or exceeded 95/180/155 mg/dl at 0/1/
2 hrs. Women with GDM were treated either with diet (n=11) or diet
plus insulin (n=10). There were no cases available of GDM before
GW34 and none of PE plus FGR after GW34.
The control group of≤ GW34 comprised women who were delivered
preterm (GW 28-34) by cesarean section, because of placenta previa
with vaginal bleeding or cervical insufficiency. Infants within the control
group had birth weights >10th percentile and normal antenatal umbili-
cal artery Doppler waveform. The control group >GW34 consisted of
healthy pregnant women, who were delivered at GW 36-41. They were
matched with the study groups for maternal age, gestational age and par-
ity. The groups were not subdivided by mode of delivery, because previ-
ous studies in our and another laboratory did not show any differences
between cesarean section and vaginal deliveries in ET-1 binding to pla-
cental tissue (Kohnen et al., 1997).
Characteristics of the entire study population are shown in Table 1.
Within both GW groups the subgroups did not significantly differ in
maternal age, BMI and gestational age. As expected with the nature of
the disease, women with FGR, PE, and PE plus FGR had lower weights
of neonates than the control women (pb 0.05, p b 0.01, p b 0.001, respec-
tively) in early third trimester. Placental weight was only lower in the
PE plus FGR group as compared to controls (p= 0.006). The weights
of neonates and placentas were significantly lower in the PE plus FGR
than in the PE group without FGR, or FGR alone (all p b 0.05). In the
late third trimester of pregnancy the birth weights of neonates and pla-
cental weights were similar (PE group) or significantly lower (FGR group)
compared to the age matched control group (pb 0.001, pb 0.05, respec-
tively). The birth weights and placental weights of the GDM group were
higher (pb 0.001) relative to the FGR group.
711
The duration of the disease could not be determined. The period
between first diagnosis and delivery was 3-6 times longer in our early
onset patients’ groups compared to the late onset group. This is in accor-
dance with the recommendations for the general management of PE ie.,
prolongation of early pregnancies to allow for fetal maturation.
The study was approved by the ethical committee of the Medical Uni-
versity of Graz and informed consents were obtained from the patients.
Placental tissue
Placentas were collected immediately after delivery and put on
ice. Six pieces of tissue, each around 0.5 g, were dissected of the cen-
tral part of the placenta near the umbilical cord, washed in ice-cold
saline, snap frozen in liquid nitrogen and stored at 80 °C. Three pieces
were put in Tri-Reagent for further RNA isolation.
Quantitative real time RT-PCR
Total RNA from placental tissue was extracted using TRI-Reagent®
(Molecular Research Center, Cincinnati, OH) according to the instruc-
tions of the manufacturer. Total RNA (1 μg) from placental tissue was
reversely transcribed to cDNA in a total volume of 20 μl, using Super-
Script II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer's instructions. Random hexamer primer were pur-
chased by Fermentas and dNTP Mix by Invitrogen. After cDNA synthesis
the reaction volume was diluted with H2O to a final volume of 100 μl
(10 ng cDNA/μl). Fifty ng cDNA (5 μl) of each reaction were subsequently
subjected to quantitative real time PCR using FAM labeled TaqMan Gene
Expression Assays (ET-1: Hs00174961_m1; ETA: Hs01003560_m1; ETB:
Hs00240747_m1, RPL30: Hs00265497_m1) and the TaqMan Universal
PCR Mastermix (Applied Biosystems, Foster City, USA). Components
were mixed according to the manufacturer's instructions and amplified
in 25 μl total volume/well (96 well plates, Applied Biosystems) using
an AB7900 TaqMan. Threshold cycle values (Ct) were defined automati-
cally by the SDS 2.2.2 program (Applied Biosystems). Copy numbers of
ET-1, ETA and ETB were calculated by the standard 2-ΔΔCt method using
the gene expression of ribosomal protein L30 (RPL30) as reference. All
reactions were performed in triplicates and controlled by nontemplate
controls. Statistical analyses used the ΔCt values.
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting
Cell membrane proteins were solubilized in Laemmli sample buff-
er (Sigma) supplemented with Complete R protease inhibitor cocktail
(Boehringer). Insoluble material was removed by centrifugation at
100,000 x g for 1 h at 4 °C. Prior to electrophoresis, samples were
mixed with Laemmli buffer (Sigma) 1:1 and boiled for 5 min at 99 °C.
SDS-PAGE and Western blotting with rabbit ETA antibody AS 444 or rab-
bit ETB antibody AS 445, respectively (generous gift of Dr. Müller-Esterl,
Johann Wolfgang Goethe University at Frankfurt, Germany) were per-
formed as described earlier (Dieber-Rotheneder et al., 2006). The mem-
branes were washed and further incubated with the secondary antibody
(goat anti-rabbit, 1:1000, Abcam, Cambridgeshire, UK) for 1 h at room
temperature. After further washings in TBS immunolabelling was visual-
ized using the chemiluminescense based SuperSignal RWest pico
Chemoluminescent Substrate system (Pierce). Membranes were ex-
posed to Hyperfilm (Amersham) and analyzed by the video documenta-
tion system GP 2000i (MWG-Biotech, Ebersberg, Germany) and Alpha
Digi Doc 1200 (Alpha Innotech, San Leandro, CA, USA) software. To com-
pare different experiments an internal reference sample (pooled lysates
from three normal term placentas) was run in each experiment and all
optical densities within each blot were normalized to the standard on
the respective blot. This method allowed between blot comparison. In
addition, for easier comparison of the figures each control group was
set to 100% and the pathologies calculated as % ETR protein relative to
the corresponding controls. Subsequently, the blots were stripped at
712 M. Dieber-Rotheneder et al. / Life Sciences 91 (2012) 710–715

Table 1
Characteristics of the Study Groups.

Gestational week (GW) ≤34 GW >34 GW

Controls FGR PE PE plus FGR Controls FGR PE GDM

Number of subjects 11 4 9 5 24 9 5 21
Maternal age (years) 32.2 ± 5.8 31.2 ± 6.1 30.0 ± 7.1 32.6 ± 5.1 30.0 ± 6.4 31.4 ± 8.8 31.4 ± 8.8 31.5 ± 5.9
BMI (kg/m²) 23.6 ± 4.0 23.0 ± 3.6 25.6 ± 3.9 24.5 ± 7.4 22.7 ± 3.0 23.9 ± 4.3 21.5 ± 4.4 25.4 ± 5.2
Gestational age (weeks) 33.0 ± 1.2 32.6 ± 1.5 32.5 ± 1.8 31.4 ± 2.4 38.6 ± 1.6 38.2 ± 1.6 39.4 ± 1.1 39.3 ± 1.3
Weight of neonate (g) 2077 ± 326 1563 ± 309* § 1564 ± 124** § 1062 ± 300*** 3089 ± 420 2296 ± 435*** 2923 ± 451 3397 ± 338 ###
Weight of placenta (g) 428 ± 118 354 ± 68 § 400 ± 93 § 232 ± 71** 565 ± 109 399 ± 85* 484 ± 53 632 ± 141 ###
Diagnosis – delivery (days) n.a. n.a. 6.0 ± 7.2 5.8 ± 6.0 n.a. 1.0 ± 1.3 2.2 ± 3.9 n.a.
Mode of delivery (CS/vaginal) 11/0 4/0 9/0 5/0 12/12 6/3 1/4 15/13

PE : preeclampsia, FGR: fetal growth restriction, GDM: gestational diabetes melitus, BMI: Body mass index (kg/m²).
Data were analyzed using ANOVA, followed by Bonferroni post hoc test and are presented as mean ± SD.
* p b 0.05; ** p b 0.01; ***p b 0.001 relative to controls; n.a.: not applicable.
In ≤ 34 GW: § p b 0.05 relative to PE plus FGR group.
In > 34 GW: ### p b 0.001 relative to FGR group.

room temperature with stripping buffer (Pierce) for 60 minutes with oc-
casional agitation. Then the blots were reprobed with a ß-actin antibody
(mouse anti ß-actin, Abcam) as a loading control. Our previous work
with the same antibodies revealed the predominant bands of both ETA
and ETB protein at 55 and 45 and 55 kDa, respectively, Therefore, these
bands were used in this study (Dieber-Rotheneder, et al., 2006).
Statistical analysis
All data are reported as mean ± standard deviation. Statistical anal-
ysis was performed by ANOVA and post hoc with Bonferroni- t-test.
Values of p b 0.05 were accepted as significant.
Results
Quantitative realtime PCR was performed to determine the expres-
sion of placental ET-1, ETA and ETB. In early third trimester placentas
(≤34GW) the expression of ET-1 (Fig. 1A) was increased in PE (3.5-
fold, pb 0.01, n= 9) and PE plus FGR (6.9-fold, pb 0.001, n = 5) com-
pared to gestational age-matched controls (n =11). The ETA expression
level (Fig. 1B) was higher in PE plus FGR (5.1-fold, pb 0.001, n =5) com-
pared to controls (n = 11). The 2.6-fold, increase in PE did not reach sig-
nificance. In the PE plus FGR group (n =5) ETA mRNA expression was
significantly increased compared to PE (n =9) or FGR alone (n =4,
both p b 0.05). ETB mRNA expression (Fig. 1C) was 3-fold upregulated
in PE (n = 9) and to a similar extent in PE plus FGR (p b 0.05, n =5).
The mRNA changes were not found at the protein level. There was only
a slight (26% versus controls) increase of ETA protein measured in early
PE (p b 0.05, n= 9).
In contrast to the early third trimester, late third trimester (>GW 34)
ET-1 and ETA expression levels were decreased (Fig. 1D and E). ET-1
mRNA was reduced (3.5-fold, p b 0.001) in PE (n =5) and GDM (3-fold,
pb 0.001, n =21). ETA expression (Fig. 1E) was reduced in placentas
from the FGR (pb 0.05, n =9), PE (1.8-fold, pb 0.05, n =5)) and GDM

Fig. 1. ETR and ET-1 mRNA expression in third trimester pregnancy pathologies. Relative expression of ET-1, ETA and ETB mRNA was determined in early (GW ≤ 34, A-C) and late
(GW > 34, D-F) third trimester pregnancy pathologies by quantitative realtime PCR. Ribosomal protein L30 was used as an internal control. FGR: fetal growth restriction, PE: pre-
eclampsia, PE + FGR: preeclampsia plus fetal growth restriction, GDM: gestational diabetes. Values are calculated relative to age matched controls (fold change 1 = 100%) and given
as mean ± standard deviation of the fold changes of two experiments per sample of n subjects of the following groups: early pathologies (A-C: Controls: n = 11, FGR: n = 4, PE:
n = 9, PE + FGR: n = 5) and late pathologies (D-F: Controls: n = 24, FGR: n = 9, PE: n = 5, GDM: n = 21). Data were analyzed using ANOVA, followed by Bonferroni post hoc test.
*p b 0.05; **p ≤0.01; ***p ≤ 0.001.
 M. Dieber-Rotheneder et al. / Life Sciences 91 (2012) 710–715 713

group (2-fold, p b 0.001, n = 21). Two-fold higher expression levels of
ETB (Fig. 1F) were detected in PE (p b 0.01, n =5) versus controls
(n =24) and versus FGR (p b 0.05, n= 9). There were no changes of
ETA protein. ETB protein was decreased in PE (pb 0.05, n =5, Fig. 2D)
by 35%. No significant changes of ETR proteins were found in FGR
samples.
Placentas from late third trimester pregnancies complicated by GDM
revealed a 33% decrease of ETB protein (pb 0.001, n = 21), (Fig. 2D). Mo-
dality of GDM treatment (diet or diet plus insulin) did not affect the re-
sults. Therefore, both groups were combined.
Discussion
One promising target molecule in the study of PE is ET-1 (George
and Granger, 2010). Based on recent research, ET-1 is regarded as key
mediator of hypertension in PE, since soluble substances secreted by
the placenta into the maternal circulation as result of placental insuf-
ficiency induce hypertension through this powerful vasoconstrictor
(George and Granger, 2011a). Because the human placenta expresses
ET-1 and its circulating levels are high in pregnancies complicated by
PE and/or FGR (Nezar, et al., 2009), the present study hypothesized an
upregulation of the ET/ETR system in pregnancies complicated by PE,
FGR and PE plus FGR. GDM was included as a further pregnancy
complication.
The results were complex and showed differences between early and
late onset pathologies. While in early onset, severe PE+FGR (≤GW 34)
ETA mRNA expression in human placentas is upregulated as compared to
gestational age matched controls, it is down regulated in late onset PE
(>GW 34). Likewise, placental ET-1 mRNA expression is elevated in
early onset PE, but downregulated in late onset PE. This difference was
not found for ETB expression indicating that both receptors are different-
ly affected by the pathology underlying late onset PE. However, there is
discordance between mRNA and protein data with few mRNA changes
found in the pathologies translating into protein differences. The under-
lying reasons are unclear, but may include mRNA and protein stability,
translation efficiency and also differences in ubiquitination and subse-
quent degradation as found for endothelin receptors (Nishimoto et al.,
2010). These differences in mRNA to protein steady state levels between
pathologies strongly argue for a complex regulation of the endothelin
system and will warrant further more molecular studies.
Despite lack of explanation at the molecular level, the results of
this study provide evidence to support the emerging concept that
early and late onset PE are different diseases (Rasmussen and Irgens,
2008; Roberts and Catov, 2008). Differences between early and late

Fig. 2. ETR protein in third trimester pregnancy pathologies. ETA and ETB protein were determined in placental lysates of early (GW ≤ 34, A-B) and late (GW > 34, C-D) third trimes-
ter pregnancy pathologies by Western blotting. The bands were analyzed by scanning densitometry as described in the methods section. Values were normalized to one reference
sample (Ref., run in all experiments), calculated relative to controls matched for gestational age (= 100%), and given as mean ± standard deviation of three experiments per sample
of n subjects of the following groups: early pathologies: (A-C: Controls: n = 11, FGR: n = 4, PE: n = 9, PE + FGR: n = 5) and late pathologies (D-F: Controls: n = 24, FGR: n = 9, PE:
n = 5, GDM: n = 21). M: molecular weight marker. Data were analyzed using ANOVA, followed by Bonferroni post hoc test.*p b 0.05; ***p ≤0.001 vs. controls, n.s.: not significant.
714 M. Dieber-Rotheneder et al. / Life Sciences 91 (2012) 710–715
onset PE have been found for several outcomes including expression of
ETA, development of later life cardiovascular disease (Catov et al., 2010;
Irgens et al., 2001), recurrence rate of PE in following pregnancies
(Caritis et al., 1998; Sibai et al., 1991), the prediction rate of some path-
ophysiological markers like anti-angiogenic factors (Levine et al., 2004)
and placental gene expression. Increased microparticle shedding
of syncytiotrophoblast in early onset PE was also not found in
late onset PE or FGR (Goswami et al., 2006). This collective evi-
dence suggests that despite sharing some common clinical features
they might have different pathogenesis (Sitras et al., 2009).
Our results differ in part from those reported by others, who found
a decreased placental expression of ETA and an unchanged expression
of ET-1 and ETB in women with PE compared to normal pregnant con-
trols (Faxen et al., 2000). That study, however, compared preeclamp-
tic placentas (GW 29-39) with healthy term placentas (GW 37-39). In
contrast to our study, the PE group was not further subdivided into
early and late onset PE. We only found the decrease of ETA expression
in placentas of the late onset (>GW 34) PE study group. Moreover,
they did not compare their study group with gestational age matched
controls, but used placentas from term pregnancies. This is of particu-
lar importance since we previously showed that ETA and ETB receptors
change their expression levels during placental development (Cervar
et al., 2000).
There is strong support for the relationship of PE and FGR (Rasmussen
and Irgens, 2008). Consistent with our data the history of FGR is more
strongly associated with severe rather than milder forms of pregnancy-
induced hypertension. In our study, the increase of placental ETA mRNA
expression in the early onset PE group was even doubled when PE was
complicated further by FGR. We were unable to obtain material from PE
plus FGR from the late third trimester pregnancies.
There are some differences between PE with and without FGR also
in amniotic fluid concentrations of anti-angiogenic proteins sFlt1, sol-
uble Endoglin, leptin and adiponectin. Their levels are significantly
higher in FGR superimposed by PE than in FGR alone (Wang et al.,
2010).
The changes in the ET/ETR mRNA in early onset PE in this study
could not be observed in normotensive FGR. These data support the
view that the placental changes in PE are not shared by normotensive
FGR.
The present study is the first to report about the ET/ETR system in
GDM placentas. Diabetes is associated with vascular dysfunction, which
may be due in part to altered vascular response to endogenous peptides
such as ET-1. A reduced sensitivity to ET-1 was demonstrated in diabetic
pregnant women (Ang et al., 2001) in spite of the normal ET-1 plasma
found in GDM (Swiderski et al., 2010; Telejko et al., 2009). We found
a downregulation of the ET/ETR system in GDM placentas in late third
trimester of pregnancy.
Conclusion
Pregnancy pathologies are associated with complex changes of the
ET/ETR system, which are mostly found at the mRNA level. Absence of
these changes in the translation product argues for differences in in-
tracellular mRNA and protein processing in the human placenta be-
tween the pathologies. Early onset PE with or without FGR is associated
with increased expression of the placental ET/ETR mRNA, while in late
onset PE, isolated FGR and GDM a opposite effect was observed. The path-
ophysiological consequences, if any, of the present findings are unclear.
Vasoconstriction is predominantly mediated by ETA. If the rise in ET-1
mRNA also resulted in increased peptide, vasoconstriction within the pla-
cental vasculature would also rise. Conversely, down regulation of ET-1/
ETA in late trimester pregnancy would be consistent with an adaptive
down regulation of the ET system. Further functional studies are needed
to identify the contribution of the observed changes to some of the clinical
features associated with the pathologies.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The work was funded by the Oesterreichische Nationalbank (Anni-
versary Fund, Project numbers: 12243 and 12601), grants of the
“Kulturamt Stadt Graz” and the Department of Science and Research,
Land Steiermark.
The authors thank Melanie Fellner for technical assistance and
Bettina Amtmann and Petra Wagner for recruiting both cases and
controls for this study. They are grateful to Dr. Werner Müller-Esterl
(Johann Wolfgang Goethe University at Frankfurt, Germany) for the
generous gift of the ETR antibodies, to Renate Michlmaier for her
assistance in collection and freezing of placental tissue, to Dr. Josef
Haas for statistical advice and Gerd Schwager for continuous support
and help with preparing the figures.
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