2014

http://informahealthcare.com/jmfISSN: 1476-7058 (print), 1476-4954 (electronic)

J Matern Fetal Neonatal Med, 2014; 27(4): 350–354! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2013.818125

ORIGINAL ARTICLE

Placental gene expression patterns of endoglin (CD105) in intrauterinegrowth restriction

Imre Szentpeteri1, Attila Rab2, Laszlo Kornya3, Peter Kovacs4, Reka Brubel5, and Jozsef Gabor Joo5

1Praxis fur Gynakologie und Geburtshilfe und allgemeine Medizin, Wehingen, Baden-Wurttemberg, Germany, 2Hospital Telki, Telki, Hungary,

Germany, 3St. Stephen’s Hospital Budapest, Hungary, Germany, 4Clinical Research Unit Hungary, Szikszo, Hungary, Germany, and 5First Department

of Obstetrics and Gynecology Semmelweis University, Budapest, Hungary, Germany

Abstract

Objective: In this study, we describe placental gene expression patterns of endoglin inpregnancies with intrauterine growth restriction (IUGR) compared to normal pregnancies.Methods: Placental samples were obtained from 101 pregnancies with IUGR using 140 normalpregnancy cases as control. Gene expression patterns and protein levels of the endoglin werecompared between the two groups. For the gene expression analysis real-time PCR wasapplied, while for the estimation of placental protein level we performed Western analysis.Results: The placental endoglin gene was significantly overexpressed in the IUGR group versusthe control group (Ln2a: 1.69). The placental endoglin protein level proved to be significantlyhigher in case of IUGR (endoglin/b-actin ratio: 13.8� 2.3) versus the control cases (5.3� 1.1).The placental gene expression as well as the protein levels of endoglin showed no significantdifference between female and male newborns. Concerning the placental gene expression andprotein level, no significant difference was justified between the more (0–5 percentile) and less(5–10 percentile) severe cases of IUGR.Conclusion: Increased placental gene expression of endoglin may result in vascular dysfunctionleading to chronic fetal hypoxia, which may induce VEGF-A to stimulate angiogenesis. This canbe explained as feed back response to restore fetal placental circulation.

Keywords

Angiogenesis, antiangiogenic activity,endoglin, gene expression, intrauterinegrowth restriction, placenta

History

Received 20 April 2013Revised 28 May 2013Accepted 12 June 2013Published online 26 August 2013

Introduction

Intrauterine growth restriction (IUGR) is defined as fetal

birthweight below the tenth percentile for fetal gender and

gestational age [1]. Although placental dysfunction is the

most common etiology of IUGR, intrauterine infections, fetal

malformations and other maternal factors are also commonly

involved.

Placental dysfunction in IUGR leads to impaired transpla-

cental transport of nutrients and oxygen compromising fetal

nutrition and oxygenization [2–4]. Despite the paramount

importance of placental dysfunction in IUGR, its molecular

level pathomechanism remains largely unexplored [5,6].

Nevertheless, it is generally assumed that impaired placental

circulation resulting from an imbalance of angiogenic and

antiangiogenic influences is a major contributor [7–10].

There are two major pathways in the formation of placental

blood vessels: vasculogenesis involves de novo blood vessel

formation from precursor cells, while angiogenesis involves

vascular growth through further differentiation of existing

blood vessels [11,12]. Both vasculogenesis and angiogenesis

are subject to the influences of several pro-angiogenic and

antiangiogenic factors. Endoglin (CD 105), a cell surface

glycoprotein discovered in 1990, is one of the several

inhibitors of angiogenesis [13]. Structurally, endoglin is a

protein consisting of 658 amino acids. It has an intracellular,

an extracellular and a transmembranous domain [14].

Although the endoglin gene is primarily expressed in

endothelial cells, its presence has also been identified in

monocytes, in the bone marrow and in syncytiotrophoblasts

[14,15]. A mutation of the endoglin gene has been found in

the autosomal dominant hereditary hemorrhagic teleangiecta-

sia (Osler-Weber-Rendu syndrome). In addition, endoglin

gene mutation is involved in the mechanism of tumor

proliferation [16]. In this latter scenario, endoglin has been

detected by multimodality imaging techniques including

contrast-enhanced ultrasonography, molecular-level MRI

and positron emission tomography [16].

Under normal circumstances, the endoglin gene has very

low activity in endothelial cells. A significant increase in gene

expression is primarily observed during embryogenesis,

inflamation or tissue injury [17,18].

Recent studies highlighted the role of endoglin in the

pathogenesis of both preeclampsia and IUGR [19–22].

Ramsay et al. proposed a hypothesis that emphasizes the

importance of vascular dysfunction in both of these disorders.

Address for correspondence: Dr. Joo Jozsef Gabor, SemmelweisEgyetem, AOK, I.Sz. Szuleszeti es No

00gyogyaszati Klinika, 1088

Budapest, Baross utca 27, Germany. E-mail: joogabor@hotmail.com

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However, whereas in preeclampsia vascular dysfunction is

systemic, in IUGR it seems to be confined to the placental

circulation [23].

Recent research has revealed that the antiangiogenic effect

of endoglin suppresses vascular endothelial growth factor

(VEGF) and placental growth factor (PlGF) activity, both of

which are recognized as pro-angiogenic factors. Ultimately,

this effect leads to the development of endothelial dysfunction

in IUGR [19,21,24].

A major underlying mechanism of persisting IUGR is

chronic fetal hypoxia. Structurally, this results from impaired

circulation within the placental villi. Yinon et al. showed that

a decreased oxygen level in the pregnant mother’s blood leads

to increased placental TGF-beta-3 (transforming growth

factor beta 3) levels. TGF-beta-3, in turn, stimulates endoglin

secretion by placental endothelial cells. Increased endoglin

secretion leads to impaired circulation through its antiangio-

genic activity [25] (Figure 1).

In this study, our primary objective was to characterize

alterations of placental endoglin (CD105) gene expression in

IUGR compared to normal pregnancies. Secondary objectives

included exploration of fetal gender dependent differences in

placental endoglin gene expression and to determine whether

differences in gene expression are affected by the degree of

growth restriction. Several clinical and demographic variables

were also assessed for clinical correlation.

Materials and methods

Patients

We obtained placental samples for characterization of

endoglin expression from all patients treated for IUGR at

the Semmelweis University, Budapest, in the study period

between January 1, 2010 and January 1, 2011, as well as 140

placental samples from cases of normal pregnancy during the

same time period used as controls. Maternal age, gestational

weight gain and gestational increase in Body Mass Index

(BMI) were also evaluated. We included the cases into our

study in which IUGR was diagnosed per standard criteria as

fetal birthweight below the tenth percentile for fetal sex and

gestational age. The IUGR group was subdivided into two

groups by the degree of growth restriction as below: less

severe growth restriction defined as birth weight of 5–10

percentiles (61 cases) versus more severe growth restriction

(0–5 percentile; 40 cases). Abdominal circumference (AC)

determined through ultrasonography was also considered

when establishing the clinical diagnosis of IUGR. AC values

in cases with clinical diagnosis of IUGR were compared to

cases with similar gestational age in the normal pregnancy

group. Placental dysfunction was considered the main

etiology of IUGR in cases where intrauterine infections,

chromosomal abnormalities, fetal malformations, malnour-

ishment of the mother during pregnancy, multiple pregnancy

and structural disorders of the placenta could be excluded on a

clinical basis.

Delivery was either vaginal or by cesarean section based

on clinical decision. In the final analysis of data, no

distinction was made with respect to the type of delivery.

Placental tissue samples were taken right after the

detachment of the placenta in a uniform manner with

approximate dimensions of 2� 2� 2 cm (8 cm3), which

were then kept at �70 �C for genetic expression testing. The

sampling of each placenta have been random, so all areas of

each placenta had an equal chance of being sampled. (In 10-

10 cases of IUGR and control cases the sampling of the

placental tissue was performed from four different points of

the placental tissue – the gene expression values have not

differed).

Maternal demographics and relevant clinical data during

pregnancy or the postnatal period were collected including

maternal and paternal age, obstetric history, genetic history,

general medical history, maternal birthweight, gestational

age, fetal gender, weight gain and BMI increase during

pregnancy, pregnancy-related pathology including disorders

of carbohydrate metabolism, neonatal weight and Apgar

score. Consent was obtained in each case from the mother

(signatures on file).

RNA isolation and cDNA synthesis

Whole placental RNA content was isolated with Quick RNA

microprep kit (Zymo Research). RNA concentration was

determined using NanoDrop spectrophotometer (NanoDrop).

Reverse transcription was performed in 20 ml target volume

using 5 mg whole RNS, 75 pmol random hexamer primer,

10 mM dNTP (Invitrogen), 20 U M-MuLV Reverse

Transciptase enzyme (MBI Fermentas) and 1x-es buffer

(MBI Fermentas). The reaction mix was incubated for 2 h at

42 �C. Subsequently, the enzyme was inactivated at 70 �C for

15 min.

Real-time PCR assay

The reverse transcriptase reaction solution was diluted three-

fold with nuclease-free water. For the real-time PCR assay,

1 ml diluted cDNS (approximately 15 ng RNA-equivalent) and

1�SYBR Green Master Mixet (Applied Biosystems,

Carlsbad, CA, USA) were used. Primers were designed

using Primer Express Software (Applied Biosystems). Primer

sequences are detailed in Table 1. Real-time PCR was

performed in 20 ml target volume using 1 ml cDNA, 1 pmol,

gene-specific forward and reverse primer and 1� SYBR

Green PCR Master mix. All real-time PCR were performed

using the MX3000 Real-time PCR (Stratagen) system with

the following settings: 40 cycles at 95 �C, denaturing process

0

0,5

1

1,5

2

endoglin gene(control: beta-

ac�n)

endoglin gene(control:GADPH)

borderline ofover expression

Placental gene expression of endoglin inIUGR placenta compared to control (Ln)

1,69 1,8

1

Figure 1. Placental gene expression pattern of endoglin in IUGRcompared to normal pregnancy (overexpression¼Ln value 41,p50.05).

DOI: 10.3109/14767058.2013.818125 Placental endoglin and the IUGR 351

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for 15 s, annealing at 60 �C, chain elongation and detection for

60 s. The PCR reactions were done in triplicates. For each

gene, relative expression was normalized using the human

b-actin and GADPH genes as controls.

Western analysis

To evaluate the endoglin content of the placental tissues, we

performed a homogenization by sonication (Ultrasonic

Processor, Tekmar, Cincinatti, OH) in four volumes of

1�Laemmli buffer (50 mM Tris-HCl, 10% Glycerol) contain-

ing 5 mM DTT, 0.5 mM PMSF (phenyl methyl sulfonyl

fluoride), 1 mM sodium vanadate and 1 ml/ml of protease

inhibitor cocktail (Protease Inhibitor Cocktail Set III,

Calbiochem, San Diego, CA). The homogenate was centri-

fuged at 13 400� g at 4 �C for 15 min. The estimation of the

protein content was performed using the Biorad essay. Each

preparations were boiled for 5 min and centrifuged for 30 s. The

proteins were tranferred to polyvinylidine fluoride membranes

(Immobilon, Millipore, Bedford, MA) at a constant current of 1

mA per cm2. Detection of the proteins was carried out after

blocking the membranes with a 5% solution of non-fat dry milk

for 1 h. Membranes were incubated with the primary endoglin

antibody for 1 h at a room temperature. After that they were

washed 3 times for 10 min each and incubated with alkaline

phosphatase conjugated secondary endoglin antibody for

30 min. Chemiluminescent detection was carried out using

the CDP-Star substrate (Boehringer, Manheim, Indianapolis,

IN) diluted 1:200 in the alkaline phosphatase buffer for 5 min.

The analysis of beta-actin was performed by the electro-

phoresis of 10 mg of the total protein on separate gels

and subjected to Western analysis and probed with a

monoclonal anti-beta-actin antibody (Clone AC-15, Sigma,

St.Louis, MO).

The bands on the developed Western blot films were

scanned by HP Laser scan (Scanjet, Hewlett Packard, Palo

Alto, CA). Densitometry was performed by the UN-SCANIT

Gel Version 4.3 digitizing software (Silk Scientific Inc.,

Orem, UT).

Statistical analysis

For gene expression studies of the endoglin gene in the IUGR

versus normal pregnancy groups, two-sample t-test was used

with 95% confidence interval (CI). Determination of degree

of freedom was performed using the Welch-Satterthwaite

correction. Values of gene expression testing were interpreted

in the following manner: (1) overexpression¼Ln value41,

p50.05; (2) underexpression¼Ln value 5�1, p50.05; (3)

normal expression¼Ln value 51,4�1, p50.05. GraphPad

Prism 3.0 (GraphPad Software Inc) software was used in all

statistical analytic procedures.

Demographics and clinical data were analyzed with SPSS

software. Logistic regression was used for dichotomous

outcomes with multiple independent variables. For continu-

ous outcomes, analysis of variance (ANOVA) and linear

regression were used as appropriate. A p value of50.05 was

accepted for statistical significance.

Results

Clinical data

A total of 101 placental samples were obtained from IUGR

pregnancies with fetal gender distribution as follows: 37

males, 64 females with a male to female ratio of 0.58. In the

control group, fetal gender distribution was 73 males and 67

females with male to female ratio of 1.09.

There was no significant difference between median values

of maternal age in the IUGR compared to the normal

pregnancy group (30.82� 4.34 years versus 31.45� 3.12

years; p40.05).

A statistically significant difference between the groups

was found both in gestational weight gain, and gestational

BMI increase. Mean gestational weight gain was 10.9 kg in

the IUGR group compared to 14.8 kg in the normal pregnancy

group (p50.05). Mean gestational BMI increase was 4.1 in

the IUGR group versus 5.3 in the normal pregnancy group

(p50.05).

The median value of the gestational age at delivery was

36� 3.02 week in case of IUGR and 38� 1.76 week in case

of the controls (p40.05).

With respect to maternal birthweight, median maternal

birthweight was significantly lower in the subgroup of IUGR

with more severe (0–5 percentile fetal birthweight) growth

restriction compared to the median maternal birthweight in

the subgroup of mothers giving birth to babies with 5–10

percentile birthweight, a less severe degree of growth

restricion (2830� 215 gram versus 3120� 260 g; p50.05).

Gene expression studies

A total of 101 placental samples were obtained for the

determination of placental endoglin gene expression in

the IUGR group versus 140 in the normal pregnancy group

(Table 2). The endoglin gene was overexpressed in IUGR

group compared to the control (b-actin and GADPH) genes

(Ln2�s�actin: 1.69; Ln2�GADPH: 1.80).

Within the IUGR group, no fetal gender-dependent differ-

ence was seen in placental EGF expression (Ln2a: �0.16;

Table 3).

There was no significant difference in placental EGF

expression between the more severe (0–5 percentile fetal

birthweight) versus less severe (5–10 percentile fetal birth-

weight) IUGR subgroups (Ln2a: �0.02; Table 4).

Table 1. Primers and sequences in real-time PCR.

Gene name and identification Forward primer Reverse primer

Endoglin (NM_000118) 50-CCACTGCACTTGGCCTACA-30 50-GCC CAC TCAAGG ATCTGG-30

b-Actin (M10277) 50-GGCACCCAGCACAATGAAG-3 50-GCCGATCCACACGGAGTACT-30

GADPH (JN038570) 50-ACCACAGTCCATGCCATCAC-30 50-TCCACCACCCTGTTGCTGT-30

352 I. Szentpeteri et al. J Matern Fetal Neonatal Med, 2014; 27(4): 350–354

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Placental endoglin levels

Placental endoglin levels were quantified after normalizing to

b-actin.

Analyzing the 101 samples of the patients with IUGR, we

justified a significantly higher placental endoglin level

compared to that of the 140 control cases. The endoglin/

b-actin ratio proved to be 13.8� 2.3 in cases of IUGR

placental samples versus the 5.3� 1.1 value of the control

cases (p50.05; Table 2).

Concerning the 64 samples from female newborns, we

found no significant difference in the placental endoglin level,

compare to the 37 samples of male newborns (12.6� 2.5

versus 14.8.3� 1.9; p40.05; Table 3).

In the 61 cases of IUGR with less severe degree (5–10

percentile), the placental endoglin level showed no significant

difference compared the 41 cases of more severe (0–5

percentile) IUGR (11.7� 2.9 versus 13.9� 2.7; p40.05;

Table 4).

Discussion

Our present findings suggest that gestational weight gain and

gestational increase in BMI are both significantly reduced in

IUGR pregnancies compared to normal pregnancy. These

findings confirm previous published results [1,6].

We also found a significant association between maternal

birthweight and the degree of growth restriction in IUGR.

Specifically, median maternal birthweight in mothers giving

birth to babies with 0–5 percentile birthweight was signifi-

cantly lower than those giving birth to less severely growth

restricted babies with a birthweight between 5 and 10

percentile. We propose that in the background of this

association, genetic factors may play a role.

The main objective of the present study was to charac-

terize alterations in placental gene expression patterns of the

endoglin gene in IUGR pregnancies. Our results suggest

that the placental endoglin gene is significantly over-

expressed in IUGR compared to normal pregnancy. Our

finding also justify, that the placental level of endoglin

protein is elevated in case of IUGR compared to normal

pregnancies. The significance of these findings is underlined

by previous studies showing that the antiangiogenic sub-

stance endoglin is present in higher concentrations in

maternal serum during IUGR pregnancy [21,22,24,26].

Similarly our further researches have verified, that the

placental gene expression of VEGF-A gene also increases in

case of IUGR, which suggests, that a connection between

these two factors (VEGF-A and endoglin) can be supposed

[27]. With our present finding, we are able to propose a

mechanism where, during IUGR, increased placental expres-

sion of endoglin would lead to impaired placental circulation

through its antiangiogenic effect. Chronic hypoxia due to

impaired placental circulation would be the signal to

increase placental VEGF-A activity as a compensatory

response. It suggests that placental endoglin and VEGF-A

act together, with the aim of creating a balance between the

angiogenetic and antiangiogenetic effects.

Impaired placental circulation is thought to be the most

common pathomechanism leading to placental dysfunction.

Our present finding suggesting increased endoglin expression

Table 4. Placental endoglin gene expression and protein level of endoglin in IUGR: more severe growth restriction (0–5 percentile birthwieght) versusless severe growth restricion (5–10 percentile birthweight).

Gene name a value� SE(a) Ln 2a p Change in gene expression

Endoglin gene �0.04� 0.63 �0.02 0.02 No significant hangeProtein name Endoglin/b-actin ratio (5–10 percentile) Endoglin/b-actin ratio (0–5 percentile) p Change in placental endoglin levelEndoglin 11.7� 2.9 13.9� 2.7 0.03 No significant change

a¼DCtA�DCtB; DCtA¼CtENG�Ctcontrol gene (5–10 percentile IUGR); DCtB¼CtENG�Ctcontrol gene (0–5 percentile IUGR); (nA¼ 61, nB¼ 40).Control gene b-actin.

Table 2. Placental gene expression pattern and protein level of endoglin in IUGR compared to normal pregnancy.

Gene name a value� SE(a) Ln 2a p Change in gene expression

Endoglin gene* 2.45� 0.78 1.69 0.04 OverexpressionEndoglin geney 3.01� 0.65 1.80 0.04 OverexpressionProtein name Endoglin/b-actin ratio (IUGR) Endoglin/b-actin ratio (control) p Change in placental endoglin levelEndoglin 13.8� 2.3 5.3� 1.1 0.03 Increase

Ncontrol¼ 140; nIUGR¼ 101; a¼DCtcontrol � DCtIUGR; *Control gene b-actin. yControl gene GADPH.

Table 3. Fetal gender dependence of placental endoglin gene expression and protein level of endoglin in IUGR: male fetuses versus female fetuses.

Gene name a value� SE(a) Ln 2a p Change in gene expression

Endoglin gene �0.24� 0.67 �0.16 0.02 No significant hangeProtein name Endoglin/b-actin ratio (female) Endoglin/b-actin ratio (male) p Change in placental endoglin levelEndoglin 12.6� 2.5 14.8.3� 1.9 0.04 No significant change

nfemale¼ 64; nmale¼ 37; a¼DCtfemale � DCtmale; Control gene b-actin.

DOI: 10.3109/14767058.2013.818125 Placental endoglin and the IUGR 353

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in IUGR, a disorder commonly arising from placental

dysfunction, reflects a potential role of the antiangiogenic

effect by endoglin in the pathomechanism. Impaired angio-

genesis in the placental villi of IUGR pregnancies in turn

leads to impaired placental circulation and chronic fetal

hypoxia [28].

It is noteworthy that the antiangiogenic effect of endoglin

has been shown to be even more pronounced in preeclampsia,

a disorder also associated with vascular dysfunction

[24,26,29].

Since in our IUGR population there was no fetal gender

dependent difference in endoglin gene expression, we

conclude that the endoglin-associated impairment of placental

circulation in IUGR may not be fetal gender dependent.

In contrast to previous findings by Laskowska et al.

published in 2012, we could not identify a correlation

between the degree of growth restriction in IUGR and

placental activity of endoglin. Our findings suggest that the

degree of growth restriction can be multifactorial and

placental endoglin expression may only be one of many

factors. Our previous results from placental VEGF-A gene

expression studies were also similar in that the degree of

growth restriction did not seem to correlate with placental

VEGF-A gene activity [27].

In summary, in placental samples obtained from IUGR

pregnancies we found increased expression of endoglin

compared to normal pregnancy controls. We propose a

mechanism in which the increased endoglin acitivity in

IUGR leads to impaired placental circulation through an

antiangiogenic effect. This results in the development of

placental vascular dysfunction and chronic fetal hypoxia. It is

chronic hypoxia that turns on VEGF-A as a compensatory

mechanism to improve fetal vascular blood supply by

promoting placental blood vessel formation. Neither fetal

gender nor the degree of growth restriction are significant

contributors to this mechanism. The results of our study

emphasize, that improving the placental circulation in IUGR

is of high priority, as the disturbance of angiogenesis is an

important factor in the etiology of this condition.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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