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Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCG and PP13 expression in BeWo cells K. Orendi a , M. Gauster a , G. Moser a , H. Meiri b , B. Huppertz a, * a Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Harrachgasse 21/VII, 8010 Graz, Austria b Diagnostic Technologies Ltd., 20692 Yoqneam, Israel article info Article history: Accepted 24 February 2010 Keywords: BeWo Beta-hCG PP13 Vitamin C Aspirin Preeclampsia abstract Preeclampsia is one of the leading causes for maternal and fetal morbidity. Placental protein 13 (PP13) is a placenta specic protein and with its decreased maternal serum levels in the rst trimester it is one of the most promising markers to predict the syndrome in early pregnancy. In clinical trials attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin, and antioxidants such as vitamins C and E. Here we investigated the effect of these agents on PP13 and beta-hCG levels using choriocarcinoma cell lines as surrogates for primary villous trophoblast. Five different cell lines were triggered with forskolin and cultured for 48 h. Amongst the ve tested cell lines BeWo cells showed the strongest increase in PP13 mRNA after forskolin treatment compared to controls. Hence these cells were used to investigate the effect of varying concentrations of vitamin C, acetylsali- cylic acid (ASA), Trolox Ò and heparin on cell fusion and PP13 and beta-hCG levels. The response to vitamin C was a dose-dependent increase in protein expression, while the other drugs showed only modest effects. Since rst trimester PP13 has been shown to be signicantly decreased in women subsequently developing preeclampsia, this data might point to a benecial effect of very early vitamin C treatment of such women already in the early rst trimester of pregnancy. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Preeclampsia remains one of the leading causes for maternal and fetal/neonatal mortality and morbidity affecting 2e5% of all pregnancies. It is characterized by new onset hypertension and proteinuria after 20 weeks of gestation in previously normotensive women [1]. However, no adequate conservative medical treatment is available, and so far the only therapeutical intervention is termination of pregnancy by inducing delivery or Caesarean section. Hence, it is becoming increasingly important to nd a way to predict and consequently prevent preeclampsia thus improving maternal and fetal health. So far, a primary prophylaxis is not available, as the underlying cause leading to this syndrome is not fully understood yet [2]. Agents that have been discussed as candidate therapeutics for secondary prophylaxis of preeclampsia include low-dose aspirin, low-molecular-weight heparin, folic acid, antioxidants such as vitamins C and E, and calcium [3]. In clinical trials the effects of some of these drugs on the development of preeclampsia have already been investigated in vivo. In randomized placebo- controlled studies administration of aspirin throughout the rst trimester or of heparin starting at 10e11 weeks of gestation low- ered the risk of developing hypertensive pregnancy complications such as preeclampsia [4,5]. However, the prophylactic benet of vitamins C and E is more controversially discussed. In a small randomized trial a benecial effect of these vitamins was suggested for women at high risk of developing preeclampsia [6]. Unfortu- nately, this nding could not be conrmed in two multicenter randomized clinical trials where administration of vitamins C and E started between 14 and 21 weeks of gestation [7,8]. Several biochemical markers have been detected in maternal peripheral blood or urine. Such markers alone or in combination with the uterine artery Doppler pulsatility index have already been tested to evaluate the risk to develop preeclampsia [9e13]. One of the most promising biochemical markers is placental protein 13 (PP13), a member of the beta-galactoside binding S-type galectin superfamily [14]. In the human PP13 is only expressed in placental tissues, and within the villous trophoblast it can only be found in the multinucleated syncytiotrophoblast [14]. PP13 is expressed and released into the intervillous space, where it enters the maternal circulation and can be detected in peripheral blood. While in normal pregnancy serum concentrations of PP13 rise from the rst to the second half of pregnancy, women who develop preeclampsia * Corresponding author. Tel.: þ43 316 380 7604; fax: þ43 316 380 9625. E-mail address: [email protected] (B. Huppertz). Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.02.017 Placenta 31 (2010) 431e438

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lable at ScienceDirect

Placenta 31 (2010) 431e438

Contents lists avai

Placenta

journal homepage: www.elsevier .com/locate/placenta

Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion, beta-hCGand PP13 expression in BeWo cells

K. Orendi a, M. Gauster a, G. Moser a, H. Meiri b, B. Huppertz a,*

a Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Harrachgasse 21/VII, 8010 Graz, AustriabDiagnostic Technologies Ltd., 20692 Yoqneam, Israel

a r t i c l e i n f o

Article history:Accepted 24 February 2010

Keywords:BeWoBeta-hCGPP13Vitamin CAspirinPreeclampsia

* Corresponding author. Tel.: þ43 316 380 7604; faE-mail address: [email protected]

0143-4004/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.placenta.2010.02.017

a b s t r a c t

Preeclampsia is one of the leading causes for maternal and fetal morbidity. Placental protein 13 (PP13) isa placenta specific protein and with its decreased maternal serum levels in the first trimester it is one ofthe most promising markers to predict the syndrome in early pregnancy. In clinical trials attempts toprevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin,and antioxidants such as vitamins C and E. Here we investigated the effect of these agents on PP13 andbeta-hCG levels using choriocarcinoma cell lines as surrogates for primary villous trophoblast. Fivedifferent cell lines were triggered with forskolin and cultured for 48 h. Amongst the five tested cell linesBeWo cells showed the strongest increase in PP13 mRNA after forskolin treatment compared to controls.Hence these cells were used to investigate the effect of varying concentrations of vitamin C, acetylsali-cylic acid (ASA), Trolox� and heparin on cell fusion and PP13 and beta-hCG levels. The response tovitamin C was a dose-dependent increase in protein expression, while the other drugs showed onlymodest effects. Since first trimester PP13 has been shown to be significantly decreased in womensubsequently developing preeclampsia, this data might point to a beneficial effect of very early vitamin Ctreatment of such women already in the early first trimester of pregnancy.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Preeclampsia remains one of the leading causes for maternaland fetal/neonatal mortality and morbidity affecting 2e5% of allpregnancies. It is characterized by new onset hypertension andproteinuria after 20 weeks of gestation in previously normotensivewomen [1]. However, no adequate conservative medical treatmentis available, and so far the only therapeutical intervention istermination of pregnancy by inducing delivery or Caesareansection. Hence, it is becoming increasingly important to find a wayto predict and consequently prevent preeclampsia thus improvingmaternal and fetal health. So far, a primary prophylaxis is notavailable, as the underlying cause leading to this syndrome is notfully understood yet [2].

Agents that have been discussed as candidate therapeutics forsecondary prophylaxis of preeclampsia include low-dose aspirin,low-molecular-weight heparin, folic acid, antioxidants such asvitamins C and E, and calcium [3]. In clinical trials the effects ofsome of these drugs on the development of preeclampsia havealready been investigated in vivo. In randomized placebo-

x: þ43 316 380 9625.(B. Huppertz).

All rights reserved.

controlled studies administration of aspirin throughout the firsttrimester or of heparin starting at 10e11 weeks of gestation low-ered the risk of developing hypertensive pregnancy complicationssuch as preeclampsia [4,5]. However, the prophylactic benefit ofvitamins C and E is more controversially discussed. In a smallrandomized trial a beneficial effect of these vitamins was suggestedfor women at high risk of developing preeclampsia [6]. Unfortu-nately, this finding could not be confirmed in two multicenterrandomized clinical trials where administration of vitamins C and Estarted between 14 and 21 weeks of gestation [7,8].

Several biochemical markers have been detected in maternalperipheral blood or urine. Such markers alone or in combinationwith the uterine artery Doppler pulsatility index have already beentested to evaluate the risk to develop preeclampsia [9e13]. One ofthe most promising biochemical markers is placental protein 13(PP13), a member of the beta-galactoside binding S-type galectinsuperfamily [14]. In the human PP13 is only expressed in placentaltissues, and within the villous trophoblast it can only be found inthe multinucleated syncytiotrophoblast [14]. PP13 is expressed andreleased into the intervillous space, where it enters the maternalcirculation and can be detected in peripheral blood. While innormal pregnancy serum concentrations of PP13 rise from the firstto the second half of pregnancy, womenwho develop preeclampsia

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Table 1Primary antibodies used for immunofluorescence.

Antigen Clone/Cat No Dilution Host/Isotype Company

Beta-hCG RB-059-A 1:50 Rabbit IgG pc ThermoScientificDesmosomal

proteinZK-31 1:400 Mouse IgG mc Sigma

Alpha-fodrin AA6 1:100 Mouse IgG mc BiotrendCytokeratin 18

neo-epitopeM30 1:50 Mouse IgG mc Roche

mc, monoclonal; pc, polyclonal.

K. Orendi et al. / Placenta 31 (2010) 431e438432

show significantly lower serum levels of PP13 in early pregnancy,then rising above normal in the second half of pregnancy [15].Serum levels of beta-hCG are also elevated in second and thirdtrimester in cases suffering from preeclampsia compared tocontrols, while in first trimester serum beta-hCG is not significantlyaltered between both groups [16]. Accordingly, alterations of PP13serum concentrations could serve to assess the risk and progressionof preeclampsia as early as mid first trimester.

Here we established a stable cell culture model to investigatehow preeclampsia prevention candidates such as vitamins C and E,aspirin and heparin affect the expression of PP13 and beta-hCG infusing trophoblastic cells. To mimic the multinucleated syncytio-trophoblast, we used several choriocarcinoma cell lines and treatedthemwith forskolin to trigger cell fusion [17e19]. Quantification ofcell fusion as well as PP13 and beta-hCG RNA and protein levelswere used to assess the agents' effects on trophoblastic cells.

2. Materials and methods

2.1. Cell culture

BeWo cells (ECACC), AC1-M59 cells (provided by H.G. Frank, Aachen, Germany)and ACH-3P cells (provided by G. Desoye, Graz, Austria) [20] were cultured at 37 �Cand 20% O2 in DMEM (Gibco, Austria) and Ham's F12K (Gibco) (ratio 1:1) supple-mented with 10% FCS, 1% penicillin/streptomycin and 1% amphotericin B. Jar cells(ATCC, USA) were cultured under the same conditions in RPMI 1640 (PAA, Austria)supplemented with 2% L-glutamine, 1% Na-pyruvate, 1% HEPES, 10% FCS, 1% peni-cillin/streptomycin and 1% amphotericin B. Jeg-3 cells (provided by H.G. Frank) werecultured as mentioned above in EMEM with L-glutamine (PAA) supplemented with10% FCS, penicillin/streptomycin and amphotericin B.

Cells were plated in 12 well dishes (BeWo: 2.3 � 105 cells/well, AC1-M59:6�104 cells/well, ACH-3P: 2�104 cells/well, Jar: 9�104 cells/well, Jeg-3: 9�104 cells/well) and incubated over night. The following day medium was replaced by freshmediumwith or without 20 mM forskolin (SigmaeAldrich, Austria) or 0.2% DMSO (asvehicle control). After 48e72h culturemediumwas collectedand centrifugedat 2300gfor 10 min at 4 �C. Supernatant was used immediately or stored at �80 �C for furtheruse. Human fibroblasts (Detroit 551; ATCC) served as negative control.

For morphological analysis and quantification of fusion, BeWo cells were seededonto chamber slides (Nunc, Austria) (1 � 105 cells/chamber) or detached by treat-ment with 120 ml/well Accutase (PAA) supplemented with 0.01 mg/ml DNase I(Roche, Germany) for 15 min at 37 �C and spun onto glass slides at 57g for 5 min(Shandon “Cytospin 2”, Histocom, Austria).

2.2. RNA-isolation

Total RNAwas isolated with Tri Reagent (Applied Biosystems, Austria) accordingto the manufacturer's instructions. The quality of total RNA was assessed byethidium bromide staining of denaturing agarose gels.

2.3. Real-time RT-PCR

Reverse transcription of total RNA was performed with High-Capacity cDNAReverse Transcription Kit (Applied Biosystems) according to the manufacturer'smanual. In brief, 1.5 mg total RNA of each sample were mixed with the kit compo-nents in a total reaction volume of 20 ml and incubated for 10min at 25 �C,120min at37 �C and 5 s at 85 �C in a thermocycler.

Real-time RT-PCR was performed using the QuantiFast SYBR Green PCR Kit(QIAGEN) and QuantiTect Primer Assays (QIAGEN) for PP13 (Hs_LGALS13_1_SGQuantiTect Primer Assay), beta-hCG (Hs_CGB_1_SG QuantiTect Primer Assay) andribosomal protein P0 (RPLP0, Hs_RPLP0_1_SG QuantiTect Primer Assay), the latterused as internal reference. 8.8 ml of cDNA (15 ng/ml) were mixed with 2.2 ml primersand 11 ml SYBR green master mix. Expression levels were analyzed in triplicates ina volume of 6 ml per well in a 384 well plate (Roche) in a Light Cycler 480 (Roche).PCR conditions comprised an initial 5 min denaturating step at 95 �C and a subse-quent two-step cycling including 10 s at 95 �C and 30 s at 60 �C for 45 cycles. Ctvalues were automatically generated by the Light Cycler 480 Software (Roche) andrelative quantification of gene expression was calculated by standard DDCt methodusing expression of RPLP0 as reference.

2.4. Exposure of BeWo cells to vitamin C, acetylsalicylic acid (ASA),Trolox� and heparin

Culture medium in presence or absence of 20 mM forskolin or 0.2% DMSO wassupplemented with increasing concentrations of vitamin C (Herba Chemosan,Austria) [30e200 mM], acetylsalicylic acid (ASA; SigmaeAldrich) [1e10 mg/ml,

equivalent to 5.6e56 mM], low-molecular-weight heparin (Fragmin�, Pfizer, Austria)[1e6 IU/ml] and the vitamin E derivative Trolox� (SigmaeAldrich) [10e100 mM].Concentration ranges of vitamin C, Trolox� and heparin were chosen according toprevious studies [6,21,22] and of ASA according to a personal communication by Dr.UweGessner (Bayer Vital GmbH, Germany). Stock solutions of vitamin C and heparinwere prepared in double distilled water, acetylsalicylic acid was dissolved in culturemedium and stock solutions of Trolox� were prepared in ethanol. Culture media ofcontrols were supplemented with respective volumes of solvents. Cells were incu-bated for 48 h or 72 h under the same conditions as mentioned above.

2.5. Cell lysis and protein determination

Cells were washed once in PBS and lysed with 80 ml/well RIPA-buffer (Sigma-eAldrich) (with Complete protease inhibitor cocktail, Roche) for 5 min on ice.Lysates were clarified by centrifugation at 16,000g for 10 min at 4 �C. Supernatantwas used immediately or stored at �80 �C for further analysis. Total proteinconcentration was assessed by Lowry protein assay.

2.6. DELFIA assay

PP13 and beta-hCG concentrations of cell lysates were determined using time-resolved fluoroimmunoassay kits (PerkinElmer, Finland). Cell lysates were diluted1:3 and 1:5 in DELFIA diluents I and II, respectively. The fluorescent signal of eachsample was converted by MultiCalc (PerkinElmer) into the respective proteinconcentration calculated from a standard curve. PP13 and beta-hCG concentrationswere normalized to total protein in cell lysates.

2.7. Cell viability assessment

Cell viability was assessed by determination of LDH in culture supernatant usinga LDH Cytotoxicity Detection Kit (Takara, Japan) according to the manufacturer'smanual.

2.8. Immunofluorescence staining and quantification of apoptosis and fusion

Prior to staining cells were fixed in acetone for 7 min. UV-Block (Lab-Vision,Thermo Scientific, Austria) was supplemented with 10% human AB-serum and usedto block non-specific background staining. Primary antibodies (Table 1) were dilutedin antibody diluent (Dako, Austria) and incubated for 30 min at room temperaturealone or in combination. Fluorescent-labeled secondary antibodies (Alexa Fluor�555goat anti-mouse IgG or Alexa Fluor�488 goat anti-rabbit IgG, 1:200; Invitrogen,Austria) were incubated for 30 min at room temperature. Nuclei were counter-stained for 5 min with DAPI (1:2000; Invitrogen). Slides were dried, mounted withProLong Gold antifade reagent (Invitrogen) and analyzed by fluorescent microscopyusing an Axiophot microscope connected to an AxioCam HRc digital camera (Zeiss,Germany) or using a Leica DM6000B microscope connected to an Olympus DP72digital camera (Olympus, Austria).

Apoptosis rates of BeWo cells were determined by analyzing M30 staining withDAPI counterstaining in a total of 15 randomly selected microscopic fields of eachcondition in three independent experiments. The ratio of M30 positive areas to totalarea of nuclei in each picture was automatically determined (Visiomorph software,Visiopharm, Denmark). The apoptosis rate of the forskolin control at 48 h was set toone and the rates of all other conditions were calculated accordingly.

BeWo cell fusion was determined by analyzing alpha-fodrin and desmosomalprotein staining in a total of at least 20 randomly selected microscopic fields of eachcondition, analyzed in three independent experiments (newCAST software, Visio-pharm). Number of nuclei in syncytia and total number of nuclei were counted andfusion index was calculated (number of nuclei in syncytia/total number ofnuclei� 100). The fusion index of the forskolin control was set to one and the indicesof the other conditions were calculated accordingly.

2.9. Statistical analysis

Real-Time RT-PCR and DELFIA data are expressed as mean � SD, fusion data arepresented as “Box-and-Whisker-Plot”. Differences between two groups (as displayed

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K. Orendi et al. / Placenta 31 (2010) 431e438 433

in Fig. 1CeD and 2) were tested with t-test or ManneWhitney Rank Sum Test, asappropriate. Differences between three or more treatment conditions (as displayedin Fig.1AeB, 3 and 5)were testedwithANOVAorKruskaleWallis Test, as appropriate,followed by Tukey post hoc tests. When comparing the control group with eachtreatment condition (as displayed in Figs. 3AeH and 5C) many-to-one testingprocedures (Dunnett's or Dunn's Test, as appropriate) were used. Statistical analysiswas performed using SigmaPlot (SigmaPlot 11.0, Systat Software, Germany). P-Valuesof <0.05 were considered significant.

3. Results

3.1. PP13 and beta-hCG mRNA and protein are increased inresponse to forskolin treatment of trophoblast derived cell lines

In five different trophoblast derived cell lines e the choriocar-cinoma cell lines BeWo, Jar and Jeg-3 and the hybridoma cell linesAC1-M59 and ACH-3P e PP13 and beta-hCG mRNA and proteinlevels were determined following treatment with 20 mM forskolin.All five cell lines significantly increased PP13 and beta-hCG mRNAafter forskolin stimulation compared to non-treated or DMSOtreated control cells (Fig. 1A and B). BeWo cells showed the stron-gest increase of PP13 and beta-hCG mRNA after forskolin stimula-tion compared to controls (Fig. 1A and B). After forskolinstimulation PP13 and beta-hCG levels significantly increased inmost of the cell lines, except for Jeg-3 cells with no changes in PP13(Fig. 1C and D). The strongest increase of PP13 was found in BeWocells. Thus, these cells were used for subsequent experiments.

3.2. In forskolin treated BeWo cells PP13 is decreased and apoptosisrates are increased after 72 h compared to 48 h

In a time-course experiment from48h to72hBeWocells showeda significantdecrease inPP13withextended forskolin exposure timewhich was not evident in the presence of vitamin C (Fig. 2A).Differences in exposure time did not lead to changes in beta-hCG(Fig. 2B). However, addition of vitamin C significantly increased

Fig. 1. Effect of 48 h forskolin treatment on PP13 and beta-hCG expression in trophoblastexpression (mean � SD). Cells were treated with culture medium only (light grey bars), 0.2%changes of non-treated controls, DMSO treated and forskolin treated cells. Significant differeDMSO treated controls are marked with a hash (#). (C, D) Protein expression of PP13 and bmeasured by DELFIA assays and related to total protein in cell lysates (mean � SD). All datatreated controls are marked with an asterisk (*). Data were obtained from three independe

beta-hCG (Fig. 2B). In the forskolin controls as well as the vitamin Ctreated cells an increase in cultivation time from 48 h to 72 h bothsignificantly increased the rate of apoptosis (Fig. 2C). Therefore anexposure time of 48 h was used for further experiments.

3.3. Candidate therapeutics affect PP13 and beta-hCG levels inBeWo cells

PP13 as assessed by DELFIA assays increased significantly aftersupplementation with physiological (�50 mM) and above physio-logical (�100 mM) serum concentrations of vitamin C in the pres-ence of forskolin compared to controls (Fig. 3A). Highest PP13values were found at 100 mM vitamin C (1.8-fold compared to for-skolin control). In the presence of forskolin vitamin C also affectedbeta-hCG levels with a significant increase of 1.9-fold at 30 mMvitamin C up to 2.4-fold at 200 mM vitamin C compared to controls(Fig. 3B).

A negative effect on PP13 was found after addition of acetylsa-licylic acid (ASA). One mg/ml ASA decreased PP13 significantly toabout 60% of the respective forskolin control. Higher concentra-tions of ASA did not decrease PP13 any further (Fig. 3C). ASA did notshow to have any effect on beta-hCG (Fig. 3D).

Supplementation of the culture medium with the vitamin Ederivative Trolox� slightly decreased PP13 in a low concentration(10 mM), whereas a significant increase of beta-hCG was only foundat above physiological concentrations (100 mM) (Fig. 3E and F). Thelow-molecular-weight heparin Fragmin� did not show any effecton PP13 and beta-hCG levels (Fig. 3GeH).

The combined treatment with vitamin C and ASA did not showa different effect compared to vitamin C alone (Fig. 3IeK). Onlyvitamin C significantly altered PP13 and beta-hCG levels while anadditive effect of both drugs could not be detected (Fig. 3IeK).

Cell viability as tested by LDH release was not affected in any ofthe conditions (data not shown).

derived cell lines. (A, B) Quantitative Real-Time RT-PCR of PP13 and beta-hCG mRNADMSO (dark grey bars) or 20 mM forskolin (black bars) for 48 h. Values represent foldnces to non-treated controls are marked with an asterisk (*), significant differences toeta-hCG in cell lysates of DMSO (grey bars) and forskolin (black bars) treated cells waswere normalized to respective DMSO treated controls. Significant differences to DMSOnt experiments.

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Fig. 2. Differences between 48 h (grey bars) and 72 h (black bars) forskolin treatment onPP13 (A) andbeta-hCG (B) expression andapoptosis (C) in BeWocells in thepresence andabsence of 100 mMvitamin C (mean� SD). (A) Expression of PP13was assessed in pg/mlby PP13 DELFIA assay and related tomg/ml total protein. (B) Expression of beta-hCGwasassessed in ng/ml by free beta-hCG DELFIA assay and related to mg/ml total protein.(C) Ratio ofM30 positive stained areas to total area of nuclei. All datawere normalized tothe forskolin control. Significant differences between 48 h and 72 h treatment aremarked with an asterisk (*). Data were obtained from three independent experiments.

K. Orendi et al. / Placenta 31 (2010) 431e438434

3.4. Vitamin C in combination with forskolin results in an increaseof beta-hCG staining

While cells treated with DMSO were mainly negative for beta-hCG in immunofluorescence staining (Fig. 4A and B), the intensityof beta-hCG staining increased in the presence of forskolin (Fig. 4Cand D). It even further increased in the presence of forskolincombined with vitamin C (Fig. 4EeH).

3.5. ASA and Trolox� affect the fusion rate of BeWo cells

Staining using an antibody against a desmosomal protein(Fig. 5A and B, red) or alpha-fodrin in combinationwith an antibody

against beta-hCG (Fig. 5A and B, green) was used to quantify fusionevents. Cell fusion analysis revealed that fusion rates in the pres-ence of 2.5 mg/mL ASA or 100 mM Trolox� increased significantlycompared to forskolin treated controls. The median of the fusionrates of forskolin controls was set to 1.0, and the medians of ASAand Trolox treated cells were 1.44 and 1.82, respectively (Fig. 5C).

4. Discussion

First we investigated the potential use of five different tropho-blast derived cell lines to serve as a surrogate for syncytialisation ofplacental trophoblast bymeans of PP13 and beta-hCG expression aswell as direct assessment of fusion. The choriocarcinoma cell linesBeWo, Jar and Jeg-3 as well as the hybridoma cell lines AC1-M59(hybrid with third trimester trophoblast; [23]) and ACH-3P (hybridwith first trimester trophoblast; [20]) were treated with forskolinto enhance differentiation and trigger cell fusion. This was per-formed to mimic the in vivo situation of cell differentiation andfusion of mononucleated cytotrophoblasts to generate and main-tain the multinucleated syncytiotrophoblast. In the presence offorskolin an increase of PP13 and beta-hCG levels in combinationwith an increased fusion rate was specifically seen in BeWo cells.Thus, this cell line was again shown to be the best-suited in vitrocell line model to study villous trophoblast differentiation andfusion.

Various candidate therapeutics have been suggested to havebeneficial effects in preeclampsia, including low-dose aspirin, low-molecular-weight heparin, folic acid, antioxidants such as vitaminsC and E, and calcium. Four of them e aspirin, heparin and vitaminsC and E �were chosen to assess their effects on PP13 and beta-hCGexpression as well as fusion of BeWo cells. Concentrations of theagents chosen for this study reflect physiological and high physi-ological plasma levels of the respective substances during treat-ment in vivo according to previous studies [6,21,22] and a personalcommunication (Dr. Uwe Gessner, Bayer Vital GmbH, Germany).

Vitamins C and E have been thought to be useful in preventingpreeclampsia. However, nowadays they are discussed controver-sially regarding their importance in the prevention of this preg-nancy related syndrome [7,24,25]. Clinical studies on the use ofanti-coagulants such as heparin or anti-platelet reagents such aslow-dose aspirin during pregnancy showed a possible benefit in theprevention of preeclampsia or other hypertensive pregnancydisorders. However, data on such drugs are considered to be onlypreliminary and need to be confirmed in large-scaled, multi-centered, randomized clinical trials [4,5].

Here we tested the impact of these four drugs on cell fusion andthe expression of two syncytiotrophoblast-specific proteins, PP13and beta-hCG. An effect of heparin could not be seen in any of theused concentrations neither on PP13 nor on beta-hCG expression.Vitamin C, low-dose acetylsalicylic acid and the vitamin E deriva-tive Trolox� affected the expression of these proteins, althoughpartly in opposite directions.Whilst the expression of both proteinswas significantly increased in the presence of vitamin C in a dose-dependent manner, the addition of low-dose aspirin resulted ina significant decrease of PP13. Beta-hCG expression was not alteredin the presence of aspirin. The effect of Trolox� was very weak witha slight increase of beta-hCG at higher concentrations. BeWo cellfusion was significantly increased only in the presence of ASA andTrolox� (Fig. 5C).

Tannetta et al. used isolated primary trophoblasts from termplacenta to study the effect of combined vitamins C and E [26].These authors described a non-significant decrease in syncytiumformation at 48 h of culture in the presence of vitamins C and Ewith no difference in hCG secretion at 48 h culture [26]. They alsosuggested that the combined treatment with vitamins C and E does

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Fig. 3. Effects of vitamin C (A, B), ASA (C, D), Trolox� (E, F), low-molecular-weight heparin (G, H) and the combination of vitamin C and ASA (I, K) on the protein expression of PP13and beta-hCG in BeWo cells after 48 h (mean � SD). Expression of PP13 (dark grey bars) was assessed in pg/ml by PP13 DELFIA assay and related to mg/ml total protein. Expressionof beta-hCG (light grey bars) was assessed in ng/ml by free beta-hCG DELFIA assay and related to mg/ml total protein. All data were normalized to the respective forskolin control.Significant differences to controls are marked with an asterisk (*). Significant differences to the treatment condition of 2.5 mg ASA/ml (Fig. 3IeK) are marked with a hash (#). Datashown here represent protein concentrations of forskolin treated cells in presence and absence of the agents. Data were obtained from three to four independent experiments.

K. Orendi et al. / Placenta 31 (2010) 431e438 435

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Fig. 4. Effect of vitamin C on cell differentiation estimated by beta-hCG expression. BeWo cells were cultured on chamber slides and treated with 0.2% DMSO (A, B), 20 mM forskolin(C, D), 30 mM vitamin C þ 20 mM forskolin (E, F) and 100 mM vitamin C þ 20 mM forskolin (G, H) for 48 h. Differentiation shown by immunofluorescence staining for beta-hCGexpression increased in the presence of vitamin C. DAPI counterstaining.

K. Orendi et al. / Placenta 31 (2010) 431e438436

not stimulate beta-hCG secretion directly but rather leads toimproved syncytiotrophoblast viability. There is striking similarityof data between the primary cells [26] and BeWo cells (this study).The vitamin C effects shown here (higher PP13 and beta-hCG withno changes in the fusion rate) point to stabilization and increased

syncytiotrophoblast differentiation rather than increased tropho-blast fusion.

Low levels of antioxidant proteins such as copper/zinc super-oxide dismutase (SOD-1) and catalase in the syncytiotrophoblastare correlated with syncytial fusion [27]. In cases with trisomy

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Fig. 5. Example of forskolin induced cell fusion (A, B) and fusion rates in the presence of forskolin and in the presence or absence of 200 mM vitamin C, 2.5 mg/mL ASA, 100 mMTrolox� or 6 IU/ml heparin (C). (A, B) Immunofluorescence staining with antibodies against a desmosomal protein (red) and beta-hCG (green). (A) DMSO treated controls remainmainly as single cells not staining for beta-hCG. (B) Forskolin treatment leads to formation of multinucleated syncytia staining positive for beta-hCG. (C) Fusion data are presented as“Box-and-Whisker-Plot”. Significant differences to controls are marked with an asterisk (*). Data were obtained from three independent experiments (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.).

K. Orendi et al. / Placenta 31 (2010) 431e438 437

21 higher levels of such proteins are related to defective trophoblastfusion [28]. It has been speculated that high activity of antioxidantproteins in the trophoblast impairs syncytial fusion. Hence, it istempting to speculate that there is a certain threshold of antioxi-dants where a beneficial concentration turns into a detrimentaleffect.

We have already used BeWo cells in combination with villousexplants to assess the effect of serum from IVF failure patients [29].In this study we could demonstrate a direct positive effect ofheparin and aspirin on the viability of villous trophoblast. Hence,we and others have shown that BeWo cells are an establishedsurrogate for villous trophoblast. However, caution is recom-mended when extrapolating such in vitro results to the in vivosituation. At the same time it is tempting to speculate on the valueof such an in vitro test system. It may be a useful tool to compare theeffect of serum from low and high risk patients on markerexpression followed by the evaluation of the impact of agents asevaluated here. May be a respective cell culture system could beused as a platform to individually test pregnant women's serum onits effects on trophoblast derived cells and which treatment is bestto minimize such effects.

Acknowledgements

Thanks to Monika Siwetz, Thomas Kroneis, Julia König andRudolf Schmied for their valuable help and expertise and thanks toGerold Schwantzer for his essential support in statistical issues.This workwas supported by a research grant of the European Union(FP6 Grant # 037244, project title Pregenesys) and the Franz-Lanyar-Foundation (Project # 331). K. Orendi is funded by the EU(FP6 Grant #037244) within the PhD program Molecular Medicineof the Medical University of Graz. G. Moser is funded by the PhDprogram Molecular Medicine of the Medical University of Graz.

References

[1] Lain KY, Roberts JM. Contemporary concepts of the pathogenesis andmanagement of preeclampsia. JAMA 2002;287:3183e6.

[2] Huppertz B. Placental origins of preeclampsia: challenging the currenthypothesis. Hypertension 2008;51:970e5.

[3] Sibai BM, Cunningham FG. Prevention of preeclampsia and eclampsia. In:Lindheimer MD, Roberts JM, Cunningham FG, editors. Chelsey's hypertensivedisorders in pregnancy. Amsterdam: Elsevier; 2009. p. 213e25.

[4] Lambers MJ, Groeneveld E, Hoozemans DA, Schats R, Homburg R, Lambalk CB,et al. Lower incidence of hypertensive complications during pregnancy in

Page 8: Effects of vitamins C and E, acetylsalicylic acid and ...royanaward.com/files12/PLacenta_Orendi_2010.pdf · Effects of vitamins C and E, acetylsalicylic acid and heparin on fusion,

K. Orendi et al. / Placenta 31 (2010) 431e438438

patients treated with low-dose aspirin during in vitro fertilization and earlypregnancy. Hum Reprod 2009;24:2447e50.

[5] Rey E, Garneau P, David M, Gauthier R, Leduc L, Michon N, et al. Dalteparin forthe prevention of recurrence of placental-mediated complications of preg-nancy in women without thrombophilia: a pilot randomized controlled trial. JThromb Haemost 2009;7:58e64.

[6] Chappell LC, Seed PT, Briley AL, Kelly FJ, Lee R, Hunt BJ, et al. Effect of anti-oxidants on the occurrence of pre-eclampsia in women at increased risk:a randomised trial. Lancet 1999;354:810e6.

[7] Aris A, Leblanc S, Ouellet A, Moutquin JM. Detrimental effects of high levels ofantioxidant vitamins C and E on placental function: considerations for thevitamins in preeclampsia (VIP) trial. J Obstet Gynaecol Res 2008;34:504e11.

[8] Poston L, Briley AL, Seed PT, Kelly FJ, Shennan AH. Vitamin C and vitamin E inpregnant women at risk for pre-eclampsia (VIP trial): randomised placebo-controlled trial. Lancet 2006;367:1145e54.

[9] D'Anna R, Baviera G, Giordano D, Russo S, Dugo N, Santamaria A, et al. Firsttrimester serum PAPP-A and NGAL in the prediction of late-onset pre-eclampsia. Prenat Diagn 2009;29:1066e8.

[10] Grill S, Rusterholz C, Zanetti-Dallenbach R, Tercanli S, Holzgreve W, Hahn S,et al. Potential markers of preeclampsia e a review. Reprod Biol Endocrinol2009;7:70.

[11] Spencer K, Cowans NJ, Chefetz I, Tal J, Kuhnreich I, Meiri H. Second-trimesteruterine artery Doppler pulsatility index and maternal serum PP13 as markersof pre-eclampsia. Prenat Diagn 2007;27:258e63.

[12] Spencer K, Cowans NJ, Chefetz I, Tal J, Meiri H. First-trimester maternalserum PP-13, PAPP-A and second-trimester uterine artery Doppler pulsatilityindex as markers of pre-eclampsia. Ultrasound Obstet Gynecol 2007;29:128e34.

[13] Cetin I, Cozzi V, Papageorghiou AT, Maina V, Montanelli A, Garlanda C, et al.First trimester PTX3 levels in womenwho subsequently develop preeclampsiaand fetal growth restriction. Acta Obstet Gynecol Scand 2009;88:846e9.

[14] Than NG, Romero R, Goodman M, Weckle A, Xing J, Dong Z, et al. A primatesubfamily of galectins expressed at the maternal-fetal interface that promoteimmune cell death. Proc Natl Acad Sci U S A 2009;106:9731e6.

[15] Huppertz B, Sammar M, Chefetz I, Neumaier-Wagner P, Bartz C, Meiri H.Longitudinal determination of serum placental protein 13 during develop-ment of preeclampsia. Fetal Diagn Ther 2008;24:230e6.

[16] Spencer K, Cowans NJ, Nicolaides KH. Low levels of maternal serum PAPP-A inthe first trimester and the risk of pre-eclampsia. Prenat Diagn 2008;28:7e10.

[17] Baczyk D, Drewlo S, Proctor L, Dunk C, Lye S, Kingdom J. Glial cell missing-1transcription factor is required for the differentiation of the human tropho-blast. Cell Death Differ 2009;16:719e27.

[18] Wice B,MentonD, Geuze H, Schwartz AL.Modulators of cyclic AMPmetabolisminduce syncytiotrophoblast formation in vitro. Exp Cell Res 1990;186:306e16.

[19] Yu C, Shen K, Lin M, Chen P, Lin C, Chang GD, et al. GCMa regulates thesyncytin-mediated trophoblastic fusion. J Biol Chem 2002;277:50062e8.

[20] Hiden U, Wadsack C, Prutsch N, Gauster M, Weiss U, Frank HG, et al. The firsttrimester human trophoblast cell line ACH-3P: a novel tool to study autocrine/paracrine regulatory loops of human trophoblast subpopulationseTNF-alphastimulates MMP15 expression. BMC Dev Biol 2007;7:137.

[21] Kharb S. Vitamin E and C in preeclampsia. Eur J Obstet Gynecol Reprod Biol2000;93:37e9.

[22] Szelke H, Harenberg J, Kramer R. Detection and neutralisation of heparin bya fluorescent ruthenium compound. Thromb Haemost 2009;102:859e64.

[23] Frank HG, Gunawan B, Ebeling-Stark I, Schulten HJ, Funayama H, Cremer U,et al. Cytogenetic and DNA-fingerprint characterization of choriocarcinomacell lines and a trophoblast/choriocarcinoma cell hybrid. Cancer Genet Cyto-genet 2000;116:16e22.

[24] Villar J, Purwar M, Merialdi M, Zavaleta N, Thi Nhu Ngoc N, Anthony J, et al.World health organisation multicentre randomised trial of supplementationwith vitamins C and E among pregnant women at high risk for pre-eclampsiain populations of low nutritional status from developing countries. BJOG2009;116:780e8.

[25] Rahimi R, Nikfar S, Rezaie A, Abdollahi M. A meta-analysis on the efficacy andsafety of combined vitamin C and E supplementation in preeclamptic women.Hypertens Pregnancy 2009;28:417e34.

[26] TannettaDS,Sargent IL, LintonEA,RedmanCW.VitaminsCandE inhibitapoptosisof cultured human term placenta trophoblast. Placenta 2008;29:680e90.

[27] Pidoux G, Guibourdenche J, Frendo JL, Gerbaud P, Conti M, Luton D, et al.Impact of trisomy 21 on human trophoblast behaviour and hormonal function.Placenta 2004;25(Suppl A):S79e84.

[28] Frendo JL, Therond P, Guibourdenche J, Bidart JM, Vidaud M, Evain-Brion D.Modulation of copper/zinc superoxide dismutase expression and activity within vitro differentiation of human villous cytotrophoblasts. Placenta2000;21:773e81.

[29] Bose P, Black S, Kadyrov M, Weissenborn U, Neulen J, Regan L, et al. Heparinand aspirin attenuate placental apoptosis in vitro: implications for earlypregnancy failure. Am J Obstet Gynecol 2005;192:23e30.