Placenta
Volume 31, Issue 4 , Pages 339-343, April 2010

Differential expression of VE-cadherin and VEGFR2 in placental syncytiotrophoblast during preeclampsia – New perspectives to explain the pathophysiology

  • T. Groten

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Ulm, Germany
    • Department of Obstetrics, Friedrich Schiller University, University Hospital Jena, Germany
    • Corresponding Author InformationCorresponding author. Department of Obstetrics, Friedrich Schiller University, University Hospital Jena, Bachstr. 18, 07743 Jena, Germany.
    • ,
  • N. Gebhard

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Ulm, Germany
    • ,
  • R. Kreienberg

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Ulm, Germany
    • ,
  • E. Schleußner

      Affiliations

    • Department of Obstetrics, Friedrich Schiller University, University Hospital Jena, Germany
    • ,
  • F. Reister

      Affiliations

    • Department of Obstetrics and Gynaecology, University of Ulm, Germany
    • ,
  • B. Huppertz

      Affiliations

    • Institute of Cell Biology, Histology and Embryology, Medical University Graz, Austria

Accepted 25 January 2010. published online 18 February 2010.

Article Outline

Abstract 

The pathophysiology of preeclampsia includes an unbalanced syncytiotrophoblast renewal from the underlying cytotrophoblast and increased necrotic/aponecrotic shedding of syncytiotrophoblast particles into the maternal circulation. These non-apoptotic syncytiotrophoblast fragments cause the maternal endothelial dysfunction underlying the syndrome of preeclampsia. In order to understand the pathophysiological changes at the fetomaternal interface in preeclampsia we studied the expression of VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR2) in preeclampsia. We show that VE-cadherin is expressed in the syncytiotrophoblast and is upregulated in fusing BeWo cells, while inhibition of VE-cadherin expression by siRNA does not block BeWo cell fusion. Our immunohistochemistry data show lower VE-cadherin expression in early onset preeclampsia compared to early controls. In late onset preeclampsia VE-cadherin was significantly more expressed compared to late controls. Concurrently VE-cadherin expression decreased significantly in control pregnancies towards term, but not in pregnancies complicated by preeclampsia. VEGFR2 expression was significantly reduced in all cases of preeclampsia compared to control placentas. Because of their close interaction in barrier function regulation we speculate that sustained expression of VE-cadherin in late onset preeclampsia could counteract VEGFR2 deficiency by enhancing survival pathway stimulation in the syncytiotrophoblast, thus preventing further decompensation of unbalanced villous trophoblast turnover.

Keywords: Preeclampsia, VE-cadherin, VEGFR2, Syncytiotrophoblast

 

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1. Introduction 

In the human placenta the syncytiotrophoblast forms the physical barrier between fetal and maternal circulation [1]. The syncytiotrophoblast is continuously renewed by fusion of cytotrophoblasts, which is counterbalanced by extrusion of apoptotic syncytial knots into the maternal circulation [2]. Excessive cytotrophoblast proliferation, increased numbers of syncytial knots, and necrotic/aponecrotic subcellular syncytial particles [3] are associated with preeclampsia [4], [5]. Such subcellular syncytial particles activate the maternal vasculature, which in turn leads to preeclampsia [6]. However, the molecular mechanisms underlying trophoblast membrane shedding are still poorly understood.

Cadherins are calcium-dependent integral membrane glycoproteins that primarily mediate cell–cell adhesion thereby controlling monolayer integrity and barrier function [7], [8]. Beyond, cadherins regulate the balance between cell survival, proliferation and migration in concert with growth factor receptors such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR2) [8]. The vascular endothelial cadherin (VE-cadherin) has been first described in the vascular endothelium [9]. Here it maintains endothelial monolayer integrity and regulates VEGF-receptor function determining its signaling to survival pathways or cell proliferation. In the presence of VE-cadherin and upon ligand binding VEGFR2 signals towards cell stabilization and cell survival. In the absence of VE-cadherin and upon ligand binding VEGFR2 signals towards cell activation due to internalization and autophosphorylation.

Increased levels of the VEGF neutralizing soluble VEGFR1 (sFlt-1) have been described in preeclampsia [10]. Hence, the resulting lowered levels of free VEGF alter VEGFR2 signaling and increase cell activation. Additionally, altered expression of VE-cadherin may further affect VEGFR2 signaling. The interplay between these altered pathways may cause an unstable trophoblast structure and may increase aponecrotic trophoblast deportation into maternal blood, thus causing preeclampsia.

We investigated VE-cadherin expression in the syncytiotrophoblast and its potential role in syncytialisation in vitro. In addition, we compared VE-cadherin and VEGFR2 expression in the syncytiotrophoblast of late and early onset preeclamptic and control placentas.

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2. Materials and methods 

2.1. Collection of placental tissues 

Following local ethical approval and informed patient consent, placentas were obtained from women delivering at the maternity unit of the Komfo Anokye Teaching Hospital in Kumasi, Ghana and at the Women's Hospital of the University of Witwatersrand, Johannesburg, South Africa. 36 normal and 21 preeclamptic placentas were collected after delivery. Pregnancies were diagnosed as preeclamptic, if blood pressure was ≥140/90 and there was ≥2+ proteinuria by dipstick determination on two occasions. Control placentas were collected from term deliveries following uncomplicated pregnancies and from preterm deliveries without preeclampsia, HELLP syndrome, hypertension, proteinuria, growth retardation or abnormal uteroplacental perfusion.

2.2. Immunohistochemistry 

Placental biopsies (2–3 per placenta) were fixed in 4% buffered formaldehyde for 24 h and stored in 0.9% saline before embedding in paraffin. Sections were cut (5 μm), de-paraffinised in xylene and rehydrated in an ethanol series. For VE-cadherin and CD34 staining, specimens were transferred into Target Retrieval Solution (DAKO, Glostrup, Denmark) and boiled in the microwave at 750 W for 15 min to unmask antigens. VE-cadherin and CD34 immunohistochemistry was performed using the catalysed signal amplification (CSA)-Kit (DAKO) following the manufacturer's protocol and visualization with AEC. VEGFR2 immunohistochemistry was performed using the polymer kit (LabVision, Astmoor, UK) and visualization with AEC (3-amino-9-ethylcarbazole). Primary antibodies used were anti-VE-cadherin clone BV6 (Millipore (formerly Chemicon), Billerica, USA, MAB1989, mab) or clone TEA1/31 (Beckman Coulter (formerly Immunotech), Fallerton, USA, PN IM1597, mab) at 1:20, anti-CD34 (Serotec, Kidlington, UK, MCAP547, mab) at 1:100 and anti-VEGFR2 (Santa Cruz, Santa Cruz, USA, sc-6251, rab) at 1:1000. Sections were counterstained with hematoxylin.

2.3. Cell culture 

BeWo cells were purchased from the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in F12 medium (Invitrogen, Carlsbad, USA) supplemented with 10% FCS (Sigma, Saint Louis, USA) and l-glutamine (Biochrom Labs, Terre Haute, USA). For forskolin treatment (48 h) medium was changed to DMEM/F12 (Sigma) containing 100 μM forskolin (Sigma).

2.4. Western blot 

For Western blot analysis cell pellets were lysed in TBS containing 0.5% Triton X-100 (Sigma) as previously described [11]. Antibodies used were: anti-VE-cadherin (BD, Franklin Lakes, USA, 610251, mab) at 1:100 and HRP-conjugated secondary antibody was goat anti-mouse (Santa Cruz) at 1:1000.

2.5. Immunocytochemistry 

For immunocytochemistry cells were grown on glass cover slips, treated with forskolin (Sigma) for 48 h and stained as previously described [11]. Antibodies used were anti-VE-cadherin (BD, 610251, mab) at 1:50, anti-β-catenin (Sigma, C2206, rab) at 1:100 with Alexa 488 or Alexa 594 conjugated secondary goat anti-mouse and goat anti-rabbit antibodies (Invitrogen) at 1:500.

2.6. RT-PCR 

RNA was isolated from cell pellets using the RNeasy Mini Kit and QIAshredder (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA was reverse transcribed using the Omniscript RT-kit (Qiagen), RNase inhibitor (Promega, Madison, USA) and Oligo(dT) primer (Promega). PCR amplification was performed using the HotstarTaq Masterkit (Qiagen). Primers used were for VE-cadherin 5′-GAGACAAACCCCGCCCCAA-3′ and 5′-GCCGCCGCAGGAAGATGA-3′.

2.7. siRNA transfection 

Cells were cultured on glass cover slips over night and transfected with siRNA duplexes at a final concentration of 10 nM with Dharmafect1 (Dharmacon, Lafayette, USA) according to manufacturer's instructions for 48 h. Pooled siRNA duplexes against human VE-cadherin and non-specific control were designed and purchased from Dharmacon.

2.8. Analysis of staining 

Analysis of immunohistochemical staining was performed following the recommendations of randomized sampling and the forbidden line rule [12]. From each paraffin block two placental sections more than five sections away from each other were stained and from each section five pictures were systematically randomly taken (newCAST software and equipment, Visiopharm, Denmark). Quantification of positive staining in the syncytiotrophoblast lining the circumference of placental villi was performed by calculating the ratio of stained surface per total surface. For each group, the 95% confidence interval was calculated and the 5% extreme low and the 5% extreme high values were excluded. Results were statistically analysed using unpaired t-test. Significance was defined as p < 0.05.

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3. Results and discussion 

Here we demonstrate differential expression of VE-cadherin and VEGFR2 during normal pregnancy and in pregnancies complicated by preeclampsia. Considering the impact of VE-cadherin on VEGFR2 function this data reveals new insights into the pathophysiology of preeclampsia undermining the impact of the syncytiotrophoblast in this syndrome.

3.1. Clinical characteristics 

Clinical characteristics of the patients studied revealed no difference between late onset preeclampsia and corresponding term controls. Average maternal age was 27.0 (±5.2, n = 24) years in the healthy control group and 25.0 (±5.0, n = 19) years in the preeclampsia group. Average gestational age at delivery was 38.6 (±2.0) weeks in controls and 36.4 (±2.6) weeks in the late onset preeclamptic cases. Average birth weight was 3040 g (±360 g) in controls and 2734 g (±660 g) in preeclampsia, both birth weight percentile values on the 50th percentile [13]. In preterm controls and early onset preeclampsia cases maternal age was 29.5 (±5.99, n = 12) years in control patients and 27.4 (±6.25, n = 12) in preeclamptic cases. Gestational age and birth weight were 26.8 weeks (±3.25) and 840 g (±387) in preterm controls and 30.3 weeks (±2.34) and 1026 g (±368) in preterm preeclampsia cases. In 83% of the early preeclampsia cases birth weight was below the 10th percentile and all were below the 25th percentile. In the preterm controls only 8% were below the 10th percentile (range: 5–50). Thus, low birth weight was overrepresented in the early onset preeclampsia group but not in late onset cases.

3.2. VE-cadherin in the syncytiotrophoblast 

In extravillous trophoblast differentiation, cadherins are differentially expressed and in the syncytiotrophoblast type II cadherin 11 has been shown to be mandatory for cell fusion [14]. Also VE-cadherin belongs to the type II classical cadherins. VE-cadherin was first described to be endothelial specific, functioning in maintaining the blood tissue barrier. Here we demonstrate that VE-cadherin is also expressed at the barrier between maternal blood and fetal tissues, the syncytiotrophoblast (Fig. 1).

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  • Fig. 1 

    In serial sections of a control placenta, term syncytiotrophoblast stained positive for VE-cadherin (A, ×40; B, ×63) and VEGFR2 (E, ×40). Immunostaining for VE-cadherin (A, ×40; B, ×63) was detected in placental endothelium as well as in the syncytiotrophoblast. Immunostaining for CD34 (C, ×40; D, ×63) only revealed labeling of placental endothelium. Negative controls for VE-cadherin/CD34 (F, ×63) and VEGFR2 (G, ×40). Quantification of VE-cadherin positive staining in the syncytiotrophoblast lining the circumference of placental villi was performed by calculating the ratio of stained surface per total surface. (H) VE-cadherin expression decreased significantly in control pregnancies towards term, but not in pregnancies complicated by preeclampsia. In early onset preeclampsia (n = 12) VE-cadherin expression was lower compared to early controls (n = 12). In late onset preeclampsia (n = 19) VE-cadherin expression was significantly higher compared to late controls (n = 24). (I) VEGFR2 expression was significantly reduced in all cases of preeclampsia compared to control placentas. This difference was more pronounced in the late onset cases than in the early cases of preeclampsia.

Expression of VE-cadherin in the cytotrophoblast has been described before [15], [16], [17], while expression of VE-cadherin in the syncytiotrophoblast has not been described so far [18], [19], [20]. We only achieved a consistent VE-cadherin detection in the syncytiotrophoblast when using the high sensitive CSA-kit from DAKO. We confirmed specificity of VE-cadherin staining in the syncytiotrophoblast using two different antibody clones (TEA1/31 and BV6) showing identical staining patterns in the syncytiotrophoblast (data not shown). Staining analysis revealed significantly different VE-cadherin expression between groups (Fig. 1A–G).

VE-cadherin expression was significantly (p = 0.002) downregulated with gestational age in control pregnancies (Fig. 1H: 24.4% positive staining in early controls vs. 9.0% in late controls) but not in pregnancies complicated by preeclampsia (early cases 14.7% vs. late cases 16.6%). In early onset preeclampsia VE-cadherin expression was lower compared to early controls (14.7% vs. 24.4%, p = 0.059). In late onset preeclampsia VE-cadherin was significantly higher compared to late controls (16.5% vs. 9.0%, p = 0.01) (Fig. 1H).

3.3. VE-cadherin in fusing BeWo cells 

Investigations on differential gene regulation in BeWo cells with fusion demonstrated upregulation of VE-cadherin gene expression [21]. Consistently, we showed upregulation of VE-cadherin protein and mRNA expression in BeWo cells stimulated to fuse (Fig. 2). In cultured BeWo cells blocking VE-cadherin expression using RNA interference did not change forskolin induced cell fusion (Fig. 2G and H). Data on β-hCG secretion confirmed these results (not shown). VE-cadherin knock down was confirmed in Western blot analysis (Fig. 2M). siRNA experiments illustrate that in distinction to cadherin 11, VE-cadherin suppression does not prevent fusion or β-hCG secretion in forskolin stimulated BeWo cells. Thus, VE-cadherin expression is not required for but occurs during/after cell fusion.

  • View full-size image.
  • Fig. 2 

    VE-cadherin protein expression was upregulated in fusing BeWo cells. (A–F) BeWo cells treated with forskolin for 48 h were stained for β-catenin (green) to mark cell borders and VE-cadherin (red). Single channels and merged pictures are displayed. (G–I) siRNA mediated VE-cadherin knock down suppressed forskolin induced VE-cadherin expression but did not prevent cell fusion. (J) No VE-cadherin expression with vehicle treatment only. (K) IgG control. (L) PCR analysis for VE-cadherin RNA expression. (M) Western blot analysis for VE-cadherin expression with and without forskolin, untreated and after siRNA treatment. Bars = 50 μm. G–K, merged pictures only.

3.4. VE-cadherin and VEGFR2 

In addition to its adhesive functions, VE-cadherin regulates cellular processes including cell proliferation and apoptosis by various outside-in signaling processes [22]. In the absence of VE-cadherin the complex consisting of VE-cadherin, β-catenin, phosphatidylinositol 3-kinase (PI3-K) and VEGFR2 fails to form. As a consequence VEGF cannot activate the serine/threonine kinase PKB/AKT or increase levels of Bcl-2, both part of the antiapoptotic machinery [23], [24]. Additionally, recent data illustrate that VE-cadherin stabilizes VEGFR2 at the cell membrane. Internalization of VEGFR2 leads to activation of phospholipase C and consequently p44/42 mitogen-activated protein kinase and subsequently cell proliferation [25]. Thus, in the presence of VE-cadherin VEGF can activate cell survival via VEGFR2, which is stabilised at the cell membrane. In the absence of VE-cadherin VEGFR2 is internalised and autophosphorylated, subsequently activating cell proliferation.

VEGFR2 expression was significantly reduced in all cases of preeclampsia compared to control placentas (26.0% vs. 38.1%, p = 0.0002). Comparing early cases of preeclampsia with early controls the difference was less pronounced (29.0% vs. 37.5%, p = 0.19) while in late cases the difference was considerably clear (24.0% vs. 38.4%, p = 0.0001) (Fig. 1I).

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4. Conclusion 

Imbalanced expression of VE-cadherin and VEGFR2 could interfere with accurate apoptotic shedding in the syncytiotrophoblast leading to more aponecrotic shedding into the maternal circulation. Sustained expression of VE-cadherin as shown here for late onset preeclampsia would stabilise VEGFR2 in the complex with PI3-K at the cell membrane enhancing survival pathways and may support accurate forming of apoptotic syncytial knots. Thus, in the context of late onset preeclampsia sustained expression of VE-cadherin could serve as a compensatory effect to maintain integrity of the syncytiotrophoblast. In early onset preeclampsia such a compensatory mechanism may fail.

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Acknowledgements 

The authors want to thank Monika Siwetz for her excellent technical assistance and her help in quantifying the immunohistochemical staining. Furthermore, the authors want to thank Vera Hoffmann and Christoph Hintzen for collecting placental samples under the supervision of Prof. Eckhart Buchmann at the University of Witwatersrand, Johannesburg, South Africa.

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References 

  1. Kingdom J, Huppertz B, Seaward G, Kaufmann P. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol. 2000;92:35–43
  2. Huppertz B, Frank HG, Kingdom JC, Reister F, Kaufmann P. Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem Cell Biol. 1998;110:495–508
  3. Huppertz B, Kingdom JC. Apoptosis in the trophoblast-role of apoptosis in placental morphogenesis. J Soc Gynecol Investig. 2004;11:353–362
  4. Arnholdt H, Meisel F, Fandrey K, Lohrs U. Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies. Virchows Arch B Cell Pathol Incl Mol Pathol. 1991;60:365–372
  5. Goswami D, Tannetta DS, Magee LA, Fuchisawa A, Redman CW, Sargent IL, et al. Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. Placenta. 2006;27:56–61
  6. Redman CW, Sargent IL. Microparticles and immunomodulation in pregnancy and pre-eclampsia. J Reprod Immunol. 2007;76:61–67
  7. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251:1451–1455
  8. Yap AS, Crampton MS, Hardin J. Making and breaking contacts: the cellular biology of cadherin regulation. Curr Opin Cell Biol. 2007;19:508–514
  9. Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Houen G, et al. A novel endothelial-specific membrane protein is a marker of cell–cell contacts. J Cell Biol. 1992;118:1511–1522
  10. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004;350:672–683
  11. Groten T, Pierce AA, Huen AC, Schnaper HW. 17 Beta-estradiol transiently disrupts adherens junctions in endothelial cells. FASEB J. 2005;19:1368–1370
  12. Mayhew TM. Taking tissue samples from the placenta: an illustration of principles and strategies. Placenta. 2008;29:1–14
  13. Voigt M, Schneider KT, Jahrig K. Analysis of a 1992 birth sample in Germany. 1: new percentile values of the body weight of newborn infants. Geburtshilfe Frauenheilkd. 1996;56:550–558
  14. Getsios S, MacCalman CD. Cadherin-11 modulates the terminal differentiation and fusion of human trophoblastic cells in vitro. Dev Biol. 2003;257:41–54
  15. Bulla R, Villa A, Bossi F, Cassetti A, Radillo O, Spessotto P, et al. VE-cadherin is a critical molecule for trophoblast–endothelial cell interaction in decidual spiral arteries. Exp Cell Res. 2005;303:101–113
  16. Challier JC, Carbillon L, Kacemi A, Vervelle C, Bintein T, Galtier M, et al. Characterization of first trimester human fetal placental vessels using immunocytochemical markers. Cell Mol Biol (Noisy-le-grand). 2001;47:Online Pub:OL79-87
  17. Tabata T, McDonagh S, Kawakatsu H, Pereira L. Cytotrophoblasts infected with a pathogenic human cytomegalovirus strain dysregulate cell–matrix and cell–cell adhesion molecules: a quantitative analysis. Placenta. 2007;28:527–537
  18. Leach L, Lammiman MJ, Babawale MO, Hobson SA, Bromilou B, Lovat S, et al. Molecular organization of tight and adherens junctions in the human placental vascular tree. Placenta. 2000;21:547–557
  19. Dye JF, Jablenska R, Donnelly JL, Lawrence L, Leach L, Clark P, et al. Phenotype of the endothelium in the human term placenta. Placenta. 2001;22:32–43
  20. Leach L, Gray C, Staton S, Babawale MO, Gruchy A, Foster C, et al. Vascular endothelial cadherin and beta-catenin in human fetoplacental vessels of pregnancies complicated by type 1 diabetes: associations with angiogenesis and perturbed barrier function. Diabetologia. 2004;47:695–709
  21. Kudo Y, Boyd CA, Sargent IL, Redman CW, Lee JM, Freeman TC. An analysis using DNA microarray of the time course of gene expression during syncytialization of a human placental cell line (BeWo). Placenta. 2004;25:479–488
  22. Vestweber D. VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol. 2008;28:223–232
  23. Nunez G, del Peso L. Linking extracellular survival signals and the apoptotic machinery. Curr Opin Neurobiol. 1998;8:613–618
  24. Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell. 1999;98:147–157
  25. Lampugnani MG, Orsenigo F, Gagliani MC, Tacchetti C, Dejana E. Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol. 2006;174:593–604

PII: S0143-4004(10)00040-8

doi:10.1016/j.placenta.2010.01.014

Placenta
Volume 31, Issue 4 , Pages 339-343, April 2010

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