Molecular Human Reproduction, Vol. 8, No. 1, 88-94,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression of vasodilator-stimulated phosphoprotein in human placenta: possible implications in trophoblast invasion
1 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Yale University School of Medicine,333 Cedar Street, New Haven, CT 06520-8063, USA and 2 Department of Histology and Embryology, Akdeniz University School of Medicine, Antalya, 07070, Turkey
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Abstract |
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The vasodilator-stimulated phosphoprotein (VASP) is a 46 kDa protein present at the leading edge of migrating cells. Because trophoblastic cell migration and invasion are critical stages for the achievement of successful implantation and development of the placenta, we investigated VASP expression in different cell types of the human placenta throughout pregnancy by immunohistochemistry and Western blot analysis. We also studied the effect of leukaemia inhibitory factor (LIF) and transforming growth factor-ß1 (TGF-ß1) on the regulation of VASP expression in first trimester placental tissue explants. We found that VASP is expressed throughout pregnancy by a variety of cells in the human placenta. The strongest VASP immunoreactivity was observed in the first trimester. In these samples, the most intense immunoreactivity was in invasive trophoblasts, namely, extravillous cells of the anchoring villi, distal extravillous trophoblasts of cell columns, and also in cells of placental fibrinoids. We also found that LIF (but not TGF-ß1) has a stimulatory effect on VASP expression in placental explants. The strong VASP immunoreactivity in invasive trophoblasts suggests that this protein may be associated with trophoblastic cell motility and may have a role in implantation and trophoblastic cell invasion. We speculate that one of the effects of LIF in successful pregnancy may be its induction of VASP expression.
extravillous trophoblast/implantation/invasion/placenta/VASP
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Introduction |
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Vasodilator-stimulated phosphoprotein (VASP), a member of Ena-VASP family, is a 46 kDa membrane-associated protein that was first described in human platelets (Halbrugge and Walter, 1989
). Members of the Ena-VASP family share common amino acid sequence patterns including an EHV1 N-terminal domain that binds to proteins containing a E/DFPPPPXD/E motif and targets family members to focal adhesion (Niebuhr et al., 1997). This family of proteins has been studied in many systems and appears to have a universal role in the control of cell motility and intracellular actin dynamics via a linear pathway from receptor–ligand interactions (Machesky, 2000).
Integrin 
IIbß3 (also known as fibrinogen receptor or platelet glycoprotein IIb–IIIa) is involved in 125 kDa focal adhesion protein (pp125FAK) interaction, and it has been shown that VASP phosphorylation correlates well with the reversible inhibition of this integrin (Horstrup et al., 1994). Cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases have been shown to phosphorylate VASP in a variety of cells. In the intracellular environment, VASP is associated with filamentous actin formation and is involved in an intracellular signalling pathway of integrin–extracellular matrix (ECM) interactions. It is thus suggested that it takes part in cell adhesion and motility (Halbrugge et al., 1992).
Trophoblastic invasion is arguably the most critical stage for the achievement of blastocyst implantation and development of the placenta. Subsequent to the differentiation of their blastocyst to inner and outer cell masses, trophoblastic cells differentiate into different phenotypes. Invasion of extravillous trophoblasts into uterine tissues and eventual replacement of the spiral artery walls by trophoblasts are the essential features of blastocyst implantation and placental development (Loke and King, 1995).
Many studies have shown that binding of trophoblasts to extracellular matrix proteins via adhesion proteins is one of the possible mechanisms for successful implantation and placental development (Cross et al., 1994; Damsky et al., 1994; Burrows et al., 1995). Although extracellular factors, and their membrane receptors, that stimulate and control trophoblast invasion into the endometrium have been studied, the intracellular signalling pathway that follows the binding of these proteins is still unclear. Since one of the possible mechanisms that may explain trophoblastic cell invasion is related to integrin–ECM interactions, we have hypothesized that VASP may take part in the regulation of intracellular dynamics in trophoblastic adhesion and invasion during implantation and placentation.
In the present study, we investigated VASP expression in different cell types of the human placenta. We also studied the effect of LIF and TGF-ß1 on VASP expression in placental explants since studies on human and rodent implantation mechanisms have shown that both are essential cytokines for trophoblastic differentiation (Stewart et al., 1992; Jokhi et al., 1997).
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Materials and methods |
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Collection of tissues
Human placental tissues from the first, second and third trimester (n = 30) were collected from clinically normal pregnancies which were either voluntarily terminated by dilation and curettage or delivered by Caesarean section before the initiation of labour, due to obstetric reasons. Written informed consent was obtained from each woman prior to surgery using consent forms and protocols approved by the Human Investigation Committee of either Yale University or Akdeniz University. Some pieces of the placenta were used for protein extraction and Western blot analysis. The remaining part of the tissue was fixed either in Bouin's fixative and embedded into paraffin or was snap-frozen in OCT (Tissue Tek; Sakura, Torrance, CA, USA) for immunohistochemistry. Thereafter, tissues were cut at 5–7 µm thicknesses and mounted on gelatin-coated slides. Cryostat sections were fixed in cold methanol for 10 min. Paraffin sections were deparaffinized and rehydrated in alcohol gradient. For the detection of VASP, immunohistochemical reactions were carried out using a standardized method based on the streptavidin–biotin technique.
Explant culture of human chorionic villi
Chorionic villous tissues (n = 5) from first trimester pregnancies were obtained under aseptic conditions from women undergoing voluntary termination of pregnancy (6–9 weeks gestation). Tissues were rinsed twice in phosphate-buffered saline (PBS), then dissected free of maternal decidua and the fetal amniotic membranes. Later, tissues were minced into 2–3 mm small pieces in Hanks' balanced salts solution (HBSS; Sigma, St Louis, MO, USA). Tissue pieces were then placed in 2 ml serum-free Medium-199 (M-199) alone, or with either LIF (1 ng/ml) or TGF-ß1 (1 ng/ml). Explants were maintained in culture dishes at 37°C in a humidified atmosphere (5% CO2 in air) for 24 h. Thereafter, some of the tissues were snap-frozen in OCT for immunohistochemistry and the remaining tissues were used for protein extraction and Western blot analysis.
Immunohistochemistry
To detect the VASP immunoreactivity, we used two different antibodies that were kindly supplied by Prof. U.Walter (Institute for Clinical Biochemistry, University of Wurzburg, Germany). Following methanol fixation or deparaffinization of sections, tissues were rinsed twice in PBS for 10 min. Endogenous peroxidase activity was quenched by 3% H2O2 (0.6 ml H2O2 and 5.4 ml methanol) for 10 min followed by a rinse in PBS–Tween-20 (0.05 % Tween-20 in PBS, PBS-T; pH 7.3). Sections were then incubated with primary antibodies [mouse anti-VASP monoclonal antibody (1/200) or rabbit anti-VASP polyclonal antibody (1/500), mouse anti-cytokeratin 7 antibody and mouse anti-vimentin antibody; ready-to-use; Dako, Glostrup, Denmark] for 60 min at room temperature or overnight at 4°C. Normal mouse IgG or rabbit pre-immune serum was applied onto negative control slides instead of the primary antibody. After several rinses in PBS-T, biotinylated goat anti-mouse or anti-rabbit IgG antibodies (Biogenex, San Ramon, CA, USA) were applied for 30 min, and following several PBS-T rinses slides were incubated with streptavidin–peroxidase complex for 30 min (Biogenex). Subsequently slides were rinsed several times in PBS-T, and then were incubated with 3-amino 9-ethyl-carbazole (AEC; Biogenex). Slides were counterstained with haematoxylin prior to permanent mounting.
The evaluations were recorded as positively stained target cells in each of four intensity categories which were recorded as: – (no staining), 1+ (weak but detectable), 2+ (distinct), and 3+ (intense) for VASP immunoreactivity. The evaluations for the mouse monoclonal antibody or rabbit antibody were carried out separately. Results presented here are from paraffin-embedded placental tissues and from frozen explant cultures.
Western blot analysis
Total proteins from the tissues were extracted by RIPA buffer [50 mmol/l Tris–HCl (pH 7.4), 150 mmol/l NaCl, 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS, 1% protease inhibitor cocktail]. The protein concentration was determined by standard DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). 20 µg protein was loaded into each lane, separated by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis using 7.5% Tris–HCI Ready Gels (Bio-Rad Laboratories) and blotted onto a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Bucks, UK). Equal loading of proteins in each lane was confirmed by staining the membrane with Ponceau 2S (Sigma). The membrane was blocked with 5% non-fat dry milk in PBS-T buffer for 1 h to inhibit the non-specific binding. The membrane was then incubated for 1 h with mouse anti-VASP or rabbit anti-VASP antibodies, which were diluted at 1/2000 and 1/3000 respectively. Membranes were then incubated for 1 h with horse anti-mouse IgG peroxidase-labelled antibody (diluted at 1/10 000; Vector Laboratories, Burlingame, CA, USA) or goat anti-rabbit IgG peroxidase-labelled antibody (diluted at 1/10000; Chemicon, Temecula, CA, USA). The immunoblot reactivity was developed using TMB peroxidase substrate kit (Vector Laboratories) or chemiluminescence substrate kit (NEN Life Science, Boston, MA, USA). Immunoblot bands were quantified using a laser densitometer (Molecular Dynamics, Sunnyvale, CA, USA).
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Expression of VASP in human placenta throughout pregnancy
Immunohistochemistry
The staining profiles of both antibodies used in this study were similar to each other. All figures in the current paper represent immunoreactivity of mouse anti-VASP monoclonal antibody. In the first trimester placental tissues, villous cytotrophoblast cells showed either no or weak immunoreactivity for VASP (Figure 1a, c, d
). The most interesting observation of this study was that distal cells of the extravillous trophoblast (EVT) cell columns showed the strongest VASP immunoreactivity while the proximal EVT cells had weak or no immunoreactivity for VASP (Figure 1a). Maternal leukocytes were also strongly immunoreactive for VASP (Figure 1a). VASP expression of the cytotrophoblasts located in the placental fibrinoids was as strong as that in distal EVT cells (Figure 1b). In villous stroma, endothelial and Hofbauer cells had strong staining for VASP and mesenchymal cells showed weaker immunoreactivity compared to these cells (Figure 1c). We also observed that both interstitial and endovascular trophoblastic cells confirmed by cytokeratin and vimentin immunoreactivity expressed strong VASP immunoreactivity in the first trimester decidual tissues, while there was no staining in decidual cells themselves where the interstitial cytotrophoblasts invade (Figure 1d–f).
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Placenta samples from the first trimester showed the strongest VASP immunoreactivity in extravillous trophoblasts, when compared to second and third trimester samples (Figure 2A
). Villous trophoblast cells showed no immunoreactivity for VASP in the second trimester and term placenta. However, some trophoblastic cells located in the intervillous space or in placental fibrinoid still had strong VASP immunoreactivity during the second trimester and at term (Figure 2B, C). VASP expression in different cells of chorionic villi is summarized according to immunohistochemical staining intensity throughout the pregnancy in Table I.
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Western blot analysis
To investigate if there is any quantitative difference of VASP expression in human placenta throughout pregnancy, Western blot analysis was carried out with placental tissues from all trimesters. Immunoblot analysis showed two different bands (46 and 50 kDa, unphosphorylated and phosphorylated forms respectively) for VASP immunoreactivity. While first trimester placental tissues showed the strongest band for the 46 kDa form, and a weak band for the 50 kDa form, the second and third trimester placenta samples had stronger bands for the 50 kDa form compared to first trimester placental samples (Figure 3
).
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Regulation of VASP in human placenta by TGF-ß1 and LIF
TGF-ß1 and LIF are known to have important roles in implantation. To study the effect of TGF-ß1 and LIF on VASP expression in placental cells, explant cultures were carried out from first trimester placental villi. Explants were treated with TGF-ß1 (1 ng/ml) and LIF (1 ng/ml) for 24 h. TGF-ß1-treated placental villi showed similar VASP immunostaining intensity to the control group (Figure 4a, b
). On the other hand, LIF had a stimulatory effect on VASP expression in extravillous and chorionic villous cells, especially in stromal cells, compared to control explants (Figure 4a, c). Moreover, the immunoblot results from the explant cultures were similar to our immunohistochemistry observations. LIF-treated samples showed a prominent increase in the expression of unphosphorylated VASP compared to the control group, while TGF-ß1-treated explants showed no increase in protein expression of VASP (Figure 5).
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Discussion |
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The VASP/Ena family of proteins has been implicated in the control of intracellular actin dynamics. Both VASP and the VASP form in mouse tissues (Mena) localize to the leading edge of motile cells and the tips of growing filopodia, where actin polymerization occurs (Rottner et al., 1999
; Lanier and Gertler, 2000). Moreover, the amount of VASP appears to correlate with the rate of filopodia protrusion (Rottner et al., 1999).
Our results are the first description of human placental villi expressing VASP throughout pregnancy. Integrins that establish focal adhesion with ECM are the most important factors in human blastocyst implantation and placentation (Albers et al., 1995; Aplin, 1996). However, regulation of the intracellular dynamics emerging from integrin–ECM binding is still poorly understood. Recent studies suggest that VASP, as a focal adhesion protein, has an important role in the signalling pathway of integrin–matrix interactions, intracellular actin bundle formation and elaboration of filamentous actin–profilin binding (Walter et al., 1993, 1995; Bachmann et al., 1999).
Since endothelial and Hofbauer cells are migratory cells in villous stroma and cell migration is the essential feature of vasculogenesis in human placenta (Demir and Erbengi, 1984; Migdal et al., 1998), our results imply that VASP may also be involved in endothelial cell migration during placental vasculogenesis.
It is well known that alterations occur in cell–cell and cell–matrix interactions as cells develop from polarized stem cytotrophoblasts attached to the villous basement membrane, to interstitially migrating cytotrophoblasts (Vicovac et al., 1995; Aplin, 1996). Remarkably, the layer of villous cytotrophoblasts expresses integrin 6ß4 in floating villi. In anchoring villi, villous cytotrophoblasts and proximal cells of the extravillous column dominantly express 6ß4 integrin, whereas on the other side distal column cytotrophoblasts switch the integrin expression prominently to 5ß1, 1ß1 and v subtypes (Aplin, 1991; Damsky et al., 1992; Zhou et al., 1997). Moreover, an unusual ECM is observed in the distal parts of the column. This part of the column is rich in fibronectin and carries a characteristic oncofetal glycopeptide (Feinberg et al., 1991). The results of the present study suggest that during the invasive pathway, one of the intracellular dynamic arrangements of the integrin–ECM interaction is carried out with VASP regulation, though it is not clear yet if this interaction includes integrins expressed by cytotrophoblast. The strongest VASP immunoreactivity in the distal extravillous trophoblast cell columns, placental fibrinoids, and interstitial trophoblast cells, invading cells of the human placenta, supports the hypothesis that this protein may be associated with trophoblastic cell motility and invasion. Of course, there are many signalling pathways that regulate cytoskeletal dynamics. Cell-specific expression of VASP in human placenta suggests that VASP may be related to specific integrins such as 5ß1 or 1ß1. Cytotrophoblasts expressing these integrins show stronger VASP immunoreactivity.
The 46 kDa unphosphorylated form of VASP turns into the 50 kDa mol. wt form when phosphorylated by cAMP and cGMP-dependent protein kinases (Halbrugge et al., 1992). The decrease in VASP expression during the second and third trimesters is also in parallel with the diminished trophoblastic invasion in these trimesters. In the immunoblot analysis, VASP showed the strongest band at 46 kDa during the first trimester, while this reactivity decreased throughout pregnancy. In contrast, the weakest band was observed at 50 kDa for VASP during the first trimester while term villi samples demonstrated the strongest expression of this form. This could be one of the reasons why trophoblastic invasion decreases as the pregnancy progresses, since phosphorylation of VASP leads to inhibition of cell migration. Similarly, Smolenski et al. have shown that phosphorylation of VASP results in loss of VASP and its binding partner, zyxin, from focal adhesions. This phosphorylation causes a 1.5–2-fold inhibition of human umbilical vein endothelial cell migration in vitro (Smolenski et al., 2000).
Studies on human and rodent implantation mechanisms have shown that LIF is one of the essential cytokines for blastocyst implantation (Bhatt et al., 1991; Stewart et al., 1992; Arici et al., 1995; Nachtigall et al., 1996). One of the mechanisms for LIF action in blastocyst implantation could be through the regulation of the trophoblastic differentiation. TGF-ß is also expressed by endometrial cells and in decidua where blastocysts implant (Arici et al., 1996; Jokhi et al., 1997). Some growth factors produced by the endometrium including tumour necrosis factor- (TNF-) (Chen et al., 1991), platelet-derived growth factor (Svalander et al., 1991) and TGF-ß (Selick et al., 1994) have been shown to induce endometrial LIF production (Arici et al., 1995). LIF and TGF-ß have been shown to stimulate trophoblast cells to differentiate towards the anchoring phenotype and invasive pathway (Feinberg et al., 1994; Nachtigall et al., 1996). However, it is also speculated that TGF-ß1 inhibits trophoblast differentiation (Morrish et al., 1991). In this study, we found that LIF has a stimulatory effect on VASP expression in stromal cells of chorionic villi and extravillous trophoblastic cells. Thus, one of the effects of LIF on trophoblast differentiation toward the invasive phenotype could be explained by the induction of VASP expression that leads to focal adhesion formation and increased filamentous actin assembly.
In conclusion, in the present study, we have shown that human placenta has a trimester- and cell-specific expression for VASP. Furthermore, the strong VASP immunoreactivity in invasive cells of the human placenta supports the hypothesis that VASP may be an important factor for blastocyst implantation and placental development. It seems that LIF induces differentiation of trophoblasts toward adhesive and invasive pathways by stimulating VASP expression. Results of the present study also imply that VASP could be related to specific integrins such as 5ß1 or 1ß1 subtypes, expressed specifically by invasive cytotrophoblasts. Further studies on integrin–VASP signal pathways are needed to understand the role of VASP in trophoblastic adhesion and motility.
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This work was supported in part by a training grant to Umit A.Kayisli from the Turkish Scientific and Technical Research Council (TUBITAK).
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3 To whom correspondence should be addressed. E-mail: aydin.arici{at}yale.edu
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Submitted on May 30, 2001;
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