Villous Syncytiotrophoblast Research Articles

Biochemical characterization and modulation of LH/CG—receptor during human trophoblast differentiation

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation.

Involvement of PPARγ in Human Trophoblast Invasion

The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily that controls the expression of a large array of genes in a ligand-dependent manner. In the human placenta, PPARγ is specifically expressed in the villous cytotrophoblast and syncytiotrophoblast as well as in the extravillous cytotrophoblastic cells (EVCT) along their invasive pathway. The present study used two cellular models, primary cultures of trophoblastic cells differentiated in vitro in extravillous trophoblastic cells and a cell line (HIPEC65), which was established from a primary culture of EVCT transformed by T-SV40. We observed that natural (15d-PGJ2) or synthetic ligands of PPARγ (rosiglitazone) inhibit cell invasion in a concentration-dependent manner, with no effect on cell proliferation. This is associated with a modulation of the expression of trophoblastic genes described to be directly involved in the control of EVCT invasiveness, such as GH-V (−20%), TGFβ2 (−30%), PAPP-A (−60%) and IL1β (+300%.). In order to identify PPARγ potential ligands at the fetomaternal interface, we purified LDL (low density lipoprotein) from human sera and oxidized them in vitro in the presence of copper. OxLDL inhibit in vitro extravillous trophoblast cell invasion, whereas native LDL have no effect. In situ OxLDL and their LOX-1 receptor, as well as PPARγ are immunodetected in trophoblasts at the maternofetal interface.

Evidence for alpha-1-antitrypsin (A1AT) polymerization in preeclampsia: A novel mechanism for endothelial cell injury

PREECLAMPSIA: A NOVEL MECHANISM FOR ENDOTHELIAL CELL INJURY IRINA BUHIMSCHI, GUOMAO ZHAO, GEORGE SAADE, CATALIN S. BUHIMSCHI, Yale University, Obstetrics/Gynecology, New Haven, Connecticut, Yale University, Ob./Gyn.R p=0.009], despite a significant increase its urinary excretion (O500 fold in sPE, p!0.001). In addition, urine and serum from women with sPE contained multiple immunoreactive forms of A1AT, with molecular masses both below (fragments) and above the 52 kDa mass of the monomeric A1AT (polymers). In the placenta, monomeric A1AT localized largely intravascular and to villous macrophages and syncytiotrophoblasts. The staining was significantly more evident in sPE compared with CTR. In all sPE cases, but in none of the CRLs, an intense ZA1AT staining was localized exclusively to the vascular endothelium. CONCLUSION: A1AT protein expression is increased in preeclamptic placentas. This is the first demonstration that polymerized A1AT is present in serum and urine of patients with preeclampsia, and that this material is bound to placental vascular endothelium. This suggests that the polymerized A1AT may play a role in the pathogenesis of endothelial damage in preeclampsia.

Distinct expression and localization of serine protease HtrA1 in human endometrium and first‐trimester placenta

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.

P53 Electron Microscopical Localisation of CD61 (Beta3 Integrin) on Placental Syncytiotrophoblast Microvilli

Introduction Neonatal alloimmune thrombocytopenia (NAIT) due to maternal antibodies to the human platelet antigen (HPA)‐1a can occur in first pregnancies. The HPA‐1 polymorphism is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin). It is hypothesised that besides fetal platelets, an additional source of fetal CD61 could cause alloimmunisation early in pregnancy. CD61 was earlier identified on placental villous trophoblasts by immunocytochemistry.Methods Whole‐mount immuno‐electron microscopy was used to study the ultrastructural localisation of CD61 on placental tissue. Fragments of first trimester (n = 5, 8–12 weeks gestational age) and term (n = 1) placental chorionic villi were incubated with anti‐CD61 or IgG1 isotype control, washed, incubated with 18 nm colloidal gold‐conjugated anti‐mouse IgG, washed, fixed in phosphate‐buffered glutaraldehyde and processed through to embedding in epoxy resin. 100 nm sections were prepared from the EM blocks; these were stained with uranyl acetate and lead citrate, and examined with a Phillips CM10 electron microscope. Positive control was term tissue stained with anti‐placental alkaline phosphatase (PLAP).Results Microvilli were observed on the apical membrane of some but not all of the villous syncytiotrophoblast (STB) surfaces. Formation of this brush border occurs during the first trimester to increase the surface area of these cells for optimal transfer of nutrients and gases from the adjacent maternal blood to the fetus. All specimens had some microvilli. After staining with anti‐CD61, gold particles were present on and between some microvilli of all specimens, either singly or in small groups. Stronger staining with more frequent occurrence of groups of gold particles was evident on microvilli of the term tissue incubated with anti‐PLAP. No gold particles were seen on the isotype control specimens.Conclusion From early on in pregnancy, CD61 is present on the surface of fetal STB in contact with maternal blood. This protein carrying the HPA‐1 polymorphism might be the cause of the initial immunising event that leads to NAIT.

Zinc Transporters in the Mouse Placenta Show a Coordinated Regulatory Response to Changes in Dietary Zinc Intake

The aim of the study was to determine if the expression of zinc transporters in the mouse placenta is regulated by dietary zinc, commensurate with regulating the supply of zinc to the fetus. Mice were fed diets differing only in the concentration of zinc (moderately zinc-restricted (ZnR) – 15 mg Zn/kg; zinc-adequate (ZnA) – 50 mg Zn/kg; zinc-supplemented (ZnS) – 150 mg Zn/kg) from the onset of pregnancy until collection of tissue at day 17. Compared with mice fed the other diets, fetal weight was reduced in the ZnR group and total non-embryonic weight gain was reduced in mice fed the ZnS diet. Transcript levels of metallothionein and the zinc transporters ZnT1, ZnT4 and ZIP1 were reduced in the placenta of mice fed both the ZnR and ZnS diets compared with mice fed the ZnA diet. Placental ZnT7 and fetal liver metallothionein transcript levels did not differ significantly between mice fed the three diets and placental ZnT5 was reduced in mice fed the ZnS compared with the ZnA diet but did not differ significantly between the ZnA and ZnR diets. The pattern of mRNA expression in placenta was reflected at the protein level for ZnT1. Levels of ZnT5 protein were also highest in mice fed the ZnA diet. Both ZnT1 and ZnT5 were detected in the human villous syncytiotrophoblast by immunohistochemistry. The data indicate that the expression of zinc transporters in mouse placenta is responsive to dietary zinc supply but this modulation of expression is insufficient to maintain optimum fetal nutrition at even a modest level of dietary zinc restriction.

Expression and localization of tumor necrosis factor receptor 1 protein in the chorionic villi in early normal and spontaneous abortion

Background Increasing tumor necrosis factor α (TNFα) expression is found to be positively associated with spontaneous abortion. TNFα acts through the binding of two types of transmembrane receptors called TNFR1 and TNFR2. Here we compare membrane TNFR1 (mTNFR1) protein expression on chorionic villi in women with spontaneous abortion (SA) and viable pregnancy. Methods In a prospective study, 31 women with SA and 30 with a viable pregnancy were studied. Chorionic villous membrane TNFR1 was detected by confocal laser scanning microscopy and immunohistochemistry. Results The mTNFR1 protein is expressed in villous stromal, vessel endothelial, syncytiotrophoblast, and cytotrophoblast cells in early pregnancy. The intensity of mTNFR1 fluorescence (mean ± S.D.) in villous stromal cells was higher in the abortion groups than in the control group (35.99 ± 6.35 versus 32.64 ± 5.01; p < 0.05). In the abortion groups, villous cytotrophoblast cells were intensely positive for mTNFR1, whereas mTNFR1 staining of vessel endothelial and syncytiotrophoblast cells was significantly lower ( p < 0.001). Conclusion Abundant mTNFR1 expression in the cytotrophoblast cells in women with SA may mediate TNFα to induce programmed cell death in the mTNFR1-expressing cytotrophoblasts to limit their growth. The over-expressing mTNFR1 in villous stromal cells, mediating TNFα effects, may cause pathological changes or tissue damage in chorionic villi locally in nonviable pregnancies.

Expression of SWAP-70 in the uterus and feto-maternal interface during embryonic implantation and pregnancy in the rhesus monkey (Macaca mulatta)

SWAP-70 is a unique signaling protein involved in multiple processes including lymphatic cell activation, migration, adhesion, and cytoskeleton organization. Its role in reproductive system remains to be unclear. In the present study, the spatial and temporal expression of SWAP-70 in the uterus during normal menstrual cycle as well as on the feto-maternal interface during pregnancy was investigated in the rhesus monkey by in situ hybridization and immunohistochemistry. It was shown that SWAP-70 was mainly expressed in glandular epithelial cells of uterine endometrium, and the level peaked at the mid-secretory stage. At the beginning of embryonic implantation, SWAP-70 was intensely expressed at the implantation site, mainly localized in glandular and luminal epithelial cells, as well as in primary trophoblasts and epithelial plaque. High level of SWAP-70 was observed in villous cytotrophoblast (VCT), syncytiotrophoblast (ST), column cytotrophoblast, trophoblast shell, interstitial trophoblast, and endovascular trophoblast during gestational days 15-25. From gestational day 50 to term, expression of SWAP-70 decreased evidently and was restricted in VCT cells. What's more, SWAP-70 co-localized with F-actin on the feto-maternal interface, especially in highly motive extravillous trophoblasts. The data indicate that SWAP-70 may be involved in regulating motility of trophoblast cells during embryonic implantation and placentation.

Monocarboxylate transporter 8 expression in the human placenta: the effects of severe intrauterine growth restriction

Thyroid hormones (THs) are essential for normal fetal development, with even mild perturbation in maternal thyroid status in early pregnancy being associated with neurodevelopmental delay in children. Transplacental transfer of maternal THs is critical, with increasing evidence suggesting a role for 3,3',5-tri-iodothyronine (T3) in development and function of the placenta itself, as well as in development of the central nervous and other organ systems. Intrauterine growth restriction (IUGR) is associated with fetal hypothyroxinaemia, a factor that may contribute to neurodevelopmental delay. The recent description of monocarboxylate transporter 8 (MCT8) as a powerful and specific TH membrane transporter, and the association of MCT8 mutations with profound neurodevelopmental delay, led us to explore MCT8 expression in placenta. We describe the expression of MCT8 in normal human placenta throughout gestation, and in normal third-trimester placenta compared with that associated with IUGR using quantitative reverse transcriptase PCR. MCT8 mRNA was detected in placenta from early first trimester, with a significant increase with advancing gestation (P=0.007). In the early third trimester, MCT8 mRNA was increased in IUGR placenta compared with normal samples matched for gestational age (P<0.05), but there was no difference between IUGR and normal placenta in the late third trimester. Western immunoblotting findings in IUGR and normal placentae were in accord with mRNA data. MCT8 immunostaining was demonstrated in villous cytotrophoblast and syncytiotrophoblast as well as extravillous trophoblast cells from the first trimester onwards with increasingly widespread immunoreactivity seen with advancing gestation. In conclusion, expression of MCT8 in placenta from early gestation is compatible with an important role in TH transport during fetal development and a specific role in placental development. Altered expression in placenta associated with IUGR may reflect a compensatory mechanism attempting to increase T3 uptake by trophoblast cells.

Differential Expression of Placental Villous Angiopoietin-1 and -2 During Early, Mid and Late Baboon Pregnancy

Although vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and Ang-2 have important roles in angiogenesis, very little is known about the regulation of these factors in the villous placenta during human pregnancy. In the present study, to investigate whether placental expression of Ang-1, Ang-2 and VEGF was altered in a cell-specific manner with advancing baboon gestation, the mRNA levels of these growth factors were determined by RT-PCR in cells isolated by Percoll gradient centrifugation from and protein localization assessed by immunocytochemistry in the villous placenta at early (day 60), mid (day 100) and late (day 170, term is 184 days) baboon gestation. Mean (±SE) Ang-1 mRNA levels, relative to 18S rRNA, in villous syncytiotrophoblast (3.92 ± 0.68) and cytotrophoblast (1.31 ± 0.31) cell fractions were highest on day 60 of gestation, then decreased by approximately 2.5-fold ( P < 0.05) to 1.39 ± 0.29 and 0.49 ± 0.07, respectively, on day 170. Moreover, Ang-1 mRNA levels in the villous stromal cells and Ang-2 mRNA levels in all placental villous cell fractions were similar on days 60, 100, and 170 of gestation. In contrast to Ang-1 and Ang-2, placental villous cytotrophoblast VEGF mRNA levels were increased 2.94-fold ( P < 0.05) between mid (0.67 ± 0.15) and late (1.97 ± 0.49) gestation. A corresponding decrease in Ang-1, absence of change in Ang-2, and increase in VEGF protein immunocytochemical expression were exhibited in placental trophoblast with advancing baboon pregnancy. Ang-1/Ang-2 and the angiopoietin Tie-2 receptor were expressed in vascular endothelial cells of the villous placenta, indicating that these blood vessel cells are a major site of ligand–receptor interaction for angiogenesis during primate pregnancy. We conclude that there is a cell-specific differential change in placental villous trophoblast expression of VEGF, Ang-1, and Ang-2 which we propose is important in regulating angiogenesis in the villous placenta during primate pregnancy.

Analysis of placental growth factor in placentas of normal pregnant women and women with hypertensive disorders of pregnancy

To investigate the expressions of placental growth factor (PLGF) in placenta with hypertensive disorders of pregnancy (HDP), 45 women with HDP and 20 normally pregnant women were studied. Among 45 women with HDP, there were 23 cases of severe preeclampsia and one case of eclampsia. The location and level of PLGF proteins was determined by immunohistochemistry and Western blot. The expression of PLGF mRNA in placenta was assessed by reverse transcriptional-polymerase chain reaction (RT-PCR). The results showed that: (1) The distribution of PLGF in placenta with HDP was similar to normal one, which was mainly in the cytoplasm of villous syncytiotrophoblast and villous stroma; (2) The expression of PLGF protein was significantly decreased in placentas with mild and severe preeclampsia compared to the normal ones (0.3 +/- 0.4 vs 0.6 +/- 0.4, 0.2 +/- 0.5 vs 0.6 +/- 0.4, P < 0.01). There were no differences between the gestational hypertension placenta and normal one (0.5 +/- 0.6 vs 0.6 +/- 0.4, P > 0.05); (3) The transcription levels of the PLGF mRNA in placentas with preeclampsia were significantly lower than in normal groups (3.33 +/- 0.39 vs 4.87 +/- 0.60, 1.97 +/- 0.29 vs 4.87 +/- 0.60, P < 0.01), and no differences were found between the gestational hypertension placenta and normal groups. These findings suggest that the abnormal expression of PLGF in placentas is related to the pathogenesis of HDP.

Expression of the High-Mobility Group Protein HMGI(Y) in Gestational Trophoblastic Diseases

The high-mobility group protein HMGI(Y) is a member of a family of nonhistone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes, and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. Recently we could show that HMGI(Y) is expressed in the normal human placenta, where it is localized in the nuclei of villous cytotrophoblast, in the anchoring villi at the implantation site and in extravillous (intermediate) trophoblast invading the maternal decidua. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. The purpose of this study was to investigate the expression pattern of HMGI(Y) in gestational trophoblastic diseases (GTD), which has not been studied so far. To analyze the expression of HMGI(Y), we performed immunohistochemistry on a total of 29 cases of GTD, including 21 hydatidiform moles and 8 choriocarcinomas. Hydatidiform moles showed a positivity for HMGI(Y) in villous cytotrophoblast and in areas of the trophoblast proliferations on the villous surface; villous syncytiotrophoblast was negative. The choriocarcinomas showed strong immunoreactivity in all cases. The expression pattern of HMGI(Y) in gestational trophoblastic diseases indicates that it might play a role in the pathogenesis of GTD and might be potentially useful as an additional diagnostic marker for such lesions.

Soluble HLA-G isoforms: technical deficiencies lead to misinterpretations

The recent report authored by Blaschitz et al. (in press) would lead the reader to believe that human trophoblast cells cannot synthesize or secrete any of the soluble isoforms derived from splice variants of the HLA-G message. They propose that all of the soluble HLA-G presumed to be produced in trophoblast cells is, in fact, cleaved membrane HLA-G1. An interesting idea. Yet several published works have presented solid, well documented evidence that argues against this position. The original article describing the molecular structure of soluble HLA-G (Fujii et al., 1994) showed that soluble protein encoded by the intron 4-containing mRNA could be clearly distinguished from membrane HLA-G1 by isoelectric focusing (IEF). Further, a class I protein (reactive with W6/32) with an IEF pattern completely overlapping with soluble HLA-G from transfected cells was demonstrated in JEG-3 choriocarcinoma cells. Also in that study, PCR primers specific for soluble HLA-G-encoding mRNA were clearly described and used to confirm the protein work. In 2002, Solier and colleagues showed that soluble, intron-4 retaining HLA-G1 (now known as HLA-G5) mRNA is present in villous cytotrophoblast cells and that the HLA-G5 protein is secreted by the cells (Solier et al., 2002). In 2003, a team working in the Geraghty laboratory reported that membrane HLA-G1 is essentially absent in placentas, whereas HLA-G5 is abundant in both villous cytotrophoblast cells and syncytiotrophoblast (Ishitani et al., 2003). HLA-G1 was identified only in migrating extravillous cytotrophoblast cells using the highly specific o1G monoclonal antibody. Also in that study, the soluble HLA-G1 specific antibody 16G1 was used to isolate protein directly from term placentas. Bound peptide was isolated from this complex and shown to have a nearly identical profile to peptide isolated from membrane HLA-G1, which was also isolated from placentas. These peptides collectively matched very precisely the peptide binding motif previously described for HLA-G (some peptides were identical), and that work left no doubt as to the source of that peptide by N-terminal sequencing of the heavy chain (Lee et al., 1995). Taken together, these facts leave no doubt that 16G1 does bind HLA-G produced in vivo, and can only be reconciled with the conclusions made by Blaschitz study if one posits that 16G1 instead binds membrane HLA-G1. That conclusion in turn contradicts several other studies including some of the results presented in the Blaschitz et al. paper (Blaschitz et al., in press). At the next level of study alternative HLA-G protein forms were investigated. Newly generated, fully characterized, isoform-specific monoclonal antibodies developed in the Hunt laboratory demonstrated that villous cytotrophoblast cells contain HLA-G5 and HLA-G6 mRNA but express only HLA-G5 protein (Morales et al., 2003). By contrast, expression of HLA-G2/G6 protein was identified exclusively in extravillous cytotrophoblast cells. Despite this strong and consistent evidence put forth by three independent laboratories, it is always important to critically examine new evidence contradicting previously held conclusions. Taking the major features of the Blaschitz paper one at a time, let us look at the evidence they present to support their conclusions.

Serine Peptidase HTRA3 Is Closely Associated with Human Placental Development and Is Elevated in Pregnancy Serum1

HTRA3 is a newly identified serine peptidase of the mammalian HTRA (high-temperature requirement factor A) family, that is upregulated dramatically during mouse placental development. The current study determined whether HTRA3 was involved in human placentation. During the menstrual cycle, HTRA3 was expressed primarily in the endometrial glands, being significantly upregulated toward the mid- to late secretory phases; prominent expression in the stroma detected only in the decidual cells in the late secretory phase. Thus, overall endometrial HTRA3 expression was highest in the late secretory phase, when the endometrium is prepared for maternal-trophoblast interaction. During the first trimester of pregnancy, both glandular and decidual HTRA3 expression increased further with the decidual upregulation being highly significant. The strong link between HTRA3 expression and endometrial stromal cell decidualization was further established in an in vitro model using primary endometrial stromal cells. HTRA3 was also expressed by certain trophoblast subtypes in the first-trimester placenta: strongly in the villous syncytiotrophoblast, trophoblast shell, and endovascular trophoblast and weakly in the distal portion of the trophoblast cell columns but not in villous cytotrophoblast, the proximal region of the cell columns, or interstitial trophoblast. Upregulation of HTRA3 expression in association with placental development was revealed by a significant elevation of this protein in the maternal serum during the first trimester. We thus propose that HTRA3 is a previously unrecognized factor closely associated with and potentially important for human placentation. This study established crucial groundwork for future investigations toward establishing the physiological roles of HTRA3 in human placentation.

Efficient Apical IgG Recycling and Apical-to-basolateral Transcytosis in Polarized BeWo Cells Overexpressing hFcRn

The human neonatal Fcgamma-receptor (hFcRn) involved in overall maternal-fetal IgG transmission is expressed in placental villous syncytiotrophoblast. However, the role of hFcRn in IgG transport and trafficking across this cell layer is poorly characterized. To gain insight into this mechanism we have overexpressed functional hFcRn in trophoblast-derived BeWo cells (BeWo/hFcRn cells) [Ellinger I, Reischer H, Lehner C, Leitner K, Hunziker W, Fuchs R. Placenta 2005;26:171-82] since parental BeWo cells endogenously express low levels of the receptor. We now demonstrate that hFcRn overexpression differentially affected apical-to-basolateral transcytosis and apical recycling: whereas IgG transcytosis was reduced by 35%, IgG recycling was stimulated by 100% as compared to parental cells, indicating that hFcRn plays a role in both processes. The endosomal compartments involved in hFcRn/IgG transport after apical IgG internalization were then analyzed by confocal immunofluorescence microscopy using compartment specific markers. hFcRn/IgG were found in apical early endosomes, in transferrin recycling compartments and in vesicles near the basolateral plasma membrane, presumably transcytotic vesicles. Neither hFcRn nor IgG was routed to the degradative pathway to lysosomes. These transport and localization data are in accordance with efficient hFcRn-mediated apical IgG recycling and basolateral directed IgG transcytosis in placental trophoblasts.

Increased immunohistochemical expression of thrombomodulin at placental perivascular myofibroblast in severe preeclampsia (PE).

The presence of pro-coagulant and anti-coagulant components of the placental vascular endothelium and syncytiotrophoblast are essential for homeostasis. Vascular endothelium prevents blood clot formation in vivo by involving a cell surface thrombin-binding glycoprotein, thrombomodulin (TM), that activates plasma anti-coagulant protein C. The TM levels increase during pregnancy, but the fibrinolytic capacity diminishes. Since vascular lesions with placental coagulation disorders can be associated with preeclampsia (PE), we hypothesized that TM expression in the stem villous vasculature and syncytiotrophoblast of the placenta are impaired in PE. Plasma and placental tissue samples were collected from PE (n=12) and normotensive pregnant patients (n=11). Patient's gestational age was 35.7+/-1.2 (normotensive) and 30.6+/-1.5 weeks (PE). Blood samples were drawn 30 min before delivery. Serum PAI-1 and PAI-2 antigens were determined by enzyme-linked immunoabsorbent assay (ELISA). A monoclonal antibody specific for TM was used for immunohistochemical tissue staining (ABC) and the staining was quantified by semi quantitative scores. Results show no intensity differences at the apical syncytiotrophoblast between the two groups. However, in preeclamptic placenta, TM expression diminished in the endothelium of the stem villi arteries and increased in the perivascular and stromal myofibroblats in cases of severe PE. TM changes were associated with an increased PAI-1/PAI-2 ratio. It is suggested that in severe PE, the decreased placental blood flow may be due to structural and functional impairment of the endothelium of the stem villi vessels and the surrounding perivascular and stromal myofibroblast, by increasing TM expression which may modulate fetal blow flow in the villous tree.

Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level

The local immune privilege of the fetus is created by the placenta. Fas ligand (FasL) expression in trophoblast has been implied as one of the mechanisms of fetal tolerance. However, the expression of membranal FasL by trophoblast has failed to explain this role of FasL. Two objections can be raised: (1) there have been contradictions considering which trophoblast cells, syncytiotrophoblast (ST) or cytotrophoblast, express FasL; (2) in vivo and in vitro studies have shown that the membranal form of FasL evokes inflammatory response and thus may promote fetal rejection. Using different assays and the FasL-specific antibody G247-4 we demonstrate beyond doubt that in vivo, (1) FasL is produced by and stored in the first trimester human ST only and (2) the human ST lacks surface membranal FasL. Instead, FasL, loaded in microvesicles, is stored in cytoplasmic granules. These results complement the recent in vitro studies of the microvesicular form of FasL secretion by cultured trophoblast cells, and suggest that placental FasL is synthesized by villous ST, stored in microvesicular form and secreted as exosomes. Secretion of the exosome-associated form of FasL may be one mechanism by which the placenta promotes a state of immune privilege. Additionally, FasL expression in Hofbauer cells is also demonstrated.

Expression pattern of the CCAAT/enhancer-binding protein C/EBP-beta in gestational trophoblastic disease.

The CCAAT/enhancer-binding protein (C/EBP) family consists of several factors that are important regulators of intracellular processes and hormone action. C/EBP-beta, the most important member of the C/EBP family, was shown recently to be expressed in the normal human placenta where it is localized in villous syncytiotrophoblast and in the extravillous (intermediate) trophoblast but not the villous cytotrophoblast. The purpose of this study was to investigate the expression pattern of C/EBP-beta in gestational trophoblastic disease (GTD) which has not been studied so far. We used immunohistochemistry on a total of 15 cases of GTD including nine complete hydatidiform moles, one placental site nodule (PSN), one placental site trophoblastic tumor (PSTT), and four choriocarcinomas. All our tested specimens showed positivity for C/EBP-beta. The strongest C/EBP-beta expression could be observed in villous syncytiotrophoblast and in the trophoblast proliferations on the villous surface of hydatidiform moles; villous cytotrophoblast was negative. The PSN also showed positive nuclear staining but the expression was not as strong as it was in the hydatidiform moles and the total amount of stained cells was the lowest of all GTD. The PSTT also showed immunoreactivity but with a weaker and more heterogeneous staining than in the choriocarcinomas. The specific expression pattern of C/EBP-beta in GTD indicate that C/EBP-beta could potentially be an additional marker of such lesions.

Endocytic and transcytotic processes in villous syncytiotrophoblast: role in nutrient transport to the human fetus.

The supply of nutrients to the developing fetus is a major function of the human hemochorial placenta, a placenta type in which the fetal chorion is in direct contact with the maternal blood. At term, nutrients have to be transported across two cell layers in chorionic villi, the syncytiotrophoblast (STB) and fetal endothelial cells. The STB is a continuous syncytium covering the entire surface of chorionic villi. This polarized epithelium is specialized in exchange processes and membrane trafficking between the apical membrane facing the maternal blood and the basal membrane facing the fetal endothelium. To meet placental and fetal requirements, the STB selectively takes up and transports a variety of nutrients, hormones, growth factors and cytokines and also transfers passive immunity to the fetus by receptor-mediated transcytosis. In this review in vivo and in vitro systems currently used to study STB functions are discussed and the potential mechanisms of transplacental IgG, iron, lipoprotein and glucose transport are presented. As revealed in this article, the placenta is a tissue where intensive cell biological research is required to unravel endocytic trafficking pathways in a highly specialized cell such as the STB.

Overexpression of the human neonatal Fc-receptor α-chain in trophoblast-derived BeWo cells increases cellular retention of β2-microglobulin

Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcγ-receptor, FcRn, are heterodimers consisting of a transmembrane α-chain non-covalently associated with β2-microglobulin (β2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express β2m and low levels of hFcRn α-chain. The contribution of FcRn α-chain in retention and subcellular distribution of β2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn α-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with β2m. In comparison to parental BeWo cells, β2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated β2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn α-chain expression retained otherwise secreted β2m. Flow cytometry revealed that β2m plasma membrane expression was unaffected by α-chain overexpression whereas by fluorescence microscopy a preferential staining of β2m in peripheral endosomes was observed.