Normal Trophoblast Research Articles

Glucose-6-phosphate Dehydrogenase is Present in Normal and Pre-eclamptic Placental Trophoblasts: Ultrastructural Enzyme-histochemical Evidence

The present study investigated the localization of glucose-6-phosphate dehydrogenase (G6PD) activity in the human placenta at various gestational ages. Placentae from patients with severe pre-eclampsia were also studied. Ultrastructural enzyme–histochemical analysis was performed by newly developed G6PD histochemistry using copper ferrocyanide as a capturing agent. Precipitates indicative of G6PD activity were markedly visible in the cytotrophoblastic cytoplasm and faintly in the syncytiotrophoblastic cytoplasm of placentae at various gestational ages, as well as those from pre-eclampsia. Frequently, precipitations were localized on the cytosolic side of the endoplasmic reticular membranes of the cytotrophoblasts. Stringent cytochemical control experiments performed also ensured the specific detection of G6PD activity. The results indicated that cytochemically detectable G6PD was localized in cytotrophoblastic cytoplasm. This enzyme may play significant roles in the carbohydrate metabolism of the human placenta, and further maintenance of villous tree architecture. Combining the previous data, the human placenta has many carbohydrate metabolizing-enzymes, similar to the adult liver.

The Expression and Localization of Neutral Endopeptidase 24.11/CD10 in Human Gestational Trophoblastic Diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-γ inducible promoter (PIV) of CIITA

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-γ (IFN-γ)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Note- worthy, this lack of IFN-γ-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

Surface Expression of HLA-C Antigen by Human Extravillous Trophoblast

In this paper definitive evidence that the classical class I product, HLA-C, is expressed on the surface of normal trophoblast cells is provided. HLA-C transcripts were sequenced from cDNA isolated from first trimester trophoblast cells obtained by flow cytometric sorting. Both paternal and maternal alleles were transcribed. HLA-C proteins were demonstrated by biochemical analysis and found on the cell surface in association with β2-microglobulin. Upregulation of cell surface HLA-C but not HLA-G expression after interferon (IFN)-γ treatment was demonstrated by flow cytometric analysis. Immunohistology has confirmed HLA-C is expressed by all extravillous subpopulations in vivo. The question of whether trophoblast HLA-C molecules interact with decidual NK cells expressing killer Ig-like receptors (KIR) has also been addressed. Our results demonstrate that extravillous trophoblast expresses at least two HLA class I molecules, HLA-G and HLA-C on the cell surface.

Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15.

The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56(bright) CD16(-). These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone-dominated secretory endometrium and early decidua.

Pre-eclampsia does not Change the Adhesion Molecule Status in the Placental Bed

During normal placentation trophoblast cells invade maternal tissues and remodel the uterine arteries into low-resistance channels. In pre-eclampsia, trophoblast invasion is impaired and this, along with endothelial dysfunction, has been suggested to play a role in the pathogenesis of pre-eclampsia. We studied the expression of adhesion molecules important for leukocyte extravasation in the placental bed with immunohistochemistry and compared the expression in pre-eclampsia to that in normal pregnancy. Our major finding was that only invasive trophoblasts expressed cutaneous lymphocyte antigen-1 (CLA-1) in the third trimester of pregnancy, whereas villous trophoblasts did not. In the first trimester both villous trophoblasts and invasive trophoblast cells in decidua remained negative for CLA-1. Pre-eclampsia did not change the expression of leukocyte–endothelium adhesion or lymphocyte homing-associated antigens, ICAM-1, ICAM-2, VCAM, P-selectin, E-selectin, L-selectin, CLA-1, CD73, VAP-1 and αEβ7 in the placental bed. Furthermore, pre-eclampsia was not associated with an aberrant accumulation of lymphocytes carrying antigens of any particular known organ-specific homing systems. The results on the unchanged pattern of adhesion molecule expression in pre-eclampsia suggests that there is no major change in the adhesive properties of the endothelium of the placental bed in pre-eclampsia.

Cytogenetic and DNA-Fingerprint Characterization of Choriocarcinoma Cell Lines and a Trophoblast /Choriocarcinoma Cell Hybrid

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Differential Gene Expression Pattern between Normal Human Trophoblast and Choriocarcinoma Cell Lines: Downregulation of Heat Shock Protein-27 in Choriocarcinoma in Vitro and in Vivo

Objective.Our purpose was to identify potential differences in gene expression between normal trophoblast and choriocarcinoma cells.Methods. The Atlas human cDNA expression array hybridization technique was used to study the gene expression pattern in normal trophoblast and choriocarcinoma cell lines. Furthermore, to confirm heat shock protein-27 (Hsp-27) expression data, reverse transcriptase-PCR (RT-PCR), Western blot, and immunohistochemical analyses were used in vitro with cell lines and in vivo with paraffin sections.Results. The expression of nine genes was strongly different comparing a normal trophoblast cell line with choriocarcinoma cells on the Atlas membranes. Compared to normal trophoblast cells, six genes were upregulated and three were downregulated in choriocarcinoma cells. Furthermore, the downregulation of Hsp-27 in choriocarcinoma cells was confirmed both in vitro with cell lines and in vivo with paraffin sections using RT-PCR, Western blot, and immunohistochemical techniques.Conclusion. cDNA expression array is a useful technique for identifying differentially expressed gene patterns in normal trophoblast and choriocarcinoma cells. The strong expression of Hsp-27 in placental villous trophoblast cells may play a role in trophoblast differentiation. The downregulation of Hsp-27 in choriocarcinoma may contribute to the extreme sensitivity of trophoblastic tumors to chemotherapy.

Expression pattern alterations of syndecans and glypican‐1 in normal and pathological trophoblast

Syndecans (syn-1, -2, -3, -4) and glypican-1 are proteoglycans expressed during development in association with changes in tissue organization and differentiation. They participate in the modulation of growth factor actions and in cell–cell and cell–matrix adhesion. The expression of syn-1, -2, -3, -4, and glypican-1 has been studied in normal human placenta and in gestational trophoblastic disease such as hydatidiform mole, invasive mole, and choriocarcinoma, using immunohistochemistry and western blots. Syndecan-3 was not expressed in normal or pathological tissues. During normal gestation, the other proteoglycans showed a specific staining pattern, which for some was modified during pregnancy. For instance, syn-1 was only expressed in syncytiotrophoblast; syn-4 was mainly localized in the villous and extravillous cytotrophoblast in the first trimester, whereas at term it was expressed in the syncytiotrophoblast. The most striking results are the altered expression patterns of syndecans and glypican-1 in pathological tissues. These proteoglycans showed a progressive decrease of immunostaining related to the increase of severity of trophoblastic disease, in particular in invasive mole and choriocarcinoma. In addition, dysregulation in the localization of the expression patterns was observed for syn-2 and -4. Because changes in syndecan expression enable cells to become more or less responsive to their micro-environment, the down-regulation and/or dysregulation of syndecans in relation to the degree of severity of trophoblastic diseases provides new insights into the progression of these pathologies. Copyright © 1999 John Wiley & Sons, Ltd.

Expression pattern alterations of syndecans and glypican-1 in normal and pathological trophoblast.

Syndecans (syn-1, -2, -3, -4) and glypican-1 are proteoglycans expressed during development in association with changes in tissue organization and differentiation. They participate in the modulation of growth factor actions and in cell-cell and cell-matrix adhesion. The expression of syn-1, -2, -3, -4, and glypican-1 has been studied in normal human placenta and in gestational trophoblastic disease such as hydatidiform mole, invasive mole, and choriocarcinoma, using immunohistochemistry and western blots. Syndecan-3 was not expressed in normal or pathological tissues. During normal gestation, the other proteoglycans showed a specific staining pattern, which for some was modified during pregnancy. For instance, syn-1 was only expressed in syncytiotrophoblast; syn-4 was mainly localized in the villous and extravillous cytotrophoblast in the first trimester, whereas at term it was expressed in the syncytiotrophoblast. The most striking results are the altered expression patterns of syndecans and glypican-1 in pathological tissues. These proteoglycans showed a progressive decrease of immunostaining related to the increase of severity of trophoblastic disease, in particular in invasive mole and choriocarcinoma. In addition, dysregulation in the localization of the expression patterns was observed for syn-2 and -4. Because changes in syndecan expression enable cells to become more or less responsive to their micro-environment, the down-regulation and/or dysregulation of syndecans in relation to the degree of severity of trophoblastic diseases provides new insights into the progression of these pathologies.

Evidence for a Novel RasGAP-Associated Protein of 105 kDa in both Mature Trophoblasts and Differentiating Choriocarcinoma Cells

A novel tyrosine-phosphorylated, RasGAP-associated protein of 105 kDa (p105) is found in normal human term placental trophoblasts, as well as in JEG-3 human choriocarcinoma cells induced to differentiate by okadaic acid (OA). This p105 RasGAP-associated protein is distinct from other RasGAP-associated proteins described so far, none of which has either a molecular size close to p105 or a trophoblastic cell origin. The p105 appears, accompanied by p120 and p100 RasGAP expression, after OA treatment of JEG-3 cells but is almost undetectable in the absence of stimulation. Moreover, the p105 is the first discovered RasGAP-associated protein bound to p100 RasGAP. The natural occurrence of the p105 in normal mature trophoblasts isolated from human term placenta suggests that it may be linked to the differentiation state of human trophoblasts. Hence, this p105 RasGAP-associated protein might be considered a marker of human trophoblast differentiation.

Signal transduction and biological function of placenta growth factor in primary human trophoblast.

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.

Regulation of activin A in cell proliferation and hormone secretion by human normal trophoblast cells

Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in serum-free system. It was shown that activin A promoted hCG and progesterone secretion in primary cultured cytotrophoblast cells as well as progesterone secretion in NPC cells, while it had no effect on cell proliferation and hCG secretion in NPC cells. Important evidence is provided for the autocrine regulatory mechanism of activin A on hormone secretion in placental trophoblast cells at early pregnancy.

Etiology and Pathogenesis of Preeclampsia

The etiology of preeclampsia is unknown. At present, 4 hypotheses are the subject of extensive investigation, as follows: (1) Placental ischemia-Increased trophoblast deportation, as a consequence of ischemia, may inflict endothelial cell dysfunction. (2) Very low-density lipoprotein versus toxicity-preventing activity-In compensation for increased energy demand during pregnancy, nonesterified fatty acids are mobilized. In women with low albumin concentrations, transporting extra nonesterified fatty acids from adipose tissues to the liver is likely to reduce albumin's antitoxic activity to a point at which very-low density lipoprotein toxicity is expressed. (3) Immune maladaptation-Interaction between decidual leukocytes and invading cytotrophoblast cells is essential for normal trophoblast invasion and development. Immune maladaptation may cause shallow invasion of spiral arteries by endovascular cytotrophoblast cells and endothelial cell dysfunction mediated by an increased decidual release of cytokines, proteolytic enzymes, and free radical species. (4) Genetic imprinting-Development of preeclampsia-eclampsia may be based on a single recessive gene or a dominant gene with incomplete penetrance. Penetrance may be dependent on fetal genotype. The possibility of genetic imprinting should be considered in future genetic investigations of preeclampsia.

Glucose-6-phosphatase is Present in Normal and Pre-eclamptic Placental Trophoblasts: Ultrastructural Enzyme-histochemical Evidence

The purpose of the present study was to localize glucose-6-phosphatase (G6Pase) activity in the human placenta at various gestational stages and to compare them to pre-eclamptic placenta activity. Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicative of G6Pase activity were observed in the endoplasmic reticulum and the nuclear envelope of the syncytiotrophoblasts in near-term placenta obtained from women with normal pregnancies. In placenta taken from women with severe pre-eclampsia, the localization pattern, enzyme activity intensity, and morphology of the endoplasmic reticulum did not differ from normal pregnancies. Stringent control experiments were performed also to ensure specific detection of G6Pase activity. The results indicate that cytochemically detectable G6Pase is present in the human placenta. This enzyme may play significant roles in carbohydrate metabolism in the human placenta.

Choriocarcinoma-trophoblast hybrid cells: Reconstructing the pathway from normal to malignant trophoblast — Concept and perspectives: A review

Extravillous trophoblast cells of normal and molar pregnancies spontaneously can transform into choriocarcinoma cells. This malignant transformation differs from that in other tissues in two respects: (a) the normal tissue of origin, extravillous trophoblast, is already invasive, and (b) both, choriocarcinoma and extravillous trophoblast, are foreign (allohaploid) to the host. Carcinogenetic transformation of trophoblast into choriocarcinoma is a multistep process in the course of which temporary intermediate geno- and phenotypes are generated; these are mixtures between trophoblast and choriocarcinoma and illustrate the process of carcinogenesis. The intermediates are either selected by the host for survival or undergo further mutations. Consequently, it remains unknown which properties are lost or acquired during the process and which of them are crucial either for control of invasion of normal extravillous trophoblast or for the malignant development of choriocarcinoma cells. In order to identify mutative trophoblast-relevant oncogenes and tumor suppressor genes, we have generated somatic cell hybrids using the choriocarcinoma cell line AC1-1 and extravillous trophoblast cells. The resulting hybrid cells were cloned, characterized cytogenetically and classified according to tumorigenicity. These hybrids represent a population of intermediates between non-tumorigenic extravillous trophoblast cells and highly tumorigenic choriocarcinoma cells, similar to those temporarily formed during carcinogenesis. The hybrids are thought to represent a suitable in vitro tool for the identification of genes and gene products involved in both control of normal invasion and malignant transformation of trophoblast cells. Analysis of the hybrid clones involves both, methods assessing phenotypic (functional) and genotypic properties.

Expression of matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and transforming growth factor-β1 (TGF-β1) in human placenta during preeclampsia

Summary Matrix metalloproteinase-2, -9 (MMP-2, -9) and tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2) possibly play important roles in the invastion of trophoblast. We noted that the abnormal shallow invasion of trophoblast observed in preeclampsia may be associated with the alteration of MMP, TIMP and its regulator for secretion. Expression of MMP-2, MMP-9, TIMP-2 and TGF-β1 in preeclampsia and normal pregnancy was examined by EIA, zymographic analysis and immunohischemistry. TIMP-1 levels in serum, placental bed and placenta in preelampsia cases were significantly elevated. TGF-β1 levels in both plasma and placental bed were higher in preeclampsia. Zymographic analysis of EIA showed increased secretion of MMP-9 in preeclampsia. TIMP-1 immunostaining in trophoblast cells invading into preeclamptic placental bed tissues was much more prominent than normal trophoblast cells. These data suggested that the increased expressions of TIMP-1 and TGF-β1 may inhibit the invasion of trophoblast into the decidua and myometrium, which is closely associated with preeclampsia.

Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies

Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl 8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal–fetal interface remain unclear.

In vitro modulation of L‐arginine transport in trophoblast cells by glucose

The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.

Etiology and pathogenesis of preeclampsia: Current concepts

The etiology of preeclampsia is unknown. At present, 4 hypotheses are the subject of extensive investigation, as follows: (1) Placental ischemia—Increased trophoblast deportation, as a consequence of ischemia, may inflict endothelial cell dysfunction. (2) Very low-density lipoprotein versus toxicity-preventing activity—In compensation for increased energy demand during pregnancy, nonesterified fatty acids are mobilized. In women with low albumin concentrations, transporting extra nonesterified fatty acids from adipose tissues to the liver is likely to reduce albumin’s antitoxic activity to a point at which very-low density lipoprotein toxicity is expressed. (3) Immune maladaptation—Interaction between decidual leukocytes and invading cytotrophoblast cells is essential for normal trophoblast invasion and development. Immune maladaptation may cause shallow invasion of spiral arteries by endovascular cytotrophoblast cells and endothelial cell dysfunction mediated by an increased decidual release of cytokines, proteolytic enzymes, and free radical species. (4) Genetic imprinting—Development of preeclampsia-eclampsia may be based on a single recessive gene or a dominant gene with incomplete penetrance. Penetrance may be dependent on fetal genotype. The possibility of genetic imprinting should be considered in future genetic investigations of preeclampsia. (Am J Obstet Gynecol 1998;179:1359-75.)