Trophoblast

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Lawrence W. Chamley - One of the best experts on this subject based on the ideXlab platform.

  • Side-Population Trophoblasts Exhibit the Differentiation Potential of a Trophoblast Stem Cell Population, Persist to Term, and are Reduced in Fetal Growth Restriction
    Stem Cell Reviews and Reports, 2020
    Co-Authors: Teena K. J. B. Gamage, Lawrence W. Chamley, Jasper J Perry, Katie Groom, Joanna L. James
    Abstract:

    Background Fetal growth restriction often results from poor placental function and is a major cause of stillbirth. Clinically, fetal growth restriction is difficult to diagnose and currently has no effective treatment. Trophoblasts are unique placental cells that form the feto-maternal interface and facilitate nutrient and gas exchange. Fetal growth restriction is linked to inadequate Trophoblast function. However, our understanding of the mechanisms underlying this dysfunction are poor, in part because of our inability to isolate and study the Trophoblast stem cells from which mature Trophoblasts arise in pathologic pregnancies. Methods Cells isolated from first-trimester placentae using the Hoechst side-population technique were propagated or differentiated into mature Trophoblasts. Side-population Trophoblasts were isolated from normal third-trimester and growth restricted placentae using the same technique. First and third-trimester side-population Trophoblasts were compared by microarray analysis. Results First-trimester side-population Trophoblasts could be propagated in an undifferentiated state or differentiated, via intermediate cytoTrophoblasts, into syncytioTrophoblast or extravillous Trophoblasts. Using the same technique, side-population Trophoblasts could be isolated from term placentae for the first time, demonstrating that while they were present at consistent levels throughout gestation (~3·5%), side-population Trophoblasts were significantly depleted in growth restricted pregnancies (0·32%). Conclusions Our novel method of isolating a population of human Trophoblast stem cell-like cells directly from human placental tissue throughout gestation provides the first insights into Trophoblast dysfunction in pregnancy pathologies. The depletion of side-population Trophoblasts in growth restricted placentae may contribute to poor placental function.

  • Side-Population Trophoblasts Exhibit the Differentiation Potential of a Trophoblast Stem Cell Population, Persist to Term, and are Reduced in Fetal Growth Restriction
    Stem cell reviews and reports, 2020
    Co-Authors: Teena Gamage, Lawrence W. Chamley, Jasper J Perry, Vicky Fan, Katie M. Groom, Joanna L. James
    Abstract:

    Fetal growth restriction often results from poor placental function and is a major cause of stillbirth. Clinically, fetal growth restriction is difficult to diagnose and currently has no effective treatment. Trophoblasts are unique placental cells that form the feto-maternal interface and facilitate nutrient and gas exchange. Fetal growth restriction is linked to inadequate Trophoblast function. However, our understanding of the mechanisms underlying this dysfunction are poor, in part because of our inability to isolate and study the Trophoblast stem cells from which mature Trophoblasts arise in pathologic pregnancies. Cells isolated from first-trimester placentae using the Hoechst side-population technique were propagated or differentiated into mature Trophoblasts. Side-population Trophoblasts were isolated from normal third-trimester and growth restricted placentae using the same technique. First and third-trimester side-population Trophoblasts were compared by microarray analysis. First-trimester side-population Trophoblasts could be propagated in an undifferentiated state or differentiated, via intermediate cytoTrophoblasts, into syncytioTrophoblast or extravillous Trophoblasts. Using the same technique, side-population Trophoblasts could be isolated from term placentae for the first time, demonstrating that while they were present at consistent levels throughout gestation (~3·5%), side-population Trophoblasts were significantly depleted in growth restricted pregnancies (0·32%). Our novel method of isolating a population of human Trophoblast stem cell-like cells directly from human placental tissue throughout gestation provides the first insights into Trophoblast dysfunction in pregnancy pathologies. The depletion of side-population Trophoblasts in growth restricted placentae may contribute to poor placental function.

  • Growing human Trophoblasts in vitro: a review of the media commonly used in Trophoblast cell culture.
    Reproduction (Cambridge England), 2020
    Co-Authors: Yohanes Nursalim, Katie M. Groom, Cherie Blenkiron, Lawrence W. Chamley
    Abstract:

    Trophoblasts are unique epithelial cells found only in the placenta. It has been possible to isolate and maintain human Trophoblasts in in vitro culture for many decades. During this period there have been a vast array of media and supplements reported for Trophoblast culture and often the reasons for using the media and specific supplements employed in any given laboratory have been lost in the 'mists of time'. After a gradual development over many years this field has recently changed, with the publication of several reports of the isolation, growth and differentiation of human Trophoblast stem or stem-like cells. This advance was made largely because of a greater understanding of the molecular pathways that control human Trophoblasts and availability of media supplements that can be used to manipulate those pathways. We have searched the literature and here summarise many of the different media and supplements and describe how and why they were developed and are used to culture human Trophoblasts.

  • The role of DNA methylation in human Trophoblast differentiation
    2018
    Co-Authors: Teena K. J. B. Gamage, Lawrence W. Chamley, William Schierding, Daniel Hurley, Robert J. Weeks, Erin C. Macaulay, Peter Tsai, Jackie L. Ludgate, Chandrakanth Bhoothpur, Joanna L. James
    Abstract:

    The placenta is a vital fetal exchange organ connecting mother and baby. Specialised placental epithelial cells, called Trophoblasts, are essential for adequate placental function. Trophoblasts transform the maternal vasculature to allow efficient blood flow to the placenta and facilitate adequate nutrient uptake. Placental development is in part regulated by epigenetic mechanisms. However, our understanding of how DNA methylation contributes to human Trophoblast differentiation is limited. To better understand how genome-wide methylation differences affect Trophoblast differentiation, reduced representation bisulfite sequencing (RRBS) was conducted on four matched sets of Trophoblasts; side-population Trophoblasts (a candidate human Trophoblast stem cell population), cytoTrophoblasts (an intermediate progenitor population), and extravillous Trophoblasts (EVT, a terminally differentiated population) each isolated from the same first trimester placenta. Each Trophoblast population had a distinct methylome. In line with their close differentiation relationship, the methylation profile of side-population Trophoblasts was most similar to cytoTrophoblasts, whilst EVT had the most distinct methylome. In comparison to mature Trophoblast populations, side-population Trophoblasts exhibited differential methylation of genes and miRNAs involved in cell cycle regulation, differentiation, and regulation of pluripotency. A combined methylomic and transcriptomic approach was taken to better understand cytoTrophoblast differentiation to EVT. This revealed methylation of 41 genes involved in epithelial to mesenchymal transition and metastatic cancer pathways, which likely contributes to the acquisition of an invasive EVT phenotype. However, the methylation status of a gene did not always predict gene expression. Therefore, while CpG methylation plays a role in Trophoblast differentiation, it is likely not the only regulatory mechanism involved in this process.

  • aspirin triggered lipoxin prevents antiphospholipid antibody effects on human Trophoblast migration and endothelial cell interactions
    Arthritis & Rheumatism, 2015
    Co-Authors: Angela M Alvarez, Lawrence W. Chamley, Melissa J Mulla, Angela P Cadavid, Vikki M Abrahams
    Abstract:

    Objective Antiphospholipid antibodies (aPL) interfere with several physiologic functions of human Trophoblasts, including reducing their ability to migrate, decreasing their production of angiogenic factors, and inducing an inflammatory response. This may provide the underlying mechanism by which aPL responses lead to recurrent pregnancy loss or preeclampsia in women with obstetric antiphospholipid syndrome (APS). Although treatment with heparin may reduce the rate of recurrent pregnancy loss, the risk of preeclampsia remains high. Therefore, alternative treatments are needed for the management of pregnant patients with APS. Since aspirin-triggered lipoxins (ATLs) have immune and angiogenic modulatory properties, the objective of this study was to determine the effects of the ATL 15-epi-lipoxin A4 on the function of aPL-altered human Trophoblasts in the first trimester of pregnancy. Methods A first-trimester human Trophoblast cell line (HTR8) was treated with mouse anti-human β2 -glycoprotein I monoclonal antibodies (aPL) in the presence or absence of the ATL 15-epi-lipoxin A4 . Trophoblast migration and interactions with endometrial endothelial cells were measured using Transwell and coculture assays. Trophoblast secretion of cytokines and angiogenic factors was measured by enzyme-linked immunosorbent assay. Results Treatment of HTR8 cells with ATL reversed the aPL-induced decrease in Trophoblast migration, an effect that appeared to be regulated through restoration of interleukin-6 production. Using a model of spiral artery transformation, aPL and sera from APS patients with pregnancy morbidity disrupted Trophoblast-endothelial cell interactions, and treatment with ATL restored the stability of the cocultures. In contrast, ATL treatment did not resolve the proinflammatory and antiangiogenic responses of Trophoblasts induced by aPL. Conclusion These findings indicate that ATLs may have some benefits in terms of preventing the effects of aPL on Trophoblast function, which raises the possibility of the use of ATLs as an adjuvant therapy in women with aPL.

Ursula Hiden - One of the best experts on this subject based on the ideXlab platform.

  • Expression of matrix metalloproteinase 12 is highly specific for non-proliferating invasive Trophoblasts in the first trimester and temporally regulated by oxygen-dependent mechanisms including HIF-1A
    Histochemistry and Cell Biology, 2018
    Co-Authors: Ursula Hiden, Gernot Desoye, Berthold Huppertz, Christian P. Eyth, Alejandro Majali-martinez, Carmen Tam-amersdorfer, Nassim Ghaffari Tabrizi-wizsy
    Abstract:

    During first trimester pregnancy, Trophoblast cells invade from the placenta into the maternal decidua where they anchor the placenta and remodel luminal structures like spiral arteries. This process depends on proteases secreted by invading Trophoblasts, which degrade extracellular matrix (ECM). We here aimed to identify proteases particularly important for Trophoblast invasion. We generated a list of proteases capable of degrading decidual ECM and Trophoblast integrins using MEROPS database and compared expression of these proteases between primary Trophoblasts isolated from first trimester placenta (FT, n  = 3), representing an invasive phenotype, vs Trophoblasts isolated from term pregnancy (TT, n  = 3), representing a non-invasive Trophoblast phenotype. Matrix metalloproteinase 12 ( MMP12 ) revealed highest expression levels in FT, with absent expression in TT. In situ hybridisation and immunofluorescence localised MMP12 specifically to extravillous Trophoblasts (evCT) whilst Ki67 co-staining revealed that proliferating Trophoblasts of the cell columns were almost negative for MMP12. Quantification revealed a decline in MMP12 positive evCT at the end of first trimester, when oxygen levels start rising. MMP12 promoter analysis identified potential binding sites for hypoxia-inducible factor (HIF-1) and other oxygen-sensitive transcription factors. Moreover, MMP12 protein was increased by low oxygen in FT in vitro and by addition of a HIF-1α activator. Collectively, MMP12 is a highly expressed protease specific for invasive evCT during the first trimester. MMP12 down regulation by increasing oxygen concentration enables temporal expression control of MMP12 and involves several mechanisms including HIF-1α. These findings suggest MMP12 involved in Trophoblast invasion during the first trimester.

  • GDM alters paracrine regulation of feto-placental angiogenesis via the Trophoblast
    Laboratory Investigation, 2017
    Co-Authors: Jelena Loegl, Gernot Desoye, Berthold Huppertz, Erika Nussbaumer, Silvija Cvitic, Ursula Hiden
    Abstract:

    Feto-placental angiogenesis and vascular development are tightly regulated by pro- and anti-angiogenic factors. Villous Trophoblast may be a major source of these factors. It forms the classical placental barrier between mother and fetus, and is thus exposed to maternal influences as well. Metabolic and hormonal derangements in gestational diabetes mellitus (GDM) affect feto-placental angiogenesis and vascular growth. Here we hypothesized that GDM alters the Trophoblast secretome, which will modulate the paracrine regulation of feto-placental angiogenesis. Primary term Trophoblasts were isolated from normal ( n =6) and GDM ( n =6) pregnancies. Trophoblast conditioned medium (CM) was used to investigate paracrine effects of normal and GDM-exposed Trophoblasts on feto-placental endothelial cells (fpECs; n =7), using functional assays for 2D network formation, wound healing, chemotaxis, and proliferation. Gene expression of 23 pro- and anti-angiogenic factors was analyzed. Four Trophoblast-derived paracrine regulators of angiogenesis were specifically measured in CM. CM from GDM Trophoblasts increased 2D network formation of fpEC by 2.4-fold ( P

  • The first trimester human Trophoblast cell line ACH-3P: A novel tool to study autocrine/paracrine regulatory loops of human Trophoblast subpopulations – TNF-α stimulates MMP15 expression
    BMC developmental biology, 2007
    Co-Authors: Ursula Hiden, Christian Wadsack, Nicole Prutsch, Martin Gauster, U. Weiss, Hans-georg Frank, Ulrike Schmitz, Christa Fast-hirsch, Markus Hengstschläger, Andy J.g. Pötgens
    Abstract:

    Background The Trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester Trophoblast cell line (ACH-3P) by fusion of primary human first trimester Trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).

  • the first trimester human Trophoblast cell line ach 3p a novel tool to study autocrine paracrine regulatory loops of human Trophoblast subpopulations tnf α stimulates mmp15 expression
    BMC Developmental Biology, 2007
    Co-Authors: Ursula Hiden, Christian Wadsack, Nicole Prutsch, Martin Gauster, U. Weiss, Hans-georg Frank, Ulrike Schmitz, Markus Hengstschläger, Christa Fasthirsch, Andy J.g. Pötgens
    Abstract:

    Background The Trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester Trophoblast cell line (ACH-3P) by fusion of primary human first trimester Trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).

  • KISSPEPTINS AND THE PLACENTA: REGULATION OF Trophoblast INVASION
    Reviews in endocrine & metabolic disorders, 2007
    Co-Authors: Ursula Hiden, Martin Bilban, Martin Knofler, Gernot Desoye
    Abstract:

    The invasion of extravillous Trophoblasts into the uterine wall is of crucial importance for placental and fetal development, and its dysregulation has been implicated in a wide spectrum of abnormal pregnancies. Mechanistically, Trophoblast invasion strongly resembles the invasion of tumour cells, but differs from it by tight regulation in time and space. This regulation is accomplished by different factors including cytokines and hormones, which are produced by both fetal as well as maternal tissues i.e., placenta and uterus, respectively. Recently, products of the KiSS-1 gene (kisspeptins) have been identified to not only inhibit metastasis in various tumours, but also to repress Trophoblast invasion via binding to the G protein-coupled receptor KiSS-1R. In the placenta, expression levels of kisspeptins and their receptor are highest in the first trimester in humans and at day 12.5 in rats, respectively. This coincides with the time when invasiveness peaks and invasion regulation is of central importance. Human kisspeptins are predominantly produced by the syncytioTrophoblast, whereas KiSS-1R is additionally expressed on the invading extravillous Trophoblasts indicating a paracrine regulation of extravillous Trophoblast invasion by the syncytioTrophoblast. In the structurally different rat placenta both KiSS-1 and its receptor are predominantly expressed by the invasive Trophoblast giant cells, thus establishing an autocrine system in the invasion regulation of this Trophoblast subpopulation. Amongst all kisspeptins the highly conserved kisspeptin Kp-10 has strongest invasion inhibiting effects suggesting its major role in regulation of Trophoblast invasion.

Andy J.g. Pötgens - One of the best experts on this subject based on the ideXlab platform.

Martin Gauster - One of the best experts on this subject based on the ideXlab platform.

  • Phospholipid scramblase 1 (PLSCR1) in villous Trophoblast of the human placenta
    Histochemistry and Cell Biology, 2015
    Co-Authors: Veronika M. Berghold, Martin Gauster, Gerit Moser, Monika Siwetz, Monika Sundl, Denise G. Hemmings, Julia Kremshofer, Berthold Huppertz
    Abstract:

    A crucial factor for effective villous Trophoblast fusion in the human placenta is the transient deregulation of plasma membrane phospholipid asymmetry leading to externalization of phosphatidylserine to the outer membrane leaflet. Screening of scramblase family members implicated in the collapse of phospholipid asymmetry revealed that phospholipid scramblase 1 (PLSCR1) is strongly expressed in villous Trophoblast. Therefore, we assessed the putative role of PLSCR1 in villous Trophoblast fusion. Spatio-temporal analysis in first trimester and term placenta showed abundant expression of PLSCR1 in syncytioTrophoblast, macrophages and endothelial cells, while it was virtually absent in villous cytoTrophoblasts. For functional studies, BeWo cells, isolated primary term Trophoblasts and first trimester villous explants were used. During forskolin-mediated BeWo cell differentiation, neither PLSCR1 mRNA nor protein levels showed significant changes. In contrast, when primary Trophoblasts were stimulated with Br-cAMP, a decrease in PLSCR1 mRNA and protein expression was observed. To elucidate a role for PLSCR1 in syncytialization, we used RNA interference and a chemical scramblase inhibitor, R5421 (ethanedioic acid). Silencing of PLSCR1 using siRNA had no effects while inhibition of scramblase activity by R5421 increased GCM - 1 mRNA expression, beta-hCG protein secretion and fusion rates of BeWo cells. In primary Trophoblasts and villous explants, no effects of siRNA or R5421 treatment on fusion were detected. This study provides data on PLSCR1 localization and general expression in the human placenta. The data make it tempting to speculate on a role of PLSCR1 in negatively regulating Trophoblast fusion.

  • The art of identification of extravillous Trophoblast
    Placenta, 2010
    Co-Authors: Gerit Moser, Martin Gauster, K. Orendi, Monika Siwetz, C. Helige, Berthold Huppertz
    Abstract:

    Immunohistochemical staining with specific markers for the respective cell type facilitates tracking and identification of cells such as extravillous Trophoblast in the uterine wall. Cytokeratin has been recommended as a marker for all kinds of Trophoblasts and is commonly used as a marker to identify interstitial as well as endovascular Trophoblast. With immunohistochemical double staining of specimens of first trimester placental bed we show that staining with anti-cytokeratin alone is not sufficient to track all routes of Trophoblast invasion. Endovascular Trophoblasts can be easily mixed up with endoglandular Trophoblasts. Thus, additional application of specific markers for extravillous Trophoblast such as anti-HLA-G is strongly recommended, ideally in combination with other markers in immunohistochemical or immunofluorescence double staining.

  • Oxygen as modulator of Trophoblast invasion.
    Journal of anatomy, 2009
    Co-Authors: Berthold Huppertz, Martin Gauster, K. Orendi, Julia König, Gerit Moser
    Abstract:

    At the time of blastocyst implantation the uterine spiral arteries have already undergone morphological changes in the absence of any extravillous Trophoblast invasion. Only 2 weeks after implantation, extravillous Trophoblast cells develop and come into first contact with decidual tissues. Invading through the decidual interstitium, extravillous Trophoblasts potentially reach and transform spiral arteries into uteroplacental arteries. Spiral arterial erosion starts at about mid-first trimester, whereas flow of maternal blood into the intervillous space is continuously established only at the beginning of the second trimester. One key regulator of the number of extravillous Trophoblasts is oxygen. The steep gradient in oxygen concentration within the first trimester placenta is diminished with the onset of maternal blood flow. This gradient is used by the Trophoblast to generate a large number of invasive cells to adapt the arterial vasculature in the placental bed to the growing needs of the fetus. Changes in oxygen concentrations or other factors leading to alterations in the rates of proliferation and/or apoptosis of extravillous Trophoblast clearly impact on the remodelling of the vessels. The respective consequences of a failure in Trophoblast invasion are growth restrictions of the baby and perhaps other pregnancy complications.

  • The first trimester human Trophoblast cell line ACH-3P: A novel tool to study autocrine/paracrine regulatory loops of human Trophoblast subpopulations – TNF-α stimulates MMP15 expression
    BMC developmental biology, 2007
    Co-Authors: Ursula Hiden, Christian Wadsack, Nicole Prutsch, Martin Gauster, U. Weiss, Hans-georg Frank, Ulrike Schmitz, Christa Fast-hirsch, Markus Hengstschläger, Andy J.g. Pötgens
    Abstract:

    Background The Trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester Trophoblast cell line (ACH-3P) by fusion of primary human first trimester Trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).

  • the first trimester human Trophoblast cell line ach 3p a novel tool to study autocrine paracrine regulatory loops of human Trophoblast subpopulations tnf α stimulates mmp15 expression
    BMC Developmental Biology, 2007
    Co-Authors: Ursula Hiden, Christian Wadsack, Nicole Prutsch, Martin Gauster, U. Weiss, Hans-georg Frank, Ulrike Schmitz, Markus Hengstschläger, Christa Fasthirsch, Andy J.g. Pötgens
    Abstract:

    Background The Trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester Trophoblast cell line (ACH-3P) by fusion of primary human first trimester Trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1).

Richard E. Leach - One of the best experts on this subject based on the ideXlab platform.

  • mta1 and mta3 regulate hif1a expression in hypoxia treated human Trophoblast cell line htr8 svneo
    Medical journal of obstetrics and gynecology, 2013
    Co-Authors: Kai Wang, Susan D Ferguson, Ying Chen, Richard E. Leach
    Abstract:

    : Hypoxia plays an important role in placental Trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in Trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental Trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in Trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental Trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous Trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in Trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in Trophoblast function and differentiation during early pregnancy.

  • MTA1 and MTA3 Regulate HIF1a Expression in Hypoxia-Treated Human Trophoblast Cell Line HTR8/Svneo.
    Medical journal of obstetrics and gynecology, 2013
    Co-Authors: Kai Wang, Susan D Ferguson, Ying Chen, Richard E. Leach
    Abstract:

    : Hypoxia plays an important role in placental Trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in Trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental Trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in Trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental Trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous Trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in Trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in Trophoblast function and differentiation during early pregnancy.

  • 5 aza dc treatment induces mesenchymal to epithelial transition in 1st trimester Trophoblast cell line htr8 svneo
    Biochemical and Biophysical Research Communications, 2013
    Co-Authors: Ying Chen, Kai Wang, Richard E. Leach
    Abstract:

    Placental Trophoblast invasion involves a cellular transition from epithelial to mesenchymal phenotype. CytoTrophoblasts undergo epithelial to mesenchymal transition (EMT) when differentiating into extravillous Trophoblasts and gaining the capacity of invasion. In this research, we investigated the role of DNA methylation in Trophoblasts during this EMT. First, using BeWo and HTR8/SVneo cell lines as models of cytoTrophoblasts and extravillous Trophoblasts, respectively, we analyzed the gene expression and DNA methylation status of the known epithelial marker genes, E-Cadherin and Cytokeratin7. We found that, in HTR8/SVneo cells, both genes were silenced and their promoters hypermethylated, as compared with the high-level gene expression and promoter hypomethylation observed in BeWo cells. This result suggests that dynamic DNA methylation of epithelial marker genes plays a critical role in the Trophoblast EMT process. To verify these results, we treated HTR8/SVneo cells with 5-aza-dC, a known inhibitor of DNA methyltransferase, for three days. Five-Aza-dC treatment significantly increased the expression of epithelial marker genes and slightly decreased the expression of mesenchymal genes, as detected by qRT-PCR, immunocytochemistry and Western blot. Furthermore, 5-aza-dC treated HTR8/SVneo cells changed their morphology from mesenchymal into epithelial phenotype, indicating that 5-aza-dC induced mesenchymal to epithelial transition. Lastly, we examined the effect of 5-aza-dC on Trophoblast migration and invasion capacity. We applied 5-aza-dC to HTR8/SVneo cells in trans-well cell migration and invasion assays and found that 5-aza-dC treatment decreased Trophoblast migration and invasion capacity. In conclusion, DNA methylation of epithelial marker genes represents a molecular mechanism for the process of Trophoblast EMT.

  • mta1 and mta3 regulate hif1a expression in hypoxia treated human Trophoblast cell line
    2013
    Co-Authors: Kai Wang, Richard E. Leach, Ying Chen, Susan D Ferguson
    Abstract:

    Hypoxia plays an important role in placental Trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in Trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental Trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in Trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental Trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous Trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in Trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in Trophoblast function and differentiation during early pregnancy.