Dora Baczyk

Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast

Mammalian placentation is a highly regulated process and is dependent on the proper development of specific trophoblast cell lineages. The two major types of trophoblast, villous and extravillous, show mitotic arrest during differentiation. In mice, the transcription factor, glial cell missing-1 (Gcm1), blocks mitosis and is required for syncytiotrophoblast formation and morphogenesis of the labyrinth, the murine equivalent of the villous placenta. The human homolog GCM1 has an analogous expression pattern, but its function is presently unknown. We studied GCM1 function in the human-derived BeWo choriocarcinoma cell line and in first trimester human placental villous and extravillous explants. The GCM1 expression was either inhibited by siRNA and antisense oligonucleotides methods or upregulated by forskolin treatment. Inhibition of GCM1 resulted in an increased rate of proliferation, but prevented de novo syncytiotrophoblast formation in syncytially denuded floating villous explants. GCM1 inhibition prevented extravillous differentiation along the invasive pathway in extravillous explants on matrigel. By contrast, forskolin-induced expression of GCM1 reduced the rate of proliferation and increased the rate of syncytialization in the floating villous explant model. Our studies show that GCM1 has a distinct role in the maintenance, development and turnover of the human trophoblast. Alterations in GCM1 expression or regulation may explain several aspects of two divergent severe placental insufficiency syndromes, namely preeclampsia and intrauterine growth restriction, which cause extreme preterm birth.

Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60-70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed 2 main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as 2 CpG sites mapping to each of 2 tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.

A Novel In Vitro Model of Trophoblast-Mediated Decidual Blood Vessel Remodeling

In vivo the extravillous trophoblasts (EVTs) penetrate the decidua and the first third of the myometrium to remodel the uterine spiral arteries and achieve the high-flow, low-resistance circulation characteristic of the intervillous space of the term placenta. Much of our understanding of these processes comes from histologic analysis of placental bed biopsies, a limited tissue source and one that can provide only a snapshot of a dynamic process. To better characterize these cellular interactions, we have developed an in vitro co-culture system in which first trimester villous explants are cultured at low oxygen tension in contact with 2-mm2 sections of decidua parietalis from the same patient. Hematoxylin eosin counterstaining of paraffin sections shows that EVT columns form at the tips of the placental villi and adhere and penetrate the decidual surface. The decidual blood vessels in the path of the EVT show morphologic disruption. Immunohistochemical analysis of the co-cultures using both an endothelial specific anti-CD31 and an anti–smooth muscle actin antibody show a disruption of the integrity of the vessel lining together with a complete loss of organized smooth muscle actin surrounding the blood vessels. In contrast control decidua samples in the absence of placental villi exhibit blood vessels with a complete endothelial lining and an organized muscular sheath. Using both an anti–cytokeratin-7 and anti–Cdx-2 antibody specific to trophoblasts, we show that these changes coincide with invasion of the vessels by endovascular trophoblasts and penetration of the decidua by interstitial EVTs. No EVTs were found in the control decidua. Thus we conclude that this in vitro model mimics the physiologic change observed in vivo during trophoblast invasion into maternal decidual tissues, and as such it may provide useful information concerning the interactions between EVTs and decidual cells and vessels during early gestation.

Translational Analysis of Mouse and Human Placental Protein and mRNA Reveals Distinct Molecular Pathologies in Human Preeclampsia

Preeclampsia (PE) adversely impacts ∼5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling

Summary.  Background: Severe preeclampsia is characterized by hypertension, renal injury and placental dysfunction. Prothrombotic disorders are discovered in 10–20% of women with preeclampsia, providing the rationale for prescribing low‐molecular‐weight heparin (LMWH) in future pregnancies. Heparin has diverse molecular actions and appears to reduce the recurrence risk of preeclampsia in women without prothrombotic disorders. The placenta‐derived anti‐angiogenic splice‐variant protein soluble vascular endothelial growth factor (VEGF) receptor‐1 (sFLT1) is strongly implicated in the pathogenesis of the underlying endothelial dysfunction. As the placental syncytiotrophoblast is the principal source of sFLT1, we tested the hypothesis that heparin suppresses placental sFLT1 secretion. Methods and Results: First trimester placental villi exposed to LMWH (0.25–25 IU mL−1) in an in vitro explant model significantly increased the expression and release of sFLT1 by the syncytiotrophoblast into culture media, reducing phosphorylation of FLT1 and KDR receptors in cultured human umbilical vein endothelial cells. This response was significantly diminished in placental villi from healthy term pregnancies. Placental villi from severely preeclamptic pregnancies had a higher baseline sFLT1 release, compared with first trimester placental villi and did not respond to LMWH treatment. LMWH promoted villous cytotrophoblast proliferation (BrdU incorporation) and impaired syncytial fusion‐differentiation, causing syncytiotrophoblast apoptosis (by caspase 3&7 activity and TUNEL staining) and necrosis (ADP/ATP ratio). Conclusion: LMWH promotes sFLT1 synthesis and release from first trimester placental villi in a manner similar to that of severely preeclamptic placental villi, which antagonizes VEGF signaling in endothelial cells. These effects in part are mediated by an interaction between heparin and the cytotrophoblasts that regenerates the overlying syncytiotrophoblast responsible for sFLT1 secretion into the maternal blood.

The Molecular Role of Connexin 43 in Human Trophoblast Cell Fusion

ABSTRACT Connexin expression and gap junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43)/gap junction A1 (GJA1) are required for cytotrophoblast fusion into the syncytium, the outer functional layer of the human placenta. Cx43 also impacts intracellular signaling through protein-protein interactions. The transcription factor GCM1 and its downstream target ERVW-1/SYNCYTIN-1 are key players in trophoblast fusion and exert their actions through the ERVW-1 receptor SLC1A5/ASCT-2/RDR/ATB(0). To investigate the molecular role of the Cx43 protein and its interaction with this fusogenic pathway, we utilized stable Cx43-transfected cell lines established from the choriocarcinoma cell line Jeg3: wild-type Jeg3, alphahCG/Cx43 (constitutive Cx43 expression), JpUHD/Cx43 (doxycyclin-inducible Cx43 expression), or JpUHD/trCx43 (doxycyclin-inducible Cx43 carboxyterminal deleted). We hypothesized that truncation of Cx43 at its C-terminus would inhibit trophoblast fusion and protein interaction with either ERVW-1 or SLC1A5. In the alphahCG/Cx43 and JpUHD/Cx43 lines, stimulation with cAMP caused 1) increase in GJA1 mRNA levels, 2) increase in percentage of fused cells, and 3) downregulation of SLC1A5 expression. Cell fusion was inhibited by GJIC blockade using carbenoxylone. Neither Jeg3, which express low levels of Cx43, nor the JpUHD/trCx43 cell line demonstrated cell fusion or downregulation of SLC1A5. However, GCM1 and ERVW-1 mRNAs were upregulated by cAMP treatment in both Jeg3 and all Cx43 cell lines. Silencing of GCM1 prevented the induction of GJA1 mRNA by forskolin in BeWo choriocarcinoma cells, demonstrating that GCM1 is upstream of Cx43. All cell lines and first-trimester villous explants also demonstrated coimmunoprecipitation of SLC1A5 and phosphorylated Cx43. Importantly, SLC1A5 and Cx43 gap junction plaques colocalized in situ to areas of fusing cytotrophoblast, as demonstrated by the loss of E-cadherin staining in the plasma membrane in first-trimester placenta. We conclude that Cx43-mediated GJIC and SLC1A5 interaction play important functional roles in trophoblast cell fusion.

Fusion Assays and Models for the Trophoblast

A healthy syncytium in the placenta is vital to a successful pregnancy. The trophoblast builds up the natural barrier between the mother and the developing fetus and is the site of gas, nutrition, and waste exchange. An inadequate formation of this tissue leads to several pathologies of pregnancy, which may result in fetal death during the second trimester or iatrogenic preterm delivery due to intrauterine growth restriction, preeclampsia, or abruption.Cytotrophoblastic cells fuse constantly with the overlying syncytiotrophoblast/syncytium to maintain the function of the trophoblast. Syncytin-1 is the only molecule known to directly induce fusion in the placental trophoblast. Many other proteins, such as gap junctions (e.g., connexin 40) and transcription factors, play a role in the molecular pathways directing the trophoblast turn over. Despite the significance of this process for successful placentation, the mechanisms regulating its activity remain poorly understood.In this chapter we present several different model systems that can be utilized to investigate the regulation of the cell fusion process in the trophoblast. We describe cell-based assays as well as tissue-related protocols. We show how fusion can be monitored in (1) BeWo cells as a trophoblast cell line model, (2) HEK239 using syncytin-1 as a fusion molecule, and (3) a floating villi explant model. Furthermore, we will present strategies to inhibit fusion in the different models. These techniques represent powerful tools to study the molecular mediators of cell fusion in the trophoblast.

Calcium signaling in placenta.

The placenta sustains the developing fetus throughout gestation and its major functions include nutrition, gas and waste exchange via a variety of passive or active mechanisms. Up to 30 g of calcium (Ca(2+)) actively crosses the trophoblast layer during human pregnancy. The Ca(2+) ion not only plays an important role for skeletal development but is also an essential second messenger. This review is intended to highlight the implications of Ca(2+) signaling during reproduction and specifically placentation. Initially, a Ca(2+) wave induces fertilization of the oocyte. The intracellular Ca(2+) concentration is key for the blastocyst implantation, proper placental development and function. Current knowledge of many proteins involved in placental Ca(2+) regulation and their function in pathologic conditions is largely limited. Recent studies, however, point to alterations in Ca(2+) homeostasis in placental pathologies such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR). A broader understanding of the role of Ca(2+) signaling during human reproduction may offer insight into impaired pregnancy outcomes.

Glial cell missing-1 mediates over-expression of tissue inhibitor of metalloproteinase-4 in severe pre-eclamptic placental villi

BACKGROUND Severe pre-eclampsia (sPE) causes significant maternal morbidity and intrauterine growth restriction as a result of severe placental dysfunction. Defects in the formation of both extra-villous and villous trophoblast are characteristic of this disease. The outer syncytiotrophoblast layer covering the placental villi develops syncytial knots and focal necrosis while reduced invasion of the extra-villous trophoblast results in a reduced maternal blood supply and ischemia of the placental villi. The transcription factor glial cell missing-1 (GCM1) regulates formation of both types of trophoblast. GCM1 expression is reduced in placental villi of women with sPE but the functional downstream consequences of reduced GCM1 expression are unknown. METHODS AND RESULTS In floating first trimester villous explants we demonstrated increased mRNA (2.5-fold, n = 12) and protein level (9.8-fold) of tissue inhibitor of metalloproteinase-4 (TIMP4) following repression of GCM1 (70 ± 7%) by small interfering-RNA, using RT-PCR and western blot, respectively. Similar increases in TIMP4 mRNA (4.2-fold, n = 7, P< 0.001 versus control) and protein levels were found following gene silencing of GCM1 in BeWo cells (<90% knock down of protein). TIMP4 protein was increased in placenta from women with sPE (3.5 ± 0.4 pg/µg, n = 8), compared with preterm (1.7 ± 0.17 pg/µg, n = 9) and term controls (1.6 ± 0.16 pg/µg, n = 9; P< 0.01; quantified by enzyme-linked immunosorbent assay and visualized using immunohistochemistry) with reduced GCM1 expression, mostly in the pathologic syncytial knots. CONCLUSIONS TIMP4 is a downstream target of GCM1 that may link the consequences of reduced GCM-1-directed trophoblast differentiation to histologic and functional components of disordered placentation in sPE.

PPAR-γ Regulates Trophoblast Differentiation in the BeWo Cell Model

Common pregnancy complications, such as severe preeclampsia and intrauterine growth restriction, disrupt pregnancy progression and impair maternal and fetal wellbeing. Placentas from such pregnancies exhibit lesions principally within the syncytiotrophoblast (SCT), a layer in direct contact with maternal blood. In humans and mice, glial cell missing-1 (GCM-1) promotes differentiation of underlying cytotrophoblast cells into the outer SCT layer. GCM-1 may be regulated by the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-γ); in mice, PPAR-γ promotes labyrinthine trophoblast differentiation via Gcm-1, and, as we previously demonstrated, PPAR-γ activation ameliorates disease features in rat model of preeclampsia. Here, we aimed to characterize the baseline activity of PPAR-γ in the human choriocarcinoma BeWo cell line that mimics SCT formation in vitro and modulate PPAR-γ activity to study its effects on cell proliferation versus differentiation. We report a novel negative autoregulatory mechanism between PPAR-γ activity and expression and show that blocking PPAR-γ activity induces cell proliferation at the expense of differentiation, while these remain unaltered following treatment with the agonist rosiglitazone. Gaining a deeper understanding of the role and activity of PPAR-γ in placental physiology will offer new avenues for the development of secondary prevention and/or treatment options for placentally-mediated pregnancy complications.