Publications (19) View all
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Article: Trophoblast invasion: assessment of cellular models using gene expression signatures.
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ABSTRACT: Invasive, extravillous trophoblasts (EVT) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular markers with their primary counterpart, it is unknown to what extent they recapitulate the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs). Analyses of approximately 14,000 commonly expressed genes revealed that EVTs most closely resemble CTBs with considerable differences to the group of choriocarcinoma cells (JEG-3, BeWo, ACH-3P) and the group of SV40 Large T Antigen-selected cell types (SGHPL-5, HTR-8/SVneo). Similarly, analyses of 912 genes discriminating EVT from CTB, or 370 EVT-specific genes did not unravel a particular cell line with close similarity to any of the primary cell types, although molecular signatures common to EVT and each group of cell lines could be identified. Considering the diversity of mRNA expression patterns it is suggested that molecular studies in trophoblast cell lines require verification of the critical steps in an appropriate primary model system.Placenta 11/2010; 31(11):989-96. · 3.69 Impact Factor -
Article: Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization.
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ABSTRACT: Decidualization, the differentiation process of maternal uterine stromal cells into secretory decidual cells, is a prerequisite for successful implantation and progression of pregnancy. For in vitro differentiation mostly primary human endometrial stromal cells (HESC) isolated from uterine samples after hysterectomy for benign gynaecological diseases are utilised. However, a continuous supply of endometrial tissue is often lacking. Hence, we analysed whether cultivated human decidual stromal cells (HDSC) prepared from first trimester pregnancy terminations may represent an alternative model system for in vitro decidualization. Moreover, based on the expression of critical marker genes these cells were compared to a previously established endometrial stromal cell line during in vitro differentiation. HDSC isolated from decidual tissue attached to first trimester placentae, and telomerase-transformed human endometrial stromal cells (THESC) were characterised by immunofluorescence and differentiated in vitro using either cyclic adenosine monophosphate (cAMP) and/or estrogen (E2)/progesterone (P4). Proliferation was measured by analyzing cumulative cell numbers. Expression of mRNAs encoding progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1) was evaluated using quantitative PCR after 3, 6, 9 and 12 days of in vitro differentiation. PRL and IGFBP-1 protein expression was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Furthermore, forkhead box O1A (FOXO1A), a critical transcription factor in decidualization, was analysed by immunofluorescence and Western blotting at two different time points of differentiation. Treatment with cAMP provoked morphological changes and growth arrest of THESC and HDSC, the latter showing loss of cells after 6 days of treatment. E2P4 stimulation did neither affect cell morphology nor proliferation of THESC and HDSC. Upon cAMP stimulation PR mRNA was suppressed in HDSC but not in THESC, whereas E2P4 did not alter transcript levels in both cell types. Protein expression of PR-A and PR-B was detectable in HDSC and diminished under cAMP, whereas THESC failed to produce the nuclear receptors. Supplementation of cAMP induced mRNA and protein expression of PRL and IGFBP-1 in both cell types at day 3, 6, 9, and 12 of treatment. In HDSC stimulation with E2P4 increased PRL and IGFBP-1 mRNA and protein production, whereas hormone treatment did not induce the two factors in THESC. E2P4 increased DKK1 mRNA at all time points in HDSC and cAMP provoked induction at day 9 and 12 of differentiation. In contrast, cAMP suppressed DKK1 mRNA in THESC, whereas E2P4 was ineffective. In both cell types combined treatments with cAMP and E2P4 provoked higher expression levels of PRL and IGFBP1 mRNA and protein as compared to cAMP stimulation alone. FOXO1A protein and its nuclear abundance were increased by cAMP in both cell types. However, reduction of its nuclear localisation upon E2P4 treatment could only be observed in HDSC. Both HDSC and THESC may represent suitable model systems for cAMP-dependent in vitro decidualization. Since cAMP decreases cell viability of HDSC after 6 days of incubation, this substance should be preferentially used in short-term experiments. Progesterone treatment of THESC might not be applicable since these cells lack progesterone response and PR protein. In contrast, stimulation of PR-expressing HDSC with E2P4 or cAMP/E2P4 may represent an appropriate protocol for human in vitro decidualization inducing and maintaining expression of critical marker genes in a time-dependent manner.Reproductive Biology and Endocrinology 12/2011; 9:155. · 2.05 Impact Factor -
Article: Endostatin suppresses IGF-II-mediated signaling and invasion of human extravillous trophoblasts.
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ABSTRACT: Endostatin, a biological active fragment of the extracellular matrix protein collagen XVIII, is known to interfere with cellular motility in the context of pathological angiogenesis. However, the physiological role of endostatin remains largely elusive. Recent evidence suggested that the inhibitor is produced in human decidual cells of early pregnancy, indicating that endostatin could be involved in diverse reproductive processes, such as implantation and/or placental differentiation. To gain more insights into the role of endostatin, we here analyzed its effects on trophoblast motility, proliferation, and signaling using purified primary trophoblasts, first-trimester villous explant cultures, and trophoblastic SGHPL-5 cells. In vitro Transwell assays demonstrated that purified endostatin inhibited both basal and IGF-II-induced migration and invasion as well as outgrowth from villous explant cultures. In contrast, basal and IGF-II-stimulated proliferation was unaffected upon addition of the inhibitor. Analyses of IGF-II-associated downstream signaling events showed that endostatin interfered with activation of various signaling kinases such as ERK1/2, protein kinase B (Akt)/mammalian target of rapamycin/p70 S6 kinase, and focal adhesion kinase. Furthermore, virus-mediated, stable gene silencing of Akt1 in SGHPL-5 cells using a micro-RNA-adapted short hairpin RNA-expressing plasmid revealed that endostatin-mediated inhibition of IGF-II-induced Akt phosphorylation was critically dependent on the expression of the particular isoform. In conclusion, the data suggest that endostatin could be a physiological inhibitor of IGF-II-dependent trophoblast cell motility by suppressing focal adhesion kinase/Akt/mammalian target of rapamycin/p70 S6 kinase signaling.Endocrinology 09/2011; 152(11):4431-42. · 4.46 Impact Factor -
Article: Transcription factor AP-2α promotes EGF-dependent invasion of human trophoblast.
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ABSTRACT: The basic helix-span-helix transcription factor activating protein (AP)-2α is critically involved in cell-specific hormone expression of syncytializing human trophoblasts. Its role in invasive trophoblast differentiation, however, remains largely elusive. Using RT-PCR, Western blotting, and immunofluorescence of first-trimester placentae, we here show that AP-2α is expressed in extravillous trophoblasts (EVTs) both in situ and in vitro as well as in invasive trophoblast cell lines. Its protein expression was increased upon supplementation of epidermal growth factor (EGF) both in primary EVTs and trophoblastic SGHPL-5 cells. Gene silencing of AP-2α using small hairpin microRNA (shRNAmir) did not affect basal invasion of SGHPL-5 cells through Matrigel-coated filters but reduced EGF-stimulated invasion. Similarly, treatment of primary EVTs with AP-2α small interfering RNA decreased EGF-dependent invasion. Proliferation of SGHPL-5 cells and primary EVTs, measured by cumulative cell numbers and 5-bromo-2'-deoxyuridine labeling, respectively, were not affected on loss of AP-2α. EGF-dependent induction of matrix metalloproteinase (MMP)-2, pro- and active form of urokinase plasminogen activator, and chorionic gonadotropin (CG)-β was noticed in shRNAmir-control cells, whereas these genes were suppressed in EGF-treated shRNAmir-AP-2α cells. Similarly, EGF-stimulated MMP-2 and CGβ protein expression was reduced in AP-2α small interfering RNA-treated primary EVTs. Knockdown of AP-2α also decreased luciferase activity of the CGβ5 promoter in SGHPL-5 cells, which was compensated upon transient overexpression of AP-2α cDNA. In conclusion, we show that AP-2α expression positively affects human trophoblast invasion under EGF-stimulated conditions, likely by inducing critical invasion-promoting genes such MMP-2, urokinase plasminogen activator, and CG.Endocrinology 02/2011; 152(4):1458-69. · 4.46 Impact Factor -
Article: Administration of betaHCG leads to dose-dependent changes of gene expression signature of endometriotic stromal cells.
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ABSTRACT: Preliminary studies have shown that systemic beta-human chorionic gonadotrophin (betaHCG) therapy alleviates endometriosis-related chronic pelvic pain. The underlying mechanism, however, is completely unknown. This study has investigated the dose-dependent alterations in the overall gene expression profile of endometriosis-derived stromal cells under increasing concentrations of betaHCG by using the Affymetrix GeneChip U133 Set. It has been previously shown that betaHCG concentrations of 0.1U/ml and higher lead to a significant and dose-dependent increase in the expression of 68 genes. This study reports on a cluster analysis which identified three clusters of genes with a comparable expression pattern in response to increasing concentrations of betaHCG. Most of the up-regulated genes encoded proteins that are involved in cell adhesion, intercellular communication, extracellular matrix remodelling, apoptosis and inflammation. Stromal monocultures from eight patients, treated with and without 50U/ml of betaHCG, were then incubated and real-time polymerase chain reaction for the highly up-regulated genes PAI2, DUSP6, PLAU and MMP1 performed in order to validate the cDNA array findings in patients with endometriosis. Taken together, this study shows that betaHCG induces dose-dependent characteristic response clusters in the gene expression profile of stromal cells obtained from endometriotic lesions which could explain the differential biological responses of betaHCG in endometriosis.Reproductive biomedicine online 05/2010; 20(5):699-706. · 2.04 Impact Factor
