Aims/hypothesis. Early intrauterine growth delay in diabetes could be caused by a reduced growth of the placenta. Our study investigates whether hyperglycaemia in vitro reduces trophoblast proliferation. Methods. First-trimester trophoblast cell models (BeWo, JAR and JEG-3 choriocarcinoma cells) were cultured for 24 and 48 h with 5.5 mmol/l d-glucose, 25 mmol/l d-glucose (hyperglycaemia) and with an osmotic control. Cell number, total protein and nucleic acid content and mitochondrial activity (tetrazolium salt assay) were measured, the cell cycle analysed (FACS, cyclin B1 levels) and apoptosis (Annexin-V) measured. Results. In BeWo cells hyperglycaemia reduced cell number, protein, nucleic acid and cyclin B1 levels. The reduced G2/M and increased G0/G1 population after 24 h reflects growth arrest at G0/G1. In JAR cells after 24 h the population was arrested in G0/G1, whereas after 48 h the G0/G1 block was abrogated and the cells were arrested at G2/M. The net effect was an unchanged cell number. In JEG-3 cells hyperglycaemia resulted in fewer cells after 24 h but not after 48 h indicating some adaptation. Mitochondrial activity was either stimulated (BeWo) or reduced (JAR, JEG-3) under hyperglycaemia. Some of these effects were also induced by hyperosmolarity alone. Conclusion/interpretation. Hyperglycaemia has the potential to inhibit the proliferation of first-trimester trophoblast cell models. The mechanisms leading to growth arrest and to changes in mitochondrial activity are complex and depend on differentiation. We hypothesise a hyperglycaemia-induced impairment of placental growth in the first trimester of a poorly controlled diabetic pregnancy. [Diabetologia (2001) 44: 209–219]

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.

International Journal of Gynecology & ObstetricsVolume 70, Issue S1 p. A72-A72 Uterine cancer Viability and adhesion of hysteroscopically disseminated endometrial cancer cells in vitro M.G. Arikan, M.G. ArikanSearch for more papers by this authorU. Weiss, U. WeissSearch for more papers by this authorO. Reich, O. ReichSearch for more papers by this authorT. Hahn, T. HahnSearch for more papers by this authorH. Pickel, H. PickelSearch for more papers by this authorK. Tamussino, K. TamussinoSearch for more papers by this authorM. Cervar, M. CervarSearch for more papers by this authorG. Desoye, G. DesoyeSearch for more papers by this author M.G. Arikan, M.G. ArikanSearch for more papers by this authorU. Weiss, U. WeissSearch for more papers by this authorO. Reich, O. ReichSearch for more papers by this authorT. Hahn, T. HahnSearch for more papers by this authorH. Pickel, H. PickelSearch for more papers by this authorK. Tamussino, K. TamussinoSearch for more papers by this authorM. Cervar, M. CervarSearch for more papers by this authorG. Desoye, G. DesoyeSearch for more papers by this author First published: 09 December 2003 https://doi.org/10.1016/S0020-7292(00)82718-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume70, IssueS12000Pages A72-A72 RelatedInformation

Endothelin (ET) secretion and expression of both ET-A and ET-B receptor subtypes have been found in a number of primary cancers. The present study tested (1) whether choriocarcinoma cells and their nonmalignant counterpart, the trophoblast, secrete ET-1 and express ET-A and ET-B receptors; (2) whether ET-1 secretion and receptor mRNA levels are regulated by the same factors in nonvascular tissues as in vascular tissues; and (3) whether such regulation is similar in malignant and nonmalignant cells. All cells secreted ET-1 in similar amounts (approximately 0.8 fmol/10(6) cells per 24 h) and secretion was unaffected by culture and treatment. Whereas ET-B accounted for almost all (>98%) ET receptor transcripts in the choriocarcinoma cells, the trophoblasts expressed about 20% ET-A receptor mRNA. During control cultures, ET-B mRNA levels rose in choriocarcinoma, with the greatest relative increase (6-fold; P < 0.05 vs 0 h) in BeWo, whereas in trophoblasts, ET-A mRNA transiently changed after 24 and 48 h. Treatment with dexamethasone and glucose did not alter the mRNA levels in all cells. Insulin induced changes (P < 0.05) in ET-B mRNA levels in BeWo (+90 and +60% after 24 and 48 h, respectively) and JEG-3 (-70%), but not in JAR and trophoblast cells. We conclude that malignant transformation affects the responsiveness of the endothelin receptor system to external stimuli and that the regulation of the endothelin system differs in vascular and nonvascular tissues.

Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33–35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development.

In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (–IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; –IP ∼70%) as identified by rigorous immunocytochemistry. Most (∼70%) non-trophoblast cells in –IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-β (hCG-β) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-β: 18-fold) in +IP than in –IP, with the exception of endothelin-1,2 (no change), angiotensin II (–70%) and 6-keto-prostaglandin-F1α (–40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in –IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.

Endothelins and their receptors are the most potent vasoconstricting system which has been identified in vascular tissues including human placenta. Over the past years the expression of this system in a variety of non-vascular tissues has suggested the existence of functions not related to the regulation of vascular tone. In the human trophoblast endothelin biosynthesis and secretion along with endothelin-binding sites were identified indicating autocrine or paracrine regulation of yet unidentified ET-effects. Changes in the ET-system of term trophoblast in preeclampsia suggest its implication in this disease, which originates from inadequate trophoblast invasion in the first trimester. Recent results with first trimester trophoblast showing a mitogenic activity, and the discovery of the novel invasion-promoting effect have opened new avenues of research in the field of trophoblast-derived endothelin-1 and its receptors.

OBJECTIVE: We hypothesized that aspirin, Mg++, and dihydralazine affect the release of vasoactive agents from cultured human placental trophoblast.STUDY DESIGN: Cytotrophoblasts isolated from placentas of preterm or term deliveries of 14 healthy control women and 15 preeclamptic women were cultured in Dulbecco's modified Eagle's medium for 5 days in the presence or absence of either 0.1 mmol/L aspirin, 3 mmol/L magnesium chloride, or 136 ngl mi dihydralazine. Vasoactive substances were quantitated by radioimmunoassay with mean +/- SEM percentage of untreated cells (= 100%) compared by the Mann-Whitney U test and analysis of variance.RESULTS: Aspirin inhibited (p < 0.01) both thromboxane and prostacyclin on days 1 and 2 in culture but not on days 3 to 5 unless the Dulbecco's modified Eagle's medium was supplemented with arachidonic acid. Aspirin inhibition was greater lo < 0.01) for thromboxane in cells cultured 24 hours after preeclamptic pregnancy (preterm 29.9% +/- 6.8%, term 20.1% +/- 5.9%) compared with normal controls (preterm 66.3% +/- 10.6%, term 68.9% +/- 11.6%). Aspirin reduced (p < 0.01) the ratio of thromboxane to prostacyclin in media of cells from preeclampsia (untreated 27.8 +/- 7.2, aspirin 13.3 +/- 4.4), but aspirin had no effect on this ratio in cultures from control normal pregnancies (untreated 6.8 +/- 2.9, aspirin 4.8 +/- 1.1). Neither magnesium chloride nor dihydralazine affected trophoblast prostanoid production, and no drug altered the media levels of angiotensin II, endothelin-1, or leukotriene B-4.CONCLUSION: Aspirin selectively inhibits trophoblast prostanoid production. This inhibition depends on the availability of arachidonic acid and the presence or absence of preeclampsia. Magnesium and dihydralazine effects in pregnancy are not related to altered release of trophoblast vasoactive compounds.

Three regions of the human placental lactogen (hPL) promoter that contain several half-site motifs that closely resemble the responsive elements for thyroid hormone (TR), all trans retinoic acid (RAR) and 1,25 dihydroxyvitamin D3 (VDR) have been identified and characterized. Transfection studies in BeWo choriocarcinoma cells indicate that site A (nt −979 to −954) is responsive to RARα but not TRβ. Site B (nt −1140 to −1170) is responsive to both RARα and TRβ, and site C (nt −550 to −580) is not responsive to either RARα or TR. These findings, together with the observation that placental cells express retinoid receptors and TRs, strongly suggest a role for these receptors in the regulation of the hPL gene. Site B on the hPL promoter is able to integrate the responses to RA and T3 through a single element.

Three regions of the human placental lactogen (hPL) promoter that contain several half-site motifs that closely resemble the responsive elements for thyroid hormone (TR), all trans retinoic acid (RAR) and 1,25 dihydroxyvitamin D3 (VDR) have been identified and characterized. Transfection studies in BeWo choriocarcinoma cells indicate that site A (nt −979 to −954) is responsive to RARα but not TRβ. Site B (nt −1140 to −1170) is responsive to both RARα and TRβ, and site C (nt −550 to −580) is not responsive to either RARα or TR. These findings, together with the observation that placental cells express retinoid receptors and TRs, strongly suggest a role for these receptors in the regulation of the hPL gene. Site B on the hPL promoter is able to integrate the responses to RA and T3 through a single element.

The aim of the study was to investigate whether pre-eclampsia is associated with an altered release of vasoactive substances from trophoblastic cells in vitro. Trophoblastic cells from 15 uncomplicated control pregnancies and 18 pre-eclamptic pregnancies at preterm (weeks 31-36; n = 12) and term (weeks 37-40; n = 21) were cultured for 5 days. The concentrations of angiotensin II (AII), endothelin-1,2 (ET-1,2), thromboxane B-2 (TXB(2)), 6-keto-prostaglandin F-1 alpha (6-keto-PGF1(alpha)) and leukotriene B-4 (LTB(4)) were measured daily in culture media for 5 days by radioimmunoassay. In pre-eclampsia, concentrations of ET-1,2 were decreased (P < 0.01) at both preterm and term, TXB(2) concentrations were increased (P < 0.05) only at preterm and the TXB(2)-6-keto-PGF(1 alpha) ratio was increased at both preterm and term (P < 0.01) as compared with the controls. Concentrations of AII, 6-keto-PGF(1 alpha) and LTB4 were similar to the controls. The data suggest that pre-eclampsia is associated with a decreased release of ET-1 and an increased release of TXB(2) from trophoblastic cells in vitro.

BACKGROUND:Endothelin-1 is a potent mitogen and stimulates cell chemokinesis, but these properties have not been investigated in the placenta. Trophoblastic cells from pre-eclamptic pregnancies have a reduced invasive potency and secrete less endothelin-1 than those from normal pregnancies. The present study tested the hypothesis that endothelin-1 affects trophoblast proliferation and invasion. METHODS:Trophoblastic cells were isolated from 37 placentae of normal human pregnancies at week 12 by dispastrypsin digestion and subsequently immunopurified to remove nontrophoblastic components. The effects of 5, 10, and 20 nmol/mL endothelin-1 on proliferation and invasion were determined after 24 and 72 hours, respectively, by fluorescence-activated pulse cytometry (FACS) analysis, by measuring DNA synthesis using two different methods and by a Matrigel invasion assay. RESULTS:All cell preparations uniformly responded to 10 nmol/mL by increased proliferation, owing to a greater proportion of cells in the S-phase of their cell cycle, and invasion. The effects were more pronounced after 24 hours than after 72 hours, by which time cell viability, as measured by the secretion of human chorionic gonadotropin (hCG-beta), had deteriorated. The nonselective receptor antagonist PD 142893 inhibited both endothelin-1 effects. CONCLUSION:Endothelin-1 is a mitogenic stimulus for first trimester trophoblastic cells in vitro. The stimulation of cellular invasion represents a novel effect of endothelin-1. We suggest the implication of endothelin-1 in proliferation and invasion of trophoblast and tumour cells and hypothesize a possible role in the etiology of pre-eclampsia.