Fetal Rat Hearts Do Not Display Acute Cardiotoxicity in Response to Maternal Doxorubicin Treatment s

ABSTRACT Anthracyclines are used to treat cancers during the second and third trimester of pregnancy. The chemotherapeutic effect of anthracyclines is associated with a dose-and time-dependent cardiotoxicity that is well described for infants and adults. However, data regarding fetal anthracycline-related cardiotoxicity after administration of chemotherapeutics during pregnancy are limited. In this study, we analyzed the acute effect of doxorubicin, an anthracycline derivative, on fetal and maternal rat myocardium. We injected 10 or 20 mg/kg i.v. doxorubicin to pregnant Wistar rats at day 18 of pregnancy; age-matched pregnant rats injected with physiologic saline served as controls. Maternal echocardiography and fetal Doppler scanning were performed before the injection and before sacrifice. Cesarean operation was performed at day 19 or 20, and maternal and fetal blood samples and heart biopsies were collected to measure apoptosis, the impact on cell proliferation, and structural cardiac damage. Acute maternal cardiotoxicity is associated with loss of body weight, moderately deteriorated left ventricular function, induction of apoptosis, and a decrease in cell turnover. Despite a 30% lower fetal body weight and elevated plasma B-type natriuretic peptide concentrations after doxorubicin administration, the fetal hearts had intact microstructure, an unaltered number of apoptotic cells, and preserved cell proliferation compared with controls. Our study suggests that acute treatment using anthracyclines during pregnancy impairs maternal cardiac function, whereas fetal hearts are protected

Genic expression of human placental inhibin and activin subunits

Thèse de doctorat en sciences biomédicales (biologie de le reproduction) (SBIM 3)--UCL, 200

Control of the human inhibin alpha chain promoter in cytotrophoblast cells differentiating into syncytium.

Inhibins are dimeric proteins consisting of a common alpha subunit linked to one of the beta subunits, beta A or beta B. During pregnancy, the placenta is the main source of inhibin A production and the in-vitro transformation of cytotrophoblast cells into syncytium is associated with an inhibin alpha subunit mRNA up-regulation. In this study, the 5' region of the human inhibin alpha gene was isolated and sequenced. Three transcription initiation sites were identified. When transiently transfected in trophoblast cells with a luciferase reporter vector, the sequence displayed promoter activity. DNase I footprinting and electrophoretic mobility shift assay (EMSA) analysis showed a specific DNA-protein interaction in the promoter when using cytotrophoblast nuclear proteins. This interaction was weaker with syncytiotrophoblast nuclear proteins. Moreover, the deletion of this DNA-protein interaction region suppressed the promoter activity. In an attempt to identify this factor, the potential binding of known factors delta EF1, AP1 and NFE2 were excluded by competition EMSA experiments. We suggest that it may correspond to an undescribed protein interaction. The identification of the human inhibin alpha promoter could help in understanding the mechanisms modulating inhibin gene transcription. Moreover, the identification of a factor, whose presence is related to the trophoblast cell differentiation state, could help in understanding the transformation of cytotrophoblast cells into syncytium

Is 8% O2 more normoxic than 21% O2 for long-term in vitro cultures of human primary term cytotrophoblasts?

STUDY QUESTION: Is 8% O2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O2 traditionally used in cell culture? SUMMARY ANSWER: Human primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels. WHAT IS KNOWN ALREADY: Cell culture is traditionally done under 21% O2, which is equal to a pO2 of ~160 mm Hg. Based on the pO2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should under 8% O2, which is equivalent to 60 mm Hg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts. STUDY DESIGN SIZE, DURATION: Cytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O2 and 8% O2 for 12 days. Several cellular parameters were assessed on days 2, 4, 8, and 12. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGβ, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H2O2 generation. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference between 21% O2 and 8% O2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O2 compared to 8% O2. The expression level of hCGβ was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O2, while PlGF expression was inhibited compared to 21% O2. This latter result indicates that 8% O2 may be more hypoxic than normoxic for in-vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H2O2 generation) in our cell cultures. LARGE-SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: The in-vitro culture of cytotrophoblasts is artificial and does not reflect the in-vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%. WIDER IMPLICATIONS OF THE FINDINGS: It is possible to culture human primary term cytotrophoblasts for at least 12 days. The O2 percentage of the air does not negatively affect in-vitro cytotrophoblast differentiation. For in-vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%. STUDY FUNDING/COMPETING INTEREST(S): This work was fully supported by 'Fetus for Life' charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper

Inhibin and activin production and subunit expression in human placental cells cultured in vitro.

Inhibins and activins are dimeric proteins, with each subunit being one of three related protein subunits (alpha, betaA or betaB). The mRNA levels of these subunits were studied quantitatively during in-vitro differentiation of human cytotrophoblast cells into syncytium, using Northern blot analysis and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The corresponding protein concentrations were determined by specific enzyme-linked immunosorbent assays for inhibin A, B, pro alphaC and activin A in cellular protein extracts and culture medium (n = 5). Immunofluorescence studies showed syncytium formation after 48 h. The alpha subunit was present before plating and increased at 48 h (P<0.001) while the betaA subunit was weak before plating and increased at 24 h. The betaB subunit was not detected. With respect to corresponding protein synthesis, inhibin A (alpha + betaA) had risen after 48 h in cellular protein extract and after 72 h in culture medium, while activin A (betaA + betaB) was detected after 24 h, with no significant variations in culture medium. There was a good correlation between inhibin A and alpha subunit expression (r = 0.736, P<0.001), as well as between activin A and betaA subunit expression (r = 0.755, P<0.001). This study showed that mRNA expression parallels protein synthesis of inhibin and activin in trophoblast cells. Inhibin A synthesis appears to be dependent on alpha subunit mRNA expression, rather than on the betaA subunit which controls activin A synthesis. This study has also shown that isolated cytotrophoblast cells do not produce dimeric inhibin. However, during the transformation of cytotrophoblast cells into syncytium, betaA subunit mRNA expression may be an indicator of cell aggregation, while alpha subunit mRNA expression may be an indicator of cell fusion

Transcription factor AP2 regulates human inhibin α subunit gene expression during in vitro trophoblast differentiation.

During human trophoblast differentiation, inhibin alpha subunit mRNA expression and protein secretion are increased. To understand how inhibin alpha subunit gene was regulated during syncytialization, we firstly cloned the inhibin alpha promoter and found a region with transcriptional activity related to the differentiation state. In this paper, we identified this protein and defined its DNA binding site. Protein purification and identification was done by DNA affinity chromatography followed by mass spectrometry and western blotting. In order to confirm the identity of the protein, characterize its DNA binding properties and to measure its expression during in vitro trophoblast differentiation, gel retardation assays and real-time polymerase chain reaction were done. We found that the cytotrophoblastic protein interacting with the inhibin alpha promoter was the transcription factor Activating Protein 2 (AP2). Western blotting using specific antibodies against AP2alpha and AP2gamma confirmed that AP2alpha was the main subtype present in trophoblast cells while AP2gamma was barely detectable. Supershift experiments indicated that these two factors were able to bind to the sequence 5'-GCCtcaAGC-3'. We also observed an increase in AP2alpha mRNA and protein during in vitro trophoblast differentiation correlated with an increase in inhibin alpha subunit gene expression. Furthermore, AP2alpha and AP2gamma overexpression in these cells was responsible for an increase in inhibin alpha subunit mRNA expression. We conclude that AP2 regulates the inhibin alpha subunit gene expression during trophoblast differentiation and may be a key regulator of syncytializatio