Histone deacetylase inhibition by trichostatin A mitigates LPS induced TNFα and IL-10 production in human placental explants.

INTRODUCTION: Cytokine expression by the placenta is known to change across pregnancy, and is altered in a number of pathologies; however the precise mechanisms of cytokine regulation in gestational tissues are not well understood. It has been previously reported that cytokine protein production in gestational tissues is regulated in a tissue-specific manner and appears to be epigenetically regulated. METHODS: In this study we investigated changes in cytokine mRNA expression and protein production by term placental explants maintained at 8% O2 in the presence or absence of lipopolysaccharide (LPS) (5 µg/mL) and the histone deacetylase inhibitor trichostatin A (TSA) (300 nM). RESULTS: As expected, exposure to LPS stimulated gene expression and protein production of the proinflammatory cytokines IL-1ß, IL-6, IL-8 and TNFα, as well as the anti-inflammatory cytokine IL-10. While TSA alone had little effect, TSA co-treatment mitigated the effects of LPS on TNFα and IL-10 protein production with an accompanying reduction in TNFα mRNA transcript levels detected. However, TSA had no significant effect on LPS induced IL-1ß, IL-1ra, IL-6 or IL-8 mRNA expression or protein production. DISCUSSION: The data from this study show that TSA selectively mitigates the stimulatory effect of LPS on TNFα mRNA expression, TNFα protein production and IL-10 protein production. As there is no compensatory effect on IL-1ß, IL-1ra, IL-6, or IL-8 mRNA expression or protein production, this results in a dysregulation of the cytokine balance. CONCLUSIONS: Insights into HDAC regulation of cytokine expression may provide novel therapeutic strategies for conditions associated with dysregulation of the cytokine network, such as preeclampsia and infection mediated preterm labor.

Passage of 4-nonylphenol across the human placenta.

4-nonylphenol (4-NP) is an estrogenic endocrine active chemical that is present in detergents and is known to contaminate food and drinking water. The aim of the present study was to evaluate whether 4-NP crosses the human placenta and if so, to what extent. Human placentae obtained from healthy term singleton pregnancies were utilized in a dual ex-vivo re-circulating model of placental perfusion. Six placentae were perfused for 180 min following addition of 4-NP (30 ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate the integrity of the circuits. 4-NP was measured by High Performance Liquid Chromatography coupled with Fluorescence Detection (HPLC-FD). After 180 min of perfusion the transfer percentages for antipyrine and 4-NP were 25.6 ± 1.4% (mean ± s.e.m, n = 6) and 22.75 ± 3.76% respectively and the transfer index for 4-NP was 0.8. We conclude that the intact form of 4-NP at environmentally relevant concentrations can transfer across the human placenta albeit at a slow rate.

Transplacental transfer and biotransformation of genistein in human placenta.

OBJECTIVE: To study transplacental transfer and biotransformation of genistein in the human placenta. STUDY DESIGN AND OUTCOMES: Human placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180min following addition of genistein (10ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC-MS). RESULTS: The transfer percentage for antipyrine and genistein was 25.6+/-1.40% and 22.1+/-1.61% respectively and the transfer index for genistein was 0.90+/-0.04 after 180min of perfusion. 12.0+/-2.40% of genistein in the fetal compartment and 7.36+/-4.73% of genistein in the maternal compartment were in the conjugated form. CONCLUSIONS: Genistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action.

Epigenetic regulation of endometrium during the menstrual cycle.

The endometrium undergoes morphological and functional changes during the menstrual cycle which are essential for uterine receptivity. These changes are driven by estrogen and progesterone and involve the fine control of many different genes-several of which have been identified as being epigenetically regulated. Epigenetic modification may therefore influence the functional changes in the endometrium required for successful implantation. There is, however, only limited information on epigenetic regulation in endometrium. We review the potential role of epigenetic regulation of key processes during the menstrual cycle and present our own findings following a preliminary study into global acetylation levels in the human endometrium. A changing epigenetic state is associated with the differentiation of stem cells into different lineages and thus may be involved in endometrial regeneration. Histone acetylation is implicated in the vascular endothelial growth factor pathway during angiogenesis, and studies using histone deacetylase inhibitors suggest an involvement in endometrial proliferation and differentiation. The processes of decidualization and implantation are also associated with epigenetic change and epigenetic modulators show variable expression across the menstrual cycle. Our own studies found that endometrial global histone acetylation, as determined by western blotting, changed throughout the menstrual cycle and correlated well with expected transcription activity during the different phases. This suggests that epigenetics may be involved in the regulation of endometrial gene expression during the menstrual cycle and that abnormal epigenetic modifications may therefore be associated with implantation failure and early pregnancy loss as well as with other endometrial pathologies.

Cyclic changes and hormonal regulation of annexin IV mRNA and protein in human endometrium.

Annexin IV (ANXA4) belongs to a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins. ANXA4 has been shown to be involved in a range of physiological functions including ion channel regulation, exocytosis and Ca(2+)-dependent signal transduction. The aims of this study were to fully characterize ANXA4 mRNA and protein in human endometrium during the menstrual cycle and to investigate the hormonal regulation of ANXA4. ANXA4 mRNA expression was quantified by real-time PCR in fresh endometrial tissue from cycling women, and protein expression was analysed by immunohistochemistry and western blotting. Hormonal regulation of ANXA4 transcription and translation was investigated using an endometrial explant system. ANXA4 mRNA was significantly up-regulated during mid-secretory (MS) and late-secretory (LS) phases compared with proliferative phases during the menstrual cycle. ANXA4 protein was localized to glandular and luminal epithelium and was present in high levels throughout the menstrual cycle except during early-secretory (ES) phase, when it was significantly reduced. Our data also show that, in proliferative explants, progesterone significantly increased the ANXA4 mRNA and protein after 48h in culture. Estrogen did not have any significant effects. This is the first study to show that ANXA4 transcription and translation are regulated by progesterone and suggests that ANXA4 may be important in regulating ion and water transport across the endometrial epithelium.