All-trans-retinoic acid regulates aquaporin-3 expression and related cellular membrane permeability in the human amniotic environment.

INTRODUCTION: The aquaporins (AQP1, 3, 8, 9 and 11) are known to be expressed, and involved in the transport of water and small molecules through fetal membranes. To exert these crucial functions, these AQPs have to be finely regulated. All-trans-retinoic acid (atRA) was previously found to regulate some genes in this environment, raising the question of whether these AQPs were regulated by atRA. METHODS: Explants, and primary and established amniotic cells were cultured to determine which AQP were transcriptionally modified by atRA, using the qRT-PCR strategy. Immunohistochemistry and glycerol uptake tests were used to determine the impact of atRA on AQP protein expression and function. Specific agonists of retinoic acid receptors were used to identify the molecular mechanisms of AQP promoter activation. A classical gene AQP promoter study was also used to identify DR5 retinoic acid receptor elements (RAREs). RESULTS: Beyond these AQPs, only one specific atRA-dependent increase in AQP3 transcripts and proteins level was established in amnion (not in chorion) and in related primary and established cells. We found three DR5-RAREs essential for inducing this transcriptional AQP3 through RARα. This transactivation of the AQP3 coding gene was functionally related to an increase of AQP3 permeability tests by a glycerol uptake assay. DISCUSSION: Our data support an atRA regulatory model of AQP3 expression leading to an increased cellular permeability in the epithelial amniotic environment. We cast new light on AF regulation in healthy pregnancy, and advance new hypotheses for obstetrical complications linked to impairment of the retinoic signaling pathway.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

[Medical indoor environment counsellor (MIEC) in Burgundy: evaluation by physicians and patients]. / Activité du conseil médical en environnement intérieur (CMEI) en Bourgogne : évaluation auprès des médecins et des patients.

UNLABELLED: Since December 2009, chest physicians and allergologists in Burgundy have been able to call upon a medical indoor environment counsellor (MIEC). The consultations are free for the patient and are undertaken following a medical referal after systematic cutaneous prick tests. AIMS: To describe the indications, the distribution of prescriptions and to measure the impact of the counsellor's visits on the first 100 patients at 6 months and on the physicians at 18 months. METHOD: Telephone interviews with the 67 physicians (whether prescribers or not) concerning their motivation and/or expectations, and with the first 100 patients concerning follow up of the recommendations. RESULTS: Seventy percent of the physicians replied (n=47). The satisfaction of prescribers (n=22) was 8.42/10. The indications were rhinitis and a poorly controlled asthma. The requests concerned the search for dust mite (50%) and moulds (46%). Eighty-four percent of the physicians discussed the MIEC's report with the patients. The patients' symptoms were rhinitis (79%), asthma (57%) and conjunctivitis (33%). The Acarex test(®), performed in cases of positive prick tests to house dust mites (n=72), was strongly positive for 67 patients. Sixteen mould samples out of 21 were above the standard concentrations. Sixty-nine patients had followed the recommendations of the MIEC. CONCLUSION: The impact of the MIEC visits was perceived as positive by the physicians and the patients. The medico-economic impact warrants further evaluation.

[Rupture of membranes: pathophysiology, diagnosis, consequences and management]. / Rupture des membranes : physiopathologie, diagnostic, conséquences et prise en charge.

Rupture of membranes (ROM) depends on mechanical stretch, extracellular matrix components imbalance and increased apoptosis. It occurs in 2 to 3% of all pregnancies before 37 weeks' gestation (WG) and in up to 10% at term. Main consequences are labor induction and risk of maternal-fetal infection. ROM is associated with one third of preterm births and about 20% of perinatal mortality. This review deals with recent knowledge concerning ROM including diagnosis and management. In many cases, ROM is easily identified by clinical examination. In other cases, the use of vaginal pH appears to be less efficient than the use of immunochromatographic strips based on IGFBP-1 or PAMG-1 detection. Before 34WG, conservative management consists in in utero transfer, antibioprophylaxis and corticosteroids. After 37WG, delivery is the most appropriate option. Between 34 and 37WG, recent studies demonstrate that induction of labour does not improve pregnancy outcomes. Therefore, expectant management can be the first option between 34 and 37WG when no active infection is suspected especially in case of unfavourable cervix.

[Therapeutic use of amniotic membranes and their derivate cells]. / Utilisations thérapeutiques des membranes fœtales et de leurs cellules dérivées.

During all the pregnancy, the foetal membranes play several functions (mechanic, anti-infectious, hormonal, regulation of the amniotic fluid homeostasis...) fundamental for an optimal development and maternal-foetal physiology. After delivery, these amniotic membranes have regained for a few years, a new interest and a second "ex-utero" life due to their therapeutic use. This use was firstly initiated experimentally in ophthalmological pathologies, around 1950. The recent understanding of molecular and cellular mechanisms enables to explain scientifically these first empiric uses. They are an interesting solution in ocular aggressions like viral attacks, chemical or temperature burns. They also represent an attractive alternative in case of corneal grafts and a biological matrix for limb cells cultures used to regenerate the corneal lesions. An industrial engineering is now in place to boost the performances of these human membranes. The isolation and identification of stem cells (mesenchymal origin) in these amniotic membranes are promising in the field of cell therapy. Recently, the first results have been published demonstrating the clinical efficiency of the stems cell during pancreatic, cardiac, lung neuronal lesions.

[Implications of retinoid pathway in human fetal membranes: study of target genes]. / Étude de la voie des rétinoïdes au sein des membranes fœtales humaines: mise en évidence de gènes cibles.

Retinoids (active derivatives of vitamin A) were already demonstrated to be important morphogenes and their implication at the placental and fetal level was already established. A new field of research is now developed in order to show their role on fetal membranes constituted by amnion and chorion. To describe the role of retinoids on these membranes, our studies were focused on target gene research. Firstly, all metabolism enzymes needed to vitamin A pathways were demonstrated to be present and active in signal transduction. Secondly, a bioinformatic analysis was performed to assess a list of potential target genes that could be classified in different biological pathways (inflammation, retinoids, hormones, vascularization, extracellular matrix and water homeostasis). Then, it was demonstrated that the gene coding for PLAT, implied in the degradation of extracellular matrix during programmed or premature rupture of membranes, is regulated by retinoids in a two steps mechanism. Finally, preliminary data showed that some aquaporins, which control water transport across membranes, are expressed and regulated by retinoids in the fetal membranes. A disregulation in pathologies like oligo or poly-hydramnios can be anticipated. Improvement of our knowledge about the retinoid implications is a key point in order to obtain a precise and complete documented cartography of the vitamin A (regulating) in amniotic membranes (regulated) that will permit the development of new diagnostic and therapeutic strategies.

Viral oncoproteins target the DNA methyltransferases.

Small DNA tumour viruses have evolved a number of mechanisms to drive nondividing cells into S phase. Virally encoded oncoproteins such as adenovirus E1A and human papillomavirus (HPV) E7 can bind an array of cellular proteins to override proliferation arrest. The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication. One of the hallmarks of tumour cells is disrupted DNA methylation patterns, highlighting the importance of the proper regulation of DNA methyltransferases in normal cell proliferation. Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity. These observations open up the possibility that this association may be used to control cellular proliferation pathways and suggest a new mechanism by which small DNA tumour viruses can steer cells through the cell cycle.

Transcriptional control of the human pregnancy-specific glycoprotein 5 gene is dependent on two GT-boxes recognized by the ubiquitous specificity protein 1 (Sp1) transcription factor.

Pregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression.

Use of transgenic mice model for understanding the placentation: towards clinical applications in human obstetrical pathologies?

The mammalian embryo and fetus are unable to develop without a well-established, functional placenta. This transitory yet indispensable structure attaches the conceptus to the uterus and establishes the vascular connections necessary for nutrient and gaseous exchange between maternal and fetal compartments. Genetic targeting strategy allows the generation of mice lacking a specific gene. Such approaches reveal: (i) the high incidence of mutant embryonic or fetal death in utero, and (ii) the extraembryonic (placental) causes of these deaths. Due to the similarities presented between mouse and human placenta, we propose to use the potential of mouse targeting experiments as a model in order to understand human obstetrical pathologies. In this paper, we first review genes that have been demonstrated to be required in mice for implantation, choriovitelline and chorioallantoic placentation. Using examples (integrins, homeoboxs, hepatocyte growth factor or epidermal growth factor receptor...) we demonstrate the reality and efficiency of such an approach. Other candidate genes (receptor of leukemia inhibitory factor, Wnt2 or retinoic acid receptor alpha...) in order to understand, prevent and treat human obstetrical pathologies.

Co-localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta development.

Pregnancy-specific glycoproteins (PSGs) are major placental proteins essential for the maintenance of normal gestation. However, little is known about their gene expression regulation during placentation. It was previously demonstrated that the human core promoter binding protein recently renamed Krüppel-like factor (KLF) 6 binds to a highly conserved sequence within the PSG promoters and is mainly expressed in human term placenta. Here, we determined the expression pattern of the 13 other KLFs during human placental development. We demonstrate that eight KLFs exhibit specific expression patterns in human placental tissues and membranes, in favor of a functional cooperation of specific KLFs during placentation. In addition, we demonstrate that KLF6, KLF4 and PSG proteins are co-expressed in same cell types of placental villi and membranes. This experimental evidence further strengthens the potential cross talk of both transcription factors for PSG gene regulation in vivo.

Esterification of vitamin A by the human placenta involves villous mesenchymal fibroblasts.

Vitamin A (retinol) and its active derivatives (retinoic acids) are essential for growth and development of the mammalian fetus. Maternally derived retinol must pass the placenta to reach the developing fetus. Despite its apparent importance, little is known concerning placental transfer and metabolism of retinol, and particularly of placental production and storage of retinyl esters. To elucidate this metabolic pathway, we incubated, in the presence of retinol, 1) human full-term placental explants and 2) primary cultures of major cells types contributing to placental function: trophoblasts and villous mesenchymal fibroblasts. We used HPLC to determine the types and concentrations of retinyl esters produced by these explants and cells. About 14% of total cellular retinol in placental explants was esterified. The most abundant esters were myristate and palmitate. Primary cell cultures showed that fibroblasts efficiently produced retinyl esters, but trophoblasts did not. In both types of experiments, no retinyl esters were detected in the culture medium, suggesting that retinyl esters were produced for storage purpose. These results suggest that villous mesenchymal fibroblasts are primary sites of retinol esterification and storage in the placenta.