culture is showed a high cell confluence and later, ten days in culture lined up. Functional ... tenance, AQP3 should require a fine regulation of its expression and.
Abstracts / Placenta 35 (2014) A1eA112
LOX-mediated cross-linking and consequently increased degradation by MMPs.
P1.110-N. IN VITRO FORMATION OF CAPILLARY TUBULES FROM STEM CELLS OF THE BOVINE YOLK SAC WITH PROSPECTS FOR THERAPEUTIC APPLICATION Celina A.F. Mançanares a, c, Vanessa C. Oliveira b, Ana Flavia lica Miglino b, Carlos Eduardo Carvalho a, Flavio V. Meirelles c, Maria Ange rio da Fundaça ~o de Ensino Ambrosio c a UNIFEOB - Centro Universita ~o vio Bastos, Sa ~o Joa ~o da Boa Vista, Brazil; b USP -Universidade de Sa Octa ~o Paulo, Pirassununga, ~o Paulo, Brazil; c FZEA - Universidade de Sa Pau, Sa Brazil Objectives: The yolk sac (Ys) is an fetal membrane responsible by formation of the primitive intestine, as well as hematopoiesis because it develops the beginnings of blood cell and part of the primitive circulatory system of the embryo. This research aimed to generate structures like blood vessels “in vitro” from mesenchymal stem cells (MSCs) from the yolk sac of bovine embryos. Were used 79 Ys of bovine embryos from 25 to 35 days of gestation. Methods: The cells of the yolk sac were cultivates in alpha men and for the formation of capillary tubules “in vitro “assay was performed using the BD Matrigel™ (BD Biosciences). The expression of VEGF (formation of capillary tubules) was evaluated by RT-PCR. Results: The analysis showed that, immediately after plating, the MSCs of the yolk sac had rounded morphology with presence of cellular colonies and after 24 hours adhered at the bottom of the plate. With five days of culture is showed a high cell confluence and later, ten days in culture lined up. Functional assay showed that yolk sac cells was aligned and organized themselves and subsequently forming tubular structures, similar to capillary tubules. In the PCR analysis showed that VEGF was expressed proving that the bovine embryo yolk sac contains hemangioblasts, progenitors cells that are able to differentiate into hematopoietic cells and vascular endothelial cells through the processes of hematopoiesis and vasculogenesis. Conclusion: Based on tests performed we conclude that yolk sac cells differentiate into tubular structures, as similar to capillaries and express the VEGF gene. Considering the importance of this embryonic annex and the ability of MSCs in generate other tissues, we believe that with the study of these cells, will be possible develop new therapeutic strategies, such as tissue regeneration and vasculogenesis “in vitro”. Finnancial support: FAPESP. Process Number: 2010/50395-3.
P1.111. ALL-TRANS RETINOIC ACID REGULATES AQUAPORIN-3 EXPRESSION AND RELATED CELLULAR MEMBRANE PERMEABILITY IN HUMAN AMNIOTIC ENVIRONMENT lie Comptour a, Damien Bouvier a, b, Geoffroy cile Prat a, Aure Ce a, b Marceau , Corinne Belville a, c, Denis Gallot a, d, Vincent Sapin a, b, Loïc Blanchon a a EA 7281 R2D2 UdA, Clermont-Ferrand, France; b Biochemistry and Molecular Department - CHU Clermont-Ferrand, Clermont-Ferrand, France; c GReD MR INSERM 1103 CNRS 6247, Clermont-Ferrand, France; d Obstetrics and Gynaecology - CHU Clermont-Ferrand, Clermont-Ferrand, France Objectives: The fetal membranes are fundamental for the maintenance of amniotic fluid (AF) homeostasis and volume, important parameters for fetal harmonious development. Expressed in the human amniotic environment, aquaporin-3 (AQP3) a trans-membranous glycoprotein facilitating water and small solutes (e.g. glycerol) flow across lipid cellular membranes has been revealed as a good candidate for the pathophysiological regulation of AF volume. To be reactive on AF homeostasis maintenance, AQP3 should require a fine regulation of its expression and function by efficient and well controlled cellular pathways. A developmentally morphological active derivative of vitamin A, i.e. all-trans retinoic acid (atRA) emerged as potential regulator of this system.
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Methods: Q-RTPCR and Western-Blot assays were realized on amniotic tissues, primary cells and related cell lines. Gene promoter assays were done to identify regulatory atRA mechanisms. Functional glycerol study was developed to achieve the physiological regulations. Results: An atRA-sensitive increase of AQP3 transcripts was identified in human amnion (but not chorion) explants, primary epithelial and established FL amniotic cells. Thereby, using directed-site mutagenesis on the promoter of AQP3 coding gene, we highlighted three DR5-Retinoic Acid Responsive Elements elocated at -812 base pairs (bp), -1349 bp and -1630 bpeessentials to retinoic-induce this transcriptional AQP3 coding gene in FL cells. Moreover, using a selective agonist of retinoic acid nuclear receptors, we demonstrated that the atRA signaling was mediated by RARa but not RARg in FL cells. Interestingly, this transactivation of AQP3 coding gene was functionally related with a protein increase resulting in an uptake of solute (radiolabelled glycerol) in both human amnion explants and FL cells. Conclusion: Our study provides new insights on the understanding of amniotic fluid regulation in healthy pregnancies and opens new hypothesis in obstetrical complications.
P1.112-N. ROLE OF EG-VEGF (ENDOCRINE GLAND DERIVED ENDOTHELIAL GROWTH FACTOR) IN THE HUMAN FETAL MEMBRANES. Thibaut Mouchet a, Wael Traboulsi a, Camille Dunand a,b, Vanessa sophie Brouillet a,b, Jean Guibourdenche c, Pascale Garnier a, HOffmann a, b, Mohamed Benharouga d, Jean-jacques Feige a, Nadia Alfaidy a, b a INSERM U1036, Grenoble, France; b Grenoble hospital, Grenoble, France; c Cochin Hospital INSERM U767, Paris, France; d University Joseph Fourier, Grenoble, France Objectives: We recently demonstrated that EG-VEGF and its receptors PROKR1 and PROKR2 are highly expressed in the human fetal membranes (FMs), with proposed protective role during late pregnancy. Here, we investigated its effects on the amnion epithelial cells and examined the effects of potential stressors such as deregulated osmolality, and infection on its expression. The aims of this study were: to 1) determine the levels of EG-VEGF in the amniotic fluid (AF) and in the hydramnios condition; 2) determine its effects on the permeability of the amniotic layer; 3) assess the effect of the osmolality on EG-VEGF and its receptors expression and 3) determine the effect of LPS (lipopolysaccharide) on its expression and that of its receptors. Methods: Amniotic fluids and sera were collected from pregnant women during the second and the third trimesters. Human amniotic cell lines, WISH an FL were also used. ELISA was employed to quantify EG-VEGF levels. RT-qPCR, western blotting and immunohistochemistry were used to determine the effect of LPS and osmolality on EG-VEGF and its receptors expression. Permeability test, western blotting and immunofluorescence were used to assess EG-VEGF effect on amnion epithelial resistance and on the expression of junctional proteins, respectively. Results: We demonstrated that i) EG-VEGF is abundantly present in the AF throughout pregnancy, ii) Its levels were 5 times higher in the AF compare to age matched sera, and it is increased in the hydramnios condition iii) EG-VEGF increases the amniotic layer permeability via PROKR1 and PROKR2 receptors, iv) hypotonicity but not hypertonicity increases PROKR1 and PROKR2 expression in amnion cells; and v) LPS significantly increased EG-VEGF, PROKR1 and PROKR2 expression. Conclusion: These results demonstrate that EG-VEGF/ PROKRs is a new regulatory system that might play key role in the homeostasis of the AF and its surrounding amniotic layer.
P1.113-N. A SCANNING ELECTRON MICROSCOPY STUDY OF MICROCOTILEDONARY DEVELOPMENT OF EQUINE PLACENTA THROUGHOUT GESTATION lica Andreza Souza a, Gustavo Winter a, Nicolas Cazales b, Maria Ange Miglino c, Rodrigo Mattos a a universidade Federal do Rio Grande do Sul, Porto Alegre/ RIO Grande do Sul, Brazil; b universidad de la Republica, ~o Paulo, Sa ~o Paulo/ Sa ~o Paulo, Montevideo, Uruguay; C universidade de Sa Brazil
