- Email: [email protected]
placental growth restricted phenotype in a mouse model
A32
Abstracts / Placenta 35 (2014) A1eA112
normal term and diabetic placentas. Some of the stromal cells showed strong cytoplasmic ‘dote kind of staining’. There was a significantly weak Syncytin2 (p<0,001) and MFSD2 (p¼0,005) immunoreactions in diabetic term placentas compared to normal term placentas by IMAGE-J analysis. Syncytin2 (p¼0,029) and MFSD2 (p¼0,006) protein expressions were found to be decreased in diabetic placentas compared to normal term placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta (p¼0,027). The presence of Syncytin1 and Syncytin2 mRNA in diabetic placentas were confirmed by RT-PCR. Conclusion: Compared to previous studies showing Syncytins in cytotrophoblast cells and the basement membrane of the syncytiotrophoblasts, here we demonstrated that Syncytins were also present in the cytoplasm of syncytiotrophoblasts and in some of the stromal cells suggesting further complex interactions of these proteins. Decreased Syncytin2 and MFSD2 expressions might cause abnormal syncytiotrophoblasts formation and possibly involve in diabetic placental pathology. Therefore, our study is of importance of showing the potential relationship between Syncytins and gestational diabetic placenta.
P1.70-N. PATERNAL DIET INDUCED OBESITY HAS A FETAL/PLACENTAL GROWTH RESTRICTED PHENOTYPE IN A MOUSE MODEL Natalie Binder a, b, Stephen Tong b, David Gardner a, Natalie Hannan b a Department of Zoology, University of Melbourne, Victoria, Australia; b Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Objectives: Worldwide, 48% of adult males are overweight or obese. Although most research has focussed on maternal obesity, there is growing data indicating the negative effect of paternal obesity. Previously, we demonstrated that paternal obesity resulted in aberrant preimplantation embryo development and significantly reduced fetal and placental weights. In the current study we hypothesise that this growth restriction is due to alterations in metabolic, cell signalling, and stress pathways. Methods: Sperm was collected from normal and obese C57BL/6 mice (as previously described (Binder et al., 2012)) and used for IVF with normal oocytes. Embryos were cultured to the blastocyst stage and either collected for gene expression analysis, or transferred to recipient females. Placentas derived from the embryos of normal and obese fathers were collected at E14. RNA was extracted from embryos and placentas, and gene expression assessed using custom RT-qPCR arrays (24 genes of interest were selected). Human placentas (34wks gestation) were collected and RNA extracted. Results: Expression of COX4i1, a nuclear encoded terminal enzyme of the mitochondrial respiratory chain, tended to be increased in blastocysts derived from obese fathers (p¼0.056). PPARa and Casp12 expression was significantly altered in male placentas from obese fathers compared to normal (p<0.05), but not female placentas. PPARa and Casp12 protein was localised within the placenta using immunohistochemistry and antibody specificity confirmed by western blot on placental lysates. Global DNA methylation was significantly increased in female placentas from obese fathers compared to normal (p<0.05), but not male placenta. Given these results, fetal growth restricted (FGR) human placenta and gestationally matched controls were assessed for PPARa and Casp12 expression. PPARa expression was confirmed in human placenta, however Casp12 could not be detected. Conclusion: These findings reinforce the negative consequences of paternal obesity prior to conception, and emphasise the need for more lifestyle advice for prospective fathers.
P1.71-N. AN ENDOTHELIAL-MESENCHYMAL TRANSITION OCCURS IN PRIMARY PLACENTAL CELLS IN VITRO e COULD THIS CONTRIBUTE TO FETAL GROWTH RESTRICTION? Stefanie Swietlik, Melissa Westwood, Edward Johnstone, John Aplin University of Manchester, Manchester, UK
Introduction: Reduced placental vascularisation is associated with fetal growth restriction (FGR). In other organ systems, loss of endothelial cells can occur via an endothelial-mesenchymal transition (EndMT). EndMT has not previously been described in placenta. Hypothesis: EndMT occurs during placental development, but becomes dysregulated in FGR. Methods: Stromal cells isolated from first trimester placentas (n¼15; 615w) were characterised over 4 passages by immunocytochemistry using mesenchymal (alpha-smooth muscle actin (aSMA)) and endothelial (CD31, vWF) markers. CD31+ cells were isolated from primary cells isolates using immunomagnetic beads (iCD31+ cells) at p1 and characterised for aSMA-positivity immediately or after 24h culture in DMEM/ FCS; iCD31+ cells were also cultured ± the TGFb-blocker SB431542 (10mM) or in endothelial medium (EGM2) on matrigel. aSMA was characterised in iCD31+ cells from normal and FGR term placentas. vWF/ aSMA expression in first trimester placenta was examined using dual immunohistochemistry Results: Perivascular cells in first trimester tissue express both vWF and aSMA. Freshly isolated first trimester cells were mainly aSMA-positive. 12% were CD31+, but CD31+ cells decreased to 1% (p<0.05) by passage 4, with no loss of cell viability. At each passage ~5% of iCD31+ cells expressed aSMA at isolation, increasing to ~35% after 24h. iCD31+ cells with abundant stress fibres contained internalised anti-CD31 beads. iCD31+ cells acquisition of aSMA was not inhibited by culture in EGM2 on matrigel, but was reduced by SB431542. Preliminary data suggest more iCD31+ cells from FGR placentas become aSMA-positive than those from normal tissue. Conclusion: Loss of endothelial markers in the mixed cell population and gain of mesenchymal markers in isolated endothelial cells shows an EndMT is occurring in vitro. The presence of cells expressing both endothelial and mesenchymal markers in vivo suggests that EndMT also occurs in tissue. A greater potential for endothelial cells to undergo EndMT could contribute to reduced vascularisation in FGR.
P1.72. IMMUNOLOCALISATION OF CYTOKERATIN 7 IN HUMAN UMBILICAL MESENCHYMAL STEM CELLS, UMBILICAL VEIN ENDOTHELIAL CELLS AND HUMAN UTERINE MICROVASCULAR ENDOTHELIAL CELLS Neven Ebrahim, Azlina Razak, Jennifer Sedcole, Olivia Volk, Lopa Leach University of Nottingham, Nottingham, UK Objectives: Cytokeratin 7 (CK 7) is an intermediate filament cytokeratin of glandular epithelium, transitional epithelium and transitional carcinoma. It has been localised in the syncytiotrophoblast, endometrial glands and extra-villous trophoblast cells (EVT). Indeed it is extensively used to indicate spiral artery remodelling by EVTs. However, the specificity of CK 7 may be questionable. Recently human amniotic mesenchymal stromal cells have been shown to express CK7 (Konig et al. 2013). In this study we expanded the search to include mesenchymal stem cells from the umbilical cord, endothelial cells from the umbilical vein (HUVEC) and human uterine microvessels (HUtMEC) at different passages. Methods: Isolated Wharton’s Jelly mesenchymal stem cells (WJMSC; P2,3 & 5) and HUVEC (P2 - 5) and commercially bought HUtMEC (P5) were grown to confluence, double labelled with anti-CK 7 monoclonal antibody (12.4 mg/ml) and the appropriate marker antibodies (endothelial VE-cadherin, CD 31) or (mesenchymal stem cell CD 29 & 105). Results: All passages of HUVEC showed >90% CK7 cell labelling. 27 to 33% of WJMSC were positive to CK 7. HUtMEC cells (29 + 4%) also showed immunopositivity to CK7. Double labelling confirmed their endothelial or stem cell phenotype. Conclusion: CK 7 have a ubiquitous localisation in endothelial and mesenchymal stem cells. The % of umbilical stem cells showing positivity to CK 7 may be related to a specific cohort or a mesenchymal-epithelial transition of these cells in culture. The endothelial localisation was curious. The co- expressions of cytokeratin 7 suggests the existence of diversity of differentiated cell types in fetal endothelial cultures or demonstrate a dedifferentiation capability of fetal endothelial cells. The smaller percentage
Abstracts / Placenta 35 (2014) A1eA112
normal term and diabetic placentas. Some of the stromal cells showed strong cytoplasmic ‘dote kind of staining’. There was a significantly weak Syncytin2 (p<0,001) and MFSD2 (p¼0,005) immunoreactions in diabetic term placentas compared to normal term placentas by IMAGE-J analysis. Syncytin2 (p¼0,029) and MFSD2 (p¼0,006) protein expressions were found to be decreased in diabetic placentas compared to normal term placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta (p¼0,027). The presence of Syncytin1 and Syncytin2 mRNA in diabetic placentas were confirmed by RT-PCR. Conclusion: Compared to previous studies showing Syncytins in cytotrophoblast cells and the basement membrane of the syncytiotrophoblasts, here we demonstrated that Syncytins were also present in the cytoplasm of syncytiotrophoblasts and in some of the stromal cells suggesting further complex interactions of these proteins. Decreased Syncytin2 and MFSD2 expressions might cause abnormal syncytiotrophoblasts formation and possibly involve in diabetic placental pathology. Therefore, our study is of importance of showing the potential relationship between Syncytins and gestational diabetic placenta.
P1.70-N. PATERNAL DIET INDUCED OBESITY HAS A FETAL/PLACENTAL GROWTH RESTRICTED PHENOTYPE IN A MOUSE MODEL Natalie Binder a, b, Stephen Tong b, David Gardner a, Natalie Hannan b a Department of Zoology, University of Melbourne, Victoria, Australia; b Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Objectives: Worldwide, 48% of adult males are overweight or obese. Although most research has focussed on maternal obesity, there is growing data indicating the negative effect of paternal obesity. Previously, we demonstrated that paternal obesity resulted in aberrant preimplantation embryo development and significantly reduced fetal and placental weights. In the current study we hypothesise that this growth restriction is due to alterations in metabolic, cell signalling, and stress pathways. Methods: Sperm was collected from normal and obese C57BL/6 mice (as previously described (Binder et al., 2012)) and used for IVF with normal oocytes. Embryos were cultured to the blastocyst stage and either collected for gene expression analysis, or transferred to recipient females. Placentas derived from the embryos of normal and obese fathers were collected at E14. RNA was extracted from embryos and placentas, and gene expression assessed using custom RT-qPCR arrays (24 genes of interest were selected). Human placentas (34wks gestation) were collected and RNA extracted. Results: Expression of COX4i1, a nuclear encoded terminal enzyme of the mitochondrial respiratory chain, tended to be increased in blastocysts derived from obese fathers (p¼0.056). PPARa and Casp12 expression was significantly altered in male placentas from obese fathers compared to normal (p<0.05), but not female placentas. PPARa and Casp12 protein was localised within the placenta using immunohistochemistry and antibody specificity confirmed by western blot on placental lysates. Global DNA methylation was significantly increased in female placentas from obese fathers compared to normal (p<0.05), but not male placenta. Given these results, fetal growth restricted (FGR) human placenta and gestationally matched controls were assessed for PPARa and Casp12 expression. PPARa expression was confirmed in human placenta, however Casp12 could not be detected. Conclusion: These findings reinforce the negative consequences of paternal obesity prior to conception, and emphasise the need for more lifestyle advice for prospective fathers.
P1.71-N. AN ENDOTHELIAL-MESENCHYMAL TRANSITION OCCURS IN PRIMARY PLACENTAL CELLS IN VITRO e COULD THIS CONTRIBUTE TO FETAL GROWTH RESTRICTION? Stefanie Swietlik, Melissa Westwood, Edward Johnstone, John Aplin University of Manchester, Manchester, UK
Introduction: Reduced placental vascularisation is associated with fetal growth restriction (FGR). In other organ systems, loss of endothelial cells can occur via an endothelial-mesenchymal transition (EndMT). EndMT has not previously been described in placenta. Hypothesis: EndMT occurs during placental development, but becomes dysregulated in FGR. Methods: Stromal cells isolated from first trimester placentas (n¼15; 615w) were characterised over 4 passages by immunocytochemistry using mesenchymal (alpha-smooth muscle actin (aSMA)) and endothelial (CD31, vWF) markers. CD31+ cells were isolated from primary cells isolates using immunomagnetic beads (iCD31+ cells) at p1 and characterised for aSMA-positivity immediately or after 24h culture in DMEM/ FCS; iCD31+ cells were also cultured ± the TGFb-blocker SB431542 (10mM) or in endothelial medium (EGM2) on matrigel. aSMA was characterised in iCD31+ cells from normal and FGR term placentas. vWF/ aSMA expression in first trimester placenta was examined using dual immunohistochemistry Results: Perivascular cells in first trimester tissue express both vWF and aSMA. Freshly isolated first trimester cells were mainly aSMA-positive. 12% were CD31+, but CD31+ cells decreased to 1% (p<0.05) by passage 4, with no loss of cell viability. At each passage ~5% of iCD31+ cells expressed aSMA at isolation, increasing to ~35% after 24h. iCD31+ cells with abundant stress fibres contained internalised anti-CD31 beads. iCD31+ cells acquisition of aSMA was not inhibited by culture in EGM2 on matrigel, but was reduced by SB431542. Preliminary data suggest more iCD31+ cells from FGR placentas become aSMA-positive than those from normal tissue. Conclusion: Loss of endothelial markers in the mixed cell population and gain of mesenchymal markers in isolated endothelial cells shows an EndMT is occurring in vitro. The presence of cells expressing both endothelial and mesenchymal markers in vivo suggests that EndMT also occurs in tissue. A greater potential for endothelial cells to undergo EndMT could contribute to reduced vascularisation in FGR.
P1.72. IMMUNOLOCALISATION OF CYTOKERATIN 7 IN HUMAN UMBILICAL MESENCHYMAL STEM CELLS, UMBILICAL VEIN ENDOTHELIAL CELLS AND HUMAN UTERINE MICROVASCULAR ENDOTHELIAL CELLS Neven Ebrahim, Azlina Razak, Jennifer Sedcole, Olivia Volk, Lopa Leach University of Nottingham, Nottingham, UK Objectives: Cytokeratin 7 (CK 7) is an intermediate filament cytokeratin of glandular epithelium, transitional epithelium and transitional carcinoma. It has been localised in the syncytiotrophoblast, endometrial glands and extra-villous trophoblast cells (EVT). Indeed it is extensively used to indicate spiral artery remodelling by EVTs. However, the specificity of CK 7 may be questionable. Recently human amniotic mesenchymal stromal cells have been shown to express CK7 (Konig et al. 2013). In this study we expanded the search to include mesenchymal stem cells from the umbilical cord, endothelial cells from the umbilical vein (HUVEC) and human uterine microvessels (HUtMEC) at different passages. Methods: Isolated Wharton’s Jelly mesenchymal stem cells (WJMSC; P2,3 & 5) and HUVEC (P2 - 5) and commercially bought HUtMEC (P5) were grown to confluence, double labelled with anti-CK 7 monoclonal antibody (12.4 mg/ml) and the appropriate marker antibodies (endothelial VE-cadherin, CD 31) or (mesenchymal stem cell CD 29 & 105). Results: All passages of HUVEC showed >90% CK7 cell labelling. 27 to 33% of WJMSC were positive to CK 7. HUtMEC cells (29 + 4%) also showed immunopositivity to CK7. Double labelling confirmed their endothelial or stem cell phenotype. Conclusion: CK 7 have a ubiquitous localisation in endothelial and mesenchymal stem cells. The % of umbilical stem cells showing positivity to CK 7 may be related to a specific cohort or a mesenchymal-epithelial transition of these cells in culture. The endothelial localisation was curious. The co- expressions of cytokeratin 7 suggests the existence of diversity of differentiated cell types in fetal endothelial cultures or demonstrate a dedifferentiation capability of fetal endothelial cells. The smaller percentage