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Impaired interactions between decidual natural killer cells and trophoblast contribute to poor spiral artery remodelling
Abstracts / Placenta 34 (2013) A1–A99
remodeling, increasing BMI, lipid transport, energy metabolism, immune and/or inflammatory responses. The qRT-PCR analysis demonstrated down-regulation of the differentially methylated genes that are located within the growth hormone and pregnancy-specific glycoprotein gene clusters. Conclusion: We find increased overall DNA methylation in placentas of obese women and direct evidence that the maternal obesogenic environment may specifically alter the placental epigenome, potentially changing placental function. http://dx.doi.org/10.1016/j.placenta.2013.06.115
P1.79. THE ROLE OF EGFL7 IN TROPHOBLAST CELLS AND ENDOTHELIAL CELLS DURING PLACENTAL DEVELOPMENT Lauretta Lacko, Heidi Stuhlmann Weill Cornell Medical College, New York, NY, USA Objective: The objective of this study is to determine the role of the partially secreted angiogenic factor, EGFL7, in placental development. Background: Placentation requires trophoblast differentiation and invasion, as well as coordinated maternal vascular remodeling and fetal vasculogenesis. Egfl7 is largely restricted to actively proliferating endothelial cells, but is also seen in embryonic stem cells and primordial germ cells. Functionally, EGFL7 has been shown to act as a chemoattractant in vitro for endothelial cells, to regulate tubulogenesis in zebrafish, and to control vascular patterning in mice. Methods: We have used Real Time RT-PCR, in situ hybridization, and immunohistochemical approaches, as well as a mouse model of preeclampsia (PE) and Egfl7 gain- and loss-of-function mice in this study. Ongoing experiments are investigating EGFL7 in the trophoblast cell lineage and its effect on placental development using lentivirus-mediated knockdown and overexpression of EGFL7 in trophoblast stem cells and blastocysts. Results: We have defined the dynamic spatiotemporal expression profile of Egfl7 transcript and protein in the mouse placenta throughout gestation. Our studies have also demonstrated, for the first time, EGFL7 localization to the non-endothelial trophoblast cell lineage in vitro in trophoblast stem cells, and in vivo in both the trophectoderm of blastocysts and in a subset of differentiated trophoblast cells in the developing placenta. We also demonstrated a downregulation of EGFL7 in placentas from the BPH/5 mouse model of PE and human PE placentas. Preliminary analyses of placentas from Egfl7-knockout mice indicated reduced fertility and patterning defects in the fetal labyrinth zone. Egfl7-overexpressing mice exhibited partial embryonic lethality and defects in junctional zone development. Conclusion: Together, these results have correlated a change in Egfl7 expression in PE placentas, uncovered a novel site of EGFL7 expression, and have indicated placental defects in Egfl7-mutant mice, suggesting an important role for EGFL7 during placental development. http://dx.doi.org/10.1016/j.placenta.2013.06.116
P1.80. IMPAIRED INTERACTIONS BETWEEN DECIDUAL NATURAL KILLER CELLS AND TROPHOBLAST CONTRIBUTE TO POOR SPIRAL ARTERY REMODELLING Alison Wallace, Amanda Host, Guy Whitley, Judith Cartwright St George's, University of London, London, UK Objective: Decidual natural killer (dNK) cells play a role in transformation of the uterine spiral arteries (SA) during pregnancy; however their interactions with fetal trophoblast remain unclear. Uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure
A39
of the extent of remodelling of the SA. Using this technique we isolated dNK cells from pregnancies with normal (normal RI) or impaired SA remodelling (high RI) and determined how they contributed to trophoblast motility, chemotaxis and invasion, important components of placentation. Methods: Decidual NK cells were isolated with local ethical approval from first trimester terminations of pregnancy screened by uterine artery Doppler ultrasound, cultured for 6 hours and conditioned media (CM) collected. The trophoblast cell line SGHPL-4 and extravillous trophoblast (EVT) outgrowths from first trimester villous tissue explants were incubated with dNK cell CM, and invasion, motility and chemotaxis of trophoblast was examined. The signalling pathways controlling these events in trophoblast were investigated using the chemical inhibitors PD98059 and LY294002. Results: Decidual NK cell CM from both normal and high RI pregnancies induced invasion of SGHPL-4 cells compared to control (p<0.01). However, high RI dNK cell CM was less able to induce chemotaxis of SGHPL-4 cells and EVT outgrowth as compared to normal RI dNK cell CM (p<0.05). SGHPL-4 cells displayed decreased ERK 1/2 (p<0.05) and Aktser473 (p<0.01) phosphorylation when incubated with high RI dNK cell CM as compared to normal RI dNK cell CM, and both these signalling pathways were required for EVT outgrowth in response to normal RI dNK cell CM. SGHPL-4 chemotaxis was dependent only on ERK 1/2 signalling. Conclusion: Using uterine artery Doppler RI screening prior to cell isolation has identified that dNK cells from high RI pregnancies have altered interactions with trophoblast, which may lead to poor placentation. http://dx.doi.org/10.1016/j.placenta.2013.06.117
P1.81. IS MITOCHONDRIAL DNA IN TROPHOBLAST DEBRIS AN INFLAMMATORY MEDIATOR RELEVANT TO PREECLAMPSIA? Kate Wauchop 1, Jonathan Warren 2, Larry Chamley 1, Lynsey Cree 1, Jo James 1 1
University of Auckland, Auckland, New Zealand; 2 St George's University of London, London, UK Introduction: Throughout pregnancy, large multinucleated structures known as syncytial nuclear aggregates (SNAs) and other trophoblast cellular debris is shed from the placenta into the maternal blood. Excessive production of trophoblast debris in response to placental stress is associated with preeclampsia. Extensive cellular stress can cause the accumulation/release of mitochondrial DNA (mtDNA) via necrosis or autophagy. Free mtDNA has been shown to induce an inflammatory response, leading to heart failure in mice1, and possibly other chronic inflammatory diseases. Increased mtDNA has been reported in maternal serum of preeclamptic patients. However, it is unclear whether this mtDNA is derived from the placenta (as a causative factor) or the maternal endothelium (as a stressresponse). We hypothesised that trophoblast debris contains mtDNA that may act as an inflammatory trigger in preeclampsia. Methods: Trophoblast debris and individual SNAs were harvested from 8 first trimester placentae and mtDNA was extracted, amplified and visualised by agarose gel electrophoresis. Beclin-1 expression in SNAs from 7 further first trimester placentae, indicated to be at increased risk of preeclampsia by uterine artery Doppler, was determined by immunohistochemistry. Results: mtDNA was detected in unfractionated trophoblast debris (n¼3 placentae) and individual SNAs (n¼25 SNAs from 5 placentae). The autophagy marker beclin-1 was detected in SNAs from 2/4 placentae at high risk of preeclampsia, but no SNAs from 3 placentae at low risk of preeclampsia. Conclusions: mtDNA is present in trophoblast debris. SNAs from placentae at increased risk of preeclampsia exhibit signs of autophagy, which may exacerbate the release of mtDNA from these structures. The clearance of trophoblast debris by maternal cells may be a mechanism that brings inflammatory mtDNA into contact with maternal immune and cardiovascular systems. 1. Oka, T, et al., Nature, 2012. 485(7397):251-5. http://dx.doi.org/10.1016/j.placenta.2013.06.118
remodeling, increasing BMI, lipid transport, energy metabolism, immune and/or inflammatory responses. The qRT-PCR analysis demonstrated down-regulation of the differentially methylated genes that are located within the growth hormone and pregnancy-specific glycoprotein gene clusters. Conclusion: We find increased overall DNA methylation in placentas of obese women and direct evidence that the maternal obesogenic environment may specifically alter the placental epigenome, potentially changing placental function. http://dx.doi.org/10.1016/j.placenta.2013.06.115
P1.79. THE ROLE OF EGFL7 IN TROPHOBLAST CELLS AND ENDOTHELIAL CELLS DURING PLACENTAL DEVELOPMENT Lauretta Lacko, Heidi Stuhlmann Weill Cornell Medical College, New York, NY, USA Objective: The objective of this study is to determine the role of the partially secreted angiogenic factor, EGFL7, in placental development. Background: Placentation requires trophoblast differentiation and invasion, as well as coordinated maternal vascular remodeling and fetal vasculogenesis. Egfl7 is largely restricted to actively proliferating endothelial cells, but is also seen in embryonic stem cells and primordial germ cells. Functionally, EGFL7 has been shown to act as a chemoattractant in vitro for endothelial cells, to regulate tubulogenesis in zebrafish, and to control vascular patterning in mice. Methods: We have used Real Time RT-PCR, in situ hybridization, and immunohistochemical approaches, as well as a mouse model of preeclampsia (PE) and Egfl7 gain- and loss-of-function mice in this study. Ongoing experiments are investigating EGFL7 in the trophoblast cell lineage and its effect on placental development using lentivirus-mediated knockdown and overexpression of EGFL7 in trophoblast stem cells and blastocysts. Results: We have defined the dynamic spatiotemporal expression profile of Egfl7 transcript and protein in the mouse placenta throughout gestation. Our studies have also demonstrated, for the first time, EGFL7 localization to the non-endothelial trophoblast cell lineage in vitro in trophoblast stem cells, and in vivo in both the trophectoderm of blastocysts and in a subset of differentiated trophoblast cells in the developing placenta. We also demonstrated a downregulation of EGFL7 in placentas from the BPH/5 mouse model of PE and human PE placentas. Preliminary analyses of placentas from Egfl7-knockout mice indicated reduced fertility and patterning defects in the fetal labyrinth zone. Egfl7-overexpressing mice exhibited partial embryonic lethality and defects in junctional zone development. Conclusion: Together, these results have correlated a change in Egfl7 expression in PE placentas, uncovered a novel site of EGFL7 expression, and have indicated placental defects in Egfl7-mutant mice, suggesting an important role for EGFL7 during placental development. http://dx.doi.org/10.1016/j.placenta.2013.06.116
P1.80. IMPAIRED INTERACTIONS BETWEEN DECIDUAL NATURAL KILLER CELLS AND TROPHOBLAST CONTRIBUTE TO POOR SPIRAL ARTERY REMODELLING Alison Wallace, Amanda Host, Guy Whitley, Judith Cartwright St George's, University of London, London, UK Objective: Decidual natural killer (dNK) cells play a role in transformation of the uterine spiral arteries (SA) during pregnancy; however their interactions with fetal trophoblast remain unclear. Uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure
A39
of the extent of remodelling of the SA. Using this technique we isolated dNK cells from pregnancies with normal (normal RI) or impaired SA remodelling (high RI) and determined how they contributed to trophoblast motility, chemotaxis and invasion, important components of placentation. Methods: Decidual NK cells were isolated with local ethical approval from first trimester terminations of pregnancy screened by uterine artery Doppler ultrasound, cultured for 6 hours and conditioned media (CM) collected. The trophoblast cell line SGHPL-4 and extravillous trophoblast (EVT) outgrowths from first trimester villous tissue explants were incubated with dNK cell CM, and invasion, motility and chemotaxis of trophoblast was examined. The signalling pathways controlling these events in trophoblast were investigated using the chemical inhibitors PD98059 and LY294002. Results: Decidual NK cell CM from both normal and high RI pregnancies induced invasion of SGHPL-4 cells compared to control (p<0.01). However, high RI dNK cell CM was less able to induce chemotaxis of SGHPL-4 cells and EVT outgrowth as compared to normal RI dNK cell CM (p<0.05). SGHPL-4 cells displayed decreased ERK 1/2 (p<0.05) and Aktser473 (p<0.01) phosphorylation when incubated with high RI dNK cell CM as compared to normal RI dNK cell CM, and both these signalling pathways were required for EVT outgrowth in response to normal RI dNK cell CM. SGHPL-4 chemotaxis was dependent only on ERK 1/2 signalling. Conclusion: Using uterine artery Doppler RI screening prior to cell isolation has identified that dNK cells from high RI pregnancies have altered interactions with trophoblast, which may lead to poor placentation. http://dx.doi.org/10.1016/j.placenta.2013.06.117
P1.81. IS MITOCHONDRIAL DNA IN TROPHOBLAST DEBRIS AN INFLAMMATORY MEDIATOR RELEVANT TO PREECLAMPSIA? Kate Wauchop 1, Jonathan Warren 2, Larry Chamley 1, Lynsey Cree 1, Jo James 1 1
University of Auckland, Auckland, New Zealand; 2 St George's University of London, London, UK Introduction: Throughout pregnancy, large multinucleated structures known as syncytial nuclear aggregates (SNAs) and other trophoblast cellular debris is shed from the placenta into the maternal blood. Excessive production of trophoblast debris in response to placental stress is associated with preeclampsia. Extensive cellular stress can cause the accumulation/release of mitochondrial DNA (mtDNA) via necrosis or autophagy. Free mtDNA has been shown to induce an inflammatory response, leading to heart failure in mice1, and possibly other chronic inflammatory diseases. Increased mtDNA has been reported in maternal serum of preeclamptic patients. However, it is unclear whether this mtDNA is derived from the placenta (as a causative factor) or the maternal endothelium (as a stressresponse). We hypothesised that trophoblast debris contains mtDNA that may act as an inflammatory trigger in preeclampsia. Methods: Trophoblast debris and individual SNAs were harvested from 8 first trimester placentae and mtDNA was extracted, amplified and visualised by agarose gel electrophoresis. Beclin-1 expression in SNAs from 7 further first trimester placentae, indicated to be at increased risk of preeclampsia by uterine artery Doppler, was determined by immunohistochemistry. Results: mtDNA was detected in unfractionated trophoblast debris (n¼3 placentae) and individual SNAs (n¼25 SNAs from 5 placentae). The autophagy marker beclin-1 was detected in SNAs from 2/4 placentae at high risk of preeclampsia, but no SNAs from 3 placentae at low risk of preeclampsia. Conclusions: mtDNA is present in trophoblast debris. SNAs from placentae at increased risk of preeclampsia exhibit signs of autophagy, which may exacerbate the release of mtDNA from these structures. The clearance of trophoblast debris by maternal cells may be a mechanism that brings inflammatory mtDNA into contact with maternal immune and cardiovascular systems. 1. Oka, T, et al., Nature, 2012. 485(7397):251-5. http://dx.doi.org/10.1016/j.placenta.2013.06.118