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Hypoxia and high glucose modulate the bioactivity of placental exosomes on endothelial cells
A4
Abstracts / Placenta 36 (2015) A1eA60
NI1.5. NECROPTOSIS: A NOVEL CONTRIBUTOR TO PLACENTAL CELL DEATH IN PREECLAMPSIA Liane Bailey 1, 3, Isabella Caniggia 1, 2. 1 Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada; 2 Department of Obstetrics and Gynecology, Mount Sinai Hospital, Toronto, Ontario, Canada; 3 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada Objectives: A recent advance in the cell death field is the characterization of a regulated form of necrosis known as necroptosis. Necroptosis occurs under caspase-8 inhibition and assembly of a protein complex called the necrosome, which includes receptor-interacting protein 1 and 3 (RIP1, RIP3). Upon necrosome activation, RIP3 phosphorylates mixed lineage kinase domain-like protein (MLKL), leading to cell membrane permeabilization and cell death. We reported that high ceramide levels in preeclampsia lead to increased trophoblast cell death. However, the contribution of necroptosis in the placenta remains unknown. Hence, our objectives are to investigate necroptotic cell death in preeclampsia and ascertain the role of ceramide in necroptosis. Methods: Choriocarcinoma JEG3 cells were exposed to the pan-caspase inhibitor Q-VD-OPh to establish necroptosis. To mimic oxidative stress and high ceramide levels of preeclampsia, JEG3 were treated with 2.5 mM sodium nitroprusside (SNP, a nitric oxide donor), 50 mM synthetic C16 ceramide or 25 mM 2-oleoylethanolamine (2-OE, an acid ceramidase inhibitor). Preeclamptic (n¼10) and normotensive control placentae (n¼10) were collected after informed consent. Necrosome protein expression was assessed by western blot analysis and immunoprecipitation. Results: Caspase-8 was inhibited in Q-VD-OPh-treated JEG3, and this was associated with significantly increased RIP1 and RIP3 protein levels compared to DMSO controls. Necrosome proteins were similarly upregulated following SNP, ceramide and 2-OE treatments, suggesting increased susceptibility to necroptosis. RIP1 and phospho-MLKL protein levels were increased in preeclamptic tissue compared to controls, while no changes were found for RIP3. Necrosome activation is dependent on RIP1/RIP3 assembly. Immunoprecipitation analysis revealed a marked increase in RIP1/RIP3 binding in preeclamptic tissue relative to controls, indicating necrosome activation. Conclusion: Our work demonstrates that key players in the necroptotic pathway are activated in preeclampsia. Furthermore, oxidative stress and ceramide may stimulate necroptosis, thereby contributing to increased trophoblast cell death found in preeclampsia. Supported by CIHR.
NI1.6. HYPOXIA AND HIGH GLUCOSE MODULATE THE BIOACTIVITY OF PLACENTAL EXOSOMES ON ENDOTHELIAL CELLS Katherin Scholz-Romero, Emma Sweeney, Grace Truong, Miharu Kobayashi, Hassendrini Peiris, Gregory Duncombe, Murray Mitchell, Gregory Rice, Carlos Salomon. Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD, Australia Background: Hyperglycaemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose sensing pathways and regulation of exosome bioactivity, however, have not been established. The aim of this study was to test the hypothesis that exosomal signalling by placental cells is responsive to extracellular glucose concentrations. Methods: Cytotrophoblast cells were isolated from first trimester chorionic villi and incubated with D-glucose (5 mM or 25 mM) under 1%, 3% or 8% O2 for 48 h. L-glucose (20 mM) was used as the osmotic control.
Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63 with the ELISA kit. The effect of exosomes on cytokine (GM-CSF, IL-2, IL-4, IL-6. IL-8, IL-10, IFN-g and TNF-a) release from endothelial cells was established using the protein solution array analysis. Results: High glucose (25 mM) does not affect the size, presence of endocytc markers and morphology of exosomes, however, significantly increased the release of exosomes from cytotrophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared to controls, p <0.001). Exosomes (100 mg exosomal protein/ml) released from cytotrophoblast cells significantly increased (p <0.05) the release of all cytokines from human umbilical vein endothelial cells (HUVEC) when compared to the control (i.e. without exosomes), with the exception of IL-2 and IL-10 (p > 0.05). Maximum cytokine release was observed when HUVEC were exposed to exosomes released by cytototrophoblast cells incubated with 25 mM D-glucose under an atmosphere of 1% O2. Conclusions: Exposure of high glucose to trophoblast cells at low oxygen tension increased their exosome production and bioactivity on endothelial cells. These effects may be recapitulated and of clinical relevance in vivo in association with maternal insulin resistance, placental insufficiency and hypoxia.
NI2.1. BLOCKING LEUKEMIA INHIBITORY FACTOR IMPAIRS TROPHOBLAST INVASION AND LEADS TO ABNORMAL PLACENTATION AND PREGNANCY LOSS IN MICE Amy Winship 1, 2, Jeanne Correia 1, Ellen Menkhorst 1, Jian-Guo Zhang 3, Nicos Nicola 3, Eva Dimitriadis 1, 2. 1 MIMR-PHI Institute, Melbourne, Australia; 2 Monash University, Melbourne, Australia; 3 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Introduction: Extravillous trophoblast (EVT) invasion is crucial to remodel uterine arteries and establish utero-placental blood flow. Leukemia inhibitory factor (LIF) is produced by EVT in women and alters EVT adhesion in vitro, suggesting a role in placentation. Though this has not been investigated in vivo. We hypothesized LIF alters EVT invasion and is required for normal placentation. We determined the effect of LIF/LIF inhibition on placentation and pregnancy outcome in mice and on primary human first trimester EVT invasion. Methods: PEGylated LIF antagonist (PEGLA) or PEG were administered 2xdaily (n¼4/gp; 500mg/injection) at E8-10, E10-13 or E10-17 and mice sacrificed on final day of treatment. Placental morphology was assessed by immunohistochemistry and gene targets by PCR arrays. Primary human first trimester placental villous explants cultured on collagen drops were treated with LIF (100ng/ml) ± PEGLA (100ng/ml) or control for 48h. Area of outgrowth (mm2) was quantified (n¼6/gp). Results & discussion: Temporally blocking LIF action during placentation impaired trophoblast invasion and spiral artery remodeling. PEGLA also dramatically altered the labyrinth, with a 35% reduction in maternal sinusoid area (isolectin-B4, p<0.01) and 38% reduction in fetal vessel area (CD31, p<0.01) compared to PEG. PEGLA altered gene targets regulating placental angiogenesis and oxidative stress and increased trophoblast apoptosis at all time points in PEGLA treated mice (Cleaved caspase-3, p<0.01). At E17, pregnancy viability was significantly compromised following PEGLA from E10-17 (PEG 8.0 implantation sites±0.4vsPEGLA 4.7±0.95, p<0.05). LIF promoted human placental villous outgrowth (144 ± 28%, p<0.01) compared to control (100%). PEGLA reduced outgrowth compared to LIF (55 ± 15%, p<0.05) and totally blocked LIF-mediated outgrowth (p<0.01). Conclusion: This is the first study to demonstrate that LIF is critical for trophoblast function and in placentation in vivo. Blocking LIF resulted in significant pregnancy loss, demonstrating that LIF is required for placentation and pregnancy viability.
Abstracts / Placenta 36 (2015) A1eA60
NI1.5. NECROPTOSIS: A NOVEL CONTRIBUTOR TO PLACENTAL CELL DEATH IN PREECLAMPSIA Liane Bailey 1, 3, Isabella Caniggia 1, 2. 1 Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada; 2 Department of Obstetrics and Gynecology, Mount Sinai Hospital, Toronto, Ontario, Canada; 3 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada Objectives: A recent advance in the cell death field is the characterization of a regulated form of necrosis known as necroptosis. Necroptosis occurs under caspase-8 inhibition and assembly of a protein complex called the necrosome, which includes receptor-interacting protein 1 and 3 (RIP1, RIP3). Upon necrosome activation, RIP3 phosphorylates mixed lineage kinase domain-like protein (MLKL), leading to cell membrane permeabilization and cell death. We reported that high ceramide levels in preeclampsia lead to increased trophoblast cell death. However, the contribution of necroptosis in the placenta remains unknown. Hence, our objectives are to investigate necroptotic cell death in preeclampsia and ascertain the role of ceramide in necroptosis. Methods: Choriocarcinoma JEG3 cells were exposed to the pan-caspase inhibitor Q-VD-OPh to establish necroptosis. To mimic oxidative stress and high ceramide levels of preeclampsia, JEG3 were treated with 2.5 mM sodium nitroprusside (SNP, a nitric oxide donor), 50 mM synthetic C16 ceramide or 25 mM 2-oleoylethanolamine (2-OE, an acid ceramidase inhibitor). Preeclamptic (n¼10) and normotensive control placentae (n¼10) were collected after informed consent. Necrosome protein expression was assessed by western blot analysis and immunoprecipitation. Results: Caspase-8 was inhibited in Q-VD-OPh-treated JEG3, and this was associated with significantly increased RIP1 and RIP3 protein levels compared to DMSO controls. Necrosome proteins were similarly upregulated following SNP, ceramide and 2-OE treatments, suggesting increased susceptibility to necroptosis. RIP1 and phospho-MLKL protein levels were increased in preeclamptic tissue compared to controls, while no changes were found for RIP3. Necrosome activation is dependent on RIP1/RIP3 assembly. Immunoprecipitation analysis revealed a marked increase in RIP1/RIP3 binding in preeclamptic tissue relative to controls, indicating necrosome activation. Conclusion: Our work demonstrates that key players in the necroptotic pathway are activated in preeclampsia. Furthermore, oxidative stress and ceramide may stimulate necroptosis, thereby contributing to increased trophoblast cell death found in preeclampsia. Supported by CIHR.
NI1.6. HYPOXIA AND HIGH GLUCOSE MODULATE THE BIOACTIVITY OF PLACENTAL EXOSOMES ON ENDOTHELIAL CELLS Katherin Scholz-Romero, Emma Sweeney, Grace Truong, Miharu Kobayashi, Hassendrini Peiris, Gregory Duncombe, Murray Mitchell, Gregory Rice, Carlos Salomon. Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD, Australia Background: Hyperglycaemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose sensing pathways and regulation of exosome bioactivity, however, have not been established. The aim of this study was to test the hypothesis that exosomal signalling by placental cells is responsive to extracellular glucose concentrations. Methods: Cytotrophoblast cells were isolated from first trimester chorionic villi and incubated with D-glucose (5 mM or 25 mM) under 1%, 3% or 8% O2 for 48 h. L-glucose (20 mM) was used as the osmotic control.
Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63 with the ELISA kit. The effect of exosomes on cytokine (GM-CSF, IL-2, IL-4, IL-6. IL-8, IL-10, IFN-g and TNF-a) release from endothelial cells was established using the protein solution array analysis. Results: High glucose (25 mM) does not affect the size, presence of endocytc markers and morphology of exosomes, however, significantly increased the release of exosomes from cytotrophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared to controls, p <0.001). Exosomes (100 mg exosomal protein/ml) released from cytotrophoblast cells significantly increased (p <0.05) the release of all cytokines from human umbilical vein endothelial cells (HUVEC) when compared to the control (i.e. without exosomes), with the exception of IL-2 and IL-10 (p > 0.05). Maximum cytokine release was observed when HUVEC were exposed to exosomes released by cytototrophoblast cells incubated with 25 mM D-glucose under an atmosphere of 1% O2. Conclusions: Exposure of high glucose to trophoblast cells at low oxygen tension increased their exosome production and bioactivity on endothelial cells. These effects may be recapitulated and of clinical relevance in vivo in association with maternal insulin resistance, placental insufficiency and hypoxia.
NI2.1. BLOCKING LEUKEMIA INHIBITORY FACTOR IMPAIRS TROPHOBLAST INVASION AND LEADS TO ABNORMAL PLACENTATION AND PREGNANCY LOSS IN MICE Amy Winship 1, 2, Jeanne Correia 1, Ellen Menkhorst 1, Jian-Guo Zhang 3, Nicos Nicola 3, Eva Dimitriadis 1, 2. 1 MIMR-PHI Institute, Melbourne, Australia; 2 Monash University, Melbourne, Australia; 3 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Introduction: Extravillous trophoblast (EVT) invasion is crucial to remodel uterine arteries and establish utero-placental blood flow. Leukemia inhibitory factor (LIF) is produced by EVT in women and alters EVT adhesion in vitro, suggesting a role in placentation. Though this has not been investigated in vivo. We hypothesized LIF alters EVT invasion and is required for normal placentation. We determined the effect of LIF/LIF inhibition on placentation and pregnancy outcome in mice and on primary human first trimester EVT invasion. Methods: PEGylated LIF antagonist (PEGLA) or PEG were administered 2xdaily (n¼4/gp; 500mg/injection) at E8-10, E10-13 or E10-17 and mice sacrificed on final day of treatment. Placental morphology was assessed by immunohistochemistry and gene targets by PCR arrays. Primary human first trimester placental villous explants cultured on collagen drops were treated with LIF (100ng/ml) ± PEGLA (100ng/ml) or control for 48h. Area of outgrowth (mm2) was quantified (n¼6/gp). Results & discussion: Temporally blocking LIF action during placentation impaired trophoblast invasion and spiral artery remodeling. PEGLA also dramatically altered the labyrinth, with a 35% reduction in maternal sinusoid area (isolectin-B4, p<0.01) and 38% reduction in fetal vessel area (CD31, p<0.01) compared to PEG. PEGLA altered gene targets regulating placental angiogenesis and oxidative stress and increased trophoblast apoptosis at all time points in PEGLA treated mice (Cleaved caspase-3, p<0.01). At E17, pregnancy viability was significantly compromised following PEGLA from E10-17 (PEG 8.0 implantation sites±0.4vsPEGLA 4.7±0.95, p<0.05). LIF promoted human placental villous outgrowth (144 ± 28%, p<0.01) compared to control (100%). PEGLA reduced outgrowth compared to LIF (55 ± 15%, p<0.05) and totally blocked LIF-mediated outgrowth (p<0.01). Conclusion: This is the first study to demonstrate that LIF is critical for trophoblast function and in placentation in vivo. Blocking LIF resulted in significant pregnancy loss, demonstrating that LIF is required for placentation and pregnancy viability.