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Wnt5a decreases sFlt1 expression in trophoblast cells
Abstracts / Placenta 35 (2014) A1eA23
allele whose GC-rich promoter is epigenetically regulated. In this study, we compared the methylation status of the upstream CpG-related promoter of C19MC between chorionic villous trophoblast (VT) and extravillous trophoblast (EVT). Methods: First-trimester placental tissues from patients who gave informed consent were aseptically obtained after legal abortions (at 8-10 weeks of gestation, n¼5). Chorionic villous tips including VT were defined to be VT; EVT growing from explanted human chorionic villi was isolated. We employed bisulfite genome sequencing for analysis of the methylation status of 31 CpG sites in the C19MC promoter. The rate of methylation change was calculated using the following formula; [(extent of methylation in EVT cells/extent of methylation in VT) -1] X 100. Results: In some patients, the levels of methylation at the 31 CpG sites in EVT were higher than those in VT. However, the other patients did not show remarkable differences in methylation between EVT and VT. The levels of methylation across the 31 CpG sites was greater by approximately 30% (the average rate of methylation change of 5 samples) in EVT compared to VT. Conclusion: These findings suggest that the observed difference in epigenetic profile reflects a disparity in C19MC miRNA expression between EVT and VT cells. The detailed epigenetic regulatory mechanisms underlying C19MC miRNA expression in trophoblast cells require further investigation.
O-108. THE ANTI-INFLAMMATORY EFFECT OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS Reizo Onishi a, b, c, d, Shunsuke Ohnishi a, Ryosuke Higashi b, Kenichi Yamahara c, Jun Yoshimatsu d, Takehiko Katsurada b, Naoto Okubo b, Koji Nakagawa b, Hiroshi Takeda b, Naoya Sakamoto a. a Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Japan; b Laboratory of Pathophysiology and Therapeutics, Hokkaido University Graduate School of Pharmacy, Japan; c Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Japan; d Division of Maternal and Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Aims: Several studies have reported that mesenchymal stem cells (MSCs) have the potential to improve damaged tissues by the secretion of antiinflammatory molecules. Recent investigations have demonstrated that amnion-derived MSCs can be easily isolated from human fetal membrane, and a large amount of cells can be obtained. We examined whether human amnion-derived MSCs (AMSCs) have the anti-inflammatory effects in vitro. Methods: Mouse macrophage RAW264.7 cells were stimulated with lipopolysaccharide (LPS) for 24h under co-incubation with AMSCs, or were cultured with the conditioned medium (CM) obtained from AMSCs. The
A23
expression levels and production of proinflammatory cytokines were measured by quantitative RT-PCR and the enzyme-linked immunosorbent assay (ELISA). HEK293 cells transfected with NF-kB reporter plasmid were treated with TNF-a or LPS for 6h under incubation with CM, and reporter gene assay was performed. RAW264.7 cells were stimulated by LPS after co-incubation for 3h with CM. Nuclear translocation of NF-kB was investigated with immunofluorescent staining. Phosphorylation of IkB was examined by Western blot analysis. Results: Coculture with AMSCs or incubation with CM obtained from AMSCs decreased the expression of proinflammatory cytokines, and CM down-regulated the secretion of TNF-alpha. AMSC coculture inhibited the NF-kB activation induced by TNF-a or LPS. Nuclear translocation of NF-kB by LPS was significantly inhibited by CM. However, Western blotting analysis demonstrated that the phosphorylation of IkB induced by TNF-a or LPS was not inhibited by the conditioned medium. Conclusion: AMSC coculture and incubation with CM downregulated the inflammatory response in cultured macrophages. CM may have suppressed the inflammatory signal of NF-kB at the step of nuclear translocation.
O-109. WNT5A DECREASES SFLT1 EXPRESSION IN TROPHOBLAST CELLS Mari Ujita, Eiji Kondoh, Kaoru Kawasaki, Yoshitsugu Chigusa, Hiroshi Takai, Hikaru Kiyokawa, Fumitomo Nishimura, Haruta Mogami, Ikuo Konishi. Department of Obstetrics and Gynecology, Kyoto University, Japan Objective: Wnt signaling is implicated in mice placentation, and human trophoblast migration and invasion. However, the role of Wnt signaling in preeclampsia（PE） has not been clarified yet. We previously reported the expression of Wnt5a, which is involved in non-beta-catenin dependent Wnt pathway, was significantly decreased in PE placenta. The aim of the present study was to elucidate the influence of Wnt5a on vascular growth factor related genes. Methods: HTR-8/SVneo trophoblast cells were cultured with or without recombinant Wnt5a（50ng, 100ng, 200ng/ml）for 24 hours. The mRNA expressions of sFlt-1, PlGF, TGF-b1, TGF-b2 and TGF-b3 were measured by quantitative RT-PCR. Moreover, we inhibited Wnt5a expression of HTR-8/ SVneo trophoblast cells using shRNA vector. Results: sFlt1 was significantly decreased by recombinant Wnt5a in a dose-dependent manner. The expression levels of PlGF, TGF-b1 and TGF-b3 were significantly increased by Wnt5a in a concentration-dependent manner. TGF-b3 was significantly decreased in HTR-8/SV neo cells via knockout of Wnt5a, while no alteration was observed in TGF-b1 and TGFb2 expression. Conclusion: Wnt5a may be involved in the regulation of vascular growth factor related genes in trophoblast cells.
allele whose GC-rich promoter is epigenetically regulated. In this study, we compared the methylation status of the upstream CpG-related promoter of C19MC between chorionic villous trophoblast (VT) and extravillous trophoblast (EVT). Methods: First-trimester placental tissues from patients who gave informed consent were aseptically obtained after legal abortions (at 8-10 weeks of gestation, n¼5). Chorionic villous tips including VT were defined to be VT; EVT growing from explanted human chorionic villi was isolated. We employed bisulfite genome sequencing for analysis of the methylation status of 31 CpG sites in the C19MC promoter. The rate of methylation change was calculated using the following formula; [(extent of methylation in EVT cells/extent of methylation in VT) -1] X 100. Results: In some patients, the levels of methylation at the 31 CpG sites in EVT were higher than those in VT. However, the other patients did not show remarkable differences in methylation between EVT and VT. The levels of methylation across the 31 CpG sites was greater by approximately 30% (the average rate of methylation change of 5 samples) in EVT compared to VT. Conclusion: These findings suggest that the observed difference in epigenetic profile reflects a disparity in C19MC miRNA expression between EVT and VT cells. The detailed epigenetic regulatory mechanisms underlying C19MC miRNA expression in trophoblast cells require further investigation.
O-108. THE ANTI-INFLAMMATORY EFFECT OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS Reizo Onishi a, b, c, d, Shunsuke Ohnishi a, Ryosuke Higashi b, Kenichi Yamahara c, Jun Yoshimatsu d, Takehiko Katsurada b, Naoto Okubo b, Koji Nakagawa b, Hiroshi Takeda b, Naoya Sakamoto a. a Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Japan; b Laboratory of Pathophysiology and Therapeutics, Hokkaido University Graduate School of Pharmacy, Japan; c Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Japan; d Division of Maternal and Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Aims: Several studies have reported that mesenchymal stem cells (MSCs) have the potential to improve damaged tissues by the secretion of antiinflammatory molecules. Recent investigations have demonstrated that amnion-derived MSCs can be easily isolated from human fetal membrane, and a large amount of cells can be obtained. We examined whether human amnion-derived MSCs (AMSCs) have the anti-inflammatory effects in vitro. Methods: Mouse macrophage RAW264.7 cells were stimulated with lipopolysaccharide (LPS) for 24h under co-incubation with AMSCs, or were cultured with the conditioned medium (CM) obtained from AMSCs. The
A23
expression levels and production of proinflammatory cytokines were measured by quantitative RT-PCR and the enzyme-linked immunosorbent assay (ELISA). HEK293 cells transfected with NF-kB reporter plasmid were treated with TNF-a or LPS for 6h under incubation with CM, and reporter gene assay was performed. RAW264.7 cells were stimulated by LPS after co-incubation for 3h with CM. Nuclear translocation of NF-kB was investigated with immunofluorescent staining. Phosphorylation of IkB was examined by Western blot analysis. Results: Coculture with AMSCs or incubation with CM obtained from AMSCs decreased the expression of proinflammatory cytokines, and CM down-regulated the secretion of TNF-alpha. AMSC coculture inhibited the NF-kB activation induced by TNF-a or LPS. Nuclear translocation of NF-kB by LPS was significantly inhibited by CM. However, Western blotting analysis demonstrated that the phosphorylation of IkB induced by TNF-a or LPS was not inhibited by the conditioned medium. Conclusion: AMSC coculture and incubation with CM downregulated the inflammatory response in cultured macrophages. CM may have suppressed the inflammatory signal of NF-kB at the step of nuclear translocation.
O-109. WNT5A DECREASES SFLT1 EXPRESSION IN TROPHOBLAST CELLS Mari Ujita, Eiji Kondoh, Kaoru Kawasaki, Yoshitsugu Chigusa, Hiroshi Takai, Hikaru Kiyokawa, Fumitomo Nishimura, Haruta Mogami, Ikuo Konishi. Department of Obstetrics and Gynecology, Kyoto University, Japan Objective: Wnt signaling is implicated in mice placentation, and human trophoblast migration and invasion. However, the role of Wnt signaling in preeclampsia（PE） has not been clarified yet. We previously reported the expression of Wnt5a, which is involved in non-beta-catenin dependent Wnt pathway, was significantly decreased in PE placenta. The aim of the present study was to elucidate the influence of Wnt5a on vascular growth factor related genes. Methods: HTR-8/SVneo trophoblast cells were cultured with or without recombinant Wnt5a（50ng, 100ng, 200ng/ml）for 24 hours. The mRNA expressions of sFlt-1, PlGF, TGF-b1, TGF-b2 and TGF-b3 were measured by quantitative RT-PCR. Moreover, we inhibited Wnt5a expression of HTR-8/ SVneo trophoblast cells using shRNA vector. Results: sFlt1 was significantly decreased by recombinant Wnt5a in a dose-dependent manner. The expression levels of PlGF, TGF-b1 and TGF-b3 were significantly increased by Wnt5a in a concentration-dependent manner. TGF-b3 was significantly decreased in HTR-8/SV neo cells via knockout of Wnt5a, while no alteration was observed in TGF-b1 and TGFb2 expression. Conclusion: Wnt5a may be involved in the regulation of vascular growth factor related genes in trophoblast cells.