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lysosome pathway cooperated with endoplasmic reticulum (ER) stress
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Abstracts / Placenta 46 (2016) 102e121
we should take care of patients in this situation. Most of the time during pregnancy, reduced blood platelets is not a significant risk but cases when it is under 50,000m3 is both rare and dangerous. The cause of such a dramatic decrease may be blood loss. This leads to problems in coagulation and thus even more blood loss. Another common cause may be complications during pregnancy or ideopacific blood platelet decrease. During pregnancy and after delivery if the number of platelets is less than 50,000m3 we should examine the placenta to help discover the reason for blood platelet loss. Materials: Pregnancy and post-delivery blood examinations over a ten year period (approximately 10,000 cases in total) Result: Out of 10,000 cases over a ten year period we found 18 cases in which blood platelets were under 50,000m3. From those 18 cases: - 8 cases were complicated with HELLP syndrome - 3 cases with PIH (Pregnancy induced hypertension) without HELLP syndrome - 1 case of Multiple Myeloma： ：MM - 1 case of Idiopathic Thrombocytopenic Purpura: ITP - 1 case of Myelodysplastic Syndromes: MDS - 4 cases of abruption and low set placenta and so on PIH and HELLP syndrome cases were complicated with decidua thrombosis, placental infarction, and evidence of previous bleeding. Furthermore low set placenta had decidua defect and acute abruption had new bleeding. Conclusion: The cause of platelet number under 50,000m3 is decidua thrombosis and the way to solve this problem is through blood examination and ultrasound prediction and placental examination.
PREECLAMPSIA (PE) SERUM DISRUPTS THE AUTOPHAGY/LYSOSOME PATHWAY COOPERATED WITH ENDOPLASMIC RETICULUM (ER) STRESS Akitoshi Nakashima 1, Tomoko Shima 1, Aiko Aoki 1, Kumiko Inada 1, Satoshi Yoneda 1, Arihiro Shiozaki 1, Osamu Yoshino 1, Surendra Sharma 2, Shigeru Saito 1. 1 Department of Obstetrics & Gynecology, Faculty of Medicine, University of Toyama, Japan; 2 Department of Pediatrics, Women and Infant hospital, Brown University, Japan Purpose: We have reported that impaired autophagy contributes to the shallow trophoblast invasion and poor vascular remodeling (1st step of PE). It has recently reported that protein aggregation is involved in dysfunction of trophoblasts on PE placenta (2nd step of PE). The aim of this study is to clarify the correlation between impaired autophagy and dysfunction of placenta. Methods: We used placental tissues and sera from PE patients with informed consent. Proteostat dye® is used for detecting aggregated proteins in placental tissues. An autophagy-deficient trophoblast cell line, HchEpC1b-Atg4Bmt, was used in vitro assays. Results: We firstly found that the number of lysosome was significantly decreased (p¼0.037), and aggregated proteins were increased (p¼0.021) in the syncytiotrophoblast of PE placenta, suggesting dysfuction of trophoblasts. Interestingly, ER stress inducers suppressed the lysosomal number and functions in a trophoblast cell line, suggesting that ER stress reduces autophagy and protein quality control. The expression of nuclear TFEB, a master regulator of autophagy and lysosome biogenesis, was significantly decreased (p¼0.005) in the PE placenta than that in normal placenta. Sera from PE patients suppressed the TFEB activation via hyperactivating mammalian target of rapamycin. In addition, trehalose, a natural alpha-linked disaccharide, activated TFEB in the trophoblast cell line. Conclusions: This study firstly showed ER stress reduce autophagy by suppressing lysosome number and function. Impaired autophagy contribute not only 1st step of PE but also 2nd step of PE. TFEB-modulated autophagy activation can be a therapuetic option for PE near future.
IDENTIFICATION EQUIVALENT
AND
ISOLATION
OF
A
MURINE
SUPPRESSYN
Jun Sugimoto 1, Danny Schust 2, So Nakagawa 3, Takaya Oda 1, Yoshihiro Jinno 1. 1 Department of Molecular Biology, University of the Ryukyus, Japan; 2 Department of Obstetrics, Gynecology and Women's Health, University of Missouri, United States; 3 Department of Molecular Life Science, Tokai University School of Medicine, Japan Suppressyn is one of the first human proteins to be identified that inhibits cell-cell fusion. Its origination from a human endogenous retrovirus (HERV), targeted expression in the placenta and ability to inhibit the function of known placental fusogens such as syncytin-1 suggests possible involvement in normal and abnormal placental development. In vivo manipulation of this protein to allow determination of physiological relevance, however, requires the establishment of a suppressyn-related animal model (e.g., a suppressyn knock-out mouse). To accomplish this, we sought to identify and isolate a murine equivalent of human suppressyn. Using existing murine placental RNAseq expression datasets, we first identified 16 candidate transcripts that originated from murine endogenous retroviral envelope regions. Additional RT- PCR analyses demonstrated that four of these murine candidates displayed were expressed in the placenta, although only one of these four was expressed only in the placenta. We stably expressed these 4 candidate genes in 293T cells, in which cell-cell fusion can be induced by the murine equivalent of human syncytin-1 (mouse syncytin-A). One of these candidates displayed antifusogenic effects in this in vitro model. We hypothesize that this endogenous retroviral envelope protein may represent the murine equivalent of human suppressyn. Further functional analyses using murine knock-out technology are underway and should help to define the role for human suppressyn in placental health and disease.
A RHO-ASSOCIATED KINASE INHIBITOR, Y-27632, IMPROVES CYTOTROPHOBLAST CULTURE EFFICIENCY BY ENHANCING ADHESION AND DIFFERENTIATION Kenichiro Motomura, Naoko Okada, Akio Matsuda, Haruhiko Sago, Hirohisa Saito, Kenji Matsumoto. National Center for Child Health and Development, Japan Objectives: In vitro studies using human syncytiotrophoblast (STB) provide invaluable evidence regarding the physiology of the placenta and the pathogenesis of placental dysfunction-related disorders. Term human placenta-derived cytotrophoblasts (CTB), which spontaneously differentiate into STB, are reportedly useful for investigating STB function. However, because individuals show variation in cell adhesion to culture plates and in cell-to-cell fusion of CTB, reproducible in vitro culture of CTB is not always easy. The Rho kinase pathway reportedly regulates various cell functions, including cell adhesion and survival. Previous reports suggested that specific inhibitors of Rho-associated coiled-coil kinase (ROCK), a component of the Rho/Rho kinase pathway, improve the culture efficiency of other primary cells. In this study, we attempted to improve the culture efficiency of CTB by using a ROCK inhibitor, Y27362. Methods: This study was approved by the ethics board of our institute. CTB were isolated from term, uncomplicated placentas by trypsineDNase IeDispase II treatment and Percoll density gradient centrifugation, followed by negative- selection using anti-HLA-class I antibody and immunomagnetic beads. Purified CTB were cultured for 96 h on non- coated tissue culture plates in medium containing 10% FBS and EGF (10 ng/ml), with and without Y-27632 (10 mg/ml). The effect of Y-27632 was assessed by q-PCR, ELISA, immunofluorescence staining and WST8 assay. Results: Y-27632 treatment significantly enhanced CTB’s adhesion to tissue culture plates, mitochondrial activity, mRNA expression of cell fusionrelated proteins, cell-to-cell fusion and hCG secretion. Conclusion: Y-27632 improved cytotrophoblast culture efficiency by enhancing adhesion and differentiation. In addition, the Rho/Rho kinase pathway may be involved in CTB differentiation in vivo.
REGULATORY MECHANISMS OF EXTRAVILLOUS TROPHOBLAST CELL INVASION: CD44 AND PLACENTA-SPECIFIC MICRORNA
Abstracts / Placenta 46 (2016) 102e121
we should take care of patients in this situation. Most of the time during pregnancy, reduced blood platelets is not a significant risk but cases when it is under 50,000m3 is both rare and dangerous. The cause of such a dramatic decrease may be blood loss. This leads to problems in coagulation and thus even more blood loss. Another common cause may be complications during pregnancy or ideopacific blood platelet decrease. During pregnancy and after delivery if the number of platelets is less than 50,000m3 we should examine the placenta to help discover the reason for blood platelet loss. Materials: Pregnancy and post-delivery blood examinations over a ten year period (approximately 10,000 cases in total) Result: Out of 10,000 cases over a ten year period we found 18 cases in which blood platelets were under 50,000m3. From those 18 cases: - 8 cases were complicated with HELLP syndrome - 3 cases with PIH (Pregnancy induced hypertension) without HELLP syndrome - 1 case of Multiple Myeloma： ：MM - 1 case of Idiopathic Thrombocytopenic Purpura: ITP - 1 case of Myelodysplastic Syndromes: MDS - 4 cases of abruption and low set placenta and so on PIH and HELLP syndrome cases were complicated with decidua thrombosis, placental infarction, and evidence of previous bleeding. Furthermore low set placenta had decidua defect and acute abruption had new bleeding. Conclusion: The cause of platelet number under 50,000m3 is decidua thrombosis and the way to solve this problem is through blood examination and ultrasound prediction and placental examination.
PREECLAMPSIA (PE) SERUM DISRUPTS THE AUTOPHAGY/LYSOSOME PATHWAY COOPERATED WITH ENDOPLASMIC RETICULUM (ER) STRESS Akitoshi Nakashima 1, Tomoko Shima 1, Aiko Aoki 1, Kumiko Inada 1, Satoshi Yoneda 1, Arihiro Shiozaki 1, Osamu Yoshino 1, Surendra Sharma 2, Shigeru Saito 1. 1 Department of Obstetrics & Gynecology, Faculty of Medicine, University of Toyama, Japan; 2 Department of Pediatrics, Women and Infant hospital, Brown University, Japan Purpose: We have reported that impaired autophagy contributes to the shallow trophoblast invasion and poor vascular remodeling (1st step of PE). It has recently reported that protein aggregation is involved in dysfunction of trophoblasts on PE placenta (2nd step of PE). The aim of this study is to clarify the correlation between impaired autophagy and dysfunction of placenta. Methods: We used placental tissues and sera from PE patients with informed consent. Proteostat dye® is used for detecting aggregated proteins in placental tissues. An autophagy-deficient trophoblast cell line, HchEpC1b-Atg4Bmt, was used in vitro assays. Results: We firstly found that the number of lysosome was significantly decreased (p¼0.037), and aggregated proteins were increased (p¼0.021) in the syncytiotrophoblast of PE placenta, suggesting dysfuction of trophoblasts. Interestingly, ER stress inducers suppressed the lysosomal number and functions in a trophoblast cell line, suggesting that ER stress reduces autophagy and protein quality control. The expression of nuclear TFEB, a master regulator of autophagy and lysosome biogenesis, was significantly decreased (p¼0.005) in the PE placenta than that in normal placenta. Sera from PE patients suppressed the TFEB activation via hyperactivating mammalian target of rapamycin. In addition, trehalose, a natural alpha-linked disaccharide, activated TFEB in the trophoblast cell line. Conclusions: This study firstly showed ER stress reduce autophagy by suppressing lysosome number and function. Impaired autophagy contribute not only 1st step of PE but also 2nd step of PE. TFEB-modulated autophagy activation can be a therapuetic option for PE near future.
IDENTIFICATION EQUIVALENT
AND
ISOLATION
OF
A
MURINE
SUPPRESSYN
Jun Sugimoto 1, Danny Schust 2, So Nakagawa 3, Takaya Oda 1, Yoshihiro Jinno 1. 1 Department of Molecular Biology, University of the Ryukyus, Japan; 2 Department of Obstetrics, Gynecology and Women's Health, University of Missouri, United States; 3 Department of Molecular Life Science, Tokai University School of Medicine, Japan Suppressyn is one of the first human proteins to be identified that inhibits cell-cell fusion. Its origination from a human endogenous retrovirus (HERV), targeted expression in the placenta and ability to inhibit the function of known placental fusogens such as syncytin-1 suggests possible involvement in normal and abnormal placental development. In vivo manipulation of this protein to allow determination of physiological relevance, however, requires the establishment of a suppressyn-related animal model (e.g., a suppressyn knock-out mouse). To accomplish this, we sought to identify and isolate a murine equivalent of human suppressyn. Using existing murine placental RNAseq expression datasets, we first identified 16 candidate transcripts that originated from murine endogenous retroviral envelope regions. Additional RT- PCR analyses demonstrated that four of these murine candidates displayed were expressed in the placenta, although only one of these four was expressed only in the placenta. We stably expressed these 4 candidate genes in 293T cells, in which cell-cell fusion can be induced by the murine equivalent of human syncytin-1 (mouse syncytin-A). One of these candidates displayed antifusogenic effects in this in vitro model. We hypothesize that this endogenous retroviral envelope protein may represent the murine equivalent of human suppressyn. Further functional analyses using murine knock-out technology are underway and should help to define the role for human suppressyn in placental health and disease.
A RHO-ASSOCIATED KINASE INHIBITOR, Y-27632, IMPROVES CYTOTROPHOBLAST CULTURE EFFICIENCY BY ENHANCING ADHESION AND DIFFERENTIATION Kenichiro Motomura, Naoko Okada, Akio Matsuda, Haruhiko Sago, Hirohisa Saito, Kenji Matsumoto. National Center for Child Health and Development, Japan Objectives: In vitro studies using human syncytiotrophoblast (STB) provide invaluable evidence regarding the physiology of the placenta and the pathogenesis of placental dysfunction-related disorders. Term human placenta-derived cytotrophoblasts (CTB), which spontaneously differentiate into STB, are reportedly useful for investigating STB function. However, because individuals show variation in cell adhesion to culture plates and in cell-to-cell fusion of CTB, reproducible in vitro culture of CTB is not always easy. The Rho kinase pathway reportedly regulates various cell functions, including cell adhesion and survival. Previous reports suggested that specific inhibitors of Rho-associated coiled-coil kinase (ROCK), a component of the Rho/Rho kinase pathway, improve the culture efficiency of other primary cells. In this study, we attempted to improve the culture efficiency of CTB by using a ROCK inhibitor, Y27362. Methods: This study was approved by the ethics board of our institute. CTB were isolated from term, uncomplicated placentas by trypsineDNase IeDispase II treatment and Percoll density gradient centrifugation, followed by negative- selection using anti-HLA-class I antibody and immunomagnetic beads. Purified CTB were cultured for 96 h on non- coated tissue culture plates in medium containing 10% FBS and EGF (10 ng/ml), with and without Y-27632 (10 mg/ml). The effect of Y-27632 was assessed by q-PCR, ELISA, immunofluorescence staining and WST8 assay. Results: Y-27632 treatment significantly enhanced CTB’s adhesion to tissue culture plates, mitochondrial activity, mRNA expression of cell fusionrelated proteins, cell-to-cell fusion and hCG secretion. Conclusion: Y-27632 improved cytotrophoblast culture efficiency by enhancing adhesion and differentiation. In addition, the Rho/Rho kinase pathway may be involved in CTB differentiation in vivo.
REGULATORY MECHANISMS OF EXTRAVILLOUS TROPHOBLAST CELL INVASION: CD44 AND PLACENTA-SPECIFIC MICRORNA