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Trophoblast specific conditional Atg7 knockout pregnant mice develop gestational hypertension
Abstracts / Placenta 36 (2015) A1eA14
in placentas with VUE are still largely unknown. We aimed to verify whether apoptosis-related factors are increased in VUE placentas. Furthermore, we determined apoptosis of villous cells. Methods: Six placentas with VUE and 3 control placentas were stained using immunohistochemistry with antibodies for CD3, CD4, CD8, CD68, CD163, perforin, granzyme B, granzyme K, and C5b-9. TUNEL assay analysis was also performed with these placentas. The percentage of cells that stained positive, CD163/CD68 ratio, percentage of C5b-9 positive area, and apoptosis index were quantified and compared between the inflammatory lesions of the VUE placentas, non-VUE inflammatory lesions of the VUE placentas, and control placentas. Results: The percentages of CD3, CD4, CD8 CD68, CD163, perforin, and granzyme B positive cells were significantly higher in the inflammatory lesions of the VUE placentas (p<0.05). The intravillous CD163/CD68 ratio was higher in the inflammatory lesions compared with the non-inflammatory lesion of the VUE placentas (p<0.05). The percentage of granzyme K-positive cells was not significantly different between the groups. C5b-9 deposition was higher in the inflammatory lesions of the VUE placentas (p<0.05). TUNEL-positive cells were significantly higher in the inflammatory lesions of the VUE placentas (p<0.05). Discussion: To the best of our knowledge, this is the first report to assess villous injury, especially from a viewpoint of villous apoptosis in VUE placentas. An activated perforin/granzyme pathway and C5b-9 are suggested as possible mechanisms of apoptosis.
JPA2015-49. PREECLAMPSIA SERUM DISRUPTS THE AUTOPHAGY COOPERATED WITH ENDOPLASMIC RETICULUM STRESS
PATHWAY
Akitoshi Nakashima 1, 2, Tomoko Shima 1, Akemi Ushijima 1, Aiko Aoki 1, Kumiko Inada 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 & Infants Hospital of Rhode Island, Brown University, Japan Purpose: We have reported that impaired autophagy contributes to the pathophysiology of preeclampsia. It is, however, unknown the mechanism. This paper shows the mechanism how autophagy is inhibited in preeclamptic placenta. Materials & methods: We used placental tissues and serums from preeclampsia patients with informed consent. In addition, an autophagydeficient extravillous trophoblast cell line is used for in vitro studies. Proteostat dye® is also used for detecting aggregated proteins. Results: Lysosome functions are necessary for the autophagy pathway, an important mechanism for protein quality control. We found the decreased expression of lysosome-associated membrane proteins (LAMP1 & LAMP2), which are related with the number of lysosome, in the syncytiotrophoblasts in the preeclampsia placenta. Meanwhile, aggregated proteins were increased in the preeclampsia placenta. Secondarily, ER stress inducers, brefeldin A and tunicamycin, suppressed the LAMP1 and LAMP2 expressions in the autophagy-deficient trophoblasts, resulting in suppressing the autophagy in trophoblasts. We then took notice of Transcriptional factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis. TFEB activates the lysosome biogenesis and functions. The expression of TFEB was significantly decreased in the preeclampsia placenta than the normal placenta. Finally, we found that preeclampsia serum activated mammalian TOR, which inhibits TFEB activity, resulting in suppressing the TFEB function. Taken together, not only ER stress but also preeclampsia serum cooperatively induced toxic protein aggregation in preeclampsia placenta via suppressing autophagy and lysosome functions. Conclusions: In this study, failure of autophagy pathway by ER stress was involved in the placental disorder in preeclampsia. Overexpression of TFEB was known to improve the symptoms in a neurodegenerative disease of mice model. TFEB, which activates autophagy, would be a new possible therapeutic option for preeclampsia.
A13
JPA2015-50. TROPHOBLAST SPECIFIC CONDITIONAL ATG7 KNOCKOUT PREGNANT MICE DEVELOP GESTATIONAL HYPERTENSION Aiko Aoki 1, 2,3, Masahito Ikawa 2, Akitoshi Nakashima 1, Tamotsu Yoshimori 3, Shigeru Saito 1. 1 Department of Obstetrics and Gynecology, University of Toyama, Japan; 2 Research Institute for Microbial Diseases, Osaka University, Japan; 3 Department of Genetics, Graduate School of Medicine, Osaka University, Japan Preeclampsia and gestational hypertension, which affect 2-8% of pregnancies, are the major causes of maternal and neonatal morbidity and mortality. Many factors have been implicated in its pathogenesis, including anti-angiogenic factors, immune responses, antioxidants, and autophagy. We have been reported that soluble endoglin suppresses autophagic activity in human trophoblast cell line, resulting in the shallow invasion in vitro. However, it is still unclear whether the impaired autophagy is the cause or the result of the pathophysiology of preeclampsia. Systemic Atg7, an autophagy-related protein, knockout mice has been reported previously to display neonatal lethality and fetal growth retardation (FGR). In this study, we found that pregnant Atg7(+/-) females carrying some Atg7(-/-) embryos did not develop hypertension or proteinuria, typical symptoms of preeclampsia, although FGR was reproducible in Atg7(-/-) pups. To further examine the contribution of placenta-specific autophagy apart from the fetal autophagy, we utilized a lenti-viral vector mediated trophoblast-specific Atg7 knockout system and investigated adverse maternal and fetal outcomes in pregnancy. Conditional mice with Atg7 knockout placentas developed gestational hypertension, and autophagydeficient placentas were significantly lighter, whereas no significant differences were observed in protein concentrations in the urine or fetal body weights. P62 immunostaining of placental sections revealed impaired autophagy in the giant trophoblasts and the spongiotrophoblast layer, which has been reported to correspond to extravillous trophoblast in humans. Trophoblast-specific antibody was stained to see the invasive trophoblasts into the maternal decidua. The rate of invasive trophoblasts was significantly reduced in autophagy-deficient placentas. In conclusion, we showed for the first time that impaired autophagy in trophoblasts was involved in the pathogenesis of gestational hypertension, and it might be one of the factors exacerbating preeclampsia by impairing the function of giant trophoblasts and the spongiotrophoblast layer, resulting in the shallow invasion of trophoblasts into the maternal decidua.
JPA2015-51. CHANGES OF NK CELLS WITH NATURAL CYTOTOXICITY RECEPTOR (NCR) IN PREECLAMPSIA Hideki Takahashi, Yusuke Kobayashi, Souichirou Kuno, Takayuki Matsuno, Takayuki Matsuno, Motomi Yamazaki, Fumihisa Chishima, Tatsuo Yamamoto. Department of Obstetrics & Gynecology, Nihon University, School of Medicine, Tokyo, Japan Objectives: NCR have been reported as the receptor to regulate NK cell cytotoxicity and cytokine production. In order to investigate the changes of NK cell in preeclampsia, we studied NK cells with NCRs in placenta decidua and peripheral blood. Methods: Placenta decidua and peripheral blood were taken from preeclampsia patients (PE) and elective cesarean section patients (NP) with informed consent. The lymphocytes were separated using density gradient centrifugation (DGC) from peripheral blood and using DGC after enzyme digestion from decidua. NK cells were stained by FITC antiCD56, Per-CP antiCD16 and Per-CP antiCD3 monoclonal antibody. NCR was stained by PE conjugated antiCD314 (NKG2D) monoclonal antibodies. The stained cells were analyzed using flow cytometry performed with a FACS Caliber (Becton Dickinson). Results: The percentages of CD56+CD16+ cells in CD3 negative and CD56 positive lymphocytes in peripheral blood between PE and NP were no significant difference. However, the percentages in decidual cells of PE were lower than those of NP. The positive cells of CD314 (NKG2D) in
in placentas with VUE are still largely unknown. We aimed to verify whether apoptosis-related factors are increased in VUE placentas. Furthermore, we determined apoptosis of villous cells. Methods: Six placentas with VUE and 3 control placentas were stained using immunohistochemistry with antibodies for CD3, CD4, CD8, CD68, CD163, perforin, granzyme B, granzyme K, and C5b-9. TUNEL assay analysis was also performed with these placentas. The percentage of cells that stained positive, CD163/CD68 ratio, percentage of C5b-9 positive area, and apoptosis index were quantified and compared between the inflammatory lesions of the VUE placentas, non-VUE inflammatory lesions of the VUE placentas, and control placentas. Results: The percentages of CD3, CD4, CD8 CD68, CD163, perforin, and granzyme B positive cells were significantly higher in the inflammatory lesions of the VUE placentas (p<0.05). The intravillous CD163/CD68 ratio was higher in the inflammatory lesions compared with the non-inflammatory lesion of the VUE placentas (p<0.05). The percentage of granzyme K-positive cells was not significantly different between the groups. C5b-9 deposition was higher in the inflammatory lesions of the VUE placentas (p<0.05). TUNEL-positive cells were significantly higher in the inflammatory lesions of the VUE placentas (p<0.05). Discussion: To the best of our knowledge, this is the first report to assess villous injury, especially from a viewpoint of villous apoptosis in VUE placentas. An activated perforin/granzyme pathway and C5b-9 are suggested as possible mechanisms of apoptosis.
JPA2015-49. PREECLAMPSIA SERUM DISRUPTS THE AUTOPHAGY COOPERATED WITH ENDOPLASMIC RETICULUM STRESS
PATHWAY
Akitoshi Nakashima 1, 2, Tomoko Shima 1, Akemi Ushijima 1, Aiko Aoki 1, Kumiko Inada 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 & Infants Hospital of Rhode Island, Brown University, Japan Purpose: We have reported that impaired autophagy contributes to the pathophysiology of preeclampsia. It is, however, unknown the mechanism. This paper shows the mechanism how autophagy is inhibited in preeclamptic placenta. Materials & methods: We used placental tissues and serums from preeclampsia patients with informed consent. In addition, an autophagydeficient extravillous trophoblast cell line is used for in vitro studies. Proteostat dye® is also used for detecting aggregated proteins. Results: Lysosome functions are necessary for the autophagy pathway, an important mechanism for protein quality control. We found the decreased expression of lysosome-associated membrane proteins (LAMP1 & LAMP2), which are related with the number of lysosome, in the syncytiotrophoblasts in the preeclampsia placenta. Meanwhile, aggregated proteins were increased in the preeclampsia placenta. Secondarily, ER stress inducers, brefeldin A and tunicamycin, suppressed the LAMP1 and LAMP2 expressions in the autophagy-deficient trophoblasts, resulting in suppressing the autophagy in trophoblasts. We then took notice of Transcriptional factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis. TFEB activates the lysosome biogenesis and functions. The expression of TFEB was significantly decreased in the preeclampsia placenta than the normal placenta. Finally, we found that preeclampsia serum activated mammalian TOR, which inhibits TFEB activity, resulting in suppressing the TFEB function. Taken together, not only ER stress but also preeclampsia serum cooperatively induced toxic protein aggregation in preeclampsia placenta via suppressing autophagy and lysosome functions. Conclusions: In this study, failure of autophagy pathway by ER stress was involved in the placental disorder in preeclampsia. Overexpression of TFEB was known to improve the symptoms in a neurodegenerative disease of mice model. TFEB, which activates autophagy, would be a new possible therapeutic option for preeclampsia.
A13
JPA2015-50. TROPHOBLAST SPECIFIC CONDITIONAL ATG7 KNOCKOUT PREGNANT MICE DEVELOP GESTATIONAL HYPERTENSION Aiko Aoki 1, 2,3, Masahito Ikawa 2, Akitoshi Nakashima 1, Tamotsu Yoshimori 3, Shigeru Saito 1. 1 Department of Obstetrics and Gynecology, University of Toyama, Japan; 2 Research Institute for Microbial Diseases, Osaka University, Japan; 3 Department of Genetics, Graduate School of Medicine, Osaka University, Japan Preeclampsia and gestational hypertension, which affect 2-8% of pregnancies, are the major causes of maternal and neonatal morbidity and mortality. Many factors have been implicated in its pathogenesis, including anti-angiogenic factors, immune responses, antioxidants, and autophagy. We have been reported that soluble endoglin suppresses autophagic activity in human trophoblast cell line, resulting in the shallow invasion in vitro. However, it is still unclear whether the impaired autophagy is the cause or the result of the pathophysiology of preeclampsia. Systemic Atg7, an autophagy-related protein, knockout mice has been reported previously to display neonatal lethality and fetal growth retardation (FGR). In this study, we found that pregnant Atg7(+/-) females carrying some Atg7(-/-) embryos did not develop hypertension or proteinuria, typical symptoms of preeclampsia, although FGR was reproducible in Atg7(-/-) pups. To further examine the contribution of placenta-specific autophagy apart from the fetal autophagy, we utilized a lenti-viral vector mediated trophoblast-specific Atg7 knockout system and investigated adverse maternal and fetal outcomes in pregnancy. Conditional mice with Atg7 knockout placentas developed gestational hypertension, and autophagydeficient placentas were significantly lighter, whereas no significant differences were observed in protein concentrations in the urine or fetal body weights. P62 immunostaining of placental sections revealed impaired autophagy in the giant trophoblasts and the spongiotrophoblast layer, which has been reported to correspond to extravillous trophoblast in humans. Trophoblast-specific antibody was stained to see the invasive trophoblasts into the maternal decidua. The rate of invasive trophoblasts was significantly reduced in autophagy-deficient placentas. In conclusion, we showed for the first time that impaired autophagy in trophoblasts was involved in the pathogenesis of gestational hypertension, and it might be one of the factors exacerbating preeclampsia by impairing the function of giant trophoblasts and the spongiotrophoblast layer, resulting in the shallow invasion of trophoblasts into the maternal decidua.
JPA2015-51. CHANGES OF NK CELLS WITH NATURAL CYTOTOXICITY RECEPTOR (NCR) IN PREECLAMPSIA Hideki Takahashi, Yusuke Kobayashi, Souichirou Kuno, Takayuki Matsuno, Takayuki Matsuno, Motomi Yamazaki, Fumihisa Chishima, Tatsuo Yamamoto. Department of Obstetrics & Gynecology, Nihon University, School of Medicine, Tokyo, Japan Objectives: NCR have been reported as the receptor to regulate NK cell cytotoxicity and cytokine production. In order to investigate the changes of NK cell in preeclampsia, we studied NK cells with NCRs in placenta decidua and peripheral blood. Methods: Placenta decidua and peripheral blood were taken from preeclampsia patients (PE) and elective cesarean section patients (NP) with informed consent. The lymphocytes were separated using density gradient centrifugation (DGC) from peripheral blood and using DGC after enzyme digestion from decidua. NK cells were stained by FITC antiCD56, Per-CP antiCD16 and Per-CP antiCD3 monoclonal antibody. NCR was stained by PE conjugated antiCD314 (NKG2D) monoclonal antibodies. The stained cells were analyzed using flow cytometry performed with a FACS Caliber (Becton Dickinson). Results: The percentages of CD56+CD16+ cells in CD3 negative and CD56 positive lymphocytes in peripheral blood between PE and NP were no significant difference. However, the percentages in decidual cells of PE were lower than those of NP. The positive cells of CD314 (NKG2D) in