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Hypoxia increased invasion of primary human extravillous trophoblast via enhance of urokinase-type plasminogen activator system
A100
Abstracts / Placenta 35 (2014) A1eA112
Results: Cytokine analysis of Cdm from PLAEC and PLVEC showed similar secretion of different pro- and antiangiogenic proteins like angiopeietin-2, IL-8 or TIMP-1 and TIMP-2. Cdm from PLAEC exhibited elevated secretion of GRO, Il-6, MCP-1, MMP-1 and uPAR compared to Cdm from PLVEC. Invasion assays revealed increased migration of ACH-3P only towards Cdm of PLAEC. Conclusion: PLAEC secrete a cytokine pattern that is responsible for successful invasion of trophoblast cells and explains why trophoblasts are more likely to invade uterine spiral arteries than uterine veins. Therefore ACH-3P display a good model for studying early placentation processes. Nevertheless the mechanism of endothelial replacement has to be further elucidated.
P2.125-N. IDENTIFICATION OF PROTEINS DIRECTLY INTERACTING WITH THE HELLP-SYNDROME ASSOCIATED LINCRNA USING CHIRP Allerdien Visser a, Roel van der Schors b, Cees Oudejans a, Marie van Dijk a a VU university medical center, amsterdam, The Netherlands; b VU university, amsterdam, The Netherlands The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. The chromosomal locus on 12q23.2 between PMCH and IGF1 with linkage to the HELLP syndrome contains a 205 kbp long intergenic noncoding RNA with (peri)nuclear expression in the extravillous trophoblast. siRNA-mediated knockdown followed by RNA-sequencing revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. Because lincRNAs predominantly function through interaction with proteins, we used a method named Chromatin Isolation by RNA Purification (ChIRP) to identify proteins directly interacting with the HELLP lincRNA. The method was optimized using the KCNQ1OT1 noncoding RNA interacting with DNMT1. Cultured SGHPL-5 extravillous trophoblast cells were crosslinked, and the lysed complexes DNAse treated. To capture the HELLP lincRNA, four biotinylated morpholino probes were used. The HELLP and negative controle morpholinos were hybridized and isolated using magnetic straptavidin beads. Extracted proteins were analysed by protein mass spectrometry. 22 proteins were only present in the ChIRP sample treated with HELLP lincRNA morpholinos. Pathway analysis of these proteins predicted involvement in RNA and protein processing. PCBP1, PCBP2 and YBX1 have so far been validated being the most interesting as literature describes PCBP1 upregulation in preeclamptic placentas, PCBP2 is, like the HELLP lincRNA itself, involved in the cell cycle. Furthermore YBX1 is associated with proliferation in cancer.
P2.126-N. HYPOXIA INCREASED INVASION OF PRIMARY HUMAN EXTRAVILLOUS TROPHOBLAST VIA ENHANCE OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR SYSTEM Taihei Tsunemi, Natsuki Koike, Juria Akasaka, Aiko Shigemitsu, Kana Iwai, Katsuhiko Naruse, Masayoshi Akasaki, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan Introduction: The process of extravillous trophoblast cell (EVT) invasion into uterus is critical for a successful pregnancy. Impaired invasion is implicated in several complications, such as preeclampsia and fetal growth restriction. EVT invasion in early pregnancy progresses under low oxygen environment. However, there have been few studies about the influence of low oxygen environment on invasiveness of primary trophoblast with alternated urokinase-type plasminogen activator (uPA) system. In this study, we investigated uPA, its receptor uPAR and inhibitor PAI-1 secretion by primary EVTs in normal or hypo/ hyper-oxia condition, and examined
whether uPA system contribute to the fair placental development in lowoxygen environment. Materials and methods: Placental samples (5-10 weeks gestation) were obtained from patients with artificial abortion after written informed consent. Cytotrophoblast was separated with Percoll-based method and cultured on Matrigel for 24 hours to obtain invasive phenotype (EVT like cell). The culture was performed under 20% oxygen, 5%, and 5% that repeated three times of hypoxic stimulation for one hour (0.1%).The invasion capacity were investigated with Matrigel-coated invasion assay. The localization of intracellular protein expression was examined by immunofluorescence staining. Expression of uPAR and PAI-1 protein on EVT was measured with Western Blot and ELISA. All data were shown as a comparison of 5% or 0.1% with 20%. Result: Invasive capacities were significantly increased under 5% and 0.1% oxygen compared to 20%. By immunofluorescence staining, uPAR protein was immunolocalized to cell membrane. The production of uPA and uPAR was increased under 5% and 0.1% oxygen condition. Induced production of uPA, uPAR on hypoxic condition by EVT cells were defined with Western Blot and ELISA. Conclusion: These results indicated that up-regulation of uPAR by hypoxia may enhanced EVT invasion on early first trimester of human pregnancy. The regulation of uPA system may contribute to fair trophoblast invasion.
P2.127-N. INVASIVENESS AND PROTEASE SYSTEM OF EARLY HUMAN EXTRAVILLOUS TROPHOBLAST IS REGULATED BY HYPOXIA INDUCED FACTOR (HIF) PATHWAY Natsuki Koike, Taihei Tsunemi, Chiharu Yoshimoto-Uekuri, Hiroshi Shigetomi, Juria Akasaka, Katsuhiko Naruse, Masayoshi Akasaki, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan Introduction: Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human body, especially in human pregnancy. We reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Materials and methods: Human placental villi of 5 to 10 weeks gestation (obtained on artificial abortion after patient’s written informed consent) was minced, digested with trypsin and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel for 24 hours with several concentrations of siRNA designed for knock down of HIF. The culture was performed under 20% and 5% oxygen condition. The invasive capacity was investigated with Matrigelcoated invasion assay. The localization of HIF was examined by immunofluorescence staining. Production of HIF, uPA and receptor uPAR protein on EVT was semi-quantified with Western Blot and measured with ELISA. Result: HIF protein was located on nuclear by immunofluorescence staining. HIF protein was significantly increased under 5% oxygen condition than 20%. Dose-dependent decrease of intracellular HIF protein by siRNA was defined by Western Blot. Invasive capacity was significantly decreased in the presence of siRNA for HIF. The production of uPA and uPAR were also decreased in the presence of the siRNA. Conclusion: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that under hypoxic conditions, which may be normal in the uterus of early pregnancy, uPA system in human trophoblast was induced by HIF and enhanced invasive capacity.
Abstracts / Placenta 35 (2014) A1eA112
Results: Cytokine analysis of Cdm from PLAEC and PLVEC showed similar secretion of different pro- and antiangiogenic proteins like angiopeietin-2, IL-8 or TIMP-1 and TIMP-2. Cdm from PLAEC exhibited elevated secretion of GRO, Il-6, MCP-1, MMP-1 and uPAR compared to Cdm from PLVEC. Invasion assays revealed increased migration of ACH-3P only towards Cdm of PLAEC. Conclusion: PLAEC secrete a cytokine pattern that is responsible for successful invasion of trophoblast cells and explains why trophoblasts are more likely to invade uterine spiral arteries than uterine veins. Therefore ACH-3P display a good model for studying early placentation processes. Nevertheless the mechanism of endothelial replacement has to be further elucidated.
P2.125-N. IDENTIFICATION OF PROTEINS DIRECTLY INTERACTING WITH THE HELLP-SYNDROME ASSOCIATED LINCRNA USING CHIRP Allerdien Visser a, Roel van der Schors b, Cees Oudejans a, Marie van Dijk a a VU university medical center, amsterdam, The Netherlands; b VU university, amsterdam, The Netherlands The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. The chromosomal locus on 12q23.2 between PMCH and IGF1 with linkage to the HELLP syndrome contains a 205 kbp long intergenic noncoding RNA with (peri)nuclear expression in the extravillous trophoblast. siRNA-mediated knockdown followed by RNA-sequencing revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. Because lincRNAs predominantly function through interaction with proteins, we used a method named Chromatin Isolation by RNA Purification (ChIRP) to identify proteins directly interacting with the HELLP lincRNA. The method was optimized using the KCNQ1OT1 noncoding RNA interacting with DNMT1. Cultured SGHPL-5 extravillous trophoblast cells were crosslinked, and the lysed complexes DNAse treated. To capture the HELLP lincRNA, four biotinylated morpholino probes were used. The HELLP and negative controle morpholinos were hybridized and isolated using magnetic straptavidin beads. Extracted proteins were analysed by protein mass spectrometry. 22 proteins were only present in the ChIRP sample treated with HELLP lincRNA morpholinos. Pathway analysis of these proteins predicted involvement in RNA and protein processing. PCBP1, PCBP2 and YBX1 have so far been validated being the most interesting as literature describes PCBP1 upregulation in preeclamptic placentas, PCBP2 is, like the HELLP lincRNA itself, involved in the cell cycle. Furthermore YBX1 is associated with proliferation in cancer.
P2.126-N. HYPOXIA INCREASED INVASION OF PRIMARY HUMAN EXTRAVILLOUS TROPHOBLAST VIA ENHANCE OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR SYSTEM Taihei Tsunemi, Natsuki Koike, Juria Akasaka, Aiko Shigemitsu, Kana Iwai, Katsuhiko Naruse, Masayoshi Akasaki, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan Introduction: The process of extravillous trophoblast cell (EVT) invasion into uterus is critical for a successful pregnancy. Impaired invasion is implicated in several complications, such as preeclampsia and fetal growth restriction. EVT invasion in early pregnancy progresses under low oxygen environment. However, there have been few studies about the influence of low oxygen environment on invasiveness of primary trophoblast with alternated urokinase-type plasminogen activator (uPA) system. In this study, we investigated uPA, its receptor uPAR and inhibitor PAI-1 secretion by primary EVTs in normal or hypo/ hyper-oxia condition, and examined
whether uPA system contribute to the fair placental development in lowoxygen environment. Materials and methods: Placental samples (5-10 weeks gestation) were obtained from patients with artificial abortion after written informed consent. Cytotrophoblast was separated with Percoll-based method and cultured on Matrigel for 24 hours to obtain invasive phenotype (EVT like cell). The culture was performed under 20% oxygen, 5%, and 5% that repeated three times of hypoxic stimulation for one hour (0.1%).The invasion capacity were investigated with Matrigel-coated invasion assay. The localization of intracellular protein expression was examined by immunofluorescence staining. Expression of uPAR and PAI-1 protein on EVT was measured with Western Blot and ELISA. All data were shown as a comparison of 5% or 0.1% with 20%. Result: Invasive capacities were significantly increased under 5% and 0.1% oxygen compared to 20%. By immunofluorescence staining, uPAR protein was immunolocalized to cell membrane. The production of uPA and uPAR was increased under 5% and 0.1% oxygen condition. Induced production of uPA, uPAR on hypoxic condition by EVT cells were defined with Western Blot and ELISA. Conclusion: These results indicated that up-regulation of uPAR by hypoxia may enhanced EVT invasion on early first trimester of human pregnancy. The regulation of uPA system may contribute to fair trophoblast invasion.
P2.127-N. INVASIVENESS AND PROTEASE SYSTEM OF EARLY HUMAN EXTRAVILLOUS TROPHOBLAST IS REGULATED BY HYPOXIA INDUCED FACTOR (HIF) PATHWAY Natsuki Koike, Taihei Tsunemi, Chiharu Yoshimoto-Uekuri, Hiroshi Shigetomi, Juria Akasaka, Katsuhiko Naruse, Masayoshi Akasaki, Hiroshi Kobayashi OB/GYN, Nara Medical University, Kashihara, Nara, Japan Introduction: Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human body, especially in human pregnancy. We reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Materials and methods: Human placental villi of 5 to 10 weeks gestation (obtained on artificial abortion after patient’s written informed consent) was minced, digested with trypsin and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel for 24 hours with several concentrations of siRNA designed for knock down of HIF. The culture was performed under 20% and 5% oxygen condition. The invasive capacity was investigated with Matrigelcoated invasion assay. The localization of HIF was examined by immunofluorescence staining. Production of HIF, uPA and receptor uPAR protein on EVT was semi-quantified with Western Blot and measured with ELISA. Result: HIF protein was located on nuclear by immunofluorescence staining. HIF protein was significantly increased under 5% oxygen condition than 20%. Dose-dependent decrease of intracellular HIF protein by siRNA was defined by Western Blot. Invasive capacity was significantly decreased in the presence of siRNA for HIF. The production of uPA and uPAR were also decreased in the presence of the siRNA. Conclusion: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that under hypoxic conditions, which may be normal in the uterus of early pregnancy, uPA system in human trophoblast was induced by HIF and enhanced invasive capacity.