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Endothelial cytokines influence trophoblast invasion during first trimester of pregnancy
A36
Abstracts / Placenta 36 (2015) A1eA60
controls. Changes in JEG-3 and HTR-8/SVneo cell proliferation and invasion were investigated after miR-141 inhibition and over-expression by MTS-, BrdU- and Matrigel assays. Results: In comparison to primary tissues, cell lines expressed significantly lower levels of miR-141. Up-regulation of miR-141 was found in the tissues from preeclampsia patients compared to normal pregnancies. Inhibition of miR-141 resulted in decreased viability only in JEG-3 cells. Whilst, invasion capability was reduced in both cell lines after treatment with miR-141inhibitor. Conclusion: Regulatory mechanisms associated to miR-141 differ in HTR8/SVneo and JEG-3 cells possibly due to differences in their cellular origin. High expression in maternal plasma, elevated levels in PE placenta and its regulatory function in trophoblast cells suggest miR-141 as novel biomarker for PE.
P1.75. CRIPTO1/3 MODULATES INVASION OF TROPHOBLAST HTR8/SV-NEO CELL LINEAGE Carla L. Bandeira 1, Mara S. Hoshida 3, Martin Knofler 2, Graciela M. Suasana Genti-Raimondi 4, Magali E. PanzettaDutari 4, 4 3 Ridano , Rossana P.V. Francisco , Estela Bevilacqua 1. 1 Dept of Cell and ~o Developmental Biology, Institute of Biomedical Sciences, University of Sa ~o Paulo, SP, Brazil; 2 Dept of Obstetrics and Fetal-Maternal Paulo, Sa Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, ~o Paulo, Sa ~o Austria; 3 Dept of Obstetrics, Medical School, University of Sa Paulo, SP, Brazil; 4 Dept of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, Cordoba, Argentina Background. CRIPTO1 (CR1) is a member of the epidermal growth factor family with specific functions during embryo development (coreceptor for Nodal in the establishment of embryonic axis) and tumor (found at high levels in colon, breast, lung, testis, ovary, pancreas and stomach cancer cells). In normal adult tissues, it has been found in the mammary glands during pregnancy and lactation. Trophoblast development resembles in many ways the oncogenic process; however, as well as in tumors, the exact regulatory mechanisms that regulate cell invasion are not clear. Previous studies have shown immunolocalization of CR1 at term healthy placenta and in placentas with invasiveness disorders, which suggested important roles in this biological process. Objectives. To determine the role of CR1 and CR3 in human trophoblastic cell invasion. Material & methods. Gene and protein levels were determined by RT-PCR and immunolocalization and Western blotting in first and third trimester EVT cells and in HTR8/SV-neo cells. Recombinant CRIPTO was used to evaluate the role of this growth factor in invasion and migration activity of the trophoblast cell lineage by using transwell/matrigel assays. Interfering RNA strategy was employed to determine the role of CR1 in HTR-8/SVneo cell invasion. Results. CR3 were highly expressed in EVT and HTR8 cells, whereas CR1 was only found in third trimester EVT cells. Recombinant CRIPTO significantly increased the expression of CRIPTO and invasion and migration of HTR8 cells in a time- and concentration-dependent manner. Interfering RNA specific for CRIPTO significantly decreased migration and invasion activities of HTR-8/SVneo cells. These findings suggest that CR3 participates in the trophoblast cell invasion-associated pathways. This study contributes to the identification of factors associated with trophoblast invasiveness in normal and pathological conditions. Supported by CAPES, CNPq & FAPESP
P1.76. PERICONCEPTIONAL ALCOHOL EXPOSURE CAUSES MID-GESTATIONAL PLACENTAL GROWTH RESTRICTION AND ALTERS TROPHOBLAST INVASION INTO THE DECIDUA. Jacinta Kalisch-Smith, Marie Pantaleon, David Simmons, Karen Moritz. The University of Queensland, Brisbane, QLD, Australia
Objectives.We have shown previously that maternal periconceptional alcohol (PC-EtOH) exposure can cause foetal growth restriction and sexspecific changes to placental morphology in late gestation. It is currently unknown how this early exposure disturbs placental morphogenesis and contributes to fetal growth restriction. Methods.Sprague Dawley dams were administered 12.5% v/v EtOH or a control diet from 4 days prior (E-4) to 4 days after, conception (E4) in a liquid diet. On E5 dams were returned to standard chow until sacrifice. A cohort at E15 (N¼3-4/treatment) was assessed for fetal and placental dimensions. Another cohort (N¼2/treatment) was taken at E20 for stereological analysis of glycogen T cell (GlyT) and spiral artery trophoblast giant cell (SpA-TGC) invasion into the decidua, using in situ hybridisation for Prl5a1. Results.PC-EtOH exposure resulted in no change to fetal body weights, but did cause reduced placental growth at E15 (P<0.05). Although no change in placental zonal weights, length or depth was found, placental width tended to decrease after PC-EtOH exposure (P¼0.06). PC-EtOH also reduced GlyT and SpA-TGC invasion depth into the decidua from the junctional zone (N¼2-4/treatment). Conclusion.Analysis of fetal and placental tissues at E15 suggests PCEtOH exposure results in fetal growth restriction after mid-gestation. A reduction in placental growth could lead to inadequate nutrient availability to facilitate fetal growth. Here we also report improper invasion of interstitial (GlyT) and endovascular (SpA-TGC) trophoblasts following PC-EtOH. Perturbations in invasion or number of GlyT cells may reduce growth signals to the fetus through altered IGF2 or glycogen release. Altered SpA-TGCs invasion may cause poor remodelling of the maternal spiral arteries, causing aberrant placental blood flow. Future analysis of SpA-TGC and GlyT cell counts will confirm whether these critical invasive and remodelling events have occurred. Results will also be analysed for sexual dimorphic responses to PCEtOH exposure.
P1.77. ENDOTHELIAL CYTOKINES INFLUENCE TROPHOBLAST DURING FIRST TRIMESTER OF PREGNANCY
INVASION
Gregor Weiss, Berthold Huppertz, Monika Siwetz, Ingrid Lang-Olip, Gerit Moser. Medical University of Graz, Graz, Austria Objectives: The interaction of endovascular trophoblasts and endothelial cells is crucial for successful trophoblast invasion in early human pregnancy. In the present study we focused on the influence of endothelial cytokines on trophoblast proliferation and mitochondrial activity as well as on the direct interaction between the two cell types. Methods: The first trimester trophoblast cell line ACH-3P and human iliac arterial (AEC) and venous endothelial cells (VEC) were used as respective cellular models. Conditioned medium (Cdm) was prepared by incubating confluent AEC/VEC with EGM for 48 h under standard culture conditions. The impact of Cdm on proliferation and mitochondrial activity of ACH-3P was assessed as well as the cytokine secretion of AEC/VEC. Furthermore co-culture experiments were performed to investigate the direct effect of ACH-3P on AEC. Results: ACH-3P cells showed a reduced proliferation rate when cultivated in AEC-Cdm (237,660 ± 51,983 cells/ml) and VEC-Cdm (330,000 ± 100,000 cells/ml) for 24 h compared to cells grown in control medium (620,000 ± 50,000 cells/ml). AEC-Cdm significantly reduced mitochondrial activity of ACH-3P (2.5% oxygen: 55.5% ± 4.7%; 8% oxygen: 63.4% ± 4.4%; 21% oxygen: 75.5% ± 8.3%) compared to control media (set to 100%). VEC-Cdm only had this effect at 2.5% oxygen (75.1% ± 10%). Cytokine analysis showed elevated levels of different pro- and antiangiogenic proteins like Angiopoietin-2, IL8 or TIMP-1. In co-culture experiments ACH-3P inhibited network formation of AEC. Conclusion: In conclusion, our results clearly demonstrate that cytokines of AEC/VEC contribute to a reduction of proliferation and mitochondrial activity of first trimester trophoblasts, without enhancing angiogenesis, which resembles the in vivo situation. This enables further insight into the interactions of extravillous trophoblast with maternal endothelial cells.
Abstracts / Placenta 36 (2015) A1eA60
controls. Changes in JEG-3 and HTR-8/SVneo cell proliferation and invasion were investigated after miR-141 inhibition and over-expression by MTS-, BrdU- and Matrigel assays. Results: In comparison to primary tissues, cell lines expressed significantly lower levels of miR-141. Up-regulation of miR-141 was found in the tissues from preeclampsia patients compared to normal pregnancies. Inhibition of miR-141 resulted in decreased viability only in JEG-3 cells. Whilst, invasion capability was reduced in both cell lines after treatment with miR-141inhibitor. Conclusion: Regulatory mechanisms associated to miR-141 differ in HTR8/SVneo and JEG-3 cells possibly due to differences in their cellular origin. High expression in maternal plasma, elevated levels in PE placenta and its regulatory function in trophoblast cells suggest miR-141 as novel biomarker for PE.
P1.75. CRIPTO1/3 MODULATES INVASION OF TROPHOBLAST HTR8/SV-NEO CELL LINEAGE Carla L. Bandeira 1, Mara S. Hoshida 3, Martin Knofler 2, Graciela M. Suasana Genti-Raimondi 4, Magali E. PanzettaDutari 4, 4 3 Ridano , Rossana P.V. Francisco , Estela Bevilacqua 1. 1 Dept of Cell and ~o Developmental Biology, Institute of Biomedical Sciences, University of Sa ~o Paulo, SP, Brazil; 2 Dept of Obstetrics and Fetal-Maternal Paulo, Sa Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, ~o Paulo, Sa ~o Austria; 3 Dept of Obstetrics, Medical School, University of Sa Paulo, SP, Brazil; 4 Dept of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, Cordoba, Argentina Background. CRIPTO1 (CR1) is a member of the epidermal growth factor family with specific functions during embryo development (coreceptor for Nodal in the establishment of embryonic axis) and tumor (found at high levels in colon, breast, lung, testis, ovary, pancreas and stomach cancer cells). In normal adult tissues, it has been found in the mammary glands during pregnancy and lactation. Trophoblast development resembles in many ways the oncogenic process; however, as well as in tumors, the exact regulatory mechanisms that regulate cell invasion are not clear. Previous studies have shown immunolocalization of CR1 at term healthy placenta and in placentas with invasiveness disorders, which suggested important roles in this biological process. Objectives. To determine the role of CR1 and CR3 in human trophoblastic cell invasion. Material & methods. Gene and protein levels were determined by RT-PCR and immunolocalization and Western blotting in first and third trimester EVT cells and in HTR8/SV-neo cells. Recombinant CRIPTO was used to evaluate the role of this growth factor in invasion and migration activity of the trophoblast cell lineage by using transwell/matrigel assays. Interfering RNA strategy was employed to determine the role of CR1 in HTR-8/SVneo cell invasion. Results. CR3 were highly expressed in EVT and HTR8 cells, whereas CR1 was only found in third trimester EVT cells. Recombinant CRIPTO significantly increased the expression of CRIPTO and invasion and migration of HTR8 cells in a time- and concentration-dependent manner. Interfering RNA specific for CRIPTO significantly decreased migration and invasion activities of HTR-8/SVneo cells. These findings suggest that CR3 participates in the trophoblast cell invasion-associated pathways. This study contributes to the identification of factors associated with trophoblast invasiveness in normal and pathological conditions. Supported by CAPES, CNPq & FAPESP
P1.76. PERICONCEPTIONAL ALCOHOL EXPOSURE CAUSES MID-GESTATIONAL PLACENTAL GROWTH RESTRICTION AND ALTERS TROPHOBLAST INVASION INTO THE DECIDUA. Jacinta Kalisch-Smith, Marie Pantaleon, David Simmons, Karen Moritz. The University of Queensland, Brisbane, QLD, Australia
Objectives.We have shown previously that maternal periconceptional alcohol (PC-EtOH) exposure can cause foetal growth restriction and sexspecific changes to placental morphology in late gestation. It is currently unknown how this early exposure disturbs placental morphogenesis and contributes to fetal growth restriction. Methods.Sprague Dawley dams were administered 12.5% v/v EtOH or a control diet from 4 days prior (E-4) to 4 days after, conception (E4) in a liquid diet. On E5 dams were returned to standard chow until sacrifice. A cohort at E15 (N¼3-4/treatment) was assessed for fetal and placental dimensions. Another cohort (N¼2/treatment) was taken at E20 for stereological analysis of glycogen T cell (GlyT) and spiral artery trophoblast giant cell (SpA-TGC) invasion into the decidua, using in situ hybridisation for Prl5a1. Results.PC-EtOH exposure resulted in no change to fetal body weights, but did cause reduced placental growth at E15 (P<0.05). Although no change in placental zonal weights, length or depth was found, placental width tended to decrease after PC-EtOH exposure (P¼0.06). PC-EtOH also reduced GlyT and SpA-TGC invasion depth into the decidua from the junctional zone (N¼2-4/treatment). Conclusion.Analysis of fetal and placental tissues at E15 suggests PCEtOH exposure results in fetal growth restriction after mid-gestation. A reduction in placental growth could lead to inadequate nutrient availability to facilitate fetal growth. Here we also report improper invasion of interstitial (GlyT) and endovascular (SpA-TGC) trophoblasts following PC-EtOH. Perturbations in invasion or number of GlyT cells may reduce growth signals to the fetus through altered IGF2 or glycogen release. Altered SpA-TGCs invasion may cause poor remodelling of the maternal spiral arteries, causing aberrant placental blood flow. Future analysis of SpA-TGC and GlyT cell counts will confirm whether these critical invasive and remodelling events have occurred. Results will also be analysed for sexual dimorphic responses to PCEtOH exposure.
P1.77. ENDOTHELIAL CYTOKINES INFLUENCE TROPHOBLAST DURING FIRST TRIMESTER OF PREGNANCY
INVASION
Gregor Weiss, Berthold Huppertz, Monika Siwetz, Ingrid Lang-Olip, Gerit Moser. Medical University of Graz, Graz, Austria Objectives: The interaction of endovascular trophoblasts and endothelial cells is crucial for successful trophoblast invasion in early human pregnancy. In the present study we focused on the influence of endothelial cytokines on trophoblast proliferation and mitochondrial activity as well as on the direct interaction between the two cell types. Methods: The first trimester trophoblast cell line ACH-3P and human iliac arterial (AEC) and venous endothelial cells (VEC) were used as respective cellular models. Conditioned medium (Cdm) was prepared by incubating confluent AEC/VEC with EGM for 48 h under standard culture conditions. The impact of Cdm on proliferation and mitochondrial activity of ACH-3P was assessed as well as the cytokine secretion of AEC/VEC. Furthermore co-culture experiments were performed to investigate the direct effect of ACH-3P on AEC. Results: ACH-3P cells showed a reduced proliferation rate when cultivated in AEC-Cdm (237,660 ± 51,983 cells/ml) and VEC-Cdm (330,000 ± 100,000 cells/ml) for 24 h compared to cells grown in control medium (620,000 ± 50,000 cells/ml). AEC-Cdm significantly reduced mitochondrial activity of ACH-3P (2.5% oxygen: 55.5% ± 4.7%; 8% oxygen: 63.4% ± 4.4%; 21% oxygen: 75.5% ± 8.3%) compared to control media (set to 100%). VEC-Cdm only had this effect at 2.5% oxygen (75.1% ± 10%). Cytokine analysis showed elevated levels of different pro- and antiangiogenic proteins like Angiopoietin-2, IL8 or TIMP-1. In co-culture experiments ACH-3P inhibited network formation of AEC. Conclusion: In conclusion, our results clearly demonstrate that cytokines of AEC/VEC contribute to a reduction of proliferation and mitochondrial activity of first trimester trophoblasts, without enhancing angiogenesis, which resembles the in vivo situation. This enables further insight into the interactions of extravillous trophoblast with maternal endothelial cells.