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Adenosine A2 regulate cell proliferation via nitric oxide synthesis in normal and gestational diabetes
Abstracts / Placenta 36 (2015) 469e521
receptors may be also involved. Mostly the PI3K but also the MAPK signaling pathways participate in the migration induced by LMW-HA.
PA.49. PARTICIPATION OF ENDOCANNABINOIDS IN PLACENTAL APOPTOSIS n 3, A. C. Aban 1, N. Martinez 1, C. Carou 2, D. Trigubo 3, G. Leguizamo Franchi 1, A. Damiano 4, M. Farina 1. 1 Laboratorio de Fisiopatología Placentaria, CEFyBO-CONICET, Facultad de Medicina, UBA, Buenos Aires, Argentina; 2 Departamento de Histología y Embriología, Facultad de n Ciencias Veterinarias, UBA, Buenos Aires, Argentina; 3 Centro de Educacio M edica e Investigaciones Clínicas, Unidad de Embarazo de Alto Riesgo, Departamento de Obstetricia y Ginecología, Buenos Aires, Argentina; 4 n, IFIBIO-CONICET, Facultad de Laboratorio de Biología de la Reproduccio Medicina, UBA, Buenos Aires, Argentina Apoptosis is an important process that plays a major role in the physiological development of human placenta. It has been observed that an aberrant cell turnover including an increased apoptosis is implicated in the pathogenesis of many disorders of pregnancy. Hypoxia inducible Factors (HIFs) act as key regulators of trophoblast invasion and differentiation through regulation of the expression of different genes. The catalytic HIF1a subunit is expressed in human placentas and can also induce genes involved in cell death. Endocannabinoids (ECs) are bioactive lipids implicated in placental physiology that exert their effect by the activation of cannabinoid receptors CB1 and CB2. ECs, together with their receptors are part of the Endocannabinoid System (ES) and could induce apoptosis in different cell types including cytotrophoblast cells. Previously, we demonstrated that the ES is present in human term placenta. Objective: To investigate the participation of ECs in human placental apoptosis generated by HIF1a. Methods: Explants obtained from normal placentas (n¼8) were cultured with Cobalt chloride (CoCl2, a hypoxia-mimetic agent), Met-AEA (a stable EC agonist) and/or AM251 (a CB1 antagonist). Explant viability was evaluated by MTT assay. Cleaved caspase-3, Bax and Bcl-2 protein expression and caspase-3 activity were determined as apoptotic parameters. Results: CoCl2 treatment induced a decrease in the viability of the placental tissue. Instead, co-incubation with AM251 blocked this effect. Incubation with CoCl2 or Met-AEA increased cleaved caspase 3 (active form) expression. Additionally, we detected an increase in the Bax/Bcl-2 ratio and caspase 3 activity. The expression and enzymatic activity of these proteins diminished after co-incubation with AM251 (p<0.05). Conclusions: Our results suggest that ECs may be involved in the apoptotic events induced by HIF, via a mechanism mediated by CB1 receptor in the human placenta. Further studies are needed to determine their role in pathological conditions.
PA.50. TROPHOBLAST STARD7 EXPRESSION IN RESPONSE TO CELL INJURY J. Flores-Martín, A. Racca, V. Rena, L. Reyna, M. Ridano, M. Cruz Del Puerto, G. Panzetta-Dutari, S. Genti-Raimondi. CIBICI-CONICET, Dpto. rdoba, Argentina Bioquímica Clínica, Facultad de Ciencias Químicas, UNC, Co StarD7 encodes an intracellular lipid transport protein initially identified as an up-regulated gene in the choriocarcinoma JEG-3 cell line. So far it is unclear how changes in the placental environment affect StarD7 expression. Objectives: This work was performed to investigate StarD7 expression in trophoblast cells: a) under hypoxic-reoxygenation conditions and b) in a nuclear factor E2-related factor 2 (Nrf2) loss- and gain-of-function studies. Methods: HTR8/SVneo cells were exposed to hypoxia for 1 h and then reoxygenated for 1, 2, or 3 h. In addition, Nrf2 loss- and gain-of-function assays were performed in JEG-3 cells by transfection with a specific siRNA or an expression vector, respectively. Western blot, immunofluorescence and qRT-PCR were used to explore protein and transcript expression. Results: StarD7 protein expression increased in response to hypoxia and reoxygenation exposure for 1, 2, or 3 h. Moreover, reoxygenation resulted
503
in the upregulation of Nrf2 at both mRNA and protein levels, and in heme oxygenase-1 mRNA expression (encoding a phase II detoxifying enzyme). Furthermore, Nrf2 cell transfection assays increased StarD7 expression. In contrast, knocking down Nrf2 led to a diminished StarD7 transcript level indicating that these genes are positively correlated. Conclusions: These results suggest that StarD7 is a target gene of oxidative stress response. Current studies are being conducted to validate StarD7 as a useful marker of placental cell injury.
PA.51. ADENOSINE A2 REGULATE CELL PROLIFERATION VIA NITRIC OXIDE SYNTHESIS IN NORMAL AND GESTATIONAL DIABETES F. Troncoso, J. Acurio, F. Ruiz, K. Herlitz, N. Mardones, J. Santos, P. Bertoglia, C. Escudero. Vascular Physiology Laboratory, Group of Investigation in Tumor Angiogenesis (GIANT), Group of Research and Innovation in Vascular Health (GRIVAS Health), Department of Basic n, Chile Sciences, Universidad del Bío-Bío, Chilla Objective: To characterize whether A2A and/or A2B adenosine receptors (AR) via the nitric oxide (NO) signaling pathway are involved in the proliferation of fetal endothelium isolated from pregnancies with gestational diabetes (GD). Methods: Analyses were performed in human umbilical vein endothelial cells (HUVEC) isolated from normal (n¼18) and GD (n¼15) pregnancies. Cell proliferation was assayed by cell counting and MTS in the presence (24 h) or absence of a non-selective AR agonist (NECA 10 mM), an A2AAR selective agonist (CGS-21680, 30 nM), and/or the antagonists ZM-241385 (10 nM) and MRS-1754 (10 nM) for A2AAR and A2BAR, respectively. NECA was also combined with the non-selective nitric oxide synthase (NOS) inhibitor L-NAME (100 mM) or the NO donor SNAP (10 pM). Finally, cells were transfected with shRNA-A2AAR and/or shRNA-A2BAR or the respective control (i.e., shRNA-A). Twenty-four hours post-transfection, cells were used for analysis of cell proliferation in the presence or absence of NECA or SNAP for additional 24 hours. All experiments were performed in the presence of adenosine deaminase (1 UI/ml). Results: GD was associated with high (~2 fold) A2AAR and A2BAR protein level. CGS-21680 and NECA increased (~1.3 and 1.5-fold) cell proliferation in both GD and normal pregnancies. The NECA-stimulatory effect observed in GD or normal cells was partially blocked by ZM-241385 or MRS-1754 separately, whereas additive inhibition was observed when both antagonists were co-incubated. Furthermore, knocking down of A2AAR, or double knowing down, but not repression of A2BAR expression blocked the stimulatory effect of NECA on cell proliferation in HUVEC from normal pregnancies, while in GD only combination of shRNA-A2AAR and shRNA-A2BAR blocked the stimulatory effect of NECA. Nevertheless, L-NAME co-incubation blocked the CGS21680 and NECA-stimulatory effects in both groups of pregnancies. Conversely, SNAP increased (~1.4 fold) cell proliferation in both GD and normal pregnancies. No additive effect was observed in cell proliferation when CGS-21680 or NECA were co-incubated with SNAP. Co-incubation with SNAP and NECA, in cells lacking A2AAR and/or A2BAR receptor exhibited a response similar to that of NECA alone in both groups of pregnancies. Conclusions: Elevated expression of both A2AAR and A2BAR might generate cell proliferation in gestational diabetes via intracellular generation of NO. Supported by FONDECYT 1140586.
PA.52. REPRODUCTIVE PROFILE OF A MURINE MODEL OF DIET-INDUCED OBESITY M.E. Tejada, M.C. Pustovrh, L.S. Salazar. Department of Morphology, School of Health, University of Valle, Santiago de Cali, Colombia Obesity is a multifactorial epidemic that involves genetic, physiological, environmental, and social factors. In obese and overweight women, significant impairment of the reproductive organs has been observed. This leads to a reduction in the number and quality of mature oocytes, and to a lower rate of fecundation when compared to women with normal BMI, thus increasing the risk of infertility.
receptors may be also involved. Mostly the PI3K but also the MAPK signaling pathways participate in the migration induced by LMW-HA.
PA.49. PARTICIPATION OF ENDOCANNABINOIDS IN PLACENTAL APOPTOSIS n 3, A. C. Aban 1, N. Martinez 1, C. Carou 2, D. Trigubo 3, G. Leguizamo Franchi 1, A. Damiano 4, M. Farina 1. 1 Laboratorio de Fisiopatología Placentaria, CEFyBO-CONICET, Facultad de Medicina, UBA, Buenos Aires, Argentina; 2 Departamento de Histología y Embriología, Facultad de n Ciencias Veterinarias, UBA, Buenos Aires, Argentina; 3 Centro de Educacio M edica e Investigaciones Clínicas, Unidad de Embarazo de Alto Riesgo, Departamento de Obstetricia y Ginecología, Buenos Aires, Argentina; 4 n, IFIBIO-CONICET, Facultad de Laboratorio de Biología de la Reproduccio Medicina, UBA, Buenos Aires, Argentina Apoptosis is an important process that plays a major role in the physiological development of human placenta. It has been observed that an aberrant cell turnover including an increased apoptosis is implicated in the pathogenesis of many disorders of pregnancy. Hypoxia inducible Factors (HIFs) act as key regulators of trophoblast invasion and differentiation through regulation of the expression of different genes. The catalytic HIF1a subunit is expressed in human placentas and can also induce genes involved in cell death. Endocannabinoids (ECs) are bioactive lipids implicated in placental physiology that exert their effect by the activation of cannabinoid receptors CB1 and CB2. ECs, together with their receptors are part of the Endocannabinoid System (ES) and could induce apoptosis in different cell types including cytotrophoblast cells. Previously, we demonstrated that the ES is present in human term placenta. Objective: To investigate the participation of ECs in human placental apoptosis generated by HIF1a. Methods: Explants obtained from normal placentas (n¼8) were cultured with Cobalt chloride (CoCl2, a hypoxia-mimetic agent), Met-AEA (a stable EC agonist) and/or AM251 (a CB1 antagonist). Explant viability was evaluated by MTT assay. Cleaved caspase-3, Bax and Bcl-2 protein expression and caspase-3 activity were determined as apoptotic parameters. Results: CoCl2 treatment induced a decrease in the viability of the placental tissue. Instead, co-incubation with AM251 blocked this effect. Incubation with CoCl2 or Met-AEA increased cleaved caspase 3 (active form) expression. Additionally, we detected an increase in the Bax/Bcl-2 ratio and caspase 3 activity. The expression and enzymatic activity of these proteins diminished after co-incubation with AM251 (p<0.05). Conclusions: Our results suggest that ECs may be involved in the apoptotic events induced by HIF, via a mechanism mediated by CB1 receptor in the human placenta. Further studies are needed to determine their role in pathological conditions.
PA.50. TROPHOBLAST STARD7 EXPRESSION IN RESPONSE TO CELL INJURY J. Flores-Martín, A. Racca, V. Rena, L. Reyna, M. Ridano, M. Cruz Del Puerto, G. Panzetta-Dutari, S. Genti-Raimondi. CIBICI-CONICET, Dpto. rdoba, Argentina Bioquímica Clínica, Facultad de Ciencias Químicas, UNC, Co StarD7 encodes an intracellular lipid transport protein initially identified as an up-regulated gene in the choriocarcinoma JEG-3 cell line. So far it is unclear how changes in the placental environment affect StarD7 expression. Objectives: This work was performed to investigate StarD7 expression in trophoblast cells: a) under hypoxic-reoxygenation conditions and b) in a nuclear factor E2-related factor 2 (Nrf2) loss- and gain-of-function studies. Methods: HTR8/SVneo cells were exposed to hypoxia for 1 h and then reoxygenated for 1, 2, or 3 h. In addition, Nrf2 loss- and gain-of-function assays were performed in JEG-3 cells by transfection with a specific siRNA or an expression vector, respectively. Western blot, immunofluorescence and qRT-PCR were used to explore protein and transcript expression. Results: StarD7 protein expression increased in response to hypoxia and reoxygenation exposure for 1, 2, or 3 h. Moreover, reoxygenation resulted
503
in the upregulation of Nrf2 at both mRNA and protein levels, and in heme oxygenase-1 mRNA expression (encoding a phase II detoxifying enzyme). Furthermore, Nrf2 cell transfection assays increased StarD7 expression. In contrast, knocking down Nrf2 led to a diminished StarD7 transcript level indicating that these genes are positively correlated. Conclusions: These results suggest that StarD7 is a target gene of oxidative stress response. Current studies are being conducted to validate StarD7 as a useful marker of placental cell injury.
PA.51. ADENOSINE A2 REGULATE CELL PROLIFERATION VIA NITRIC OXIDE SYNTHESIS IN NORMAL AND GESTATIONAL DIABETES F. Troncoso, J. Acurio, F. Ruiz, K. Herlitz, N. Mardones, J. Santos, P. Bertoglia, C. Escudero. Vascular Physiology Laboratory, Group of Investigation in Tumor Angiogenesis (GIANT), Group of Research and Innovation in Vascular Health (GRIVAS Health), Department of Basic n, Chile Sciences, Universidad del Bío-Bío, Chilla Objective: To characterize whether A2A and/or A2B adenosine receptors (AR) via the nitric oxide (NO) signaling pathway are involved in the proliferation of fetal endothelium isolated from pregnancies with gestational diabetes (GD). Methods: Analyses were performed in human umbilical vein endothelial cells (HUVEC) isolated from normal (n¼18) and GD (n¼15) pregnancies. Cell proliferation was assayed by cell counting and MTS in the presence (24 h) or absence of a non-selective AR agonist (NECA 10 mM), an A2AAR selective agonist (CGS-21680, 30 nM), and/or the antagonists ZM-241385 (10 nM) and MRS-1754 (10 nM) for A2AAR and A2BAR, respectively. NECA was also combined with the non-selective nitric oxide synthase (NOS) inhibitor L-NAME (100 mM) or the NO donor SNAP (10 pM). Finally, cells were transfected with shRNA-A2AAR and/or shRNA-A2BAR or the respective control (i.e., shRNA-A). Twenty-four hours post-transfection, cells were used for analysis of cell proliferation in the presence or absence of NECA or SNAP for additional 24 hours. All experiments were performed in the presence of adenosine deaminase (1 UI/ml). Results: GD was associated with high (~2 fold) A2AAR and A2BAR protein level. CGS-21680 and NECA increased (~1.3 and 1.5-fold) cell proliferation in both GD and normal pregnancies. The NECA-stimulatory effect observed in GD or normal cells was partially blocked by ZM-241385 or MRS-1754 separately, whereas additive inhibition was observed when both antagonists were co-incubated. Furthermore, knocking down of A2AAR, or double knowing down, but not repression of A2BAR expression blocked the stimulatory effect of NECA on cell proliferation in HUVEC from normal pregnancies, while in GD only combination of shRNA-A2AAR and shRNA-A2BAR blocked the stimulatory effect of NECA. Nevertheless, L-NAME co-incubation blocked the CGS21680 and NECA-stimulatory effects in both groups of pregnancies. Conversely, SNAP increased (~1.4 fold) cell proliferation in both GD and normal pregnancies. No additive effect was observed in cell proliferation when CGS-21680 or NECA were co-incubated with SNAP. Co-incubation with SNAP and NECA, in cells lacking A2AAR and/or A2BAR receptor exhibited a response similar to that of NECA alone in both groups of pregnancies. Conclusions: Elevated expression of both A2AAR and A2BAR might generate cell proliferation in gestational diabetes via intracellular generation of NO. Supported by FONDECYT 1140586.
PA.52. REPRODUCTIVE PROFILE OF A MURINE MODEL OF DIET-INDUCED OBESITY M.E. Tejada, M.C. Pustovrh, L.S. Salazar. Department of Morphology, School of Health, University of Valle, Santiago de Cali, Colombia Obesity is a multifactorial epidemic that involves genetic, physiological, environmental, and social factors. In obese and overweight women, significant impairment of the reproductive organs has been observed. This leads to a reduction in the number and quality of mature oocytes, and to a lower rate of fecundation when compared to women with normal BMI, thus increasing the risk of infertility.