- Email: [email protected]
Morphological analysis of the pancreatic beta cells of diabetic female rats at different ages of life
490
Abstracts / Placenta 36 (2015) 469e521
pregnancy morbidity plus vascular thrombosis (PM/VT), pregnancy morbidity without aPL (PM/aPL-) or healthy women (NHS). Drugs were added at the same time with IgG at previous established concentrations. After 24 h, the inserts were removed and trophoblast cell migration across the Matrigel coated membrane was determined using a colorimetric assay. The relative cell invasion value was assessed by comparing the optical densities with no treatment control. STAT-3 activation was determined at same conditions of invasion assays using Western blots. Results: IgGs from patients with APS-associated pregnancy morbidity (PM, PM/VT) induce a significantly decreased invasion in comparison with IgGs from the NHS and PM/aPL- groups. Enoxaparin, aspirin and ATL restore the aPL-decreased invasion and, moreover, ATL has a receptormediated effect as demonstrated by application of its antagonist Boc-2, which reverses the ATL-restored invasion. Additionally, phosphorylation of STAT-3 is reduced by IgGs from PM patients. Conclusion: We show here that some of drugs used in APS-related pregnancy morbidity treatment are involved in the modulation of trophoblast function. They possibly restore some processes essential for normal trophoblast invasion. Moreover, ATL effects may explain the benefits of aspirin treatment observed in patients with obstetric APS. Since STAT-3 is an intracellular signalling molecule involved in migration and invasion processes, our results support the view that aPL from patients with pregnancy morbidity alter the proper function of this pathway. Financial support: CODI- Universidad de Antioquia Grant 91515; Estra n 2014-2015; Programa de tegia de sostenibilidad Grupo Reproduccio n Científica Internacional-Proyectos de Intercambio: ColcienCooperacio cias-Colombia Convocatoria 614-2013, y CONICYT-Chile PCCI 130017. AMA is a fellow of Colciencias.
PA.7. MORPHOLOGICAL ANALYSIS OF THE PANCREATIC BETA CELLS OF DIABETIC FEMALE RATS AT DIFFERENT AGES OF LIFE F.Q. Gallego 1, Y.K. Sinzato 1, B. Dallaqua 1, I.L. Iessi 1, R.L. Amorim 2, C.E. Fonseca 2, I.M.P. Calderon 1, M.V.C. Rudge 1, D.C. Damasceno 1. 1 Laboratory of Experimental Research on Gynecology and Obstetrics, Graduate Program on Gynecology, Obstetrics and Mastology, Botucatu Medical School, Brazil; 2 Department of Pathology, Botucatu Veterinary and Zootechnology Medical ~o Paulo, Brazil School, Univ. Estadual Paulista_Unesp, Botucatu, Sa Background: Recent studies have shown regeneration of pancreatic islets in physiological and pathological circumstances. We hypothesized that diabetes disturbs insulin-producing b-cell regeneration in laboratory animals. Aim: The objective was to understand the proliferation and death mechanism of pancreatic b-cells in non-diabetic and neonatally streptozotocininduced mild diabetic rats at different stages of life and during pregnancy. Methods: The rats were randomly assigned into the following groups: Non-diabetic (Control¼C, glycemia<120 mg/dL) or Mild diabetes (MD) (n¼15/group). The mild diabetes group was induced at birth by the b-cell cytotoxic agent e Streptozotocin (STZ - 100 mg/kg b.w., sc.). On postnatal days (PND) 5, 15 and 90 and on day 18.5 of pregnancy, the different groups of rats were killed to collect blood samples for insulin and glycated hemoglobin measurements; pancreas samples were processed for histological and immunohistochemical analysis for cell proliferation, apoptosis and insulin. In addition, on day 18.5 of pregnancy, the uterine corns were exposed for evaluation of reproductive outcomes. p<0.05 was considered as the significant statistical limit. Results: On PND5, the diabetic rats showed pancreatic islet morphological alterations as decreased insulin and apoptosis immunostaining, and increased cell proliferation. On PND15, cell proliferation was lower than on PND5 in diabetic rats. In adulthood (PND90 and pregnancy), diabetic rats presented increased glycated hemoglobin levels and glucose intolerance, and no changes in pancreatic islets morphology were observed in relation to the control group. On day 18.5 of pregnancy, MD dams presented an increased number of embryonic death and rate of preimplantation losses when compared with the control group. Conclusion: Our results showed that after administration of streptozotocin pancreatic b-cells were able to regenerate partially by an increase in proliferation and a decrease in apoptosis. However, this was insufficient to
reverse the diabetic status in adulthood leading to impairment of the reproductive function in diabetic mothers. Acknowledgement: FAPESP/Brazil (Process Numbers: 2011/18519-7; 2011/15606-6).
PA.8. LEPTIN IS INVOLVED IN HUMAN TROPHOBLAST MIGRATION AND INVASION rez-Pe rez 3, B. Maskin 4, V. Sa nchezA. Toro 1, R. Sampayo 2, A. Pe gica, FCENMargalet 3, M. Simian 2, C. Varone 1. 1 Dpto. de Química Biolo de UBA, IQUIBICEN CONICET, Buenos Aires, Argentina; 2 Area Investigaciones, Instituto de Oncología “Angel H. Roffo”, Buenos Aires, Argentina; 3 Dpto. de Bioquímica M edica y Biología Molecular. Universidad de Sevilla, Sevilla, Spain; 4 Hospital Nacional “A. Posadas”, Buenos Aires, Argentina Cytotrophoblast (CTB) cells of the human placenta proliferate, migrate and invade the uterus during pregnancy to allow implantation and placentation. To successfully become invasive, CTB cells undergo a transition from the epithelial to the highly invasive mesenchymal phenotype, called EMT (epithelial-mesenchymal transition). EMT is characterized by up-regulation of motility promoting molecules such as the adhesion molecule b1 Integrin and the loss of epithelial markers such as E-cadherin. We have previously demonstrated that leptin, which is produced by placenta, promotes proliferation and survival of trophoblast cells. Objective: We aimed to study a possible role of leptin in cell migration and invasion by evaluating its effect on b1 Integrin and E-cadherin expression. Methods: The human CTB cell line from first trimester Swan-71 cells was used. Cells were treated with different leptin concentrations (50, 100 and 250 ng/ml) for 48h. To study whether leptin effect was reversible, after leptin treatment, the medium with leptin was replaced with fresh medium for 24 h. We also investigated whether metalloproteinases (MMPs) are necessary for leptin action. To this end, cells were cultured with a MMPs inhibitor (GM6001, 10mM) for 2 h before leptin treatment. b1 Integrin and E-cadherin expression were analyzed by qRT-PCR or/and WB assays. To evaluate leptin effect on cell migration, wound healing assays were performed. Results: Leptin significantly reduced E-cadherin expression at mRNA level and in a dose-dependent manner at protein level. This effect was reversible and MMPs activity was necessary. b1 Integrin protein levels were̴ 2 fold increased after stimulation with leptin (p<0.01), but leptin treatment did not modify b1 Integrin mRNA levels. Leptin action on b1 Integrin was reversible when cells were pretreated with 50 and 100 ng/ml of leptin. The wound healing assays demonstrated that 100 ng/ml of leptin promotes cell migration (p<0.05). Conclusions: All these findings suggest that leptin promotes cell migration and invasiveness of trophoblast cells.
PA.9. PARTICIPATION OF THE ENDOCANNABINOID SYSTEM (ECS) IN THE WEIGHT-GAIN AND SENSITIZATION TO LPS EXPOSURE IN A MATERNAL OBESITY MODEL M.V. Bariani, A.P. Domínguez Rubio, J. Aisemberg, A.M. Franchi. Laboratory of Physiopathology of Pregnancy and Labor, Center for Pharmacological and Botanical Studies (CEFYBO, CONICET), Buenos Aires, Argentina The endocannabinoid system (ECS) participates in the control of energy balance and regulates reproductive functions. Objectives: a) To evaluate whether a high fat diet (HFD) intake promotes the development of obesity in female CD1 wild type (WT) and cannabinoid receptor type 1 knockout (CB1KO) mice. b) To analyze whether HDFinduced maternal obesity alters gestation, pups development and sensitization to LPS exposure. Methods: Female CD1 WT and CB1KO mice were fed with standard food (control) or HFD (standard food+30% fat). Animals were weighed and food intake was measured. After 3 months of feeding, abdominal adipose tissue was weighed. Serum glucose and cholesterol levels were also
Abstracts / Placenta 36 (2015) 469e521
pregnancy morbidity plus vascular thrombosis (PM/VT), pregnancy morbidity without aPL (PM/aPL-) or healthy women (NHS). Drugs were added at the same time with IgG at previous established concentrations. After 24 h, the inserts were removed and trophoblast cell migration across the Matrigel coated membrane was determined using a colorimetric assay. The relative cell invasion value was assessed by comparing the optical densities with no treatment control. STAT-3 activation was determined at same conditions of invasion assays using Western blots. Results: IgGs from patients with APS-associated pregnancy morbidity (PM, PM/VT) induce a significantly decreased invasion in comparison with IgGs from the NHS and PM/aPL- groups. Enoxaparin, aspirin and ATL restore the aPL-decreased invasion and, moreover, ATL has a receptormediated effect as demonstrated by application of its antagonist Boc-2, which reverses the ATL-restored invasion. Additionally, phosphorylation of STAT-3 is reduced by IgGs from PM patients. Conclusion: We show here that some of drugs used in APS-related pregnancy morbidity treatment are involved in the modulation of trophoblast function. They possibly restore some processes essential for normal trophoblast invasion. Moreover, ATL effects may explain the benefits of aspirin treatment observed in patients with obstetric APS. Since STAT-3 is an intracellular signalling molecule involved in migration and invasion processes, our results support the view that aPL from patients with pregnancy morbidity alter the proper function of this pathway. Financial support: CODI- Universidad de Antioquia Grant 91515; Estra n 2014-2015; Programa de tegia de sostenibilidad Grupo Reproduccio n Científica Internacional-Proyectos de Intercambio: ColcienCooperacio cias-Colombia Convocatoria 614-2013, y CONICYT-Chile PCCI 130017. AMA is a fellow of Colciencias.
PA.7. MORPHOLOGICAL ANALYSIS OF THE PANCREATIC BETA CELLS OF DIABETIC FEMALE RATS AT DIFFERENT AGES OF LIFE F.Q. Gallego 1, Y.K. Sinzato 1, B. Dallaqua 1, I.L. Iessi 1, R.L. Amorim 2, C.E. Fonseca 2, I.M.P. Calderon 1, M.V.C. Rudge 1, D.C. Damasceno 1. 1 Laboratory of Experimental Research on Gynecology and Obstetrics, Graduate Program on Gynecology, Obstetrics and Mastology, Botucatu Medical School, Brazil; 2 Department of Pathology, Botucatu Veterinary and Zootechnology Medical ~o Paulo, Brazil School, Univ. Estadual Paulista_Unesp, Botucatu, Sa Background: Recent studies have shown regeneration of pancreatic islets in physiological and pathological circumstances. We hypothesized that diabetes disturbs insulin-producing b-cell regeneration in laboratory animals. Aim: The objective was to understand the proliferation and death mechanism of pancreatic b-cells in non-diabetic and neonatally streptozotocininduced mild diabetic rats at different stages of life and during pregnancy. Methods: The rats were randomly assigned into the following groups: Non-diabetic (Control¼C, glycemia<120 mg/dL) or Mild diabetes (MD) (n¼15/group). The mild diabetes group was induced at birth by the b-cell cytotoxic agent e Streptozotocin (STZ - 100 mg/kg b.w., sc.). On postnatal days (PND) 5, 15 and 90 and on day 18.5 of pregnancy, the different groups of rats were killed to collect blood samples for insulin and glycated hemoglobin measurements; pancreas samples were processed for histological and immunohistochemical analysis for cell proliferation, apoptosis and insulin. In addition, on day 18.5 of pregnancy, the uterine corns were exposed for evaluation of reproductive outcomes. p<0.05 was considered as the significant statistical limit. Results: On PND5, the diabetic rats showed pancreatic islet morphological alterations as decreased insulin and apoptosis immunostaining, and increased cell proliferation. On PND15, cell proliferation was lower than on PND5 in diabetic rats. In adulthood (PND90 and pregnancy), diabetic rats presented increased glycated hemoglobin levels and glucose intolerance, and no changes in pancreatic islets morphology were observed in relation to the control group. On day 18.5 of pregnancy, MD dams presented an increased number of embryonic death and rate of preimplantation losses when compared with the control group. Conclusion: Our results showed that after administration of streptozotocin pancreatic b-cells were able to regenerate partially by an increase in proliferation and a decrease in apoptosis. However, this was insufficient to
reverse the diabetic status in adulthood leading to impairment of the reproductive function in diabetic mothers. Acknowledgement: FAPESP/Brazil (Process Numbers: 2011/18519-7; 2011/15606-6).
PA.8. LEPTIN IS INVOLVED IN HUMAN TROPHOBLAST MIGRATION AND INVASION rez-Pe rez 3, B. Maskin 4, V. Sa nchezA. Toro 1, R. Sampayo 2, A. Pe gica, FCENMargalet 3, M. Simian 2, C. Varone 1. 1 Dpto. de Química Biolo de UBA, IQUIBICEN CONICET, Buenos Aires, Argentina; 2 Area Investigaciones, Instituto de Oncología “Angel H. Roffo”, Buenos Aires, Argentina; 3 Dpto. de Bioquímica M edica y Biología Molecular. Universidad de Sevilla, Sevilla, Spain; 4 Hospital Nacional “A. Posadas”, Buenos Aires, Argentina Cytotrophoblast (CTB) cells of the human placenta proliferate, migrate and invade the uterus during pregnancy to allow implantation and placentation. To successfully become invasive, CTB cells undergo a transition from the epithelial to the highly invasive mesenchymal phenotype, called EMT (epithelial-mesenchymal transition). EMT is characterized by up-regulation of motility promoting molecules such as the adhesion molecule b1 Integrin and the loss of epithelial markers such as E-cadherin. We have previously demonstrated that leptin, which is produced by placenta, promotes proliferation and survival of trophoblast cells. Objective: We aimed to study a possible role of leptin in cell migration and invasion by evaluating its effect on b1 Integrin and E-cadherin expression. Methods: The human CTB cell line from first trimester Swan-71 cells was used. Cells were treated with different leptin concentrations (50, 100 and 250 ng/ml) for 48h. To study whether leptin effect was reversible, after leptin treatment, the medium with leptin was replaced with fresh medium for 24 h. We also investigated whether metalloproteinases (MMPs) are necessary for leptin action. To this end, cells were cultured with a MMPs inhibitor (GM6001, 10mM) for 2 h before leptin treatment. b1 Integrin and E-cadherin expression were analyzed by qRT-PCR or/and WB assays. To evaluate leptin effect on cell migration, wound healing assays were performed. Results: Leptin significantly reduced E-cadherin expression at mRNA level and in a dose-dependent manner at protein level. This effect was reversible and MMPs activity was necessary. b1 Integrin protein levels were̴ 2 fold increased after stimulation with leptin (p<0.01), but leptin treatment did not modify b1 Integrin mRNA levels. Leptin action on b1 Integrin was reversible when cells were pretreated with 50 and 100 ng/ml of leptin. The wound healing assays demonstrated that 100 ng/ml of leptin promotes cell migration (p<0.05). Conclusions: All these findings suggest that leptin promotes cell migration and invasiveness of trophoblast cells.
PA.9. PARTICIPATION OF THE ENDOCANNABINOID SYSTEM (ECS) IN THE WEIGHT-GAIN AND SENSITIZATION TO LPS EXPOSURE IN A MATERNAL OBESITY MODEL M.V. Bariani, A.P. Domínguez Rubio, J. Aisemberg, A.M. Franchi. Laboratory of Physiopathology of Pregnancy and Labor, Center for Pharmacological and Botanical Studies (CEFYBO, CONICET), Buenos Aires, Argentina The endocannabinoid system (ECS) participates in the control of energy balance and regulates reproductive functions. Objectives: a) To evaluate whether a high fat diet (HFD) intake promotes the development of obesity in female CD1 wild type (WT) and cannabinoid receptor type 1 knockout (CB1KO) mice. b) To analyze whether HDFinduced maternal obesity alters gestation, pups development and sensitization to LPS exposure. Methods: Female CD1 WT and CB1KO mice were fed with standard food (control) or HFD (standard food+30% fat). Animals were weighed and food intake was measured. After 3 months of feeding, abdominal adipose tissue was weighed. Serum glucose and cholesterol levels were also