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Inflammation-induced fetal growth restriction is associated with increased placental HIF-1α accumulation
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Abstracts / Placenta 45 (2016) 63e133
proteins is reduced in a maternal nutrient restriction (MNR) baboon model of IUGR. Methods: Pregnant baboons were fed a control ad libitum (n¼3) or MNR diet (n¼6, 70% of control diet) from gestation day (GD) 30. At GD 165 (0.9 gestation) placentas were collected and homogenized, and microvillous membrane (MVM) and basal membrane (BM) were isolated. Protein expression of fatty acid binding proteins (FABP) 1, 3, 4, and 5 (homogenate), and fatty acid transport proteins (FATP) 2, 4, 6, and CD36 (MVM, BM) were determined using Western blot. Statistical differences were assessed using student’s t test. Results: Placental weights were reduced (-17%, p¼ 0.01) and fetal weights trended lower (-10%, p¼ 0.06) in MNR compared to control group. We found no significant difference between control and MNR groups in expression of FATP2, 4, 6, or CD36 in MVM or BM. FATP2 (p¼ 0.003) and FATP6 (p¼ 0.008) expression was higher in MVM than BM in MNR but not controls. There was no significant difference in expression of FABP4 in homogenates; however, expression of FABP1 (p¼ 0.04), FABP3 (p¼ 0.048), and FABP5 (p¼ 0.01) was increased in MNR compared to control. Conclusions: Rather than a decrease in fatty acid transport capacity, we found increased fatty acid binding capacity in the placenta of MNR baboons, indicating a potential for increased fatty acid transfer to support fetal brain development in the growth restricted fetus. Despite major differences between species in fetal fat deposition these findings are similar to the changes in fatty acid transport proteins in human IUGR we reported previously. Funded by P01HD21350. P1.98 THE EFFECT OF DIFFERENT PLACENTAL GLUCOCORTICOID RECEPTOR ISOFORMS ON THE EXPRESSION OF GLUCOCORTICOID REGULATED GENES IN PLACENTAE OF SMALL-FOR-GESTATIONAL AGE PREGNANCIES Vicki Clifton, Zarqa Saif. Mater Research Institute-UQ, Brisbane, QLD, Australia Introduction: We have recently identified there are 8 known isoforms of the glucocorticoid receptor (GR) in the human placental trophoblast that vary with fetal sex, gestational age at delivery, maternal asthma and maternal betamethasone exposure. The current study was designed to determine if there are differences in placental glucocorticoid regulated pathways in association with the expression of different placental GR isoforms in relation to birthweight. Methods: Placentae (n¼43) were analysed by Western blot to determine their GR protein profile. Placental RNA was extracted and GR regulated pathways were examined using a GR PCR array (n¼84 genes). Data was analysed based on birthweight at delivery. A binary analysis was conducted to examine the influence of each individual GR isoform on GR regulated gene expression. Results: In the presence of small for gestational age (SGA, n¼21) fetus relative to an appropriately grown fetus (AGA, n¼22), there were differences in the expression and localisation of GRa A, GRa C, GRa D1, GR A and GR P. Overall we observed significant alterations in GR regulated genes in placentae of SGA pregnancies that included increased expression of ANXA4, CEBPA, FKBP5 and decreased expression of IL6R, LOX, PDP1,PLD1, RASA3, SPSB1 and TINFAIP3 (t test, P<0.05). GRa A and GR P had the greatest influence on gene expression in SGA placentae relative to AGA placentae. Discussion: This work suggests that glucocorticoid regulation of placental genes is dependent on the GR isoform pattern of expression with some GR isoforms being more influential in regulating gene expression than others. P1.99 INFLAMMATION-INDUCED FETAL GROWTH RESTRICTION IS ASSOCIATED WITH INCREASED PLACENTAL HIF-1a ACCUMULATION Tiziana Cotechini, Kevin P.L. Robb, Camille Allaire, Arissa Sperou, Charles H. Graham. Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada Introduction: Hypoxia-inducible factor 1-alpha (HIF-1a) is the oxygensensitive subunit of the transcription factor HIF-1, and its expression is increased in placentas from pregnancies complicated by pre-eclampsia (PE).
Fetal growth restriction (FGR) and PE often share a common pathophysiology; however, it is unknown whether increased placental HIF-1a occurs in FGR. Previous work revealed that aberrant maternal inflammation is associated with FGR and can give rise to altered utero-placental perfusion, which could in turn favour HIF-1a up-regulation. We hypothesize that abnormal maternal inflammation leading to FGR is linked to placental HIF-1a accumulation. Methods: Levels of inflammatory factors in maternal plasma were measured using a multiplex assay after an injection of low-dose lipopolysaccharide (LPS) to rats on gestational day (GD) 13.5. Following three additional days of LPS injections, GD17.5 placentas were harvested for HIF1a immunolocalisation; serial sections were also stained for the hypoxia marker pimonidazole. A subset of rats received LPS along with the nitric oxide (NO) mimetic glyceryl trinitrate (GTN) from GD12.5-GD17.5. Results: Within two hours of LPS administration, levels of maternal proinflammatory cytokines were increased compared with saline-treated controls. GD17.5 placentas of FGR fetuses exhibited increased HIF-1a accumulation; however, no differences in pimonidazole staining were observed and HIF-1a was not localized around areas of pimonidazole staining. GTN attenuated the LPS-mediated increase in maternal inflammatory cytokine levels and placental HIF-1a accumulation. Discussion: Our results demonstrate that, although inflammation-induced FGR is associated with increased placental HIF-1a accumulation, expression of this oxygen-sensitive transcription factor may not correlate with regions of hypoxia in late-gestation placentas. P1.100 MODERATE EXERCISE ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION AND ASSOCIATED MATERNAL AND FETAL MORBIDITIES IN PREGNANT RATS Tiziana Cotechini 1, Karina T. Kasawara 1, 2, Shannyn K. Macdonaldao L. Pinto e Silva 2, Chandrakant Goodfellow 1, Fernanda G. Surita 2, Jo~ Tayade 1, Maha Othman 1, Terrence R.S. Ozolins 1, Charles H. Graham 1. 1 Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada; 2 Department of Obstetrics and Gynaecology, University of Campinas, Campinas, SP, Brazil Fetal growth restriction (FGR) and coagulopathies are often associated with aberrant maternal inflammation. Moderate-intensity exercise during pregnancy has been shown to increase utero-placental blood flow and to enhance fetal nutrition as well as fetal and placental growth. Furthermore, exercise is known to reduce inflammation. To evaluate the effect of moderate-intensity exercise on inflammation associated with the development of maternal coagulopathies and FGR, Wistar rats were subjected to an exercise regime before and during pregnancy. To model inflammationinduced FGR, pregnant rats were administered daily intraperitoneal injections of E. coli lipopolysaccharide (LPS) on gestational days (GD) 13.5 e 16.5 and sacrificed at GD 17.5. Control rats were injected with saline. Maternal hemostasis was assessed by thromboelastography. Moderateintensity exercise prevented LPS-mediated increases in white blood cell counts measured on GD 17.5 and improved maternal hemostasis profiles. Importantly, our data reveal that exercise prevented LPS-induced FGR. Moderate-intensity exercise initiated before and maintained during pregnancy may decrease the severity of maternal and perinatal complications associated with abnormal maternal inflammation. P1.101 MELATONIN SUPPLEMENTATION DURING PREGNANCY INCREASES FETAL ABDOMINAL CIRCUMFERENCE AND UMBILICAL ARTERY RELAXATION IN A MOUSE MODEL OF FETAL GROWTH RESTRICTION Lewis Renshall, Mark Wareing, Elizabeth Cowley, Elizabeth Cottrell, Colin Sibley, Susan Greenwood, Mark Dilworth. University of Manchester, Manchester, UK Objective: To assess whether the neurohormone and antioxidant melatonin altered fetal growth and umbilical artery reactivity in a well-characterised mouse model of Fetal Growth Restriction (FGR), the placental specific Igf2 knockout mouse (P0+/-). Methods: C57Bl6/J (wild-type, WT) females were mated with P0+/- males, resulting in mixed litters of WT and P0+/- fetuses. Melatonin was
Abstracts / Placenta 45 (2016) 63e133
proteins is reduced in a maternal nutrient restriction (MNR) baboon model of IUGR. Methods: Pregnant baboons were fed a control ad libitum (n¼3) or MNR diet (n¼6, 70% of control diet) from gestation day (GD) 30. At GD 165 (0.9 gestation) placentas were collected and homogenized, and microvillous membrane (MVM) and basal membrane (BM) were isolated. Protein expression of fatty acid binding proteins (FABP) 1, 3, 4, and 5 (homogenate), and fatty acid transport proteins (FATP) 2, 4, 6, and CD36 (MVM, BM) were determined using Western blot. Statistical differences were assessed using student’s t test. Results: Placental weights were reduced (-17%, p¼ 0.01) and fetal weights trended lower (-10%, p¼ 0.06) in MNR compared to control group. We found no significant difference between control and MNR groups in expression of FATP2, 4, 6, or CD36 in MVM or BM. FATP2 (p¼ 0.003) and FATP6 (p¼ 0.008) expression was higher in MVM than BM in MNR but not controls. There was no significant difference in expression of FABP4 in homogenates; however, expression of FABP1 (p¼ 0.04), FABP3 (p¼ 0.048), and FABP5 (p¼ 0.01) was increased in MNR compared to control. Conclusions: Rather than a decrease in fatty acid transport capacity, we found increased fatty acid binding capacity in the placenta of MNR baboons, indicating a potential for increased fatty acid transfer to support fetal brain development in the growth restricted fetus. Despite major differences between species in fetal fat deposition these findings are similar to the changes in fatty acid transport proteins in human IUGR we reported previously. Funded by P01HD21350. P1.98 THE EFFECT OF DIFFERENT PLACENTAL GLUCOCORTICOID RECEPTOR ISOFORMS ON THE EXPRESSION OF GLUCOCORTICOID REGULATED GENES IN PLACENTAE OF SMALL-FOR-GESTATIONAL AGE PREGNANCIES Vicki Clifton, Zarqa Saif. Mater Research Institute-UQ, Brisbane, QLD, Australia Introduction: We have recently identified there are 8 known isoforms of the glucocorticoid receptor (GR) in the human placental trophoblast that vary with fetal sex, gestational age at delivery, maternal asthma and maternal betamethasone exposure. The current study was designed to determine if there are differences in placental glucocorticoid regulated pathways in association with the expression of different placental GR isoforms in relation to birthweight. Methods: Placentae (n¼43) were analysed by Western blot to determine their GR protein profile. Placental RNA was extracted and GR regulated pathways were examined using a GR PCR array (n¼84 genes). Data was analysed based on birthweight at delivery. A binary analysis was conducted to examine the influence of each individual GR isoform on GR regulated gene expression. Results: In the presence of small for gestational age (SGA, n¼21) fetus relative to an appropriately grown fetus (AGA, n¼22), there were differences in the expression and localisation of GRa A, GRa C, GRa D1, GR A and GR P. Overall we observed significant alterations in GR regulated genes in placentae of SGA pregnancies that included increased expression of ANXA4, CEBPA, FKBP5 and decreased expression of IL6R, LOX, PDP1,PLD1, RASA3, SPSB1 and TINFAIP3 (t test, P<0.05). GRa A and GR P had the greatest influence on gene expression in SGA placentae relative to AGA placentae. Discussion: This work suggests that glucocorticoid regulation of placental genes is dependent on the GR isoform pattern of expression with some GR isoforms being more influential in regulating gene expression than others. P1.99 INFLAMMATION-INDUCED FETAL GROWTH RESTRICTION IS ASSOCIATED WITH INCREASED PLACENTAL HIF-1a ACCUMULATION Tiziana Cotechini, Kevin P.L. Robb, Camille Allaire, Arissa Sperou, Charles H. Graham. Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada Introduction: Hypoxia-inducible factor 1-alpha (HIF-1a) is the oxygensensitive subunit of the transcription factor HIF-1, and its expression is increased in placentas from pregnancies complicated by pre-eclampsia (PE).
Fetal growth restriction (FGR) and PE often share a common pathophysiology; however, it is unknown whether increased placental HIF-1a occurs in FGR. Previous work revealed that aberrant maternal inflammation is associated with FGR and can give rise to altered utero-placental perfusion, which could in turn favour HIF-1a up-regulation. We hypothesize that abnormal maternal inflammation leading to FGR is linked to placental HIF-1a accumulation. Methods: Levels of inflammatory factors in maternal plasma were measured using a multiplex assay after an injection of low-dose lipopolysaccharide (LPS) to rats on gestational day (GD) 13.5. Following three additional days of LPS injections, GD17.5 placentas were harvested for HIF1a immunolocalisation; serial sections were also stained for the hypoxia marker pimonidazole. A subset of rats received LPS along with the nitric oxide (NO) mimetic glyceryl trinitrate (GTN) from GD12.5-GD17.5. Results: Within two hours of LPS administration, levels of maternal proinflammatory cytokines were increased compared with saline-treated controls. GD17.5 placentas of FGR fetuses exhibited increased HIF-1a accumulation; however, no differences in pimonidazole staining were observed and HIF-1a was not localized around areas of pimonidazole staining. GTN attenuated the LPS-mediated increase in maternal inflammatory cytokine levels and placental HIF-1a accumulation. Discussion: Our results demonstrate that, although inflammation-induced FGR is associated with increased placental HIF-1a accumulation, expression of this oxygen-sensitive transcription factor may not correlate with regions of hypoxia in late-gestation placentas. P1.100 MODERATE EXERCISE ATTENUATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION AND ASSOCIATED MATERNAL AND FETAL MORBIDITIES IN PREGNANT RATS Tiziana Cotechini 1, Karina T. Kasawara 1, 2, Shannyn K. Macdonaldao L. Pinto e Silva 2, Chandrakant Goodfellow 1, Fernanda G. Surita 2, Jo~ Tayade 1, Maha Othman 1, Terrence R.S. Ozolins 1, Charles H. Graham 1. 1 Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada; 2 Department of Obstetrics and Gynaecology, University of Campinas, Campinas, SP, Brazil Fetal growth restriction (FGR) and coagulopathies are often associated with aberrant maternal inflammation. Moderate-intensity exercise during pregnancy has been shown to increase utero-placental blood flow and to enhance fetal nutrition as well as fetal and placental growth. Furthermore, exercise is known to reduce inflammation. To evaluate the effect of moderate-intensity exercise on inflammation associated with the development of maternal coagulopathies and FGR, Wistar rats were subjected to an exercise regime before and during pregnancy. To model inflammationinduced FGR, pregnant rats were administered daily intraperitoneal injections of E. coli lipopolysaccharide (LPS) on gestational days (GD) 13.5 e 16.5 and sacrificed at GD 17.5. Control rats were injected with saline. Maternal hemostasis was assessed by thromboelastography. Moderateintensity exercise prevented LPS-mediated increases in white blood cell counts measured on GD 17.5 and improved maternal hemostasis profiles. Importantly, our data reveal that exercise prevented LPS-induced FGR. Moderate-intensity exercise initiated before and maintained during pregnancy may decrease the severity of maternal and perinatal complications associated with abnormal maternal inflammation. P1.101 MELATONIN SUPPLEMENTATION DURING PREGNANCY INCREASES FETAL ABDOMINAL CIRCUMFERENCE AND UMBILICAL ARTERY RELAXATION IN A MOUSE MODEL OF FETAL GROWTH RESTRICTION Lewis Renshall, Mark Wareing, Elizabeth Cowley, Elizabeth Cottrell, Colin Sibley, Susan Greenwood, Mark Dilworth. University of Manchester, Manchester, UK Objective: To assess whether the neurohormone and antioxidant melatonin altered fetal growth and umbilical artery reactivity in a well-characterised mouse model of Fetal Growth Restriction (FGR), the placental specific Igf2 knockout mouse (P0+/-). Methods: C57Bl6/J (wild-type, WT) females were mated with P0+/- males, resulting in mixed litters of WT and P0+/- fetuses. Melatonin was