- Email: [email protected]
[273-POS]
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Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 53–156
Objectives: In our quest to contribute to the definition of a molecular preeclamptic signature we encountered acid beta glucosidase (GBA, encoding for the enzyme glucocerebrosidase) as a gene that is up regulated in preeclamptic placentas. GBA deficiency causes Gaucher’s disease, a lysosomal storage disease. GBA hydrolyzes glucosylceramide to free glucose and ceramide. Ceramide is a bioactive signaling molecule involved in the regulation of cell movement, differentiation, survival and apoptosis. Purified GBA from placenta extracts was used to treat Gaucher patients with enzyme replacement therapy before the recombinant protein became available. The reason for the abundant expression in placenta and its role in the (patho) physiology of pregnancy is a complete enigma. Methods: We used multiple molecular techniques such as real time polymerase chain reaction, a lysosomal enzyme activity assay, 50 race to detect alternatively spliced variants, transfection of different variants in HEK-293 cells and Western blot analysis, in situ hybridization and immunofluorescence assays to determine cellular localization and microarray analysis to determine correlation of GBA expression to expression levels of other genes in placenta. Results: GBA is up regulated and there is increased lysosomal activity in the preeclamptic placenta. In placenta multiple variants are present but only the full-length GBA protein possesses classical lysosomal activity indicating its role in the lysosomal pathway in placenta. GBA is located in the syncytiotrophoblast layer of the placenta and immunofluorescence is suggestive of lysosomal localization. 158 genes correlate either positively or negatively with GBA expression. Gene enrichment analysis confirms the lysosomal pathway in placenta. Conclusions: The increased levels of GBA are most probably a result of the increased cell turnover in the preeclamptic placenta. However since we expect higher levels of ceramide in those cases it may also put ceramide forward as a novel etiological factor in the pathophysiology of preeclampsia. Disclosures: J.M. Jebbink: None. R.G. Boot: None. R. Keijser: None. P.D. Moerland: None. J. Aten: None. G.J. Veenboer: None. M. van Wely: None. M. Buimer: None. E. Ver Loren van Themaat: None. J.M. Aerts: None. J.A. van der Post: None. G.B. Afink: None. C. Ris-Stalpers: None. doi:10.1016/j.preghy.2014.10.277
[272-POS] The role of autophagy in poor placement of preeclampsia Shigeru Saito, Akitoshi Nakashima (University of Toyama, Toyama, Japan) Objectives: Autophagy is an intracellular degradation system for energy production under stress. We have studied the role of autophagy in extravillous trophoblast (EVT) invasion and vascular remodeling under hypoxia. Methods: EVT cell lines, HTR8/ SVneo and HchEpC1b, were used. We have established autophagy deficient EVT cell lines by infection of ATG4BC74A mutant expression vector. Autophagy was evaluated by the detection of LC3 dot
formation by immunohistochemistry and L3C-IIby western blotting. Vascular remodeling was evaluated by tube formation assay using EVT cells and endothelial cells co-culture system. This study was approved by ethics committee of University of Toyama. Results: Autophagy was observed in EVT cells under hypoxia or CoCl2 treatment. In wild type EVT cells, the invasions of EVT were enhanced under hypoxic condition. On the other hand, the invasion and vascular remodeling were significantly reduced in autophagy deficient EVT cells compared with wild type EVT cells under hypoxia. Importantly, soluble endoglin (sEng) suppressed EVT invasion and vascular remodeling under hypoxia by inhabitation of autophagy formation. Soluble Flt-1 and TNFa did not affect the autophagy formation under hypoxia. CoCl2, which induces hypoxia inducible factor 1a (HIF-1a) over-expression, activated autophagy in EVT cells even in normoxic condition. Cell invasion in autophagy deficient EVT cells were reduced by CoCl2 treatment through the suppression of MMP-9 level and cellular ATP levels. Decreased invasiveness was neutralized by ATP supplementation. P62 which is selectively degraded by autophagy accumulated in interstitial EVT and vascular EVT in placental biopsy samples of preeclampsia (n = 10), but not in FGR cases, suggesting that autophagy is impaired in EVT of preeclampsia, but not in FGR. Conclusions: Impaired autophagy by sEng, through decreased EVT invasion and vascular remodeling, is one of the etiologies of poor placentation in preeclampsia. Disclosures: S. Saito: None. A. Nakashima: None. doi:10.1016/j.preghy.2014.10.278
[273-POS] Sulfasalazine reduces the toxins of preeclampsia soluble Flt1 and soluble endoglin and quenches endothelial dysfunction in primary human tissues: A novel potential therapeutic Fiona C. Brownfoot, Stephen Tong, Natalie Hannan, Roxanne Hastie, Ping Cannon, Tu’uhevaha J. Kaitu’uLino (University of Melbourne, Heidelberg, Australia) Objectives: Sulfasalazine is an anti-inflammatory and immune modulating drug. Its mode of action has remained elusive, however recent evidence suggests it induces potent antioxidant enzyme heme-oxygenase1 (HO1) via nuclear erythroid-2-related factor 2 (Nrf2). Given its anti-oxidant capacity and that it is safe in pregnancy, we examined the potential for sulfasalazine as a novel therapeutic for preeclampsia. In particular, we examined its ability to quench anti-angiogenic factors sFlt1 and soluble endoglin (sEng) and reverse endothelial dysfunction in vitro. Methods: Increasing doses of sulfasalazine were administered to primary human umbilical vein endothelial cells (HUVECs) and sFlt1 and sEng release assessed. To induce endothelial dysfunction, HUVECs were treated with TNFa and the effect of sulfasalazine on monocyte adhesion determined. Finally the effect of sulfasalazine on HUVEC migration and proliferation was assessed.
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 53–156
Results: Excitingly, we observed a significant dose dependent reduction in both sFlt1 and sEng release. Importantly, mRNA expression of newly described human and placental specific variant sFlt1-e15a mRNA was significantly decreased, as was MMP14 mRNA expression (MMP14 is the protease that produces sEng). As expected, sulfasalazine also significantly increased mRNA expression of HO-1. Treatment of HUVECs with TNFa induced significant upregulation of VCAM which was potently reversed by Sulfasalazine, indicating its ability to quench endothelial dysfunction. Furthermore, sulfalsazine significantly reduced monocyte adhesion to HUVECs treated with TNFa, again supporting its anti-inflammatory properties. Finally we demonstrated that sulfasalazine could reverse the negative effects of sFlt1 on VEGF-stimulated HUVEC migration, and enhance HUVEC proliferation. Conclusions: Sulfasalazine is a novel agent, safe in pregnancy that significantly quenches sFlt1 and sEng release from human endothelial cells. In addition, it significantly reduces endothelial dysfunction and enhances endothelial cell migration and proliferation. This provides strong evidence to suggest that sulfasalazine may be able to quench the endothelial dysfunction of preeclampsia and could be an effective treatment for preeclampsia. Disclosures: F.C. Brownfoot: None. S. Tong: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. T.J. Kaitu’u-Lino: None. doi:10.1016/j.preghy.2014.10.279
[274-POS] Placental apoptotic signaling is augmented in preeclampsia: An adverse impact on the offspring Mary Antonette M. Co, Dean Leonard, Lena Perger, Vinayak P. Govande, Madhava R. Beeram, Richard O. Jones, Steven R. Allen, Thomas J. Kuehl, Mohammad N. Uddin (Scott & White Healthcare/TAMHSC, Temple, TX, USA) Objectives: Preeclampsia (preE), a syndrome of hypertension and proteinuria in pregnancy, is thought to be initiated with alterations of placental function. Hypoxia and oxidative stress can lead to placental apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring that may predispose to a pathological response later in life. We assessed apoptotic signaling in placentas and umbilical cords from patients with or without preE. We also evaluated the pregnancy outcomes. Methods: In this study, we recruited 20 normal pregnant (NP) and 20 preE consenting patients in an IRB approved prospective study. Inclusion criteria of preE patients include blood pressure >140/90 and proteinuria >300 mg of protein/ 24 h urine. Samples of placenta and umbilical cord were collected after deliveries. Pro-apoptotic Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 protein, caspase 9 and pro-inflammatory protein cyclooxygenase-2 (Cox-2) expression were assayed both by western blot and immunohisto-
137
chemistry. The p38 MAPK phosphorylation was evaluated by western blot. Comparisons were performed using ANOVA with Duncan’s post-hoc test. Results: The ratio of Bax/Bcl-2 expression (Placenta: 1.2 fold, Cord: 1.5 fold), Cox-2 (Placenta: 0.8 fold, Cord: 2.5 fold), Caspase 9 (Placenta: 1.5 fold, Cord: 1.8 fold), and p38 phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold) were up-regulated (p < 0.05) in placenta and umbilical cords of preE compared to NP patients. We did follow up examination of the babies for both groups of patients to assess the outcomes. Average hospital stay for PreE babies were significantly longer than NP babies (3.4 vs 6.1). There were no complications has been reported for the NP babies. However, all of preE babies had multiple medical complications. Conclusions: Apoptotic signaling is augmented in preE which alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus. Disclosures: M.M. Co: None. D. Leonard: None. L. Perger: None. V.P. Govande: None. M.R. Beeram: None. R.O. Jones: None. S.R. Allen: None. T.J. Kuehl: None. M.N. Uddin: None. doi:10.1016/j.preghy.2014.10.280
[275-POS] The alterations of villous DNA methylation associated with the pathogenesis of preeclampsia Keiko Koide (Showa University School of Medicine, Tokyo, Japan) Objectives: We investigated placental DNA methylation in preeclampsia (PE) compared with normal case to understand epigenetic pathogenesis of PE. Methods: Villi were obtained from artificially aborted women after fetal heart movement were detected by ultrasonography and pregnant women delivered by cesarean section. Samples from pregnant women with 6–11 weeks, normal third trimester, and PE were selected for this study. Placental DNA methylation profiles of 6 weeks, 10– 11 weeks, normal third trimester, and PE were made using Illumina HumanMethylation450 BeadChipÒ, and they were compared among the groups. Results: Before comparison, differences among individuals were minimized using criteria of IQR75. In consequence, 473,864 CpG sites were obtained as a dataset. Of these, 43,937 CpG sites were detected as ones whose DNA methylation changed during normal pregnancy. 1317 CpG sites were differentially methylated in PE compared with normal third trimester, and 83% of them were consistent with the CpG sites extracted as differences during normal pregnancy. Among 1317 CpG sites, 246 were on promoter. The cluster analysis for all samples was done using the results of these 246 CpG sites. In consequence, not only normal third trimester and PE, but also 6 weeks and 10–11 weeks were classified accurately. Conclusions: It is suggested that the alterations of villous DNA methylation cause the pathogenesis of PE in the villous trophoblasts.
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 53–156
Objectives: In our quest to contribute to the definition of a molecular preeclamptic signature we encountered acid beta glucosidase (GBA, encoding for the enzyme glucocerebrosidase) as a gene that is up regulated in preeclamptic placentas. GBA deficiency causes Gaucher’s disease, a lysosomal storage disease. GBA hydrolyzes glucosylceramide to free glucose and ceramide. Ceramide is a bioactive signaling molecule involved in the regulation of cell movement, differentiation, survival and apoptosis. Purified GBA from placenta extracts was used to treat Gaucher patients with enzyme replacement therapy before the recombinant protein became available. The reason for the abundant expression in placenta and its role in the (patho) physiology of pregnancy is a complete enigma. Methods: We used multiple molecular techniques such as real time polymerase chain reaction, a lysosomal enzyme activity assay, 50 race to detect alternatively spliced variants, transfection of different variants in HEK-293 cells and Western blot analysis, in situ hybridization and immunofluorescence assays to determine cellular localization and microarray analysis to determine correlation of GBA expression to expression levels of other genes in placenta. Results: GBA is up regulated and there is increased lysosomal activity in the preeclamptic placenta. In placenta multiple variants are present but only the full-length GBA protein possesses classical lysosomal activity indicating its role in the lysosomal pathway in placenta. GBA is located in the syncytiotrophoblast layer of the placenta and immunofluorescence is suggestive of lysosomal localization. 158 genes correlate either positively or negatively with GBA expression. Gene enrichment analysis confirms the lysosomal pathway in placenta. Conclusions: The increased levels of GBA are most probably a result of the increased cell turnover in the preeclamptic placenta. However since we expect higher levels of ceramide in those cases it may also put ceramide forward as a novel etiological factor in the pathophysiology of preeclampsia. Disclosures: J.M. Jebbink: None. R.G. Boot: None. R. Keijser: None. P.D. Moerland: None. J. Aten: None. G.J. Veenboer: None. M. van Wely: None. M. Buimer: None. E. Ver Loren van Themaat: None. J.M. Aerts: None. J.A. van der Post: None. G.B. Afink: None. C. Ris-Stalpers: None. doi:10.1016/j.preghy.2014.10.277
[272-POS] The role of autophagy in poor placement of preeclampsia Shigeru Saito, Akitoshi Nakashima (University of Toyama, Toyama, Japan) Objectives: Autophagy is an intracellular degradation system for energy production under stress. We have studied the role of autophagy in extravillous trophoblast (EVT) invasion and vascular remodeling under hypoxia. Methods: EVT cell lines, HTR8/ SVneo and HchEpC1b, were used. We have established autophagy deficient EVT cell lines by infection of ATG4BC74A mutant expression vector. Autophagy was evaluated by the detection of LC3 dot
formation by immunohistochemistry and L3C-IIby western blotting. Vascular remodeling was evaluated by tube formation assay using EVT cells and endothelial cells co-culture system. This study was approved by ethics committee of University of Toyama. Results: Autophagy was observed in EVT cells under hypoxia or CoCl2 treatment. In wild type EVT cells, the invasions of EVT were enhanced under hypoxic condition. On the other hand, the invasion and vascular remodeling were significantly reduced in autophagy deficient EVT cells compared with wild type EVT cells under hypoxia. Importantly, soluble endoglin (sEng) suppressed EVT invasion and vascular remodeling under hypoxia by inhabitation of autophagy formation. Soluble Flt-1 and TNFa did not affect the autophagy formation under hypoxia. CoCl2, which induces hypoxia inducible factor 1a (HIF-1a) over-expression, activated autophagy in EVT cells even in normoxic condition. Cell invasion in autophagy deficient EVT cells were reduced by CoCl2 treatment through the suppression of MMP-9 level and cellular ATP levels. Decreased invasiveness was neutralized by ATP supplementation. P62 which is selectively degraded by autophagy accumulated in interstitial EVT and vascular EVT in placental biopsy samples of preeclampsia (n = 10), but not in FGR cases, suggesting that autophagy is impaired in EVT of preeclampsia, but not in FGR. Conclusions: Impaired autophagy by sEng, through decreased EVT invasion and vascular remodeling, is one of the etiologies of poor placentation in preeclampsia. Disclosures: S. Saito: None. A. Nakashima: None. doi:10.1016/j.preghy.2014.10.278
[273-POS] Sulfasalazine reduces the toxins of preeclampsia soluble Flt1 and soluble endoglin and quenches endothelial dysfunction in primary human tissues: A novel potential therapeutic Fiona C. Brownfoot, Stephen Tong, Natalie Hannan, Roxanne Hastie, Ping Cannon, Tu’uhevaha J. Kaitu’uLino (University of Melbourne, Heidelberg, Australia) Objectives: Sulfasalazine is an anti-inflammatory and immune modulating drug. Its mode of action has remained elusive, however recent evidence suggests it induces potent antioxidant enzyme heme-oxygenase1 (HO1) via nuclear erythroid-2-related factor 2 (Nrf2). Given its anti-oxidant capacity and that it is safe in pregnancy, we examined the potential for sulfasalazine as a novel therapeutic for preeclampsia. In particular, we examined its ability to quench anti-angiogenic factors sFlt1 and soluble endoglin (sEng) and reverse endothelial dysfunction in vitro. Methods: Increasing doses of sulfasalazine were administered to primary human umbilical vein endothelial cells (HUVECs) and sFlt1 and sEng release assessed. To induce endothelial dysfunction, HUVECs were treated with TNFa and the effect of sulfasalazine on monocyte adhesion determined. Finally the effect of sulfasalazine on HUVEC migration and proliferation was assessed.
Abstracts / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 5 (2015) 53–156
Results: Excitingly, we observed a significant dose dependent reduction in both sFlt1 and sEng release. Importantly, mRNA expression of newly described human and placental specific variant sFlt1-e15a mRNA was significantly decreased, as was MMP14 mRNA expression (MMP14 is the protease that produces sEng). As expected, sulfasalazine also significantly increased mRNA expression of HO-1. Treatment of HUVECs with TNFa induced significant upregulation of VCAM which was potently reversed by Sulfasalazine, indicating its ability to quench endothelial dysfunction. Furthermore, sulfalsazine significantly reduced monocyte adhesion to HUVECs treated with TNFa, again supporting its anti-inflammatory properties. Finally we demonstrated that sulfasalazine could reverse the negative effects of sFlt1 on VEGF-stimulated HUVEC migration, and enhance HUVEC proliferation. Conclusions: Sulfasalazine is a novel agent, safe in pregnancy that significantly quenches sFlt1 and sEng release from human endothelial cells. In addition, it significantly reduces endothelial dysfunction and enhances endothelial cell migration and proliferation. This provides strong evidence to suggest that sulfasalazine may be able to quench the endothelial dysfunction of preeclampsia and could be an effective treatment for preeclampsia. Disclosures: F.C. Brownfoot: None. S. Tong: None. N. Hannan: None. R. Hastie: None. P. Cannon: None. T.J. Kaitu’u-Lino: None. doi:10.1016/j.preghy.2014.10.279
[274-POS] Placental apoptotic signaling is augmented in preeclampsia: An adverse impact on the offspring Mary Antonette M. Co, Dean Leonard, Lena Perger, Vinayak P. Govande, Madhava R. Beeram, Richard O. Jones, Steven R. Allen, Thomas J. Kuehl, Mohammad N. Uddin (Scott & White Healthcare/TAMHSC, Temple, TX, USA) Objectives: Preeclampsia (preE), a syndrome of hypertension and proteinuria in pregnancy, is thought to be initiated with alterations of placental function. Hypoxia and oxidative stress can lead to placental apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring that may predispose to a pathological response later in life. We assessed apoptotic signaling in placentas and umbilical cords from patients with or without preE. We also evaluated the pregnancy outcomes. Methods: In this study, we recruited 20 normal pregnant (NP) and 20 preE consenting patients in an IRB approved prospective study. Inclusion criteria of preE patients include blood pressure >140/90 and proteinuria >300 mg of protein/ 24 h urine. Samples of placenta and umbilical cord were collected after deliveries. Pro-apoptotic Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 protein, caspase 9 and pro-inflammatory protein cyclooxygenase-2 (Cox-2) expression were assayed both by western blot and immunohisto-
137
chemistry. The p38 MAPK phosphorylation was evaluated by western blot. Comparisons were performed using ANOVA with Duncan’s post-hoc test. Results: The ratio of Bax/Bcl-2 expression (Placenta: 1.2 fold, Cord: 1.5 fold), Cox-2 (Placenta: 0.8 fold, Cord: 2.5 fold), Caspase 9 (Placenta: 1.5 fold, Cord: 1.8 fold), and p38 phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold) were up-regulated (p < 0.05) in placenta and umbilical cords of preE compared to NP patients. We did follow up examination of the babies for both groups of patients to assess the outcomes. Average hospital stay for PreE babies were significantly longer than NP babies (3.4 vs 6.1). There were no complications has been reported for the NP babies. However, all of preE babies had multiple medical complications. Conclusions: Apoptotic signaling is augmented in preE which alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus. Disclosures: M.M. Co: None. D. Leonard: None. L. Perger: None. V.P. Govande: None. M.R. Beeram: None. R.O. Jones: None. S.R. Allen: None. T.J. Kuehl: None. M.N. Uddin: None. doi:10.1016/j.preghy.2014.10.280
[275-POS] The alterations of villous DNA methylation associated with the pathogenesis of preeclampsia Keiko Koide (Showa University School of Medicine, Tokyo, Japan) Objectives: We investigated placental DNA methylation in preeclampsia (PE) compared with normal case to understand epigenetic pathogenesis of PE. Methods: Villi were obtained from artificially aborted women after fetal heart movement were detected by ultrasonography and pregnant women delivered by cesarean section. Samples from pregnant women with 6–11 weeks, normal third trimester, and PE were selected for this study. Placental DNA methylation profiles of 6 weeks, 10– 11 weeks, normal third trimester, and PE were made using Illumina HumanMethylation450 BeadChipÒ, and they were compared among the groups. Results: Before comparison, differences among individuals were minimized using criteria of IQR75. In consequence, 473,864 CpG sites were obtained as a dataset. Of these, 43,937 CpG sites were detected as ones whose DNA methylation changed during normal pregnancy. 1317 CpG sites were differentially methylated in PE compared with normal third trimester, and 83% of them were consistent with the CpG sites extracted as differences during normal pregnancy. Among 1317 CpG sites, 246 were on promoter. The cluster analysis for all samples was done using the results of these 246 CpG sites. In consequence, not only normal third trimester and PE, but also 6 weeks and 10–11 weeks were classified accurately. Conclusions: It is suggested that the alterations of villous DNA methylation cause the pathogenesis of PE in the villous trophoblasts.