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Deletion of PAPP-A2 does not exacerbate the phenotype of a mouse model of preeclampsia
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Abstracts / Placenta 57 (2017) 225e335
Objective: Placental fibrosis is known in pathologic analysis in preeclampsia (PE). TGFb has been known as the central profibrogenic cytokine and over expression of TGFb is thought to induce tissue injury. We focused on the relationship between pathogenesis of PE and placental fibrosis induced by TGFb. Methods: The mRNA expression levels of the fibrosis related factors (FRFs) were evaluated by real-time RT-PCR. Collagen gel contraction assay was used to evaluate the fibroblasts activation. Phosphorylation of Smad2 was evaluated by immunoblotting. Protein secretion of fibronectin (FN) and TGFb in the culture medium was assessed by ELISA. Changes for using the small interfering RNA (siRNA) specific for TGFBR1 in PE and under stimulation of TGFb in normal pregnancy (NP) were determined. This study was approved by the ethics committee in our institute. Results: The mRNA levels of FRFs in PE were higher than those in NP. The fibroblasts from PE cases showed a higher ability to contract than those from NP cases by collagen gel assay. FN and TGFb concentrations in the conditioned medium of fibroblasts were higher in PE cases. The inhibition of TGFBR1 suppressed the expression of FRFs in PE, while TGFb stimuli accentuate them in NP cases. Conclusion: Fibroblasts from PE placentas were more sensitive to TGFb stimulation than those of NP placentas. Consistent with the increased levels of TGFb in villous stromal of PE might be affect to pathogenesis of PE. http://dx.doi.org/10.1016/j.placenta.2017.07.293
P2.80. THE ROLES OF METFORMIN AND PRAVASTATIN ON PLACENTAL ENDOPLASMIC RETICULUM STRESS AND PLACENTAL GROWTH FACTOR IN HUMAN VILLOUS-LIKE TROPHOBLASTIC BEWO CELLS Masahiro Suzuki 1, Masahito Mizuuchi 1, Tsuyoshi Baba 1, Shinichi Ishioka 1, Graham Burton 2, Tsuyoshi Saito 1. 1 Dept. of Obstetrics and Gynecology, Sapporo Medical University, Sapporo, Hokkaido, Japan; 2 Centre of Trophoblast Research, University of Cambridge, Cambridge, UK Objectives: Placental growth factor (PlGF) is reduced in the maternal circulation in early onset preeclampsia (PE). We previously reported that the unfolded protein response (UPR) activated by ER stress can down-regulate PlGF protein through the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway. Recent evidence indicates that metformin and statins may suppress endoplasmic reticulum (ER) stress, and we predicted that these drugs could prevent placental ER stress and up-regulate PlGF protein expression in trophoblast-like cells. The aim of this study was to establish the effects of these drugs on PlGF and regulating factors of PERK pathway; expression of activating transcription factor 4 (ATF4) and phosphorylation of eukaryotic initiation factor 2 subunit a (p-eIF2a). Methods: ER stress was induced in human choriocarcinoma BeWo cells by thapsigargin (Tg), a chemical ER stress inducer. Metformin or pravastatin were administered in increasing doses, and levels of PlGF mRNA and protein, p-eIF2a and ATF4 were estimated using quantitative RT-PCR (qPCR) and western blotting (WB). Results: When we administered metformin under ER stress induced by Tg, qPCR showed increased PlGF mRNA compared with treatment with thapsigargin alone, and that the effect was dose-dependent. Under ER stress, WB showed high levels of ATF4 and p-eIF2a but low levels of PlGF protein. By contrast, compared with thapsigargin alone, ATF4 and p-eIF2a levels were low and PlGF levels were high when metformin and thapsigargin were given. Again, these effects were dependent on metformin concentrations. WB also confirmed that pravastatin attenuated ER stress and increased PlGF protein in trophoblast-like cells in a way similar to that observed for metformin. Conclusion: Metformin and pravastatin suppressed ER stress and activation of the PERK pathway, and restored PlGF levels in trophoblast-like cells. These results indicate that metformin and pravastatin have a potential for preventing or treating PE. http://dx.doi.org/10.1016/j.placenta.2017.07.294
P2.81. DELETION OF PAPP-A2 DOES NOT EXACERBATE THE PHENOTYPE OF A MOUSE MODEL OF PREECLAMPSIA Julian Christians, Kendra Lennie. Simon Fraser University, Burnaby, BC, Canada Objectives: Pregnancy-associated plasma protein-A2 (PAPP-A2) is expressed at high levels in the placenta and is upregulated in pregnancies complicated by preeclampsia and fetal growth restriction. However, the role of PAPP-A2 in the etiology of pregnancy complications is unknown. PAPP-A2 is a protease of insulin-like growth factor-binding protein-5 (IGFBP-5), and thus is expected to increase the bioavailability of insulinlike growth factors (IGFs). We hypothesized that elevated levels of PAPPA2 in preeclamptic pregnancies represent a compensatory response to ameliorate placental dysfunction. Methods: To test this hypothesis, we deleted the PAPP-A2 gene in a mouse model of preeclampsia, deletion of matrix metalloproteinase 9 (MMP9). We predicted that, if the elevated expression of PAPP-A2 in human preeclampsia reflects a compensatory response, then deletion of PAPP-A2 in mice would exacerbate the effects of MMP9 deletion, i.e., mice null for both PAPP-A2 and MMP9 would show a more severe phenotype than mice null for MMP9 only. We collected a total of 41 females at E16.5. Results: The number of fetuses, average fetal weight, average placental weight, and the ratio of fetal to placental weight were not affected by MMP9 deletion or by PAPP-A2 deletion, and there was no interaction between the effects of MMP9 and PAPP-A2 deletion. Among 20 pregnancies segregating for MMP9 deletion, the ratio of homozygous deletion fetuses to heterozygotes did not differ from the expected 1:1 ratio. In these pregnancies, fetuses homozygous for the deletion were lighter and had lighter placentae than heterozygotes (P ¼ 0.001 in both cases). However, there was no effect of PAPP-A2 deletion, and there was no interaction between the effects of MMP9 and PAPP-A2 deletion, i.e., deletion of PAPPA2 did not exacerbate the effect of MMP9 deletion. Conclusion: Our results do not support the hypothesis that PAPP-A2 plays an important role in compensating for placental deficiencies. http://dx.doi.org/10.1016/j.placenta.2017.07.295
P2.82. THE EFFECT OF CLUSTERIN AND GLUTATHIONE-S-TRANSFERASE IN HIV ASSOCIATED PRE-ECLAMPSIA Frederick Odun-Ayo, Jagidesa Moodley, Thajasvarie Naicker. University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa Objectives: Pre-eclampsia (PE) is a major cause of maternal and neonatal morbidity and mortality world-wide. However, the frequency of PE may be affected by immunosuppressive conditions such as HIV infection (Moodley, 2013). The role of clusterin (CLU) and glutathione-s-transferase (GST) proteins in HIV infected PE is unknown; the expression of these proteins may be associated with PE though. This study determines the effect of CLU and GST in the urine of pre-eclamptic pregnant women with HIV infection. Material and Methods: Following institutional ethical approval and written informed consent by patient, urine samples were obtained from normotensive pregnant (n ¼ 38) and pre-eclamptic (n ¼ 38) women attending a regional hospital women further stratified by HIV status. The urine concentration for clusterin and glutathione-s-transferase were estimated using the Bio-Plex® ProTM (Human Kidney Toxicity Panel 1) immunoassay. Result: Irrespective of the HIV status, a significantly higher urinary CLU with an increased trend in GST levels was shown in the pre-eclamptic pregnant women. In contrast to pregnancy type, the level of urinary CLU and GST was reduced in the HIV infected compared to HIV uninfected group; albeit non-significant (p ¼ 0.414 and p ¼ 0.124 respectively). Based on the pregnancy type and HIV status, a significant down-regulation of CLU (p ¼ 0.039) with a reduced trend in GST (p ¼ 0.118) was noted in HIV infected PE.
Abstracts / Placenta 57 (2017) 225e335
Objective: Placental fibrosis is known in pathologic analysis in preeclampsia (PE). TGFb has been known as the central profibrogenic cytokine and over expression of TGFb is thought to induce tissue injury. We focused on the relationship between pathogenesis of PE and placental fibrosis induced by TGFb. Methods: The mRNA expression levels of the fibrosis related factors (FRFs) were evaluated by real-time RT-PCR. Collagen gel contraction assay was used to evaluate the fibroblasts activation. Phosphorylation of Smad2 was evaluated by immunoblotting. Protein secretion of fibronectin (FN) and TGFb in the culture medium was assessed by ELISA. Changes for using the small interfering RNA (siRNA) specific for TGFBR1 in PE and under stimulation of TGFb in normal pregnancy (NP) were determined. This study was approved by the ethics committee in our institute. Results: The mRNA levels of FRFs in PE were higher than those in NP. The fibroblasts from PE cases showed a higher ability to contract than those from NP cases by collagen gel assay. FN and TGFb concentrations in the conditioned medium of fibroblasts were higher in PE cases. The inhibition of TGFBR1 suppressed the expression of FRFs in PE, while TGFb stimuli accentuate them in NP cases. Conclusion: Fibroblasts from PE placentas were more sensitive to TGFb stimulation than those of NP placentas. Consistent with the increased levels of TGFb in villous stromal of PE might be affect to pathogenesis of PE. http://dx.doi.org/10.1016/j.placenta.2017.07.293
P2.80. THE ROLES OF METFORMIN AND PRAVASTATIN ON PLACENTAL ENDOPLASMIC RETICULUM STRESS AND PLACENTAL GROWTH FACTOR IN HUMAN VILLOUS-LIKE TROPHOBLASTIC BEWO CELLS Masahiro Suzuki 1, Masahito Mizuuchi 1, Tsuyoshi Baba 1, Shinichi Ishioka 1, Graham Burton 2, Tsuyoshi Saito 1. 1 Dept. of Obstetrics and Gynecology, Sapporo Medical University, Sapporo, Hokkaido, Japan; 2 Centre of Trophoblast Research, University of Cambridge, Cambridge, UK Objectives: Placental growth factor (PlGF) is reduced in the maternal circulation in early onset preeclampsia (PE). We previously reported that the unfolded protein response (UPR) activated by ER stress can down-regulate PlGF protein through the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway. Recent evidence indicates that metformin and statins may suppress endoplasmic reticulum (ER) stress, and we predicted that these drugs could prevent placental ER stress and up-regulate PlGF protein expression in trophoblast-like cells. The aim of this study was to establish the effects of these drugs on PlGF and regulating factors of PERK pathway; expression of activating transcription factor 4 (ATF4) and phosphorylation of eukaryotic initiation factor 2 subunit a (p-eIF2a). Methods: ER stress was induced in human choriocarcinoma BeWo cells by thapsigargin (Tg), a chemical ER stress inducer. Metformin or pravastatin were administered in increasing doses, and levels of PlGF mRNA and protein, p-eIF2a and ATF4 were estimated using quantitative RT-PCR (qPCR) and western blotting (WB). Results: When we administered metformin under ER stress induced by Tg, qPCR showed increased PlGF mRNA compared with treatment with thapsigargin alone, and that the effect was dose-dependent. Under ER stress, WB showed high levels of ATF4 and p-eIF2a but low levels of PlGF protein. By contrast, compared with thapsigargin alone, ATF4 and p-eIF2a levels were low and PlGF levels were high when metformin and thapsigargin were given. Again, these effects were dependent on metformin concentrations. WB also confirmed that pravastatin attenuated ER stress and increased PlGF protein in trophoblast-like cells in a way similar to that observed for metformin. Conclusion: Metformin and pravastatin suppressed ER stress and activation of the PERK pathway, and restored PlGF levels in trophoblast-like cells. These results indicate that metformin and pravastatin have a potential for preventing or treating PE. http://dx.doi.org/10.1016/j.placenta.2017.07.294
P2.81. DELETION OF PAPP-A2 DOES NOT EXACERBATE THE PHENOTYPE OF A MOUSE MODEL OF PREECLAMPSIA Julian Christians, Kendra Lennie. Simon Fraser University, Burnaby, BC, Canada Objectives: Pregnancy-associated plasma protein-A2 (PAPP-A2) is expressed at high levels in the placenta and is upregulated in pregnancies complicated by preeclampsia and fetal growth restriction. However, the role of PAPP-A2 in the etiology of pregnancy complications is unknown. PAPP-A2 is a protease of insulin-like growth factor-binding protein-5 (IGFBP-5), and thus is expected to increase the bioavailability of insulinlike growth factors (IGFs). We hypothesized that elevated levels of PAPPA2 in preeclamptic pregnancies represent a compensatory response to ameliorate placental dysfunction. Methods: To test this hypothesis, we deleted the PAPP-A2 gene in a mouse model of preeclampsia, deletion of matrix metalloproteinase 9 (MMP9). We predicted that, if the elevated expression of PAPP-A2 in human preeclampsia reflects a compensatory response, then deletion of PAPP-A2 in mice would exacerbate the effects of MMP9 deletion, i.e., mice null for both PAPP-A2 and MMP9 would show a more severe phenotype than mice null for MMP9 only. We collected a total of 41 females at E16.5. Results: The number of fetuses, average fetal weight, average placental weight, and the ratio of fetal to placental weight were not affected by MMP9 deletion or by PAPP-A2 deletion, and there was no interaction between the effects of MMP9 and PAPP-A2 deletion. Among 20 pregnancies segregating for MMP9 deletion, the ratio of homozygous deletion fetuses to heterozygotes did not differ from the expected 1:1 ratio. In these pregnancies, fetuses homozygous for the deletion were lighter and had lighter placentae than heterozygotes (P ¼ 0.001 in both cases). However, there was no effect of PAPP-A2 deletion, and there was no interaction between the effects of MMP9 and PAPP-A2 deletion, i.e., deletion of PAPPA2 did not exacerbate the effect of MMP9 deletion. Conclusion: Our results do not support the hypothesis that PAPP-A2 plays an important role in compensating for placental deficiencies. http://dx.doi.org/10.1016/j.placenta.2017.07.295
P2.82. THE EFFECT OF CLUSTERIN AND GLUTATHIONE-S-TRANSFERASE IN HIV ASSOCIATED PRE-ECLAMPSIA Frederick Odun-Ayo, Jagidesa Moodley, Thajasvarie Naicker. University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa Objectives: Pre-eclampsia (PE) is a major cause of maternal and neonatal morbidity and mortality world-wide. However, the frequency of PE may be affected by immunosuppressive conditions such as HIV infection (Moodley, 2013). The role of clusterin (CLU) and glutathione-s-transferase (GST) proteins in HIV infected PE is unknown; the expression of these proteins may be associated with PE though. This study determines the effect of CLU and GST in the urine of pre-eclamptic pregnant women with HIV infection. Material and Methods: Following institutional ethical approval and written informed consent by patient, urine samples were obtained from normotensive pregnant (n ¼ 38) and pre-eclamptic (n ¼ 38) women attending a regional hospital women further stratified by HIV status. The urine concentration for clusterin and glutathione-s-transferase were estimated using the Bio-Plex® ProTM (Human Kidney Toxicity Panel 1) immunoassay. Result: Irrespective of the HIV status, a significantly higher urinary CLU with an increased trend in GST levels was shown in the pre-eclamptic pregnant women. In contrast to pregnancy type, the level of urinary CLU and GST was reduced in the HIV infected compared to HIV uninfected group; albeit non-significant (p ¼ 0.414 and p ¼ 0.124 respectively). Based on the pregnancy type and HIV status, a significant down-regulation of CLU (p ¼ 0.039) with a reduced trend in GST (p ¼ 0.118) was noted in HIV infected PE.