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Characterization of a human trophoblast model to evaluate novel therapeutics for preeclampsia
Abstracts / Placenta 45 (2016) 63e133
Conclusions: This present study suggests that sildenafil citrate, through its ability to promote nitric oxide synthesis, has effects on the vasculature in preeclamptic L-NAME rat model both peripherally and in the placenta (as evidenced in this study). The results also demonstrate that sildenafil citrate can be used as a possible therapeutic agent in treating preeclampsia as our results indicated improved lacunae diameter and perfusion. But the mechanism through which the sildenafil citrate acts in placenta and kidney still needs to be investigated. P2.72 CHARACTERIZATION OF A HUMAN TROPHOBLAST MODEL TO EVALUATE NOVEL THERAPEUTICS FOR PREECLAMPSIA Jiawu Zhao 1, Rebecca McLeese 1, Michelle Hookham 2, Timothy Lyons 1, Jeremy Yu 1. 1 Queen's University Belfast, Belfast, UK; 2 Clinical Biochemistry, Royal Victoria Hospital, Belfast, UK Objectives: Preeclampsia (PE), a leading complication of pregnancy, affects 3-5% of pregnancies in general, and ~20% of women with high risk conditions such as diabetes. Currently, there is no effective therapy other than symptomatic management or delivery. There is an urgent need to develop new therapeutic interventions, and this requires reliable cell-based assays. It is generally understood that PE results from inadequate perfusion of placenta. Placental hypoxia causes release of anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt1) into the maternal bloodstream. sFlt1 neutralizes vascular endothelial growth factor and placental growth factor, resulting in vascular injury, clinically manifest as hypertension and proteinuria. We aimed to test sFlt1 secretion by the common human trophoblast cell line, BeWo, when exposed to hypoxic stress, as model to screen pharmacologic agents for PE. Methods: Cultured BeWo cells were treated under hypoxia vs. normoxia (1 vs. 20% O2) for 24hrs. Transcriptional expression of the two major sFlt1 isoforms (i13 and e15a) and protein secretion were measured by RT-PCR and ELISA, respectively. The potential involvement of the HIF pathway and the effects of pravastatin, Trolox and resveratrol were evaluated. Results: Hypoxia significantly increased sFlt1 mRNA expression (both i13 and e15a, 2.5-3.8 fold, n¼6, p<0.01) and secretion (2 fold, n¼6, p¼0.01) from cultured BeWo cells. This effect could be mimicked by DMOG (10 mM, n¼6, p<0.05), and was inhibited by the HIF inhibitor chetomin (3-10 nM, n¼3, p<0.05). Trolox (500-1000 mM) and resveratrol (50 mM), but not pravastatin (100-1000 mM), significantly reduced the hypoxia-induced sFlt1 release (n¼3, p<0.05). Conclusion: The data suggest that the BeWo cells can be used as a clinically-relevant surrogate model for PE. This model brings potential to evaluate novel therapeutics for a common and serious complication of pregnancy. P2.73 WHEN IS A PROSTAGLANDIN NOT A PROSTAGLANDIN? Hassendrini N. Peiris, Kanchan Vaswani, Yong Qin Koh, Leon Oh, Sarah Reed Murray, D. Mitchell. The University of Queensland, Centre for Clinical Research, Brisbane, Queensland, Australia Introduction: An increase in intrauterine prostaglandin production is critical for the onset and progression of labour in women and indeed all mammalian species studied. Endocannabinoids can act as substrates for enzymes of the prostaglandin biosynthetic pathways and can be utilized to generate other related compounds such as prostamides and prostanoids. The related end products are indistinguishable by radioimmunoassay. Aim: To use mass spectrometry to identify products of endocannabinoid and eicosanoid biosynthetic pathways produced by amnion upon exposure to inflammatory stimuli (interleukin 1beta; IL-1b) with and without the addition of substrate (Anandamide; AEA.) Methods: Human amnion explants from term placentae (delivery by elective caesarean section due to cephalopelvic disproportion) were treated with IL-1b (0.2 ng/mL), AEA (10mM) or a combination of IL-1b and AEA. The concentrations of eicosanoids (PGE2-EA, and PGE2) generated by
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amnion explants (in explant culture media) were measured by LC-MS/MS in positive and negative modes. Results: Amnion explants produced significantly more prostaglandins (PGE2) when treated with an inflammatory agent (IL-1b). The addition of anandamide substrate significantly increased the production of PGE2-EA (p<0.05) compared to the inflammatory agent alone. In the absence of anandamide substrate prostaglandins (PGE2) represent >90% of PGHSderivatives. However, in the presence of anandamide substrate prostamides PGE2-EA represent >90% of PGHS-derivatives. Conclusion: We provide evidence that there is differential regulation of prostaglandin and prostamide biosynthesis in human in amnion in response to inflammatory stimuli and substrate (AEA). Our data demonstrate that amnion responds by a differential drive through the prostaglandin biosynthetic pathways. Moreover, and importantly, this has been shown using the “gold standard” of measurement by mass spectrometric means. The possibility is raised that separation of these products might reduce variability in results and lead to potential uses for their measurement in the diagnosis of preterm labour. P2.74 IDENTIFICATION OF EXOSOMAL MIRNA BIOMARKERS AT EARLY GESTATION (<18 WEEKS) IN ASYMPTOMATIC WOMEN AT EARLY GESTATION (<18 WEEKS) WHO SUBSEQUENTLY DEVELOP SPONTANEOUS PRETERM BIRTH Vyjayanthi kinhal 1, Dominic Guanzon 1, Katherin Scholz-Romero 1, Sherri Stephen Fortunato 2, Ramkumar Menon 3, Gregory Longo 2, Rice 1, 2, Carlos Salomon 1, 2. 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD, Australia; 2 Department of Obstetrics and Gynecology, Ochsner Baptist Hospital, New Orleans, Louisiana, USA; 3 Division of Maternal-Fetal Medicine & Perinatal Research, Department of Obstetrics & Gynecology, The University of Texas Medical Branch at Galveston, Galveston, Texas, USA Objectives: Currently, several biomarkers are regularly used clinically for predicting spontaneous preterm labor (SPTB); however, these factors are either non specified or detected too late. The aim of this study was to determine the exosomal miRNA profile in asymptomatic women at early gestation (<18 weeks) who subsequently develop SPTB. Methods: Plasma samples (n¼6 per group) were obtained at early gestation (<18 weeks) from the Ochsner Medical Center (New Orleans, USA) and classified according to pregnancy outcomes in normal and SPTB (delivery <37 weeks). Exosomes were isolated from plasma by differential and buoyant density centrifugation and characterised by size distribution, enrichment of CD63 and morphology using nanoparticle tracking analysis (NanoSight), Western Blot and electron microscopy, respectively. Total exosomal RNA was isolated by miRNAeasy (Qiagen) according to manufacturer’s' instructions. Illumina TrueSeq Small RNA kit was used to construct a small RNA library. The resulting sequencing FASTQ file was analysed using miRDeep2, a program specifically designed to identify both known and novel microRNA’s. Finally, miRNAs differential expression was predicted using Ingenuity Pathway Analysis (IPA) software. Results: We identified common (258) and unique (108 and 75 for normal and SPTB, respectively) miRNAs for each condition. Comparison analysis using IPA identified 10 miRNAs up-regulated in exosomes from SPTB compared to normal pregnancy, including: miR-143-3p, miR-203a3p, miR-34a-5p, miR-889-3p, miR-30c-5p, miR-30a-3p, miR-125b-2-3p, miR-411-5p, miR-199a-5p and miR-344-3p. Interestingly, we identified also 10 miRNAs down-regulated in exosomes from SPTB compared to normal pregnancy, including: miR-584-5p, miR-6724-5p, miR-4433b5p, miR-328-3p, miR423-5p, miR146a-5p, miR-92a-3p and miR-16-5p. Predicted miRNA targets were associated with immune response and inflammation. Conclusions: we suggest that the exosomal miRNA in the blood of asymptomatic women at early gestation may be of utility as an early biomarker of disease onset in SPTB.
Conclusions: This present study suggests that sildenafil citrate, through its ability to promote nitric oxide synthesis, has effects on the vasculature in preeclamptic L-NAME rat model both peripherally and in the placenta (as evidenced in this study). The results also demonstrate that sildenafil citrate can be used as a possible therapeutic agent in treating preeclampsia as our results indicated improved lacunae diameter and perfusion. But the mechanism through which the sildenafil citrate acts in placenta and kidney still needs to be investigated. P2.72 CHARACTERIZATION OF A HUMAN TROPHOBLAST MODEL TO EVALUATE NOVEL THERAPEUTICS FOR PREECLAMPSIA Jiawu Zhao 1, Rebecca McLeese 1, Michelle Hookham 2, Timothy Lyons 1, Jeremy Yu 1. 1 Queen's University Belfast, Belfast, UK; 2 Clinical Biochemistry, Royal Victoria Hospital, Belfast, UK Objectives: Preeclampsia (PE), a leading complication of pregnancy, affects 3-5% of pregnancies in general, and ~20% of women with high risk conditions such as diabetes. Currently, there is no effective therapy other than symptomatic management or delivery. There is an urgent need to develop new therapeutic interventions, and this requires reliable cell-based assays. It is generally understood that PE results from inadequate perfusion of placenta. Placental hypoxia causes release of anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt1) into the maternal bloodstream. sFlt1 neutralizes vascular endothelial growth factor and placental growth factor, resulting in vascular injury, clinically manifest as hypertension and proteinuria. We aimed to test sFlt1 secretion by the common human trophoblast cell line, BeWo, when exposed to hypoxic stress, as model to screen pharmacologic agents for PE. Methods: Cultured BeWo cells were treated under hypoxia vs. normoxia (1 vs. 20% O2) for 24hrs. Transcriptional expression of the two major sFlt1 isoforms (i13 and e15a) and protein secretion were measured by RT-PCR and ELISA, respectively. The potential involvement of the HIF pathway and the effects of pravastatin, Trolox and resveratrol were evaluated. Results: Hypoxia significantly increased sFlt1 mRNA expression (both i13 and e15a, 2.5-3.8 fold, n¼6, p<0.01) and secretion (2 fold, n¼6, p¼0.01) from cultured BeWo cells. This effect could be mimicked by DMOG (10 mM, n¼6, p<0.05), and was inhibited by the HIF inhibitor chetomin (3-10 nM, n¼3, p<0.05). Trolox (500-1000 mM) and resveratrol (50 mM), but not pravastatin (100-1000 mM), significantly reduced the hypoxia-induced sFlt1 release (n¼3, p<0.05). Conclusion: The data suggest that the BeWo cells can be used as a clinically-relevant surrogate model for PE. This model brings potential to evaluate novel therapeutics for a common and serious complication of pregnancy. P2.73 WHEN IS A PROSTAGLANDIN NOT A PROSTAGLANDIN? Hassendrini N. Peiris, Kanchan Vaswani, Yong Qin Koh, Leon Oh, Sarah Reed Murray, D. Mitchell. The University of Queensland, Centre for Clinical Research, Brisbane, Queensland, Australia Introduction: An increase in intrauterine prostaglandin production is critical for the onset and progression of labour in women and indeed all mammalian species studied. Endocannabinoids can act as substrates for enzymes of the prostaglandin biosynthetic pathways and can be utilized to generate other related compounds such as prostamides and prostanoids. The related end products are indistinguishable by radioimmunoassay. Aim: To use mass spectrometry to identify products of endocannabinoid and eicosanoid biosynthetic pathways produced by amnion upon exposure to inflammatory stimuli (interleukin 1beta; IL-1b) with and without the addition of substrate (Anandamide; AEA.) Methods: Human amnion explants from term placentae (delivery by elective caesarean section due to cephalopelvic disproportion) were treated with IL-1b (0.2 ng/mL), AEA (10mM) or a combination of IL-1b and AEA. The concentrations of eicosanoids (PGE2-EA, and PGE2) generated by
125
amnion explants (in explant culture media) were measured by LC-MS/MS in positive and negative modes. Results: Amnion explants produced significantly more prostaglandins (PGE2) when treated with an inflammatory agent (IL-1b). The addition of anandamide substrate significantly increased the production of PGE2-EA (p<0.05) compared to the inflammatory agent alone. In the absence of anandamide substrate prostaglandins (PGE2) represent >90% of PGHSderivatives. However, in the presence of anandamide substrate prostamides PGE2-EA represent >90% of PGHS-derivatives. Conclusion: We provide evidence that there is differential regulation of prostaglandin and prostamide biosynthesis in human in amnion in response to inflammatory stimuli and substrate (AEA). Our data demonstrate that amnion responds by a differential drive through the prostaglandin biosynthetic pathways. Moreover, and importantly, this has been shown using the “gold standard” of measurement by mass spectrometric means. The possibility is raised that separation of these products might reduce variability in results and lead to potential uses for their measurement in the diagnosis of preterm labour. P2.74 IDENTIFICATION OF EXOSOMAL MIRNA BIOMARKERS AT EARLY GESTATION (<18 WEEKS) IN ASYMPTOMATIC WOMEN AT EARLY GESTATION (<18 WEEKS) WHO SUBSEQUENTLY DEVELOP SPONTANEOUS PRETERM BIRTH Vyjayanthi kinhal 1, Dominic Guanzon 1, Katherin Scholz-Romero 1, Sherri Stephen Fortunato 2, Ramkumar Menon 3, Gregory Longo 2, Rice 1, 2, Carlos Salomon 1, 2. 1 Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, QLD, Australia; 2 Department of Obstetrics and Gynecology, Ochsner Baptist Hospital, New Orleans, Louisiana, USA; 3 Division of Maternal-Fetal Medicine & Perinatal Research, Department of Obstetrics & Gynecology, The University of Texas Medical Branch at Galveston, Galveston, Texas, USA Objectives: Currently, several biomarkers are regularly used clinically for predicting spontaneous preterm labor (SPTB); however, these factors are either non specified or detected too late. The aim of this study was to determine the exosomal miRNA profile in asymptomatic women at early gestation (<18 weeks) who subsequently develop SPTB. Methods: Plasma samples (n¼6 per group) were obtained at early gestation (<18 weeks) from the Ochsner Medical Center (New Orleans, USA) and classified according to pregnancy outcomes in normal and SPTB (delivery <37 weeks). Exosomes were isolated from plasma by differential and buoyant density centrifugation and characterised by size distribution, enrichment of CD63 and morphology using nanoparticle tracking analysis (NanoSight), Western Blot and electron microscopy, respectively. Total exosomal RNA was isolated by miRNAeasy (Qiagen) according to manufacturer’s' instructions. Illumina TrueSeq Small RNA kit was used to construct a small RNA library. The resulting sequencing FASTQ file was analysed using miRDeep2, a program specifically designed to identify both known and novel microRNA’s. Finally, miRNAs differential expression was predicted using Ingenuity Pathway Analysis (IPA) software. Results: We identified common (258) and unique (108 and 75 for normal and SPTB, respectively) miRNAs for each condition. Comparison analysis using IPA identified 10 miRNAs up-regulated in exosomes from SPTB compared to normal pregnancy, including: miR-143-3p, miR-203a3p, miR-34a-5p, miR-889-3p, miR-30c-5p, miR-30a-3p, miR-125b-2-3p, miR-411-5p, miR-199a-5p and miR-344-3p. Interestingly, we identified also 10 miRNAs down-regulated in exosomes from SPTB compared to normal pregnancy, including: miR-584-5p, miR-6724-5p, miR-4433b5p, miR-328-3p, miR423-5p, miR146a-5p, miR-92a-3p and miR-16-5p. Predicted miRNA targets were associated with immune response and inflammation. Conclusions: we suggest that the exosomal miRNA in the blood of asymptomatic women at early gestation may be of utility as an early biomarker of disease onset in SPTB.