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Decreased VDR expression contributes to reduced feto-placental growth in idiopathic human fetal growth restriction
Abstracts / Placenta 34 (2013) A1–A99
placental development and pregnancy outcome has not yet been fully explored. It is postulated that hypomorphic expression of placental SIRT1 may compromise normal placental development and fetal growth. Methods: Human placenta biopsies were collected from 8 IUGR and 8 gestationally-age matched healthy control pregnancies. SIRT1 expression patterns were evaluated using immunohistochemistry and SIRT1 staining intensity quantified using ImageJ. Morphometric analysis of mid-line sections of placenta collected from SIRT1 knockout mice (SIRT1-/-) was performed at E15.5. Cross-sectional area and depth of the labyrinth exchange region were measured using Aperio Scanscope software (N¼4-8 placentae/genotype). Results: SIRT1 immunoreactivity was observed within the syncytiotrophoblast of all human placenta biopsies. A significant reduction in SIRT1 immunoreactivity was observed in all IUGR placentae (P<0.05, t-test). In the mouse, SIRT1-/- placentae demonstrated a significant reduction in midline cross-sectional area and depth of the labyrinth (P<0.05; t-test) compared to their wild-type littermates. These placentae also demonstrated evidence of hypovascularity. The SIRT1-mediated placental phenotype was dose-dependent, as heterozygous mice demonstrated an intermediated placental phenotype. Conclusion: Findings of abnormal SIRT1 expression patterns in cases of human IUGR along with evidence of compromised placental development and fetal growth restriction in the SIRT1 null mouse point to an important role for SIRT1 in placentation. Therapeutically targeting the SIRT1 pathway may prove to be a promising strategy aimed at improving fetal growth in pregnancies at risk of IUGR. http://dx.doi.org/10.1016/j.placenta.2013.06.200
P2.33. THE FUNCTIONAL EFFECT OF SYNDECAN 1 DEFICIENCY IN BEWO CELLS AND ITS IMPLICATIONS IN FETAL GROWTH RESTRICTION Tilini Gunatillake 1, 2, Amy Chui 1, Padma Murthi 2, Vera Ignjatovic 3, 4, Paul Monagle 3, 4, John Whitelock 5, Shaun Brennecke 2, Joanne Said 1 1
Department of Medicine, The University of Melbourne, St Albans, Victoria, Australia; 2 Departmenf of Obstetrics and Gynaecology, The University of Melbourne, Parkville, Victoria, Australia; 3 Department of Paediatrics, The University of Melbourne, Parkville, Victoria, Australia; 4 Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia; 5 Graduate School of Biomedical Engineering, The University of New South Wales, Kensington, New South Wales, Australia Objectives: Pregnancies complicated by fetal growth restriction (FGR) demonstrate increased placental fibrin deposition and thrombotic vasculopathy. Syndecans are proteoglycans expressed on the cell surface and are involved in cell growth and differentiation. We have previously demonstrated reduced expression of syndecan 1 (SDC1) in placentas affected by FGR. However, their exact role in the pathogenesis of FGR remains obscure. This study investigated the functional consequences of reduced SDC1 expression in a syncytiotrophoblast-like cell line. Methods: SDC1 downregulation was achieved in a BeWo cell line using short interference RNA (siRNA) transfection. Following transfection, BeWo cell proliferation was examined using the xCELLigence system. The differentiation potential was determined by investigating the mRNA expression of differentiation markers, syncytin, b-HCG and 3bHSD, using real time PCR. Finally, thrombin generation potential was determined using the Calibrated Automated Thrombogram system (Diagnostica Stago, France). Results: Following siRNA transfection, the mRNA and protein expression of SDC1 was significantly reduced after 72h. There were no significant differences in BeWo cell proliferation following SDC1 downregulation. However, the mRNA expression of the differentiation markers syncytin (Mock:0.850.32; NC:0.620.24 vs. S1: 0.100.04; S2:0.170.06, n¼6, p¼0.0003, ANOVA), b-HCG (Mock:2.941.11; NC:3.711.40 vs. S1:0.540.20; S2:0.8231, n¼6, p¼0.0023, ANOVA) and 3bHSD (Mock:1.280.48; NC:1.160.44 vs. S1:0.160.06; S2:0.060.02, n¼6, p¼0.0002, ANOVA) were significantly decreased following siRNA
A67
treatment. The thrombin generation potential of BeWo cells was significantly increased in SDC1 S2 downregulated cells compared to controls (Mock:1623.55811.78 vs. S1:1796.17898.08, p¼0.0506; S2:1826.67 913.33, n¼3, p¼0.0066, TTEST). Conclusion: Downregulation of SDC1 in BeWo cells results in significant reductions in cell differentiation and a significant increase in thrombin generation. Hence, it is plausible that reduced SDC1 expression may contribute to the pathogenesis of FGR by interfering with cellular differentiation and contributing to the development of thrombotic lesions within the placenta. http://dx.doi.org/10.1016/j.placenta.2013.06.201
P2.35. DECREASED VDR EXPRESSION CONTRIBUTES TO REDUCED FETOPLACENTAL GROWTH IN IDIOPATHIC HUMAN FETAL GROWTH RESTRICTION Tejasvy Cholangi, Hannah Yong, Thy Nguyen, Anthony Borg, Shaun Brennecke, Padma Murthi Department of Perinatal Medicine Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Melbourne, Victoria, Australia Objectives: Fetal growth restriction (FGR) affects up to 5% of all pregnancies worldwide and is associated with a number of significant perinatal complications including stillbirth and prematurity. The micronutrient vitamin D is critical for optimal fetal growth [1]. Vitamin D influences cellular growth through its receptor, VDR. Despite its established clinical importance in pregnancy, little is known of the molecular pathways by which vitamin D regulates feto-placental growth in FGR. Therefore, we hypothesised that altered VDR expression in FGR contributes to reduced placental growth. Methods: Placentae were collected from idiopathic FGR (n¼25) and gestation-matched control pregnancies (n¼25) using strict clinical criteria [2]. VDR mRNA was determined using real-time PCR [2]. Functional consequences of altered VDR expression on trophoblast growth were measured using an xCELLigence system in two independent trophoblastderived cell lines, BeWo and HTR8-SV-neo. Results: Real time PCR demonstrated a significant decrease in VDR mRNA in FGR placentae compared with controls (0.18 0.04, FGR (n¼25) vs 1.79 0.35, Control (n¼25), p<0.05). Vitamin D (vit D, 10nM) significantly increased VDR mRNA in BeWo (19.3813.71, vit D (n¼3) vs 1.040.52, vehicle control (n¼3), p¼0.01). Gene inactivation using siRNA for VDR in BeWo showed a significant increase in differentiation and apoptosis (p<0.01). VDR gene inactivation significantly increased the expression of a marker of apoptosis, c-JUN mRNA in HTR8-SV-neo compared to control (95.4342.68 siRNA (n¼3) vs 1.040.52, controls, (n¼3), p ¼0.05). Conclusion: This study is the first to show that decreased VDR expression in FGR may contribute to reduced placental growth that is characteristic of idiopathic FGR. Treatment of cultured trophoblast cells with vitamin D increased VDR expression, suggesting that vitamin D may have a beneficial effect on feto-placental growth in FGR. 1. Robinson, C.J., et al., AJOG, 2011. 204(6): p. 556.e1-556.e4. 2. Murthi, P., et al., AJP, 2006. 168(2): p. 511-518. http://dx.doi.org/10.1016/j.placenta.2013.06.202
P2.36. PHLDA2 – AN IMPRINTED GROWTH RESTRICTING GENE THAT DRIVES METABOLIC PROGRAMMING Simon Tunster 1, Mathew Van de Pette 1, Ben Tycko 2, Rosalind John 1 1
Cardiff University, Cardiff, Wales, UK; 2 Columbia University, New York, USA
placental development and pregnancy outcome has not yet been fully explored. It is postulated that hypomorphic expression of placental SIRT1 may compromise normal placental development and fetal growth. Methods: Human placenta biopsies were collected from 8 IUGR and 8 gestationally-age matched healthy control pregnancies. SIRT1 expression patterns were evaluated using immunohistochemistry and SIRT1 staining intensity quantified using ImageJ. Morphometric analysis of mid-line sections of placenta collected from SIRT1 knockout mice (SIRT1-/-) was performed at E15.5. Cross-sectional area and depth of the labyrinth exchange region were measured using Aperio Scanscope software (N¼4-8 placentae/genotype). Results: SIRT1 immunoreactivity was observed within the syncytiotrophoblast of all human placenta biopsies. A significant reduction in SIRT1 immunoreactivity was observed in all IUGR placentae (P<0.05, t-test). In the mouse, SIRT1-/- placentae demonstrated a significant reduction in midline cross-sectional area and depth of the labyrinth (P<0.05; t-test) compared to their wild-type littermates. These placentae also demonstrated evidence of hypovascularity. The SIRT1-mediated placental phenotype was dose-dependent, as heterozygous mice demonstrated an intermediated placental phenotype. Conclusion: Findings of abnormal SIRT1 expression patterns in cases of human IUGR along with evidence of compromised placental development and fetal growth restriction in the SIRT1 null mouse point to an important role for SIRT1 in placentation. Therapeutically targeting the SIRT1 pathway may prove to be a promising strategy aimed at improving fetal growth in pregnancies at risk of IUGR. http://dx.doi.org/10.1016/j.placenta.2013.06.200
P2.33. THE FUNCTIONAL EFFECT OF SYNDECAN 1 DEFICIENCY IN BEWO CELLS AND ITS IMPLICATIONS IN FETAL GROWTH RESTRICTION Tilini Gunatillake 1, 2, Amy Chui 1, Padma Murthi 2, Vera Ignjatovic 3, 4, Paul Monagle 3, 4, John Whitelock 5, Shaun Brennecke 2, Joanne Said 1 1
Department of Medicine, The University of Melbourne, St Albans, Victoria, Australia; 2 Departmenf of Obstetrics and Gynaecology, The University of Melbourne, Parkville, Victoria, Australia; 3 Department of Paediatrics, The University of Melbourne, Parkville, Victoria, Australia; 4 Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia; 5 Graduate School of Biomedical Engineering, The University of New South Wales, Kensington, New South Wales, Australia Objectives: Pregnancies complicated by fetal growth restriction (FGR) demonstrate increased placental fibrin deposition and thrombotic vasculopathy. Syndecans are proteoglycans expressed on the cell surface and are involved in cell growth and differentiation. We have previously demonstrated reduced expression of syndecan 1 (SDC1) in placentas affected by FGR. However, their exact role in the pathogenesis of FGR remains obscure. This study investigated the functional consequences of reduced SDC1 expression in a syncytiotrophoblast-like cell line. Methods: SDC1 downregulation was achieved in a BeWo cell line using short interference RNA (siRNA) transfection. Following transfection, BeWo cell proliferation was examined using the xCELLigence system. The differentiation potential was determined by investigating the mRNA expression of differentiation markers, syncytin, b-HCG and 3bHSD, using real time PCR. Finally, thrombin generation potential was determined using the Calibrated Automated Thrombogram system (Diagnostica Stago, France). Results: Following siRNA transfection, the mRNA and protein expression of SDC1 was significantly reduced after 72h. There were no significant differences in BeWo cell proliferation following SDC1 downregulation. However, the mRNA expression of the differentiation markers syncytin (Mock:0.850.32; NC:0.620.24 vs. S1: 0.100.04; S2:0.170.06, n¼6, p¼0.0003, ANOVA), b-HCG (Mock:2.941.11; NC:3.711.40 vs. S1:0.540.20; S2:0.8231, n¼6, p¼0.0023, ANOVA) and 3bHSD (Mock:1.280.48; NC:1.160.44 vs. S1:0.160.06; S2:0.060.02, n¼6, p¼0.0002, ANOVA) were significantly decreased following siRNA
A67
treatment. The thrombin generation potential of BeWo cells was significantly increased in SDC1 S2 downregulated cells compared to controls (Mock:1623.55811.78 vs. S1:1796.17898.08, p¼0.0506; S2:1826.67 913.33, n¼3, p¼0.0066, TTEST). Conclusion: Downregulation of SDC1 in BeWo cells results in significant reductions in cell differentiation and a significant increase in thrombin generation. Hence, it is plausible that reduced SDC1 expression may contribute to the pathogenesis of FGR by interfering with cellular differentiation and contributing to the development of thrombotic lesions within the placenta. http://dx.doi.org/10.1016/j.placenta.2013.06.201
P2.35. DECREASED VDR EXPRESSION CONTRIBUTES TO REDUCED FETOPLACENTAL GROWTH IN IDIOPATHIC HUMAN FETAL GROWTH RESTRICTION Tejasvy Cholangi, Hannah Yong, Thy Nguyen, Anthony Borg, Shaun Brennecke, Padma Murthi Department of Perinatal Medicine Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Melbourne, Victoria, Australia Objectives: Fetal growth restriction (FGR) affects up to 5% of all pregnancies worldwide and is associated with a number of significant perinatal complications including stillbirth and prematurity. The micronutrient vitamin D is critical for optimal fetal growth [1]. Vitamin D influences cellular growth through its receptor, VDR. Despite its established clinical importance in pregnancy, little is known of the molecular pathways by which vitamin D regulates feto-placental growth in FGR. Therefore, we hypothesised that altered VDR expression in FGR contributes to reduced placental growth. Methods: Placentae were collected from idiopathic FGR (n¼25) and gestation-matched control pregnancies (n¼25) using strict clinical criteria [2]. VDR mRNA was determined using real-time PCR [2]. Functional consequences of altered VDR expression on trophoblast growth were measured using an xCELLigence system in two independent trophoblastderived cell lines, BeWo and HTR8-SV-neo. Results: Real time PCR demonstrated a significant decrease in VDR mRNA in FGR placentae compared with controls (0.18 0.04, FGR (n¼25) vs 1.79 0.35, Control (n¼25), p<0.05). Vitamin D (vit D, 10nM) significantly increased VDR mRNA in BeWo (19.3813.71, vit D (n¼3) vs 1.040.52, vehicle control (n¼3), p¼0.01). Gene inactivation using siRNA for VDR in BeWo showed a significant increase in differentiation and apoptosis (p<0.01). VDR gene inactivation significantly increased the expression of a marker of apoptosis, c-JUN mRNA in HTR8-SV-neo compared to control (95.4342.68 siRNA (n¼3) vs 1.040.52, controls, (n¼3), p ¼0.05). Conclusion: This study is the first to show that decreased VDR expression in FGR may contribute to reduced placental growth that is characteristic of idiopathic FGR. Treatment of cultured trophoblast cells with vitamin D increased VDR expression, suggesting that vitamin D may have a beneficial effect on feto-placental growth in FGR. 1. Robinson, C.J., et al., AJOG, 2011. 204(6): p. 556.e1-556.e4. 2. Murthi, P., et al., AJP, 2006. 168(2): p. 511-518. http://dx.doi.org/10.1016/j.placenta.2013.06.202
P2.36. PHLDA2 – AN IMPRINTED GROWTH RESTRICTING GENE THAT DRIVES METABOLIC PROGRAMMING Simon Tunster 1, Mathew Van de Pette 1, Ben Tycko 2, Rosalind John 1 1
Cardiff University, Cardiff, Wales, UK; 2 Columbia University, New York, USA