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EGF decreases sFlt1 secretion and endothelial dysfunction: A potential therapeutic for preeclampsia
Abstracts / Placenta 36 (2015) A1eA60
P2.63. VASCULAR ENDOTHELIAL GROWTH FACTOR-RECEPTOR-3 IN PLACENTA OF HIV ASSOCIATED NORMOTENSIVE AND PRE-ECLAMPTIC PREGNANCIES Onankoy Atshakala Onyangunga, Jagidesa Moodley, Naicker. University of KwaZulu-Natal, Durban, South Africa
Thajasvarie
The last decade has seen important progress in understanding the pathogenesis of pre-eclampsia. Indeed pre-eclampsia, a human pregnancy specific condition is a result of an imbalance between angiogenic and antiangiogenic factors1. Anti-angiogenic factors are highly elevated in preeclampsia. Pre-eclampsia is characterised by a circulating pro-lymphangiogenic state as evident by a decrease in soluble VEGF-R3, slightly decreased soluble VEGF-R2, increased VEGF-C and dramatically lower ratio of soluble VEGF-R2+R3/VEGF-C. HIV infection is a systemic infection affecting all tissues including lymphatic endothelial cells. Current knowledge of the effects of HIV associated hyper permeability is limited to disruption of the integrity of vascular structures and/or enhancement of inflammatory reactions.The co-morbidity of HIV infection and preeclampsia on this lymphangiogenic disparity is poorly understood. Objective: In the present study, we investigated the expression of the cell membrane receptor tyrosine kinase VEGF-R3 in HIV associated normotensive and pre-eclamptic placenta. Methods: We analysed VEGF-R3 in term placenta in normotensive and pre-eclamptic women from Prince Mshiyeni Memorial hospital, KwaZuluNatal. Groups were further stratified by CD4 count and early or late presentation of elevated blood pressure. Immunocytochemistry was performed using mouse anti-human antibody (dilution1:10). Sections were viewed on the Zeiss Axioscope A1 microscope. Results: VEGFR-3 was expressed within the invasive extravillous cell population and VEGFR-2 to the syncytiotrophoblast and cytotrophoblast cell populations in all villi types across all study groups. The endothelium lining all capillaries and large placental blood vessels were stained. The placenta was used as a positive control. The negative control showed no staining. Conclusion: This study demonstrates a qualitative lower expression of the receptor protein levels in pre-eclampsia compared with levels in the normotensive group. However morphometric image analysis is underway to elucidate differences between groups. These findings demonstrate a dysregulation of placental expression of VEGFR-3, a novel finding in the pathological maternal co-morbidity of HIV and preeclampsia.
P2.64. PLACENTAL LYMPHATIC VASCULAR ENDOTHELIAL RECEPTOR-1 IN HIV ASSOCIATED PRE-ECLAMPSIA Onankoy Atshakala Onyangunga, Jagidesa Moodley, Naicker. University of KwaZulu-Natal, Durban, South Africa
HYALURONIC
Thajasvarie
The lymphatic vascular system serves as a conduit for interstitial fluid extravasated from blood vessels. It plays an important role in diseases resulting from over-reactive (pre-eclampsia) and under-reactive (HIV) immune responses. Lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) is a selective marker for lymphatic endothelium and a homolog of CD44, the hyaluronan receptor. Objective: The study aims to examine lymphangiogenesis in immunostained placenta with LYVE-1 in HIV associated normotensive versus preeclamptic pregnancies. Methods: Post institutional ethics approval, term placental tissue was obtained at Prince Mshiyeni Memorial district hospital in KwaZulu-Natal. Samples were divided into normotensive (HIV+ve and HIV-ve) and preeclamptic (HIV+ve and HIV-ve) cohorts and further stratified according to CD4 count 350 or 350 as well as early (<34 weeks gestation) or late presentation (>34weeks gestation). Pre-eclampsia was defined as new onset hypertension (>140/90mmHg) with proteinuria. Immuno-histochemistry was performed using the Envision kit (DAKO) with the antihuman mouse LYVE-1 (1:160; R&D Systems) antibody. Slides were viewed
A57
on the Zeiss Axioscope A1 microscope interfaced with the Axiovision software for image analysis. Results: Positive staining of LYVE-1 was present in the exchange (terminal, intermediate) and the conducting (stem) villous fetal endothelium. LYVE-1 staining was predominantly absent in the syncytiotrophoblast layer of the placenta, however, focal areas were occasionally positive. Morphometric analysis of the intensity of LYVE-1 expression is currently being performed. LYVE-1 was not found within the villous mesenchymal core. Positive controls of lymph nodes displayed staining whilst substitution of the primary antibody with a non-immune serum of the same IgG class produced no staining. Conclusion: This study demonstrates a decline in LYVE-1 staining in the HIV+ pre-eclamptic group compared to the other groups. The presence of LYVE-1 in the trophoblasts and villous fetal endothelial cells indicate that LYVE-1 may perform special functions in the placenta beyond those described in lymphatic endothelial cells.
P2.65. CHARACTERIZING THE HEMATOPOIETIC STEM/PROGENITOR CELLS IN THE PLACENTA AND UMBILICAL CORD BLOOD FROM NORMAL AND PREECLAMPTIC SUBJECTS Zahra Masoumi 1, Mary Familari 2, Mattias Magnusson 3, Eva Mezey 4, Stefan Hansson 1. 1 Division of Obstetrics and Gynecology, Department of Clinical Sciences, Lund University Hospital, Lund University, Lund, Sweden; 2 Department of Zoology, University of Melbourne, Melbourne, Australia; 3 Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 4 Adult Stem Cell Section, CSDB, NIDCR, National Institutes of Health, Bethesda, MD, USA Objective: Preeclampsia (PE) is a pregnancy-related syndrome affecting about 3e8% of pregnancies, causing high maternal and fetal mortality. Several groups have reported a significant increase in free fetal hemoglobin (HbF) in the placenta and maternal blood in PE. The placenta is a hematopoietic organ during development from where at a certain embryonic stage the hematopoietic stem/progenitor cells (HSPCs) migrate to the fetal liver and bone marrow. Looking for the source of increased HbF levels in PE, we studied the HSPCs in the placenta and umbilical cord blood (UCB). Methods: HSPCs (CD34+ CD45low) were isolated from the placenta and UCB from both normal and PE pregnancies. The surface expressions of specific adhesion molecules (AMs) associated with cell adhesion and differentiation were evaluated by flow cytometry. Two-tailed Student’s T-test was used and p values 0.05 were considered significant. Results: We detected significantly (p 0.05) higher expression of CD11a (LFA-1), CD49d (VLA-4) and CD44 on HSPCs from normal UCB compared to placenta. These markers are important in cell commitment and myeloid and/or erythroid differentiation. Interestingly, levels of these AMs were not significantly different among HSPCs from placenta vs. UCB in PE. However, comparing the UCB and placenta HSPCs in both PE and normal patients indicated a significantly (p 0.001) higher expression of CD62L (L-selectin) among the UCB HSPCs. Moreover, comparing placenta HSPCs from PE and normal patients demonstrated a trend (p 0.05) toward higher expression of CD11a, CD44 and CD49e in PE HSPCs. Conclusion: HSPCs from normal UCB seem to be more committed towards myeloid and/or erythroid lineages when compared to normal placenta HSPCs. Higher CD62L on both PE and normal UCB HSPCs suggest their higher engraftment capacities compared to placenta HSPCs. Comparing normal and PE placenta HSPCs demonstrates a commitment towards myeloid and/or erythroid lineages in PE.
P2.66. EGF DECREASES SFLT1 SECRETION AND ENDOTHELIAL DYSFUNCTION: A POTENTIAL THERAPEUTIC FOR PREECLAMPSIA Stephen Tong 1, 2, Ping Cannon 1, 2, Fiona Roxanne Hastie 1, 2, Brownfoot 1, 2, Natalie Hannan 1, 2, Tu’uhevaha Kaitu’u-Lino 1, 2. 1 University
A58
of Melbourne, Victoria, Australia; Australia
Abstracts / Placenta 36 (2015) A1eA60 2
Mercy Hospital for Women, Vicotira,
Epidermal Growth Factor (EGF) signalling plays an important role in pregnancy. EGF levels rise during pregnancy, its receptor (EGFR) is highly expressed by the syncytiotrophoblast, and EGF deficiency is embryonically lethal in mice. We propose that EGF may be a novel therapeutic for preeclampsia, a disease characterised by elevation of placental secretion of the anti-angiogenic factor sFlt-1 and significant endothelial dysfunction. Objective: To assess whether EGF could reduce sFlt1 secretion and quench endothelial dysfunction in primary human tissues. Methods: mRNA expression of EGFR was assessed in a cohort of severe early onset pre-eclamptic placentas (n ¼ 17) and gestationally matched controls (n ¼ 19). Primary trophoblast cells were purified, treated with EGF and sFlt1 secretion assessed by ELISA. Four endothelial dysfunction assays were used to measure the capacity of EGF to quench dysfunction in primary HUVECs. Outputs included VCAM and ET1 mRNA expression, monocyte adhesion and HUVEC tube formation. Results: EGFR mRNA expression was significantly (p < 0.05) up-regulated in severe early onset pre-eclamptic placentas. Exposure of trophoblast cells lines to EGF increased cell proliferation. Excitingly, EGF induced a significant dose dependent reduction in sFlt1 protein release from primary trophoblasts, with a reduction of 35% at just 10ng/mL, and a significant reduction in mRNA transcript for the major placental sFlt1 isoform, sFlt1e1a. EGF reduced the expression of endothelial cell VCAM-1 and ET1 following exposure to TNFa or pre-eclamptic serum, and reduced monocyte adhesion to endothelial cells. In the presence of TNFa, HUVEC tube formation was significantly reduced, and this effect was reversed by treatment with EGF. Conclusion: This is the first data to demonstrate that EGF significantly reduces sFlt1 secretion from placental cells and reduces endothelial dysfunction in primary HUVECs. We propose that EGF may have potential as a novel therapeutic to treat preeclampsia.
P2.67. INSR RS2059806 POLYMORPHISM AND THE RISK OF PREECLAMPSIA Prabha Andraweera 1, Gustaaf Dekker 1, 2, Kathy Gatford 1, Shalem Lesley McCowan 3, Roahn Jayasekara 4, Vajira Leemaqz 1, Dissanayake 4, Claire Roberts 1. 1 School of Paediatrics and Reproductive Health, The Robinson Research Institute, University of Adelaide, Adelaide, Australia; 2 Division of Women's Health, Lyell McEwin Hospital, Elizabeth Vale, Australia; 3 Discipline of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand; 4 Human Genetics Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Introduction: Preeclampsia is a pregnancy specific disease that occurs in 2e8% of pregnancies and is a leading cause of maternal morbidity and mortality. Increasing evidence suggests that the effects of preeclampsia on a woman’s health are not restricted to the pregnancy but that preeclampsia could represent a risk factor for later life vascular and metabolic diseases. The INSR rs2059806 single nucleotide polymorphism (SNP) is a risk factor for essential hypertension, type 2 diabetes and metabolic syndrome. We investigated the association of this polymorphism with preeclampsia. Methods: The association of the INSR rs2059806 SNP with preeclampsia was tested in 123 Caucasian preeclamptic women and 1185 controls and replicated in an independent cohort of 175 Sinhalese preeclamptic women and 171 controls. The Caucasian women were recruited from the SCOPE study in Adelaide and Auckland and the Sinhalese women were recruited in Colombo, Sri Lanka. Preeclampsia was diagnosed using international guidelines. The controls consisted of women who had uncomplicated pregnancies. DNA was extracted from peripheral blood collected from women and was genotyped using the Sequenom MassARRAY system. The genotype frequencies of the preeclamptic women were compared with controls using chi squared test.
Results: In the Caucasian cohort, the prevalence of the INSR rs2059806 AA genotype was increased among preeclamptic women [OR(95% CI) ¼ 3.1(1.6e5.8), p ¼ 0.0003]. In the Sinhalese cohort, the prevalence of the INSR rs2059806 AA genotype was increased among preeclamptic women who delivered small for gestational infants [OR(95% CI) ¼ 2.8(1.0e7.4), p ¼ 0.03]. Conclusion: The INSR rs2059806 SNP previously associated with adult vascular and metabolic diseases is also associated with preeclampsia in two independent cohorts. These findings suggest that genetic susceptibility may be implicated in the link between preeclampsia and subsequent vascular and metabolic diseases.
P2.68. FETAL HEMOGLOBIN, a1-MICROGLOBULIN AND HEMOPEXIN ARE SPECIFIC PREDICTIVE FIRST AND SECOND TRIMESTER BIOMARKERS OF PREECLAMPSIA Ulrik Dolberg Anderson 1, Magnus Gram 3, Basky Thilaganathan 2, Jonas € m 3, Stefan R. Hansson 1. 1 Section for Obstetrics Ranstam 4, Bo Åkerstro and Gynecology, Dept. of clinical sciences, Lund University, Lund, Sweden; 2 Fetal medicine unit, St Georges University Hospital, London, UK; 3 Section for Infection medicine, Dept. of clinical sciences, Lund University, Lund, Sweden; 4 Department of Clinical Sciences, RC Syd, Lund University, Sweden, Lund, Sweden Objective: To study cell-free fetal hemoglobin (HbF) and the endogenous Hb/heme scavenging systems at the end of the first trimester and to evaluate them as predictive biomarkers for preeclampsia (PE) in combination with uterine artery Doppler ultrasound (UtAD). Study design: A case-control study using sera from 519 women in early pregnancy (mean 13.7 weeks’ gestation) of which 346 were uncomplicated, 86 complicated by PE, 10 by pregnancy-induced hypertension (PIH), 7 by intrauterine growth restriction (IUGR) and 65 by spontaneous preterm delivery. The serum concentration of cell-free HbF, total cell-free Hb, the heme-scavenger hemopexin (Hpx), the Hb-scavenger haptoglobin (Hp) and the heme- and radical-scavenger a1-microglobulin (A1M) were measured. All patients were examined with UtAD e Pulsatility index (PI) and notching was recorded. Logistic regression models were developed which included the biomarkers, ultrasound indices and maternal risk factors. Results: Significantly higher serum concentrations of cell-free HbF and A1M were observed in patients who later developed PE. Furthermore, a significantly lower serum concentration of Hpx was seen in those who later developed PE. UtAD showed significantly higher PI values in the PE group. Conclusions: Increased amounts of cell-free HbF, early in the pregnancy, may strain the physiological heme- and Hb scavenging mechanisms in women who subsequently develope PE. HbF in combination with Hpx and /or A1M are potentially predictive serum biomarkers for PE e either alone or in combination with maternal risk factors and/or UtAD. Keywords: a1-microglobulin/ A1M, hemopexin, Doppler ultrasound, fetal hemoglobin, prediction and preeclampsia.
P2.69. ATYPICAL E2F PREECLAMPSIA
CONTRIBUTES
TO
PATHOPHYSIOLOGY
OF
Yoshinori Moriyama 1, Tomomi Kotani 1, Masako Sawada 1, Rika Miki 2, Fumitaka Kikkawa 1. 1 Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 2 Bell Research Center, Nagoya, Aichi, Japan Objectives: Atypical E2F7 and E2F8 (E2F7/8) have been reported to be essential for murine embryonic development, and also important for placental development using the placental lineage-specific knockout mouse of E2F7/8. E2F7/8 is known to be a regulator of the mammalian endocycle. Polyploid extravillous trophoblast (EVT) is found in human placenta, but its function remains unclear. Thus, we investigated whether E2F7/8 is involved in the pathological mechanisms of preeclampsia,
P2.63. VASCULAR ENDOTHELIAL GROWTH FACTOR-RECEPTOR-3 IN PLACENTA OF HIV ASSOCIATED NORMOTENSIVE AND PRE-ECLAMPTIC PREGNANCIES Onankoy Atshakala Onyangunga, Jagidesa Moodley, Naicker. University of KwaZulu-Natal, Durban, South Africa
Thajasvarie
The last decade has seen important progress in understanding the pathogenesis of pre-eclampsia. Indeed pre-eclampsia, a human pregnancy specific condition is a result of an imbalance between angiogenic and antiangiogenic factors1. Anti-angiogenic factors are highly elevated in preeclampsia. Pre-eclampsia is characterised by a circulating pro-lymphangiogenic state as evident by a decrease in soluble VEGF-R3, slightly decreased soluble VEGF-R2, increased VEGF-C and dramatically lower ratio of soluble VEGF-R2+R3/VEGF-C. HIV infection is a systemic infection affecting all tissues including lymphatic endothelial cells. Current knowledge of the effects of HIV associated hyper permeability is limited to disruption of the integrity of vascular structures and/or enhancement of inflammatory reactions.The co-morbidity of HIV infection and preeclampsia on this lymphangiogenic disparity is poorly understood. Objective: In the present study, we investigated the expression of the cell membrane receptor tyrosine kinase VEGF-R3 in HIV associated normotensive and pre-eclamptic placenta. Methods: We analysed VEGF-R3 in term placenta in normotensive and pre-eclamptic women from Prince Mshiyeni Memorial hospital, KwaZuluNatal. Groups were further stratified by CD4 count and early or late presentation of elevated blood pressure. Immunocytochemistry was performed using mouse anti-human antibody (dilution1:10). Sections were viewed on the Zeiss Axioscope A1 microscope. Results: VEGFR-3 was expressed within the invasive extravillous cell population and VEGFR-2 to the syncytiotrophoblast and cytotrophoblast cell populations in all villi types across all study groups. The endothelium lining all capillaries and large placental blood vessels were stained. The placenta was used as a positive control. The negative control showed no staining. Conclusion: This study demonstrates a qualitative lower expression of the receptor protein levels in pre-eclampsia compared with levels in the normotensive group. However morphometric image analysis is underway to elucidate differences between groups. These findings demonstrate a dysregulation of placental expression of VEGFR-3, a novel finding in the pathological maternal co-morbidity of HIV and preeclampsia.
P2.64. PLACENTAL LYMPHATIC VASCULAR ENDOTHELIAL RECEPTOR-1 IN HIV ASSOCIATED PRE-ECLAMPSIA Onankoy Atshakala Onyangunga, Jagidesa Moodley, Naicker. University of KwaZulu-Natal, Durban, South Africa
HYALURONIC
Thajasvarie
The lymphatic vascular system serves as a conduit for interstitial fluid extravasated from blood vessels. It plays an important role in diseases resulting from over-reactive (pre-eclampsia) and under-reactive (HIV) immune responses. Lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) is a selective marker for lymphatic endothelium and a homolog of CD44, the hyaluronan receptor. Objective: The study aims to examine lymphangiogenesis in immunostained placenta with LYVE-1 in HIV associated normotensive versus preeclamptic pregnancies. Methods: Post institutional ethics approval, term placental tissue was obtained at Prince Mshiyeni Memorial district hospital in KwaZulu-Natal. Samples were divided into normotensive (HIV+ve and HIV-ve) and preeclamptic (HIV+ve and HIV-ve) cohorts and further stratified according to CD4 count 350 or 350 as well as early (<34 weeks gestation) or late presentation (>34weeks gestation). Pre-eclampsia was defined as new onset hypertension (>140/90mmHg) with proteinuria. Immuno-histochemistry was performed using the Envision kit (DAKO) with the antihuman mouse LYVE-1 (1:160; R&D Systems) antibody. Slides were viewed
A57
on the Zeiss Axioscope A1 microscope interfaced with the Axiovision software for image analysis. Results: Positive staining of LYVE-1 was present in the exchange (terminal, intermediate) and the conducting (stem) villous fetal endothelium. LYVE-1 staining was predominantly absent in the syncytiotrophoblast layer of the placenta, however, focal areas were occasionally positive. Morphometric analysis of the intensity of LYVE-1 expression is currently being performed. LYVE-1 was not found within the villous mesenchymal core. Positive controls of lymph nodes displayed staining whilst substitution of the primary antibody with a non-immune serum of the same IgG class produced no staining. Conclusion: This study demonstrates a decline in LYVE-1 staining in the HIV+ pre-eclamptic group compared to the other groups. The presence of LYVE-1 in the trophoblasts and villous fetal endothelial cells indicate that LYVE-1 may perform special functions in the placenta beyond those described in lymphatic endothelial cells.
P2.65. CHARACTERIZING THE HEMATOPOIETIC STEM/PROGENITOR CELLS IN THE PLACENTA AND UMBILICAL CORD BLOOD FROM NORMAL AND PREECLAMPTIC SUBJECTS Zahra Masoumi 1, Mary Familari 2, Mattias Magnusson 3, Eva Mezey 4, Stefan Hansson 1. 1 Division of Obstetrics and Gynecology, Department of Clinical Sciences, Lund University Hospital, Lund University, Lund, Sweden; 2 Department of Zoology, University of Melbourne, Melbourne, Australia; 3 Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 4 Adult Stem Cell Section, CSDB, NIDCR, National Institutes of Health, Bethesda, MD, USA Objective: Preeclampsia (PE) is a pregnancy-related syndrome affecting about 3e8% of pregnancies, causing high maternal and fetal mortality. Several groups have reported a significant increase in free fetal hemoglobin (HbF) in the placenta and maternal blood in PE. The placenta is a hematopoietic organ during development from where at a certain embryonic stage the hematopoietic stem/progenitor cells (HSPCs) migrate to the fetal liver and bone marrow. Looking for the source of increased HbF levels in PE, we studied the HSPCs in the placenta and umbilical cord blood (UCB). Methods: HSPCs (CD34+ CD45low) were isolated from the placenta and UCB from both normal and PE pregnancies. The surface expressions of specific adhesion molecules (AMs) associated with cell adhesion and differentiation were evaluated by flow cytometry. Two-tailed Student’s T-test was used and p values 0.05 were considered significant. Results: We detected significantly (p 0.05) higher expression of CD11a (LFA-1), CD49d (VLA-4) and CD44 on HSPCs from normal UCB compared to placenta. These markers are important in cell commitment and myeloid and/or erythroid differentiation. Interestingly, levels of these AMs were not significantly different among HSPCs from placenta vs. UCB in PE. However, comparing the UCB and placenta HSPCs in both PE and normal patients indicated a significantly (p 0.001) higher expression of CD62L (L-selectin) among the UCB HSPCs. Moreover, comparing placenta HSPCs from PE and normal patients demonstrated a trend (p 0.05) toward higher expression of CD11a, CD44 and CD49e in PE HSPCs. Conclusion: HSPCs from normal UCB seem to be more committed towards myeloid and/or erythroid lineages when compared to normal placenta HSPCs. Higher CD62L on both PE and normal UCB HSPCs suggest their higher engraftment capacities compared to placenta HSPCs. Comparing normal and PE placenta HSPCs demonstrates a commitment towards myeloid and/or erythroid lineages in PE.
P2.66. EGF DECREASES SFLT1 SECRETION AND ENDOTHELIAL DYSFUNCTION: A POTENTIAL THERAPEUTIC FOR PREECLAMPSIA Stephen Tong 1, 2, Ping Cannon 1, 2, Fiona Roxanne Hastie 1, 2, Brownfoot 1, 2, Natalie Hannan 1, 2, Tu’uhevaha Kaitu’u-Lino 1, 2. 1 University
A58
of Melbourne, Victoria, Australia; Australia
Abstracts / Placenta 36 (2015) A1eA60 2
Mercy Hospital for Women, Vicotira,
Epidermal Growth Factor (EGF) signalling plays an important role in pregnancy. EGF levels rise during pregnancy, its receptor (EGFR) is highly expressed by the syncytiotrophoblast, and EGF deficiency is embryonically lethal in mice. We propose that EGF may be a novel therapeutic for preeclampsia, a disease characterised by elevation of placental secretion of the anti-angiogenic factor sFlt-1 and significant endothelial dysfunction. Objective: To assess whether EGF could reduce sFlt1 secretion and quench endothelial dysfunction in primary human tissues. Methods: mRNA expression of EGFR was assessed in a cohort of severe early onset pre-eclamptic placentas (n ¼ 17) and gestationally matched controls (n ¼ 19). Primary trophoblast cells were purified, treated with EGF and sFlt1 secretion assessed by ELISA. Four endothelial dysfunction assays were used to measure the capacity of EGF to quench dysfunction in primary HUVECs. Outputs included VCAM and ET1 mRNA expression, monocyte adhesion and HUVEC tube formation. Results: EGFR mRNA expression was significantly (p < 0.05) up-regulated in severe early onset pre-eclamptic placentas. Exposure of trophoblast cells lines to EGF increased cell proliferation. Excitingly, EGF induced a significant dose dependent reduction in sFlt1 protein release from primary trophoblasts, with a reduction of 35% at just 10ng/mL, and a significant reduction in mRNA transcript for the major placental sFlt1 isoform, sFlt1e1a. EGF reduced the expression of endothelial cell VCAM-1 and ET1 following exposure to TNFa or pre-eclamptic serum, and reduced monocyte adhesion to endothelial cells. In the presence of TNFa, HUVEC tube formation was significantly reduced, and this effect was reversed by treatment with EGF. Conclusion: This is the first data to demonstrate that EGF significantly reduces sFlt1 secretion from placental cells and reduces endothelial dysfunction in primary HUVECs. We propose that EGF may have potential as a novel therapeutic to treat preeclampsia.
P2.67. INSR RS2059806 POLYMORPHISM AND THE RISK OF PREECLAMPSIA Prabha Andraweera 1, Gustaaf Dekker 1, 2, Kathy Gatford 1, Shalem Lesley McCowan 3, Roahn Jayasekara 4, Vajira Leemaqz 1, Dissanayake 4, Claire Roberts 1. 1 School of Paediatrics and Reproductive Health, The Robinson Research Institute, University of Adelaide, Adelaide, Australia; 2 Division of Women's Health, Lyell McEwin Hospital, Elizabeth Vale, Australia; 3 Discipline of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand; 4 Human Genetics Unit, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka Introduction: Preeclampsia is a pregnancy specific disease that occurs in 2e8% of pregnancies and is a leading cause of maternal morbidity and mortality. Increasing evidence suggests that the effects of preeclampsia on a woman’s health are not restricted to the pregnancy but that preeclampsia could represent a risk factor for later life vascular and metabolic diseases. The INSR rs2059806 single nucleotide polymorphism (SNP) is a risk factor for essential hypertension, type 2 diabetes and metabolic syndrome. We investigated the association of this polymorphism with preeclampsia. Methods: The association of the INSR rs2059806 SNP with preeclampsia was tested in 123 Caucasian preeclamptic women and 1185 controls and replicated in an independent cohort of 175 Sinhalese preeclamptic women and 171 controls. The Caucasian women were recruited from the SCOPE study in Adelaide and Auckland and the Sinhalese women were recruited in Colombo, Sri Lanka. Preeclampsia was diagnosed using international guidelines. The controls consisted of women who had uncomplicated pregnancies. DNA was extracted from peripheral blood collected from women and was genotyped using the Sequenom MassARRAY system. The genotype frequencies of the preeclamptic women were compared with controls using chi squared test.
Results: In the Caucasian cohort, the prevalence of the INSR rs2059806 AA genotype was increased among preeclamptic women [OR(95% CI) ¼ 3.1(1.6e5.8), p ¼ 0.0003]. In the Sinhalese cohort, the prevalence of the INSR rs2059806 AA genotype was increased among preeclamptic women who delivered small for gestational infants [OR(95% CI) ¼ 2.8(1.0e7.4), p ¼ 0.03]. Conclusion: The INSR rs2059806 SNP previously associated with adult vascular and metabolic diseases is also associated with preeclampsia in two independent cohorts. These findings suggest that genetic susceptibility may be implicated in the link between preeclampsia and subsequent vascular and metabolic diseases.
P2.68. FETAL HEMOGLOBIN, a1-MICROGLOBULIN AND HEMOPEXIN ARE SPECIFIC PREDICTIVE FIRST AND SECOND TRIMESTER BIOMARKERS OF PREECLAMPSIA Ulrik Dolberg Anderson 1, Magnus Gram 3, Basky Thilaganathan 2, Jonas € m 3, Stefan R. Hansson 1. 1 Section for Obstetrics Ranstam 4, Bo Åkerstro and Gynecology, Dept. of clinical sciences, Lund University, Lund, Sweden; 2 Fetal medicine unit, St Georges University Hospital, London, UK; 3 Section for Infection medicine, Dept. of clinical sciences, Lund University, Lund, Sweden; 4 Department of Clinical Sciences, RC Syd, Lund University, Sweden, Lund, Sweden Objective: To study cell-free fetal hemoglobin (HbF) and the endogenous Hb/heme scavenging systems at the end of the first trimester and to evaluate them as predictive biomarkers for preeclampsia (PE) in combination with uterine artery Doppler ultrasound (UtAD). Study design: A case-control study using sera from 519 women in early pregnancy (mean 13.7 weeks’ gestation) of which 346 were uncomplicated, 86 complicated by PE, 10 by pregnancy-induced hypertension (PIH), 7 by intrauterine growth restriction (IUGR) and 65 by spontaneous preterm delivery. The serum concentration of cell-free HbF, total cell-free Hb, the heme-scavenger hemopexin (Hpx), the Hb-scavenger haptoglobin (Hp) and the heme- and radical-scavenger a1-microglobulin (A1M) were measured. All patients were examined with UtAD e Pulsatility index (PI) and notching was recorded. Logistic regression models were developed which included the biomarkers, ultrasound indices and maternal risk factors. Results: Significantly higher serum concentrations of cell-free HbF and A1M were observed in patients who later developed PE. Furthermore, a significantly lower serum concentration of Hpx was seen in those who later developed PE. UtAD showed significantly higher PI values in the PE group. Conclusions: Increased amounts of cell-free HbF, early in the pregnancy, may strain the physiological heme- and Hb scavenging mechanisms in women who subsequently develope PE. HbF in combination with Hpx and /or A1M are potentially predictive serum biomarkers for PE e either alone or in combination with maternal risk factors and/or UtAD. Keywords: a1-microglobulin/ A1M, hemopexin, Doppler ultrasound, fetal hemoglobin, prediction and preeclampsia.
P2.69. ATYPICAL E2F PREECLAMPSIA
CONTRIBUTES
TO
PATHOPHYSIOLOGY
OF
Yoshinori Moriyama 1, Tomomi Kotani 1, Masako Sawada 1, Rika Miki 2, Fumitaka Kikkawa 1. 1 Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 2 Bell Research Center, Nagoya, Aichi, Japan Objectives: Atypical E2F7 and E2F8 (E2F7/8) have been reported to be essential for murine embryonic development, and also important for placental development using the placental lineage-specific knockout mouse of E2F7/8. E2F7/8 is known to be a regulator of the mammalian endocycle. Polyploid extravillous trophoblast (EVT) is found in human placenta, but its function remains unclear. Thus, we investigated whether E2F7/8 is involved in the pathological mechanisms of preeclampsia,