Expression of The Efflux Transporters MRP1 and BCRP in Healthy and Preeclamptic placentas

Abstracts / Placenta 57 (2017) 225e335

Conclusion: autophagy has a role in trophoblasts’ cell death and cellular survival mechanism and involved in pathological pregnancy such as preeclampsia. Keywords: Autophagy, cell death spectrum, cellular survival, placentation defect, preeclampsia http://dx.doi.org/10.1016/j.placenta.2017.07.178

P1.92. GENETIC ASSOCIATION OF COMPLEMENT RECEPTORS PREECLAMPSIA REVEALED BY TARGETED EXOMIC SEQUENCING

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A. Inkeri Lokki 1, Michael Triebwasser 3, Emma Daly 2, Liisa Uotila 1, Susanna Fagerholm 1, Kirsi Auro 1, 4, Markus Perola 1, 4, FINNPEC Study1Mitja Kurki 2, 5, Mark Daly 2, 5, John P. Atkinson 3, Seppo Meri 1, Hannele Laivuori 1. 1 University of Helsinki, Helsinki, Finland; 2 Broad Institute of MIT, Cambridge, MA, USA; 3 Washington University School of Medicine, St.Louis, MO, USA; 4 National Institute for Health and Welfare, Helsinki, Finland; 5 Massachusetts General Hospital and Harvard Medical School, Cambridge, MA, USA Preeclampsia is a common pregnancy-specific vascular disorder typically characterized by new-onset hypertension and proteinuria during the second half of pregnancy. Etiology of the disease is unknown but predisposition to preeclampsia is heritable. The complement system is the frontline of innate immunity with capacity to discriminate between self and non-self structures and to cause inflammation and tissue destruction on one hand but to promote clean-up and initiate adaptive immune responses on the other. Inadequate regulation of the complement system may result in poor placentation and predispose to preeclampsia. We analyzed whether sequence variants of the complement system genes predispose to preeclampsia. First, we performed targeted exomic sequencing on 500 preeclamptics and 190 controls from the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort. The second cohort was composed of 122 women with and 1905 without a history of preeclampsia; these women had exome sequence data from the national FINRISK study. Fisher’s exact association test of allele frequency differences between cases and controls was done for combined data. The most significantly associated sequence variants were found in receptors or inhibitors of the complement system. Among those were three missense variants in genes for C3 receptors, two in CR4 (ITGAX) (p<2.76E4) and one in CR3 (ITGAM; p¼4.27E-4). ITGAX and ITGAM encode for the CD11c and CD11b (Mac-1) chains of the integrin CD11/CD18 receptors, respectively. They are expressed on dendritic cells and macrophages. The protective effects of missense variants in C3 receptors may be reflective of the interactions between complement C3 and phagocytic and antigen presenting cells. The variants in CR3 may influence the efficiency of complement mediated clean-up of apoptotic structures, which are abundant in the preeclamptic placenta. Also, the variations may influence the functions of platelets in the coagulation system. Overall, the results suggest that complement C3 receptor modifications influence susceptibility to preeclampsia. http://dx.doi.org/10.1016/j.placenta.2017.07.179

P1.94. PERSISTENCE OF RISK FACTORS ASSOCIATED WITH MATERNAL CARDIOVASCULAR DISEASE FOLLOWING ABERRANT INFLAMMATION IN RAT PREGNANCY Shannyn Macdonald-Goodfellow 1, Takafumi Ushida 1, 2, 1 1 Quadri , M. Yat Tse , Louise Winn 1, Stephen Pang 1, Tomomi Kotani 2, Fumitaka Kikkawa 2, Adams 1, Graham 1. 1 Queen's University, Kingston, Ontario, Canada; University, Nagoya, Japan

Allegra Michael Charles 2 Nagoya

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Introduction: Preeclampsia is associated with increased risk of subsequent cardiovascular and metabolic disease in the affected mothers. While aberrant inflammation contributes to the pathophysiology of preeclampsia, it is unclear whether maternal inflammation contributes to the increased risk of disease. Here we determined the effect of aberrant inflammation in pregnancy on cardiovascular and metabolic disease risk factors. Methods: Wistar rats were administered low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 to induce inflammation. Controls included pregnant rats treated with saline and non-pregnant rats treated with LPS or saline. We previously showed that LPS-treated pregnant rats exhibit key features of preeclampsia. Echocardiographic parameters, heart weight, blood pressure, blood lipids, pulse-wave velocity and glucose tolerance were assessed at 16 weeks post-partum. Messenger RNA levels of transcription factors associated with cardiac growth were measured in left ventricular tissue; histone modifications and global DNA methylation were determined in hearts and livers at GD 17.5 and at 16 weeks post-partum. Results: Compared with saline-treated pregnant rats and non-pregnant rats treated with LPS or saline, LPS-treated pregnant rats exhibited left ventricular hypertrophy and increased blood cholesterol and low-density lipoprotein levels at 16 weeks post-delivery. LPS-treated rats had increased left ventricular mRNA levels of hypertrophy-associated transcription factors at GD 17.5 and increased levels of modified histones in hearts and livers at GD 17.5 and 16 weeks post-partum. Other parameters remained unchanged. Conclusion: Aberrant inflammation during pregnancy results in persistent alterations in maternal physiological parameters and epigenetic modifications that could contribute to the pathophysiology of cardiovascular disease. http://dx.doi.org/10.1016/j.placenta.2017.07.180

P1.95. EXPRESSION OF THE EFFLUX TRANSPORTERS MRP1 AND BCRP IN HEALTHY AND PREECLAMPTIC PLACENTAS Rabab Al-Lahham, Marjan Afrouzian, Vsevolod Popov, Wayne Fischer, Mahmoud Ahmed, Tatiana Nanovskaya. University of Texas Medical Branch, Galveston, USA Objectives: The data from our recently completed pilot study of 10 mg pravastatin in a cohort of pregnant women at high-risk for preeclampsia suggest its preliminary efficacy for prevention of preeclampsia. However, the molecular basis of pravastatin trans-placental transfer is still not clearly understood. Due to its hydrophilic properties, the low transfer of pravastatin across human placenta is expected. However, data obtained in our laboratory revealed that 18±4% of the drug was transferred to the fetal circuit of the dually perfused placental lobule, thus suggesting the involvement of human placental transporters. Recent data from our laboratory identified MRP1 and BCRP as major placental efflux transporters involved in the bio-disposition of pravastatin. While the efflux transporters localized on the apical membrane of the syncytiotrophoblast limit the transfer of pravastatin across the placenta, efflux transporters localized on the basal membranes extrude them to the fetal circulation. Therefore, the aim of this study was to determine placental localization of these transporters and the effect of preeclampsia on their expression. Methods: Localization of transporters on apical and basal membranes of cytotrophoblasts and syncytiotrophoblasts as well as fetal endothelial cells was determined by Immunohistochemistry, and Immunogold staining of placental tissues using Electron Microscopy. Grading on IHC staining was done by a pathologist. Results: Our data revealed that the efflux transporters MRP1 and BCRP are expressed on the apical membranes of both cytotrophoblast and syncytiotrophoblast cells, with higher expression in cytotrophoblasts. MRP1 was shown to have more pronounced difference in expression between these two types of trophoblast cells in preeclamptic than in control placentas, while BCRP had the reverse pattern. Our data also showed the expression of both MRP1 and BCRP in fetal endothelial cells. Conclusions: The expression of the apical efflux transporters MRP1 and BCRP that facilitate transfer of pravastatin from the fetal to the maternal direction,

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Abstracts / Placenta 57 (2017) 225e335

i.e. limit fetal exposure to these compounds, is higher in cytotrophoblasts than syncytiotrophoblasts, and the difference in expression between these two types of trophoblast cells is higher or lower in preeclamptic compared to control healthy placentas for MRP1 and BCRP, respectively. Support was provided by the Obstetric-Fetal Pharmacology Research Center network of NICHD; U54 HD047891. http://dx.doi.org/10.1016/j.placenta.2017.07.181

P1.96. ASSOCIATION OF RETROPLACENTAL BLOOD WITH BASAL PLATE MYOFIBERS Rachel Wyand 1, Stewart Cramer 1, Assaf Oshri 2, Debra Heller 3. 1 Rochester General Hospital, University of Rochester School of Medicine, Rochester, NY, USA; 2 University of Georgia, Athens, GA, USA; 3 Rutgers-New Jersey Medical School, Newark, NJ, USA Objectives: It is generally presumed that retroplacental hemorrhage is due to abruption, and that separate blood clots submitted with the placenta are of retroplacental origin. Clinicians at Rochester General Hospital submit separate blood clots with placental specimens when their clinical diagnosis is abruption. However, some of these cases have been found to be associated with basal plate myofibers (BPMF), consistent with morbidly adherent placenta. The purpose of this study was to assess the relationship of retroplacental blood clot to BPMF. Methods: This was a retrospective study of cases received by one pathologist (SFC) over a 2 month period. Cases with significant retroplacental blood, with or without separate submitted clot, were reviewed. Cases felt to be abruptions, based on indentation of parenchyma or submission of separate clot, were compared with cases where there were BPMF consistent with morbidly adherent placenta. The two groups were compared by independent t-test analysis of mean differences. Results: 31 of 154 total cases (20%) had significant retroplacental blood. 21/154 (14%) cases had separate clots submitted (10 with BPMF, 11 without BPMF). Of 10 cases without separate clots, 8 had BPMF (of which 2 had indentation of the parenchyma by clot) and 2 had indentation of the placenta without BPMF. In total, 13 cases were felt to represent abruption, based on either separate clot received, or placental indentation on the slides without BPMF. 18 cases had BPMF. 2 abruptions with separate clots also had discrete gross retroplacental hematomas (7 and 8 cm), while 1 marginal retroplacental hematoma (4.5 cm) was associated with BPMF. There was no difference between the groups for maternal age, gestational age, or placental weight. There was a tendency for the weight of separate blood clot to be greater in the abruption group [t¼ 2.02 (df¼19) - p¼0.058 ] Conclusion: Since BPMF may confer a risk for accreta in a subsequent pregnancy, we suggest that retroplacental blood, whether separate or attached to the placenta, may be an indication to evaluate for basal plate myofibers. http://dx.doi.org/10.1016/j.placenta.2017.07.182

P1.97. INTRASYNCYTIAL PASSAGE TIME OF POST-PROLIFERATIVE VILLOUS TROPHOBLAST IS ELONGATED IN PLACENTAS OF PREGNANCIES WITH INTRAUTERINE GROWTH RESTRICTION Eva Haeussner 1, Christoph Schmitz 1, David Grynspan 2, Franz Edler von Koch 3, Hans-Georg Frank 1. 1 Ludwig-Maximilians-University, Department of Anatomy II, Munich, Germany; 2 University of Ottawa, Department of Pathology and Laboratory Medicine, Ottawa, Canada; 3 Clinic for Obstetrics and Gynecology Dritter Orden, Munich, Germany The syncytiotrophoblast ensures the materno-fetal exchange during pregnancy and is key to placental function and efficiency. The syncytiotrophoblast is continuously renewed by a process of proliferation, syncytial integration and shedding. Proliferation is assumed to be rate limiting

for the whole process. This is the background of the interest in trophoblast proliferation in conditions like IUGR which show compromised placental transport efficiency. Most analyses of proliferation of villous trophoblast are based on cell cycle marker indices determined from thin histological sections and can thus not easily be mapped topologically or allocated in 3D on the villous surface. We hypothesized that 3D topological analysis of proliferation of villous trophoblast will reveal the contribution of proliferation to syncytial maintenance in IUGR placentas and in normal placentas. The 3D topological microscopic analysis revealed that the proliferation of the villous trophoblast is not altered in IUGR pregnancies. Instead, the density (number of nuclei per unit of villous surface area) of post-proliferative trophoblast nuclei was significantly increased in IUGR. These findings are not in favor of proliferation as rate-limiting step of syncytial maintenance. In contrast, the findings can easily be interpreted as a consequence of elongated intrasyncytial passage time of villous trophoblast in IUGR. This could correspond to an increased senescence of villous trophoblast in IUGR. In the present study, kinetic regulation of syncytial structure and function occurs during the intrasyncytial phase and independent of unmodified trophoblast proliferation. http://dx.doi.org/10.1016/j.placenta.2017.07.183

P1.98. DIFFERENTIAL RESPONSES INDUCED BY PATHOGENIC AND NONPATHOGENIC INFLAMMATORY STIMULI AT THE MATERNAL-FETAL INTERFACE Cyntia Duval 1, 3, Bernadette Baker 2, 4, Rebecca Jones 2, 4, Sylvie e de Montr eal, Montreal, Quebec, Canada; 2 University Girard 1, 3. 1 Universit of Manchester, Manchester, UK; 3 Sainte-Justine Hospital Research Center, Montreal, Quebec, Canada; 4 Maternal and Fetal Health Research Center, Manchester, UK Inflammation is strongly linked to preterm birth and contributes to placental dysfunction. Infections (pathogen-associated molecular patterns, PAMPs) are a major cause of inflammation but endogenous mediators known as damage-associated molecular patterns (DAMPs) are increasingly associated to pregnancy complications and could also contribute to inflammation. PAMPs and DAMPs have to be studied together to compare their effects on the placenta. Objectives: We aimed to study the effect of a PAMP, bacterial lipopolysaccharide (LPS), compare to a known DAMP, the pro-inflammatory cytokine interleukin (IL)-1, on term human placenta. Methods: We treated explants with IL-1b (10ng/mL) or LPS (1mg/ml), the latter with/without IL-1 receptor antagonist (IL-1Ra, 100ng/mL), and we quantified cytokine mRNA and protein expression (RT-qPCR, ELISA) and used immunohistochemistry to quantify the number of CD45+ immune cells and rate of apoptosis (M30+). Results: LPS and IL-1 induced IL-6 secretion (1646 and 1630 pg/mL respectively vs 217.8 pg/mL in control, p<0.001) but only LPS induced TNFa (1009 vs 0.094 pg/mL, p<0.001). Furthermore LPS, but not IL-1, exposure led to elevated secretion of anti-inflammatory cytokines, IL-10 (140.9pg/mL vs 7.21 pg/mL in control, p<0.0001) and IL-1Ra (2706 vs 51.31 pg/mL in control, p<0.001). Although LPS induced IL-1b (74.98 vs 0.22 pg/mL in control, p<0.0001), blocking the IL-1 pathway with IL-1Ra only partially abrogated the actions of LPS, including secretion of TNFa, but preserved the anti-inflammatory effects. Both IL-1 and LPS induced increased percentage of CD45+ immune cells (8.7 and 12.3% respectively vs 3.9% in control, p<0.001) (<0.05) and these were mainly of the inflammatory (M1) phenotype. Conclusions: In summary, PAMPs/DAMPs induced distinct inflammatory profiles in the term placenta with the pathogen (LPS) stimulating both pro and anti-inflammatory responses as compared to the non-pathogenic stimuli (IL-1). Future work will focus on the mechanistic pathways that are implicated in these differences and leading to immune cells proliferation. http://dx.doi.org/10.1016/j.placenta.2017.07.184