Necrotic Trophoblast Research Articles

Antiphospholipid Antibodies Alter Cell-Death-Regulating Lipid Metabolites in First and Third Trimester Human Placentae.

Antiphospholipid antibodies (aPL) are maternal autoantibodies that increase the risk of a woman developing preeclampsia 10-fold. aPL are internalized into the syncytiotrophoblast and increase extrusion of necrotic trophoblast debris into the maternal blood. This necrotic trophoblast debris may trigger endothelial cell dysfunction contributing to the pathogenesis of preeclampsia. We hypothesize that aPL directly affect placental metabolism, leading to increased syncytiotrophoblast death. First and third trimester human placental explants were cultured with aPL, a control antibody, or media only, and placental conditioned culture media was examined by mass spectroscopy. Molecular targets of interest were investigated using qRTPCR and immunohistochemistry. The levels of 79 and 132 metabolites, respectively, were altered due to the treatment of first and third trimester placental explants with aPL. These included ceramides and diacylglycerols, which play important roles in cell death regulatory pathways. Antiphospholipid antibodies also decreased the expression of protein kinase C-epsilon (PRKCE) in placental explants, possibly due to the disrupted balance between ceramides and diacylglycerols caused by aPL. One mechanism by which aPL cause aberrant cell death in the syncytiotrophoblast in the first and third trimester is by disruption of placental lipid signaling and decreased expression of PRKCE.

Birthweight –BMI or glucose. a human in vivo study

s / Placenta 35 (2014) A1eA112 A68 Background: The presence of antiphospholipid antibodies (aPL) in the maternal circulation is a strong risk factor for preeclampsia, however the underlying mechanism is not known. Antiphospholipid antibodies are internalised into the human syncytiotrophoblast via an antigen-dependent, receptor-mediated process. Once inside the syncytiotrophoblast, aPL cause aberrant cell death leading to the extrusion of increased amounts of necrotic trophoblast debris into the maternal blood, which is believed to cause endothelial cell dysfunction and contribute to the pathogenesis of preeclampsia. Objective: To identify candidate metabolites that could link aPL to trophoblast cell death, using an unbiased metabolomics approach. Methods: First and third trimester human placental explants were cultured with aPL, a control antibody, or media only, and the placental metabolic footprints (conditioned culture media) of the explants were examined by mass spectroscopy. Molecular targets of metabolites of interest were investigated using qRTPCR and immunohistochemistry. Results: The levels of 79 (first trimester explants) and 132 metabolites (third trimester explants) were altered in response to treatment with aPL. These metabolites included ceramides and diacylglycerols, which play important roles in cell death regulatory pathways. Antiphospholipid antibodies also decreased the expression of protein kinase C-epsilon (PRKCE) in placental explants, a response that may be a result of the disrupted balance between ceramides and diacylglycerols caused by aPL. Conclusion: These data suggest that one mechanism by which aPL cause aberrant cell death in the syncytiotrophoblast is by disruption of placental lipid signalling, which may influence the placental expression of PRKCE, a key molecular regulator of cell life and death. Alterations in syncytiotrophoblast death mechanisms could lead to the extrusion of necrotic trophoblast debris, triggering endothelial dysfunction, a hallmark feature of preeclampsia. P2.21-N. BIRTHWEIGHT eBMI OR GLUCOSE. A HUMAN IN VIVO STUDY Ane Moe Holme , Trond M. Michelsen , Maia Blomhoff Holm , Gun Lisbet Opheim , Guttorm Haugen , Tore Henriksen a,b Oslo University Hospital, Department of Obstetrics, Oslo, Norway; University of Oslo, Oslo, Norway Background: Maternal BMI is a robust determinant of birthweight, also after adjusting for maternal glucose. The mechanism by which BMI affects fetal growth is largely unknown. We asked whether it may alter fetal glucose metabolism, and studied associations between maternal BMI and birthweight, uteroplacental glucose supply through the umbilical vein (fetal supply) and fetal glucose consumption (mmol/min). Methods: A cross-sectional study of 98 fasting women with uncomplicated pregnancies undergoing planned caesarean section at Oslo University Hospital. We obtained blood samples from the radial artery, the umbilical artery (UA) and vein (UV) during delivery. We measured internal vessel diameter (D) and time-averaged maximum velocity (TAMX) in the intra-abdominal UV by ultrasound at the day of delivery (n1⁄468). Blood flow (Q) 1⁄4 0.5$(D/ 2)2$p$TAMX. Fetal glucose supply and consumption were calculated by multiplying Q by the glucose concentration in the UV and the UV-UA glucose difference, respectively. Results:Mean (SD) UV flowwas 198.3 (81.1) ml/min and mean UV glucose concentrationwas 3.93 (0.47) mmol/L, giving a fetal glucose supply of 0.79 (0.36) mmol/min. Glucose concentration in the UAwas 3.37 (0.47) mmol/L. Paired t-test showed a significant fetal glucose consumption of 0.11 (0.11) mmol/min, p<0.001. Birthweight was correlated with maternal BMI (r1⁄40.37, p<0.001), gestational weight gain (GWG) (r1⁄40.30, p1⁄40.004) and fat% at term (r1⁄40.28, p1⁄40.006), but not with maternal arterial glucose concentration. Birthweight was also associated with fetal glucose supply (r1⁄40.49, p<0.001) and consumption (r1⁄40.26, p1⁄40.030). Maternal BMI, GWG and fat% were neither associated with fetal glucose supply nor consumption. However, maternal arterial glucose concentration and fetal glucose supply were associated (r1⁄40.27, p<0.034). Conclusion: Maternal BMI was correlated with birthweight, but not with fetal glucose supply or consumption. The independent effect of maternal BMI on fetal growth appears to be mediated through other factors (e.g. amino acids and lipids) than glucose supply. P2.22. A NOVEL ENERGY SOURCE FOR THE FETO-PLACENTAL UNIT e CREATINE Hayley Dickinson , Stacey Ellery , Paul Della Gatta , Lobna Ghattas , Syed Baharom, Miranda Davies-Tuck , Euan Wallace , Joanne Mockler , Rod Snow, David Walker a,b a The Ritchie Centre, MIMR-PHI Institute of Medical Research, Victoria, Australia; Department of Obstetrics & Gynaecology, Monash University, Victoria, Australia,; Centre for Physical Activity and Nutrition, Deakin University,

Antiphospholipid antibodies alter cell-death-regulating lipid metabolites in first and third trimester human placentae

Problem Antiphospholipid antibodies (aPL) are maternal autoantibodies that increase the risk of a woman developing preeclampsia 10-fold. aPL are internalized into the syncytiotrophoblast and increase extrusion of necrotic trophoblast debris into the maternal blood. This necrotic trophoblast debris may trigger endothelial cell dysfunction contributing to the pathogenesis of preeclampsia. We hypothesize that aPL directly affect placental metabolism, leading to increased syncytiotrophoblast death. Methods of study First and third trimester human placental explants were cultured with aPL, a control antibody, or media only, and placental conditioned culture media was examined by mass spectroscopy. Molecular targets of interest were investigated using qRTPCR and immunohistochemistry. Results The levels of 79 and 132 metabolites, respectively, were altered due to the treatment of first and third trimester placental explants with aPL. These included ceramides and diacylglycerols, which play important roles in cell death regulatory pathways. Antiphospholipid antibodies also decreased the expression of protein kinase C-epsilon (PRKCE) in placental explants, possibly due to the disrupted balance between ceramides and diacylglycerols caused by aPL. Conclusion One mechanism by which aPL cause aberrant cell death in the syncytiotrophoblast in the first and third trimester is by disruption of placental lipid signaling and decreased expression of PRKCE.

Antiphospholipid antibodies, necrotic trophoblasts and preeclampsia? (1084.3)

Preeclampsia is characterized by de novo hypertension, proteinuria and systemic inflammatory response during pregnancy which is preceded by endothelial cell activation. It is triggered by a placental factor(s), one of which may be necrotic trophoblast debris that is shed from the placenta into the maternal blood. Clearance of necrotic trophoblast debris results in endothelial cell activation in vitro and results in a relative increase in blood pressure in vivo. There are also maternal risk factors for preeclampsia, the strongest of which are antiphospholipid antibodies (aPL). We present a two part model observing the interaction between aPL and necrotic trophoblast debris on blood pressure, markers of endothelial cell activation and urinary protein.Pregnant female Wistar rats instrumented with radio‐telemetry blood pressure devices were received necrotic trophoblasts and aPL (n=10) or control antibodies (n=5). Fifteen further rats received only aPL (n=10), or control antibodies (n=5), in the absence of necrotic trophoblasts.Mean arterial blood pressure was not different between rats that received control antibodies or aPL with necrotic trophoblasts. However, heart rate was lower and urinary protein levels were elevated in rats that received necrotic trophoblasts and aPL. Serum levels of interleukin‐6, the proposed spreading factor for endothelial cell activation, were also elevated in rats receiving necrotic trophoblasts.In conclusion, antiphospholipid antibodies, the strongest maternal risk factor in preeclampsia, may interact with necrotic trophoblasts to heighten the systemic inflammatory response which precedes the clinical onset of hypertension in preeclampsia.

Antiphospholipid antibodies internalised by human syncytiotrophoblast cause aberrant cell death and the release of necrotic trophoblast debris

Antiphospholipid antibodies (aPL) are the strongest maternal risk factor for pre-eclampsia, a hypertensive disease of human pregnancy. Pre-eclampsia is triggered by a toxic factor released from the placenta that activates the maternal endothelium. Antiphospholipid antibodies cause the release of necrotic trophoblast debris from the placental syncytiotrophoblast and this debris can activate endothelial cells. In this study, we investigated how aPL affects syncytiotrophoblast death and production of necrotic trophoblast debris by examining the interaction between aPL and human first trimester placental explants. Human polyclonal and murine monoclonal aPL, but not control antibodies, were rapidly internalised by the syncytiotrophoblast. Inhibitors of endocytosis or the low-density lipoprotein receptor (LDLR) family, but not toll-like receptors, decreased the internalisation of aPL and prevented the release of necrotic trophoblast debris from the syncytiotrophoblast. Once internalised, aPL increased inner mitochondrial membrane leak and Cytochrome c release while depressing oxidative flux through Complex IV of the electron transport system in syncytiotrophoblast mitochondria. These data suggest that the human syncytiotrophoblast internalises aPL by antigen-dependent endocytosis involving LDLR family members. Once internalised by the syncytiotrophoblast, aPL affects the death-regulating mitochondria, causing extrusion of necrotic trophoblast debris which can activate maternal endothelial cells thereby contributing to the pathogenesis of pre-eclampsia.

Circulating fragmented mitochondria induce maternal hypertension, placental inflammation and apoptosis in pregnant rats

Mitochondrial fragments released from necrotic cells (mitochondrial damage‐associated molecular patterns, mitDAMPs) have pro‐inflammatory and immunogenic properties. Placentas from pregnancies with preeclampsia (PE) show exaggerated rates of cell death. We tested the hypothesis that circulating fragmented mitochondria would increase maternal blood pressure and induce placental inflammation and apoptosis in pregnant rats. Mitochondria were isolated from rat liver and sonication was used to disrupt their integrity. Pregnant rats were injected with fragmented mitochondria (mit‐treated; 4 mg/tissue) or saline (control) on gestational day (gd) 15. On gd17, systolic blood pressure, as measured with the tail cuff method, was increased in mit‐treated compared to saline‐treated dams (mit‐treated vs. control: 140±3 vs. 114±6 mmHg, p&lt;0.05). Protein levels of cyclooxygenase 1 (COX1) and 2 (COX2) and pro‐apoptotic protein BAX were increased in placentas from mit‐treated rats (mit‐treated vs. control: COX1, 6.3±1.8 vs. 1.9±1.3; COX2, 2.6±0.2 vs. 1.7±0.3; BAX: 9.1±1.3 vs. 4.4±0.02, p&lt;0.05). Systemic treatment with fragmented mitochondria increases maternal blood pressure, placental inflammation and apoptosis in pregnant rats. MitDAMPs released from necrotic trophoblasts may be the source of maternal hypertension and placental dysfunction in pregnancies with PE.Support: NIH, SWHR

Necrotic trophoblast debris increases blood pressure during pregnancy

Preeclampsia is a major disease of human pregnancy characterised by hypertension and proteinuria. These signs are preceded by systemic maternal endothelial dysfunction. Hypertension in preeclampsia appears to be triggered by a placental factor, which leads to endothelial activation/dysfunction. One potential placental trigger for preeclampsia is necrotic trophoblast cell debris shed from the placenta into maternal blood. The larger trophoblast debris is trapped in the maternal pulmonary vessels and is hypothesised to be cleared by endothelial cells. Phagocytosis of necrotic but not apoptotic trophoblast debris by endothelial cells leads to their activation in vitro. We hypothesised that intravenous injection of necrotic trophoblast debris would induce hypertension in pregnant rats. Virgin female Wistar rats were surgically implanted with telemetry devices to monitor arterial blood pressure and chronic intravenous catheters to allow delivery of necrotic trophoblast debris. After recovery, the rats were mated and, from day 6 of gestation until parturition, they were given five consecutive daily injections per week of 5×106 necrotic Jeg-3 cells per kilo bodyweight. Control rats received vehicle injections. The normalised mean arterial blood pressure of rats receiving injections of necrotic trophoblast debris was higher than control rats during the third week of gestation, while mean arterial blood pressure decreased less from the pre-pregnancy baseline compared to control rats. These results suggest that necrotic trophoblast debris has a hypertensive effect which manifests in late gestation in Wistar rats and supports the theory that necrotic trophoblast debris may trigger the symptoms of preeclampsia.

Calcium channel blockers prevent endothelial cell activation in response to necrotic trophoblast debris: possible relevance to pre-eclampsia

Pre-eclampsia is characterized by endothelial activation, which is triggered by placental factor(s). One such factor may be trophoblastic debris that is shed into the maternal blood to become trapped against the maternal pulmonary endothelium. Phagocytosis of necrotic trophoblastic debris (NTD) induces endothelial cell activation with increased secretion of interleukin-6 (IL-6) and transforming growth factor β1 (TGFβ1), which may induce systemic endothelial cell activation. In addition to its effects on vascular smooth muscle, evidence suggests that nifedipine may also affect the endothelium, contributing to the therapeutic benefits of the drug. We investigated whether nifedipine could reverse the endothelial cell activation induced by NTD. Trophoblastic debris was collected from placental explants and exposed to endothelial cells with or without nifedipine, verapamil, or a nitric oxide (NO) donor for 24 h. Endothelial cell activation was measured by cell-surface intracellular adhesion molecule-1 and E-selectin, as well as monocyte adhesion. The activation of endothelial cells exposed to NTD or sera from pre-eclamptic women was significantly reduced by nifedipine or verapamil. In addition, the increases in the levels of IL-6 or TGFβ1 in conditioned media from endothelial cells following phagocytosis of NTD were significantly reduced by nifedipine. These actions of nifedipine were reversed by the NO synthetase inhibitor l-NAME and mimicked by a NO donor. Our results suggest that calcium channel blockers may have a direct effect upon endothelial cells, reducing the endothelial cell activation that is a key pathogenic feature of pre-eclampsia. This action may be mediated, in part, by NO.

PP168. The role of calcium supplementation in prevention endothelial cell activation, and possible relevance to preeclampsia

Preeclampsia remains a leading causing of maternal and perinatal mortality and morbidity. Preeclampsia is currently thought to be primarily a disease of endothelial activation and inflammation. The deportation of trophoblast debris form the placenta was first linked to the pathogenesis of preeclampsia over a hundred years ago and it is hypothesised that deportation of necrotic trophoblast debris may contribute to maternal endothelial cell activation in preeclampsia. We have previously shown that treating placental explants with IL-6 results in shedding of more necrotic trophoblast debris from placental explants and that this debris when phagocytosed by endothelial cells results in activation of the endothelial cells. Although delivery remains the only definitive cure for preeclampsia a number of studies suggest that calcium supplementation may reduce the risk of developing preeclampsia by up to 50% but the protective mechanism of calcium supplementation is unclear. The aim of this work was to determine whether calcium supplementation affects either the production of necrotic trophoblast debris from the placenta or influences endothelial cell activation. First trimester placental explants were cultured with IL-6 in the presence or absence of increasing concentrations of calcium (CaCl2) for 24h. Trophoblastic debris was collected from the explants and then exposed to monolayers of endothelial cell for 24h and endothelial cell activation measured by ICAM-1 ELISA. In other experiments, endothelial cells were treated with IL-6 or necrotic trophoblastic debris in the presence of increasing concentrations of CaCl2, ranging from 230μg/mL to 700μg/mL, for 24h. In some experiments, ebdothelial cells were treated with low concentration of CaCl2, ranging from 0μg/mL to 230μg/mL for 24h. Endothelial cell activation was measured by quantifying cell-surface ICAM-1 levels by ELISA. Our results demonstrate that calcium levels are important to endothelial cell activation and supplemental calcium may reverse the activation of the endothelium induced by proinflammatory mediators while having no effect on the production of trophoblast debris. These results may in part help to explain the benefits of calcium supplementation in the reduction of risk for developing preeclampsia.

Transcriptomic analysis of placenta affected by antiphospholipid antibodies: following the TRAIL of trophoblast death

Antiphospholipid antibodies, a maternal risk factor for preeclampsia, increase shedding of necrotic trophoblast debris from the placenta, leading to endothelial dysfunction. Using Affymetrix HGU133 Plus 2 microarrays we found changes in the transcriptome of placental explants treated with antiphospholipid antibodies, including mRNAs BCL2L1, MCL1, PDCD2L, FASLG, SEMA6A, PRKCE and TRAIL that are involved in the regulation of apoptosis. Quantitative real-time RT-PCR and immunohistochemistry confirmed a reduction in TRAIL expression in response to antiphospholipid antibodies. These results may help to understand how antiphospholipid antibodies affect trophoblast cell death and how the antibodies could contribute to the pathogenesis of preeclampsia.

Spreading endothelial cell dysfunction in response to necrotic trophoblasts. Soluble factors released from endothelial cells that have phagocytosed necrotic shed trophoblasts reduce the proliferation of additional endothelial cells

The pathogenesis of preeclampsia is not clear but the disease is characterised by systemic endothelial cell dysfunction that is considered to be triggered by a placental factor. Necrotic trophoblastic debris that is deported in the maternal blood is one possible placental trigger for preeclampsia. Syncytial knots were first associated with preeclampsia over 100 years ago. However, syncytial knots are very large and most are trapped in the pulmonary capillaries making it difficult to envisage how they could lead to widespread systemic endothelial cell dysfunction. This study was undertaken to examine whether conditioned medium from endothelial cells that have phagocytosed necrotic trophoblastic debris could adversely affect the proliferation or survival of fresh endothelial cells. Trophoblastic cellular debris, harvested from placental explants was added to endothelial cell monolayers directly or after induction of necrosis by freeze–thawing. Conditioned medium from the endothelial cell cultures was exposed to fresh endothelial cells and their proliferation measured by Alamar Blue, and CyQUANT ®NF cell proliferation assays. Endothelial cell death was examined by a fluorogenic caspase-3 activity assay and LDH release. Conditioned medium from endothelial cells that had phagocytosed necrotic but not apoptotic trophoblastic debris significantly inhibited the proliferation of fresh endothelial cells but did not induce their death. The conditioned medium also reduced cell-surface endoglin expression by fresh endothelial cells. These results confirm that phagocytosis of necrotic trophoblastic debris by endothelial cells results in the secretion of soluble factors that might explain how necrotic trophoblastic debris trapped in the pulmonary capillaries could induce systemic endothelial cell dysfunction.

The role of autocrine TGFβ1 in endothelial cell activation induced by phagocytosis of necrotic trophoblasts: a possible role in the pathogenesis of pre‐eclampsia

Pre-eclampsia is a disorder of pregnancy characterized by hypertension and endothelial cell dysfunction. The causes of pre-eclampsia are unclear but it is proposed that a factor released from the placenta triggers the maternal symptoms. One possible triggering factor is dead trophoblasts that are shed from the placenta, then deported to become trapped in the maternal pulmonary capillaries. It is hypothesized that trophoblasts die by apoptosis in normal pregnancy, but by necrosis in pre-eclampsia. Deported trophoblasts may be phagocytosed by the pulmonary endothelial cells and we have previously shown that phagocytosis of necrotic trophoblasts leads to the activation of endothelial cells, accompanied by the release of interleukin-6 from these cells. However, the mechanistic pathway linking phagocytosis of necrotic trophoblasts and endothelial cell activation is unknown. Here we show that, after phagocytosis of necrotic, but not apoptotic, trophoblasts, endothelial cells secrete TGFbeta1. Using recombinant endoglin to inhibit the function of TGFbeta1 we have shown that the TGFbeta1 does not directly activate endothelial cells but rather it induces endothelial IL-6 secretion. The IL-6 then induces endothelial cell activation. Inhibiting either TGFbeta1 or IL-6 prevented endothelial cell activation in response to phagocytosing necrotic trophoblasts, but inhibiting IL-6 did not prevent secretion of TGFbeta1, confirming the order of signalling. IL-6 also reduced endothelial cell-surface endoglin but increased the amount of soluble endoglin released from placental explants. These interactions between the IL-6 and TGFbeta1 pathways in both the endothelium and placenta may help to regulate the maternal response to deported trophoblasts in pregnancy.

106. IL-6 INCREASES THE SHEDDING OF NECROTIC TROPHOBLASTS FROM PLACENTAL EXPLANTS

Preeclampsia (PE) is characterised by elevated maternal blood pressure, preceded by endothelial cell dysfunction. Dead trophoblasts, shed from the placenta may be one of the factors that trigger PE. Women with PE frequently have elevated serum levels of inflammatory markers such as, IL-6 and TNF a but their functional significance is unclear. In this study we investigated whether these or other cytokines can alter trophoblast shedding from placental explants. Placental explants were treated with 9 different cytokines for 72 hours. Shed trophoblasts then were harvested using our published method1. The numbers of trophoblasts shed were quantified by automated cell counter. Expression of active of caspases 3&amp;7 by the shed trophoblasts was determined using a FLICA kit. The trophoblasts shed from cytokine-treated or control explants were exposed to endothelial cell monolayers and endothelial activation determined by ELISA for cell surface ICAM-1. Treatment of explants with IL-6 caused a 50% increase (p=0.001), while TNF a and TGF b 1, caused smaller significant increases in the numbers of trophoblasts shed. Trophoblasts shed from explants treated with IL-6, TGF b 1, or TGF b 3 expressed significantly less active caspases 3&amp;7 than controls or trophoblasts shed from explants treated with other cytokines. Exposing trophoblasts shed from IL-6- or TGF b 1-treated explants to endothelial cells caused a significant (P&lt;0.001) increase in endothelial activation. Normally trophoblasts shed from the placenta die by an apoptosis-like process and their phagocytosis by endothelial cells is silent but a shift to shedding of necrotic trophoblasts can lead to endothelial cell activation 2. However, it remains unclear what might trigger a shift from apoptotic to necrotic trophoblast death. This study suggests that IL-6 and possibly other cytokines can alter both the number and the nature of shed trophoblasts such that the trophoblast are more necrotic and their phagocytosis by maternal endothelial cells could contribute to the pathogenesis of preeclampsia.

Vitamin C Enhances Phagocytosis of Necrotic Trophoblasts by Endothelial Cells and Protects the Phagocytosing Endothelial Cells from Activation

Preeclampsia is a pregnancy-specific disease characterised by maternal hypertension that is preceded by endothelial cell activation and an inappropriate inflammatory response. The exact cause of preeclampsia is unclear but this disease is known to be induced by a placental factor and it is hypothesised that oxidative stress may also contribute to its pathogenesis. We have shown that dead trophoblasts shed from the placenta can be phagocytosed by endothelial cells and that phagocytosis of necrotic, but not apoptotic, trophoblasts leads to endothelial cells activation. Since phagocytosis may be accompanied by an oxidative burst which may lead to damage/activation of the phagocyte, in this study we have investigated whether the antioxidant vitamin C can protect endothelial cells that phagocytose necrotic trophoblasts from activation. We demonstrate that treatment of phagocytosing endothelial cells with vitamin C induced an increase in the phagocytosis of necrotic trophoblasts but that activation of the phagocytosing endothelial cells was prevented. Treatment of phagocytosing endothelial cells with vitamin C also prevented the increase in IL-6 secretion that normally accompanies phagocytosis of necrotic trophoblasts. Thus treatment of endothelial cells with vitamin C appears to modify both the phagocytosis of necrotic trophoblasts and the response of the endothelial cells to the necrotic trophoblastic material.

A role for interleukin‐6 in spreading endothelial cell activation after phagocytosis of necrotic trophoblastic material: implications for the pathogenesis of pre‐eclampsia

Pre-eclampsia is characterized by systemic maternal endothelial dysfunction that precedes the onset of clinical symptoms. The cause of the dysfunction is not clear but the number and the nature of trophoblasts shed from the placenta may be altered in pre-eclamptic pregnancies. These dead trophoblasts become trapped in the pulmonary capillaries and may then be phagocytosed by endothelial cells. Phagocytosis of necrotic, but not apoptotic, trophoblasts results in endothelial cell activation. We have explored the hypothesis that activation can subsequently spread to other endothelial cells via soluble factors without the need for direct contact with shed trophoblasts. Conditioned medium from endothelial cells that had phagocytosed necrotic, but not apoptotic, trophoblasts was shown to activate fresh endothelial cells due, in large part, to IL-6 secreted into the conditioned medium. The amount of IL-6 secreted in response to phagocytosis of necrotic trophoblasts was similar to the levels of IL-6 found by others in the blood of pre-eclamptic women and was substantially more than the level of IL-6 which has been reported to induce symptoms of pre-eclampsia in pregnant rats. We demonstrated that phagocytosis of both a trophoblast cell line as well as trophoblasts shed from human placentae, had this effect on two different types of endothelial cells. The role of IL-6 in endothelial cell activation was confirmed using recombinant IL-6 and neutralizing antibodies against IL-6 and the IL-6 receptor. Thus, IL-6 secreted by pulmonary endothelial cells after they have phagocytosed necrotic trophoblasts that are trapped in the pulmonary capillaries could activate endothelial cells in other remote vascular beds, contributing to the systemic activation of the endothelium that is a hallmark of pre-eclampsia.

432. How do antiphospholipid antibodies contribute to preeclampsia?

Preeclampsia is characterised by elevated maternal blood pressure which is preceded by endothelial activation. The cause of this endothelial cell dysfunction is unclear but it appears to be triggered by a placental factor. One of the risk factors for developing preeclampsia is the presence of antiphospholipid antibodies (aPL) in the maternal blood but exactly how aPL predispose women to developing preeclampsia is unclear. A second feature known to be associated with preeclampsia is excessive shedding and deportation of dead trophoblasts. We have previously shown that shed trophoblasts are phagocytosed by endothelial cells and that phagocytosis of necrotic trophoblasts leads to endothelial cell activation1. In this study we examined the hypothesis that aPL alter the number or nature of trophoblasts shed from the placenta resulting in endothelial cell activation. Using our published model of trophoblast shedding 2 human first trimester placental explants were treated with monoclonal aPL, IIC5 or ID2, or control antibody CD45 for 72 h. Shed trophoblasts then were harvested and counted using a Cellometer AutoT4 automated cell counter. The activity of caspases 3&amp;7 was analysed in all treated shed trophoblasts using a FLICA™ kit. The treated shed trophoblasts also were exposed to the endothelial cell line HMEC-1 for 24 h. The level of ICAM-1 by HMEC-1 was determined by cell-based ELISA. The number of trophoblasts shed from placental explants was increased 2 fold following aPL treatment whereas, treatment with CD45 resulted in only a 1.3 fold increase in shedding. Trophoblasts shed from aPL-treated explants contained less active caspases 3 &amp; 7 compared with control shed trophoblasts. Moreover, phagocytosis of trophoblasts shed from aPL-treated explants induced significantly increased expression of ICAM-1 compared with controls. aPL treatment affected the number and nature of trophoblasts shed from placentae in such a way that phagocytosing endothelium become activated. These findings suggest that aPL treatment may have shifted the type of cell death that shed trophoblasts are undergoing from apoptosis to a more necrotic or aponecrotic mechanism. This type of shedding of trophoblasts in vivo might contribute to the endothelial cell activation which is a hallmark feature of preeclampsia. (1) Chen Q, Stone PR, McCowan LM et al. Phagocytosis of necrotic but not apoptotic trophoblasts induces endothelial cell activation. Hypertension. 2006;47:116–121. (2) Abumaree MH, Stone PR, Chamley LW. An in vitro model of human placental trophoblast deportation/shedding. Mol Hum Reprod. 2006;12:687–694.

An in vitro model of human placental trophoblast deportation/shedding

Deportation of trophoblast shed from the placenta into the maternal circulation was first described over 100 years ago. Despite this, little is known about the quantity or nature of the shed and deported trophoblasts. Neither do we have a clear understanding of the fate of deported trophoblasts nor do we have a clear understanding of their effects on the maternal physiology. This deficiency is largely due to the inaccessibility of deported trophoblasts in vivo. This study aimed to produce a model that would allow us to study deported trophoblasts. We devised a system for culturing placental explants of 12-week gestation in cell culture inserts with a stainless steel mesh bottom that allowed the ready harvesting of shed/deported trophoblasts. Immunohistochemical and morphologic investigations demonstrated that these in vitro shed/deported trophoblasts are similar to those found in vivo and that apoptotic, necrotic and viable trophoblasts were shed from the explants. Inhibiting caspases induced a change from predominantly apoptotic to predominantly necrotic trophoblast shedding. We have devised an in vitro model that allows the collection of shed/deported trophoblasts which will significantly enhance our ability to study these cells. Our preliminary investigations confirm that apoptosis plays an important role in trophoblast shedding/deportation.

The effect of experimental Bothrops jararaca envenomation on pregnant mice

The injury caused by the intramuscular injection of a single dose of Bothrops jararaca venom (0.24 mg/kg body weight) to mice on day 8 of pregnancy and examined on day 9 was investigated. Macroscopic and histological examination showed that the bothropic venom caused an increase in the incidence of fetal resorptions. Histologically, a characteristic involution of mature decidua was noticed in saline-treated mice; however, necrotic trophoblast giant cells and decidual cells were also present in this region of mice treated with B. jararaca venom, mainly close to the embryo. Hemorrhagic areas were also observed at maternal–fetal interface, which contained maternal erythrocytes and polymorphonuclears. Plasma fibrinogen levels were lower in envenomed group ( p≤0.0001), but prothrombin time and activated partial thromboplastin time remained unaltered. Total and differential white blood cell counts were not statistically different between groups. Thus, B. jararaca venom causes injuries not only to the fetus, but also to decidual tissue and blood coagulation of pregnant mice. It is not clear, nonetheless, whether disturbances during the development of pregnancy are due to a direct effect of venom on uterus/fetus or to homeostatic changes in dams, such as clotting disturbances, or to both of them.

Phagocytosis of Necrotic but Not Apoptotic Trophoblasts Induces Endothelial Cell Activation

It is hypothesized that preeclampsia is caused by factors from the placenta that induce endothelial cell activation. Trophoblasts are cells that may be shed from the placenta, then deported in the maternal blood, and finally become trapped in the pulmonary capillaries. The ultimate fate of deported trophoblasts is unknown, but to prevent clogging of the pulmonary circulation they must be cleared from the capillary beds. We examined the hypothesis that endothelial cells phagocytose deported trophoblasts and also examined the consequent effects of the trophoblasts on endothelial cells. Fluorescently labeled trophoblast-derived choriocarcinoma cells were induced to become apoptotic or necrotic and exposed to endothelial cell monolayers. Confocal microscopy demonstrated uptake of both apoptotic and necrotic trophoblasts, and this phagocytosis could be inhibited by cytochalasin B. Phagocytosis of necrotic but not apoptotic trophoblasts induced increased endothelial intercellular adhesion molecule 1 (ICAM-1) expression, as well as increased adhesion of monocytes to endothelial cell monolayers. Inhibiting the phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase pathways blocked both expression of ICAM-1 and phagocytosis, whereas inhibition of the P42/44 mitogen-activated protein kinase pathway blocked only ICAM-1 expression. This work suggests that endothelial cells can phagocytose deported trophoblasts and that the mechanism of trophoblast death (apoptotic or necrotic) could have major effects on the maternal vascular response to shed trophoblasts.

208. Exposing necrotic trophoblasts to endothelial cells in vitro causes increased adhesion of monocytes

A number of studies suggest that there is a generalized endothelial cell activation and inflammatory response in preeclampsia, which may be caused by factors released from the placenta including deported trophoblasts. Trophoblasts are the placental cells that are bathed in maternal blood during pregnancy and as they become aged or damaged trophoblasts are shed from the placenta and deported into the maternal circulation. The fate of deported trophoblasts is unknown but we have found that endothelial cells can phagocytose dead trophoblasts. The aim of this study was to examine the effects of phagocytosing dead trophoblasts on endothelial cell–monocyte interactions. Methods: The trophoblast-derived cell lines Jar and Jeg-3 were induced to undergo necrotic death by freeze/thawing or apoptotic death by exposure to UV light. HMEC-1 endothelial cells were labeled with green fluorescent cell tracker stain and then exposed to necrotic or apoptotic trophoblasts for 3 or 24 h. U937 (monocyte) cells were labeled with red fluorescent stain and incubated with the HMEC-1 monolayers for 3 or 24 h. The adhesion of the U937 cells to the HMEC-1 monolayers was quantified by flow cytometry and compared to the adhesion of U937 cells to untreated HMEC-1 monolayers. Results: Exposing the HMEC-1 cells to necrotic, but not apoptotic, trophoblasts induced an approximately two-fold increase in the adhesion of U937 cells to the HMEC-1 monolayers (P = 0.01). The findings were consistent regardless of whether the HEMC-1 cells were exposed to the dead trophoblasts for 3 or 24 h. Conclusions: We have previously shown that endothelial cells phagocytose both apoptotic and necrotic trophoblasts. The results of the current study suggest that shedding necrotic trophoblasts from the placenta could induce endothelial cells to become activated resulting in increased leucocyte adhesion. Thus, dead trophoblasts may be one of the factors released from the placenta that induce preeclampsia.