Chez A. Viall

Effect of hydroxychloroquine on antiphospholipid antibody-induced changes in first trimester trophoblast function

Women with antiphospholipid syndrome (APS) are at risk for pregnancy complications. Antiphospholipid antibodies (aPL) alter trophoblast function by triggering an inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting migration. While patients with APS are often treated with hydroxychloroquine (HCQ), its effect on trophoblast function is poorly understood.

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.

Anti-phospholipid Antibodies Increase Non-apoptotic Trophoblast Shedding: A Contribution to the Pathogenesis of Pre-eclampsia in Affected Women?

Pre-eclampsia is associated with trophoblast shedding-deportation and endothelial cell dysfunction. Anti-phospholipid autoantibodies increase a womens risk factor of developing pre-eclampsia. In this study we examined the hypothesis that anti-phospholipid antibodies alter the number and nature of trophoblasts shed from the placenta, and that phagocytosis of these altered trophoblasts results in endothelial cell activation. To investigate this we used a placental explant model in which explants were treated with anti-phospholipid antibodies. This treatment resulted in a doubling of the amount of trophoblast shed from the explants. Furthermore, the trophoblasts shed from anti-phospholipid antibody-treated explants were more readily phagocytosed by endothelial cells and subsequently caused the activation of the endothelial cells, as indicated by increased expression of endothelial cell surface ICAM-1 determined by cell-based ELISA, and monocyte adhesion as determined by flow cytometry. Confocal microscopy analysis of trophoblasts shed from anti-phospholipid antibody-treated or control explants demonstrated that anti-phospholipid antibodies, but not control antibodies, were internalised within trophoblasts shed from the explants, and this was accompanied by a reduction in the activity of caspases 3 and 7 in the shed trophoblasts as indicated by FLICA. These results suggest that anti-phospholipid antibodies are selectively transported into trophoblasts where they affect the regulation of the cell cycle leading to excess and aberrant death (necrotic or aponecrotic) and shedding of trophoblasts. If reflected in vivo this might explain, at least in part, how anti-phospholipid antibodies contribute to the pathogenesis of pre-eclampsia.

Review: Where is the maternofetal interface?

Ask where the maternofetal interface is and placental biologists will tell you, the syncytiotrophoblast and extravillous cytotrophoblasts. While correct, this is not full extent of the maternofetal interface. Trophoblast debris that is extruded into the maternal blood in all pregnancies expands the maternofetal interface to sites remote from the uterus. Trophoblast debris ranges from multinucleated syncytial nuclear aggregates to subcellular micro- and nano-vesicles. The origins of trophoblast debris are not clear. Some propose trophoblast debris is the end of the life-cycle of the trophoblast and that it results from an apoptosis-like cell death, but this is not universally accepted. Knowing whether trophoblast debris results from an apoptosis-like cell death is important because the nature of cell death that produced trophoblast debris will influence the maternal responses to it. Trophoblast debris is challenging to isolate from maternal blood making it difficult to study. However, by culturing placental explants in Netwells™ we can readily harvest trophoblast debris from beneath the Netwells™ which is very similar to debris that has been isolated from pregnant women. We have found that trophoblast debris from normal placentae shows markers of apoptosis and is phagocytosed by macrophages or endothelial cells, producing a tolerant phenotype in the phagocyte. Whereas, when we culture normal placental explants with factors such as antiphospholipid antibodies (a strong maternal risk factor for preeclampsia), or IL-6 (which is found at increased levels in the sera of preeclamptic women), the death process in the syncytiotrophoblast changes, such that the trophoblast debris becomes more necrotic. Phagocytosis of this necrotic debris leads to activation of endothelial cells. Trophoblast debris greatly expands the maternofetal interface and the nature of that debris is likely to strongly influence the responses of the maternal vascular and immune systems to the debris.

Antiphospholipid antibodies bind syncytiotrophoblast mitochondria and alter the proteome of extruded syncytial nuclear aggregates.

INTRODUCTION Antiphospholipid antibodies (aPL) are autoantibodies that increase the risk of women developing the hypertensive disorder pre-eclampsia. aPL are internalised by the syncytiotrophoblast and increase extrusion of necrotic multinucleated syncytial nuclear aggregates (SNAs), which may trigger endothelial dysfunction in pre-eclampsia. The mechanisms by which aPL alter death processes in the syncytiotrophoblast leading to extrusion of SNAs are unknown. METHODS First trimester human placentae (n = 10) were dissected into explants and cultured either with aPL (50 μg/mL), isotype-matched control antibody (50 μg/mL), or media for 24 h. Harvested SNAs underwent iTRAQ proteomic analysis. Mitochondria in syncytiotrophoblast treated with aPL labelled with FluoroNanogold were visualised using transmission electron microscopy (TEM). RESULTS aPL altered the expression of 72 proteins in SNAs. Thirteen proteins were involved in mitochondrial function. TEM demonstrated that aPL bind to mitochondria in the syncytiotrophoblast and may cause mitochondrial swelling. DISCUSSION aPL disrupt mitochondria increasing the extrusion of SNAs with an altered proteome from the syncytiotrophoblast. These altered SNAs may trigger endothelial dysfunction and pre-eclampsia in these pregnancies.

Human extravillous trophoblasts bind but do not internalize antiphospholipid antibodies

INTRODUCTION Obstetric morbidity in women with antiphospholipid antibodies (aPLs) may reflect the adverse effects of aPLs on placental cells such as extravillous trophoblasts and the syncytiotrophoblast. Antiphospholipid antibodies may affect the syncytiotrophoblast after being internalised by members of the Low-density lipoprotein receptor (LDLR) family and the antigen of aPLs, β2 glycoprotein I. AIM This study aimed to determine whether aPL internalization was a mechanism by which aPLs adversely affect extravillous trophoblasts. METHOD of STUDY Fluorescently-labelled monoclonal aPLs IIC5 or ID2 were incubated with first trimester extravillous trophoblast outgrowths and visualized by microscopy. The subcellular expression of β2 glycoprotein I and LDLR family members was investigated by live/permeabilised immunocytochemistry. RESULTS Unlike the syncytiotrophoblast of anchoring villi, monoclonal aPLs were not internalised by extravillous trophoblasts, which expressed LDLR family members intracellularly. The aPL IIC5 bound to the surface of extravillous trophoblasts in a pattern similar to the extracellular expression of β2 glycoprotein I. CONCLUSIONS The mechanisms of action of aPLs are different in extravillous trophoblasts and the syncytiotrophoblast. The interaction of aPLs with the extravillous trophoblast surface, which may involve β2 glycoprotein I, is consistent with reports that aPLs trigger intracellular signaling cascades through cell-surface receptors.

Development of a simple, cost-effective, semi-correlative light and electron microscopy method to allow the immunoelectron localisation of non-uniformly distributed placental proteins

Immunoelectron microscopy is wrought with technical limitations that complicate its use. However, advances in correlative light and electron microscopy have recently lead to improvements in this field. We report the development of a semi-correlative approach to investigate the ultrastructural location of an antiphospholipid antibody within the syncytiotrophoblast. This method offers several advantages over existing methodologies, since it preserves antigenicity, shows good immunolabel penetrability and does not require specialized equipment. The use of a pre-embedding screen has also allowed us to target individual placental villi and overcome sampling limitations of the electron microscope. This simple, cost-effective method is likely to find widespread application in placental research.

Reply: In vitro culture conditions, antiphospholipid antibodies and trophoblast function

Sir, We thank Drs Clark and Laskin for their interest in our recently published systematic review of the effects of antiphospholipid antibodies on cultured placental cells in vitro. We agree with many of the authors’ comments and their suggestions for future investigations. However, in suggesting that trophoblast plugs prevent antiphospholipid antibodies accessing the placenta in early gestation, we believe that Drs Clark and Laskin have misinterpreted the reports of trophoblast plugs in the uterine spiral arteries. Firstly, it has been shown that potentially as few as 20% of spiral arteries are fully plugged during the first trimester of human pregnancy (Meekins et al., 1997), which if true, means maternal blood-borne antibodies would readily access early gestation placentae. Secondly, trophoblast plugs in the spiral arteries are widely agreed to be only loosely cohesive (Boyd and Hamilton, 1970; Ramsey and Donner, 1980). This loose cohesion means that while the plugs prevent the passage of maternal red blood cells into the intervillous space, maternal plasma may still pass through the plugs to access the placenta. Supporting this, hysteroscopic examination reveals that placental villi are bathed in a clear fluid prior to 12 weeks of gestation (Jaffe et al., 1997) and histologic specimens of implantation sites from as early as 50 days gestation show clear tracks of fluid leading from the spiral arteries to the intervillous space (Burton et al., 1999). The flow of maternal plasma to and from the placenta is corroborated by the finding of substantial numbers of placenta-derived extracellular vesicles (syncytial nuclear aggregates, microand nanovesicles) in the maternal peripheral blood from as early as 6 weeks of gestation (Covone et al., 1984; Knight et al., 1998; Askelund and Chamley, 2011; Salomon et al., 2014). Thus, unlike maternal red blood cells, soluble factors including antiphospholipid antibodies have access to the first trimester placenta despite the presence of trophoblast plugs in the spiral arteries. Therefore, these autoantibodies may have direct deleterious effects on the placenta from the beginning of placental development.