Syncytiotrophoblast Microparticles Research Articles

Decrease in lipid levels of syncytiotrophoblast micro-particles reduced their potential to inhibit endothelial cell proliferation

Preeclampsia is characterized by damage to the maternal endothelium that has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. Previously, we have shown that STBM, prepared by three different methods: mechanical dissection, in vitro placental explants culture and perfusion of placenta, can inhibit endothelial cell proliferation. Only mechanically prepared STBM induced apoptosis in the endothelial cells. Now, we have examined lipid levels in the three STBM preparations and their differential responses on endothelial cells. We examined the lipid levels in the three STBM preparations using thin layer chromatography. Furthermore, the effects of reduced lipid levels in the three STBM preparations using the pharmacological agent methyl-beta-cyclodextrin were examined on endothelial cell proliferation and apoptosis. Among the three STBM preparations, mechanical STBM contained highest levels of lipids. The reduction in lipid levels in mechanical STBM reduced their potential to inhibit human umbilical vein endothelial cells (HUVEC) proliferation and blocked their potential to induce apoptosis. No similar effect was observed following lipid reduction in the two other STBM preparations. As it has been suggested that mechanically derived STBM may more closely resemble placental micro-particles generated in preeclampsia, our data suggest that lipid content may play a role in the anti-endothelial defects present in this disease.

Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles

Systemic inflammatory priming in normal pregnancy and preeclampsia: the role of circulating syncytiotrophoblast microparticles

Systemic Inflammatory Priming in Normal Pregnancy and Preeclampsia: The Role of Circulating Syncytiotrophoblast Microparticles

Systemic inflammatory responsiveness was studied in normal human pregnancy and its specific inflammatory disorder, pre-eclampsia. Compared with nonpregnancy, monocytes were primed to produce more TNF-alpha throughout normal pregnancy, more IL-12p70 in the first and second trimesters, and more IL-18 in the first trimester only. Intracellular cytokine measurements (TNF-alpha and IL12p70) showed little change by comparison. IFN-gamma production was suppressed in all three trimesters. In pre-eclampsia, IL-18 secretion was increased. Secreted but not intracellular measures of TNF-alpha and IL-12p70 were also further enhanced compared with normal pregnancy. Inhibition of IFN-gamma production was lost and involved both CD56(+) NK and CD56(-) lymphocyte subsets. We determined whether circulating syncytiotrophoblast microparticles (STBM) could contribute to these inflammatory changes. Unbound STBM could be detected in normal pregnancy by the second trimester and increased significantly in the third. They were also bound in vivo to circulating monocytes. Women with pre-eclampsia had significantly more circulating free but not cell-bound STBMs. STBMs prepared by perfusion of normal placental lobules stimulated production of inflammatory cytokines (TNF-alpha, IL12p70, and IL-18 but not IFN-gamma) when cultured with PBMCs from healthy nonpregnant women. Inflammatory priming of PBMCs during pregnancy is confirmed and is established by the first trimester. It is associated with early inhibition of IFN-gamma production. The inflammatory response is enhanced in pre-eclampsia with loss of the IFN-gamma suppression. Circulating STBMs bind to monocytes and stimulate the production of inflammatory cytokines. It is concluded that they are potential contributors to altered systemic inflammatory responsiveness in pregnancy and pre-eclampsia.

Oxidative Stress Alters the Integrity of Cell-Free mRNA Fragments Associated with Placenta-Derived Syncytiotrophoblast Microparticles

Objectives: Cell-free fetal RNA from placental origin is present in the peripheral blood of pregnant women. Therefore, its qualitative analysis might provide insights into the physiological condition of the placenta. Here, we examine whether oxidative stress affects the integrity of placentally derived mRNA in vitro. Methods: Placental explants were cultured under normal or oxidative conditions, and the levels of placental and syncytiotrophoblast microparticle associated glyceraldehyde-3-phosphate dehydrogenase 3′ versus 5′ mRNA fragments were analyzed by real-time reverse-transcriptase polymerase chain reaction. Result: The relative ratio of 3′ to 5′ mRNA fragments associated with placental microparticles was significantly altered upon culture under oxidative stress. Conclusions: Our data suggest that oxidative stress reduces the levels of full-length, particle-associated glyceraldehyde-3-phosphate dehydrogenase mRNA transcripts released by the placenta. Therefore, analysis of the microparticle-coupled mRNA integrity in pregnant women might prove useful to diagnose disorders such as preeclampsia, where placental oxidative stress is involved.

Occurrence of Neutrophil Extracellular DNA Traps (NETs) in Pre‐Eclampsia

Manifest pre-eclampsia is associated with activation of peripheral neutrophils as well as elevations in maternal cell-free DNA. For this reason, we were very intrigued by recent reports indicating that activated circulatory neutrophils secrete nuclear DNA to generate extracellular DNA lattices, termed NETs (neutrophil extracellular traps). Our preliminary data indicate that placental syncytiotrophoblast microparticles, which are released in elevated amounts in pre-eclampsia, can induce NETs in isolated neutrophils. Furthermore, we found evidence for the increased presence of NETs directly in the intervillous space of pre-eclamptic placentae. Therefore, these newly discovered entities may be implicated in the underlying etiology of this disorder.

Induction of Neutrophil Extracellular DNA Lattices by Placental Microparticles and IL-8 and Their Presence in Preeclampsia

Preeclampsia, a severe pregnancy-related disorder, involves an overt activation of the maternal innate immune system, proposed to result from the elevated release of inflammatory syncytiotrophoblast microparticles (STBM) and cytokines from underlying placental anomaly involving abnormal trophoblast differentiation. Activation of circulating neutrophils has recently been shown to lead to the generation of fibrous extracellular lattices containing DNA, termed NETs (neutrophil extracellular traps). Therefore, we examined whether placentally derived factors activated peripheral neutrophils to generate NETs, and whether NETs formation was increased in preeclamptic placenta. Activation of isolated circulatory neutrophils on treatment with placentally derived factors (interleukin-8 and STBM) was assessed by measuring CD11b expression using Flow cytometry. Furthermore, NETs generation by these activated neutrophils in vitro and in vivo in the placental tissue sections were examined using electron microscopy and fluorescence microscopy. We report that placentally-derived interleukin-8 and STBM efficiently activated neutrophils and triggered NETs formation. Large numbers of NETs were present directly in the intervillous space of preeclamptic placentae. NETs, therefore, appear to be an integral part of neutrophil activation, and their increased presence in preeclampsia suggests that NETs may play a role in the underlying pathology.

Syncytiotrophoblast micro-particles do not induce apoptosis in peripheral T lymphocytes, but differ in their activity depending on the mode of preparation

Recent studies have suggested that pre-eclampsia may result from endothelial cell damage and overt immune activity triggered by the elevated release of syncytiotrophoblast-derived micro-particles (STBM). In this context, STBM have been reported to inhibit lymphocyte proliferation and induce Jurkat T cell apoptosis. In this study, STBM were prepared by three different in vitro methods (mechanical dissection, villous explant culture, and placental perfusion) and their functional properties were tested on T lymphocytes enriched from peripheral blood samples. Mechanically- and villous explant-derived STBM significantly inhibited activation, proliferation and cytokine release by T lymphocytes, while placental perfusion-derived STBM significantly induced T cell proliferation and a slight increase in IFNγ release. None of the STBM preparations caused T cell apoptosis. Therefore, STBM prepared by different methods in vitro exhibit different effects on circulating T cells, a feature that will have to be taken into account when considering their potential role in normal pregnancy and pre-eclampsia.

Microparticle-Free Placentally Derived Soluble Factors Downmodulate the Response of Activated T Cells

Soluble placental factors may have immunoregulatory properties and have been demonstrated to inhibit T-lymphocyte proliferation in vitro. On the other hand, placentally derived syncytiotrophoblast microparticles and crude placental homogenates have been demonstrated to inhibit proliferation of mixed lymphocytes in vitro. Because previous studies on placentally derived soluble factors may have been contaminated by the presence of trophoblast-derived microparticles, we prepared microparticle-free placental supernatants. Such supernatants reduced the activation response of T cells, as well as their proliferation and the production of cytokines such as interleukin-2 and interferon gamma, in a dose-dependent manner. This reduction in T-cell proliferation does not appear to be caused by indoleamine 2,3-dioxygenase (IDO) because it was not reversed by the addition of l-tryptophan or an inhibitor of IDO (1-methyl- dl-tryptophan). No evidence was found for the presence of IDO in these supernatants when we used a biochemical assay measuring tryptophan catabolism. We conclude that the placenta produces currently unknown soluble factors that reduce T-cell activation, proliferation, and cytokine production.

Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction

Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive intrauterine growth restriction (nIUGR), but in the absence of maternal disease. To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia. A prospective case-control study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET < 34 week), 2) women with late-onset pre-eclampsia (LOPET > or = 34 week), 3) women with nIUGR), 4) matched normal pregnant women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p < 0.05. STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median): 41 ng/ml, n = 15) compared with matched normal pregnancy (16 ng/ml, n = 15; Wilcoxon p = 0.005). LOPET (50 ng/ml, n = 10) and nIUGR (18 ng/ml, n = 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, n = 15, and 36 ng/ml, n = 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, n = 10. Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the pathogenesis of the maternal syndrome.

Detection of Fetal DNA and RNA in Placenta-Derived Syncytiotrophoblast Microparticles Generated in Vitro

Fetal DNA and RNA can be readily detected in maternal plasma samples (1)(2)(3)(4). Most of this material appears to be of placental origin (5), and it appears to be in a predominantly cell-free form (2), whereas circulatory mRNA is membrane-encapsulated (6). Pregnancy is associated with the release of microparticles by the syncytiotrophoblast membrane into the maternal circulation (7). These particles, frequently termed STBM, are released by turnover of the syncytiotrophoblast monolayer covering the entire villous tree (8)(9)(10)(11). This process of normal physiologic syncytiotrophoblast turnover involves the release of apoptotic material into the maternal circulation by the extrusion of syncytial knots and the associated release of STBM (8)(9)(10)(11). The amount of material that is released by apoptotic shedding of syncytial knots (and STBM) is several grams per day (9), and the circulating concentrations are increased significantly in preeclampsia (11). STBM particles have been suggested to evoke the mild maternal inflammatory response accompanying normal pregnancies (12), and increased release has been proposed to play a role in the etiology of preeclampsia by triggering maternal endothelial cell damage (13)(14). As these particles are difficult to detect and prepare from maternal blood samples, use is frequently made of in vitro-prepared particles to study their physiologic activity (13). In this context, we have recently extensively examined three different modes of STBM preparation: mechanical dissection of fresh placental villous tissues; in vitro cultures of villous explants; and perfusion of single placental cotyledons (15). All three preparations lead to the production of STBM as confirmed by the presence of the syncytiotrophoblast-specific protein placental alkaline phosphatase, physiologic activity on human endothelial cell cultures, and their morphology, as seen by scanning electron microscopy (15). …

A comparative study of the effect of three different syncytiotrophoblast micro-particles preparations on endothelial cells

Pre-eclampsia is a pregnancy-associated multi-system disorder of unknown etiology, characterized by damage to the maternal endothelium. The latter facet has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. In this study, we have examined STBM prepared by three different methods: mechanical dissection, in vitro placental explant culture and perfusion of placental cotyledons. All three preparations yielded morphologically similar STBM, as confirmed by scanning electron microscopy, and all contained syncytiotrophoblast-specific proteins as determined by the presence of placental alkaline phosphatase. The functional properties of the three STBM preparations were examined on human umbilical vein endothelial cells (HUVEC), where the mechanically prepared particles were found to inhibit proliferation to the greatest extent. Furthermore, only mechanically prepared STBM lead to the detachment and apoptosis of HUVEC cells. Our study, therefore, suggests that STBM prepared from placental perfusion or in vitro explant culture are biologically different from mechanically prepared ones, and may provide a better approximation of physiologically produced placental micro-particles.