Preeclampsia (PE), a condition during pregnancy that involves high blood pressure and proteinuria, is potentially fatal to both mother and child. PE currently has no known etiology or cure but has been tied to poor placental trophoblast cell migration. Increased levels of the toxic metal cadmium (Cd) have been associated with increased risk of developing PE, as well as miRNA-associated regulation of the transforming growth factorbeta (TGF-β) pathway. Signal reprogramming of the TGF-β pathway via epigenetic mechanisms is hypothesized to modify placental trophoblast function. In the present study we investigated the role of increased and decreased signaling of the TGF-β pathway in relation to Cd-induced reduction in cellular migration in JEG3 trophoblast cells. Furthermore, the role of a miR-26a as a molecular mediator of placental trophoblast migration was confirmed. The results demonstrate that increased expression of miR-26a and decreased signaling of the TGF-β pathway increase placental cell migration. These findings have relevance for mechanistic understanding of the underpinnings of poor placentation associated with PE.
During pregnancy, maternal uterine spiral arteries (SAs) are remodelled from minimal-flow, high-resistance vessels into larger diameter vessels with low resistance and high flow. Fetal extravillous trophoblasts (EVT) have important roles in this process. Decidual natural killer cells (dNK cells) are the major maternal immune component of the decidua and accumulate around SAs before trophoblast invasion. A role for dNK cells in vessel remodelling is beginning to be elucidated. This review examines the overlapping and dissimilar mechanisms used by EVT and dNK cells in this process and how this may mirror another example of tissue remodelling, namely cancer development. The published literature was searched using Pubmed focusing on EVT, dNK cells and SA remodelling. Additional papers discussing cancer development are also included. Similarities exist between actions carried out by dNK cells and EVT. Both interact with vascular cells lining the SA, as well as with each other, to promote transformation of the SA. EVT differentiation has previously been likened to the epithelialmesenchymal transition in cancer cells, and we discuss how dNKEVT interactions at the maternalfetal interface can also be compared with the roles of immune cells in cancer. The combined role that dNK cells and EVT play in SA remodelling suggests that these interactions could be described as a partnership. The investigation of pregnancy as a multicellular system involving both fetal and maternal components, as well as comparisons to similar examples of tissue remodelling, will further identify the key mechanisms in SA remodelling that are required for a successful pregnancy.
The immunologic paradigm of pregnancy led to the conceptualization of pregnancy as an organ transplant that requires, for its success, suppression of the maternal immune system. Growing scientific evidence suggests that in many ways the placenta functions as a tumor rather than a transplant and the immune regulation of the maternal-fetal interface is the result of the coordinated interaction between all its cellular components, including bacteria. Examining the role of microbiota in reproduction is in its infancy, but there is growing literature that supports its relevance. We discuss a potential normal function of bacteria in the establishment of immune tolerance and compelling evidence that a viral infection might be the underlying cause of perturbation of homeostasis. There is compelling evidence that many infectious diseases of human beings are caused by >1 microorganism and are defined as polymicrobial infections. We propose that pregnancy complications, such as preterm birth, are the result of polymicrobial infections. We examine the potential cellular and molecular mechanisms by which a viral infection of the placenta might disrupt the normal interaction between the cellular component of the implantation site and bacteria. As we better understand the normal homeostasis among the maternal immune system, placenta, and commensal, we will be able to elucidate pathogenic conditions and design better approaches to treat pregnancy complications associated with infection.
Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. NA.
Citation Atay S, Gercel‐Taylor C, Suttles J, Mor G, Taylor DD. Trophoblast‐derived exosomes mediate monocyte recruitment and differentiation. Am J Reprod Immunol 2011; 65: 65–77 Introduction Trophoblast cells have been demonstrated to regulate monocyte migration and differentiation, leading to pro‐inflammatory profiles. Because trophoblast cells release exosomes with immunoregulatory properties, trophoblast‐derived exosomes are proposed to ‘educate’ monocytes, creating a pro‐inflammatory environment. Method of study Exosomes were isolated from conditioned media of Swan71 cells by ultrafiltration and ultracentrifugation. Exosome‐induced migration was assessed using a two‐chamber system. Cytokine profiles were defined using cytokine arrays, and mRNA levels of affected cytokines were examined by qRT‐PCR and ELISA. Results Within 20 min, 8–10% of monocytes took up labeled exosomes isolated from Swan71 cells. Trophoblast‐derived exosomes increased monocyte migration in a dose‐dependent manner and produced significant increases in production of interleukin (IL)‐1β, IL‐6, Serpin‐E1, granulocyte colony‐stimulating factor, granulocyte/monocyte colony‐stimulating factor, and tumor necrosis factor‐α. Conclusion This study presents the initial demonstration that trophoblast‐derived exosomes are capable of recruiting and ‘educating’ monocytes to produce pro‐inflammatory cytokine/chemokine profiles in a cell‐contact‐independent manner.
BACKGROUND Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. METHODS First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. RESULTS EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% ± 7% (mean ± SEM; n = 10, range 6–11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. CONCLUSIONS Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.
Background Maternal alcohol abuse leading to fetal alcohol spectrum disorder (FASD) includes fetal growth restriction (FGR). Ethanol (EtOH) induces apoptosis of human placental trophoblast cells, possibly disrupting placentation and contributing to FGR in FASD. EtOH facilitates apoptosis in several embryonic tissues, including human trophoblasts, by raising intracellular Ca2+. We previously found that acute EtOH exposure increases trophoblast apoptosis due to signaling from both intracellular and extracellular Ca2+. Therefore, nifedipine, a Ca2+ channel blocker that is commonly administered to treat preeclampsia and preterm labor, was evaluated for cytoprotective properties in trophoblast cells exposed to alcohol. Methods Human first‐trimester chorionic villous explants and the human trophoblast cell line HTR‐8/SVneo (HTR) were pretreated with 12.5 to 50 nM of the Ca2+ channel blocker nifedipine for 1 hour before exposure to 50 mM EtOH for an additional hour. Intracellular Ca2+ concentrations were monitored in real time by epifluorescence microscopy, using fluo‐4‐AM. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), accumulation of cytoplasmic cytochrome c, and cleavage rates of caspase 3 and caspase 9. Results The increase in intracellular Ca2+ upon exposure to EtOH in both villous explants and HTR cells was completely blocked (p < 0.05) when pretreated with nifedipine, accompanied by inhibition of EtOH‐induced release of cytochrome c, caspase activities, and TUNEL. Conclusions This study indicates that nifedipine can interrupt the apoptotic pathway downstream of EtOH exposure and could provide a novel strategy for future interventions in women with fetuses at risk for FASD.
This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na /K ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na /K ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.
Several aquaporins (AQPs) are expressed in extravillous (EVT) and villous trophoblast cells. Among them, AQP3 is the most abundant AQP expressed in chorionic villi samples from first trimester, followed by AQP1 and AQP9. Although AQP3 expression persists in term placentas, it is significantly decreased in placentas from preeclamptic pregnancies. AQP3 is involved in the migration of different cell types, however its role in human placenta is still unknown. Here, we evaluated the role of AQP3 in the migration of EVT cells during early gestation. Our results showed that Swan 71 cells expressed AQP1, AQP3 and AQP9 but only the blocking of AQP3 by CuSO or the silencing of its expression by siRNA significantly attenuates EVT cell migration. Our work provides evidence that AQP3 is required for EVT cell migration and suggests that an altered expression of placental AQP3 may produce failures in placentation such as in preeclampsia.
Abstract Introduction Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. Methods This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3 ), and its precursor, 25-hydroxyvitamin D3 (25-D3 ), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0–10 nM) or 25-D3 (0–100 nM) for 48–60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel® were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT. Results EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3 , both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged. Discussion These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia.