Extravillous Trophoblast Cell Migration Research Articles

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3)

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 μg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

Cyclosporin A Promotes Invasion and Migration of Extravillous Trophoblast Cells Derived from Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells.

Extravillous trophoblast (EVT) cells play an essential role in the maternal-fetal interaction. Although abnormal development and function of EVT cells, including impaired migration and invasion capability, are believed to be etiologically linked to severe pregnancy disorders including pre-eclampsia, the associated molecular mechanisms are not clear due to the lack of an appropriate cell model in vitro. Cyclosporin A (CsA) is a macrolide immunosuppressant and also used in clinic to improve pregnancy outcomes. However, whether CsA has any effects on the function of EVT cells has not been well investigated. In this study, we induced differentiation of human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) into EVT cells (hiPSC-EVT and hESC-EVT cells, respectively) by Y27632, neuregulin-1 (NRG1), A83-01, and matrigel, and collected these derived EVT cells by flow cytometry for sorting cells positive for double human leukocyte antigen-G (HLA-G) and Cytokeratin7 (KRT7), both of which are EVT markers. We then investigated the effects of CsA on the invasion and migration of these derived EVT cells. We found that the hiPSC-EVT and hESC-EVT cells expressed high levels of the EVT markers such as KRT7, integrin alpha 5 (ITGA5), and HLA-G but low levels of OCT4, a stem cell marker, and that CsA significantly promoted the invasion and migration of hiPSC-EVT and hESC-EVT cells compared with HTR-8/SVneo cells. These results represent a possible cell model for studying the function of EVT cells and mechanism of pregnancy-related disorders associated with EVT. In addition, CsA may be used to treat pregnancy complications in clinic associated with deficient EVT function.

Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species

IntroductionPlacental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. Materials and MethodsKLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. ResultsKLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DiscussionPresent data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization.

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin−1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP−2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

MNSFβ Promotes the Proliferation and Migration of Human Extravillous Trophoblast Cells and the Villus Expression Level of MNSFβ Is Decreased in Recurrent Miscarriage Patients

Aims: The invasion of extravillous trophoblast (EVT) cells into maternal decidua is essential for the establishment and maintenance of pregnancy. Derangement of EVT cell invasion might cause pregnancy complications including recurrent miscarriage (RM). We previously reported that deficiency of monoclonal nonspecific suppressor factor beta (MNSFβ) led to the early pregnancy failure in mice and the decidual MNSFβ expression level in RM patients was significantly decreased, but the underlying molecular mechanism of the role that MNSFβ played at the maternal-fetal interface remains unclear. Thus, in the present study, we determined effects of downregulated MNSFβ expression on human EVT cell activities. Methods: The MNSFβ expression in first-trimester human decidual and placental villus tissues was detected, respectively, by immunofluorescence or immunohistochemical analyses. The MNSFβ expression level in the immortalized first-trimester human EVT cell line HTR8/SVneo was downregulated by transfecting the small interfering RNA against MNSFβ and upregulated by transfecting the recombinant pDsRed-MNSFβ plasmids. The proliferation, migration, invasion, and apoptosis activities of HTR8/SVneo cells were, respectively, determined by cytometry assay, scratch test, transwell assay, and FITC/PI staining. The expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9 in HTR8/SVneo cells, as well as the expression levels of MNSFβ and RhoA in placental villi of RM patients and physically normal pregnant women (NP), were examined by Western blot analysis. Results: MNSFβ protein signals were observed in first-trimester human villus and extravillous trophoblast cells. The downregulated MNSFβ expression significantly attenuated the proliferation, migration, and invasion abilities of HTR8/SVneo cells, accompanied with the obviously decreased expression levels of P53, RhoA, Bcl-2, Bax, and MMP-9, whereas the upregulated MNSFβ expression in HTR8/SVneo cells represented the inverse effects. Furthermore, expression levels of MNSFβ and RhoA in first-trimester human placental villus tissues of RM patients were significantly decreased compared to that of NP women. Conclusion: These data suggested that MNSFβ promotes proliferation and migration of human EVT cells, probably via the P53 signaling pathway, and the deficiency of MNSFβ in placental villi might lead to early pregnancy loss by reducing proliferation and invasion activities of EVTs.

MiR-150-5p mediates extravillous trophoblast cell migration and angiogenesis functions by regulating VEGF and MMP9

IntroductionmiR-150-5p is involved in placenta function. Matrix metalloproteinases (MMPs) play important roles in migration and invasion of cells, while VEGF is the major contributing factor in angiogenesis, and they are related to miR-150-5p. However, the mechanism by which miR-150-5p regulates placental functions is not known. Thus, we investigated the influence of miR-150-5p on extravillous trophoblast function and the underlying epigenetic mechanism. MethodsReal-time PCR were used to detect the miR-150-5p in the placenta of patients with preeclampsia and normal pregnant women. HTR-8/SVneo and JEG-3 cells were transfected with miR-150-5p inhibitor. Furthermore, we used CoCl2 to establish the hypoxia cell model. qRT-PCR and Western blot analysis were performed to detect VEGF and MMP9 expression levels in the transfected cells. Cell Counting Kit-8 assay was conducted to evaluate the proliferation ability. Wound-healing and transwell assays were used to detect the migration and invasion capacities. Tube formation assay was performed to evaluate the angiogenesis ability. ResultsmiR-150-5p was up-regulated in the placenta of patients with preeclampsia. Inhibition of miR-150-5p significantly enhanced migration, invasion, and angiogenesis ability of both JEG-3 and HTR-8/SVneo cells. Similar results were seen in the hypoxic HTR-8/SVneo cell model. Moreover, mRNA and protein expression levels of VEGF and MMP9 were upregulated by the inhibition of miR-150-5p. DiscussionmiR-150-5p impacts placental cellular abilities, including migration, invasion, and angiogenesis of extravillous trophoblast cells. More importantly, miR-150-5p regulates VEGF and MMP9 expression. The results suggest that miR-150-5p may have an impact on placenta functions.

Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-β-cyclodextrin (MβCD) to disrupt caveolae. We found that after MβCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MβCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Thrombin stimulates gene expression and secretion of IL-11 via protease-activated receptor-1 and regulates extravillous trophoblast cell migration

Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times.Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.

Prostaglandin F2α stimulates adhesion, migration, invasion and proliferation of the human trophoblast cell line HTR-8/SVneo

IntroductionThe amount of prostaglandin F2α (PGF2α) in the uterine lumen increases during the window of implantation in many mammals, including humans. We hypothesized that PGF2α regulates processes related to human embryo implantation. MethodsThe effect of PGF2α was studied using an in vitro model of human extravillous trophoblast (EVT) cell line (HTR-8/SVneo). Adhesion, proliferation, invasion and migration assays, zymography for metalloproteinases (MMP) activity, and gene/protein expression analyses were applied. Doses of 100 nM and/or 1 μM of PGF2α and fluprostenol were used. PGF2α receptor (PTGFR), MMP9 and MMP2 proteins in the human first trimester placenta were localized by immunohistochemistry and immunofluorescence. ResultsThis study is the first reporting the expression of PTGFR protein in the first trimester placenta, as well as in HTR-8/SVneo cells. PGF2α and fluprostenol increased HTR-8/SVneo cell proliferation and adhesion to extracellular matrix protein (P < 0.05). This effect was abolished by mitogen activated protein kinases (MAPK) inhibitor. PGF2α induced phosphorylation of focal adhesion kinase and MAPK1/3 (P < 0.05). PGF2α increased mRNA content and protein activity of MMP9, and gene and protein expression of interleukin-6 (P < 0.05). EVT cell migration and invasiveness were stimulated by PGF2α (P < 0.05). The PGF2α effect on cell invasion was reduced by inhibitors of MMP2, MMP9 and mTOR. In all experiments, the stimulatory effects of PGF2α were diminished by using a PTGFR antagonist. DiscussionOur findings suggest a significant role for PGF2α in mechanisms associated with implantation. PGF2α acting by PTGFR in HTR-8/SVneo cells stimulates their adhesion and proliferation through the MAPK signaling pathway and increases invasiveness inducing MMP proteolytic activity and mTOR signaling.

Vascular endothelial growth factor C participates in regulation of maspin in extravillous trophoblast cell migration and invasion.

Mammary serine protease inhibitor (maspin ; also known as serpin family B member 5 (SERPINB5)) plays a vital role in regulating the biological functions of extravillous trophoblast (EVT) cells, but the mechanism remains unclear. Vascular endothelial growth factor (VEGF ) C is a signature angiogenic molecule expressed and secreted by first-trimester trophoblasts, and bioinformatics analyses has revealed upregulation of VEGFC in pre-eclampsia. The aim of this study was to explore whether maspin regulates EVT cells by regulating the expression of VEGFC . Reverse transcription-polymerase chain reaction and western blotting were used to investigate the effects of hypoxia on the expression of VEGFC in EVT cells. Cells were treated with recombinant (r) maspin and decitabine (to selectively inhibit DNA methyltransferases and then upregulate maspin gene expression), and the effects on VEGFC expression evaluated. In addition, the effects of rVEGFC on the biological functions of EVT cells invitro were evaluated using cell migration and invasion assays. Hypoxia increased the expression of VEGFC in EVT cells. rMaspin upregulated the expression of VEGFC in normoxic EVT cells, and downregulated the expression of VEGFC in hypoxic EVT cells at 24h. Decitabine increased VEGFC expression in normoxic EVT cells, but had no significant effect on VEGFC expression in hypoxic EVT cells. rVEGFC promoted the migration and invasion of normoxic EVT cells and inhibited the invasion of hypoxic EVT cells. These results suggest that VEGFC is involved in the regulation of maspin in EVT cell migration and invasion. However, other molecular mechanisms may be involved and require further investigation.

Total flavonoids from Semen Cuscutae target MMP9 and promote invasion of EVT cells via Notch/AKT/MAPK signaling pathways

Miscarriage is a common condition during pregnancy and its mechanisms remain largely unknown. Extravillous trophoblast (EVT) cell invasion is required to maintain normal pregnancy and its malfunction has been proposed as a major cause for miscarriage. Homeostasis of matrix metalloproteinase 9 (MMP9) is a key to regulate EVT cell invasion. Total flavonoids from Semen Cuscutae (TFSC) have been applied clinically used for preventing or treating miscarriage in the past. Given its potential clinical benefit on preventing miscarriage, this study aims at examining the therapeutic effect of TFSC in the prevention of premature birth by upregulating MMP9 and promote EVT cell invasion. HTR-8 cells migration and invasion functions were analyzed using wound healing and transwell assays. The regulatory effect of TFSC on MMP9 expression and relevant signaling pathways were analyzed by Western Blot. The results show compared to control group, TFSC significantly promoted the migration of EVT cells in a dose and time-dependent manner. The migration and invasion of EVT cells were maximized at the highest dosage of 5 μg/ml of TFSC. The expression of MMP9 in EVT cells was significantly increased after TFSC treatment. Furthermore, cells treated with TFSC significantly upregulated protein expressions in Notch, AKT and p38/MAPK signaling pathways. We believe TFSC can promote the migration and invasion of EVT cells by increasing MMP9 expression, and prevent miscarriage by activating Notch, AKT, and MAPK signaling pathways.

SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Migration of placental extravillous trophoblast (EVT) cells into uterine decidua facilitates the establishment of blood circulation between mother and fetus and is modulated by EVT-decidual cell interaction. Poor or excessive EVT migration is associated with pregnancy complications such as preeclampsia or placenta accreta. Glial cells missing 1 (GCM1) transcription factor is essential for placental development, and decreased GCM1 activity is detected in preeclampsia. To study whether GCM1 regulates trophoblast cell migration, here we showed that GCM1 promotes BeWo and JAR trophoblast cell migration through a novel target gene, WNT10B. Moreover, WNT10B signaling stimulated cytoskeletal remodeling via Rac1 and frizzled 7 (FZD7) was identified as the cognate receptor for WNT10B to up-regulate cell migration. We further showed that secreted frizzled-related protein 3 (SFRP3) is expressed in uterine decidual cells by immunohistochemistry and that SFRP3 expression in telomerase-transformed human endometrial stromal cells (T-HESCs) is elevated under decidualization stimuli and further enhanced by bone morphogenetic protein 2 via SMAD1. SFRP3 blocked the interaction between FZD7 and WNT10B to decrease BeWo cell migration, which corroborated the elevated BeWo cell migration when cocultured with decidualized and SFRP3-knockdown T-HESC monolayer. Our results suggest that GCM1 up-regulates EVT cell migration through WNT10B and FZD7, which is negatively modulated by decidual SFRP3.-Wang, L.-J., Lo, H.-F., Lin, C.-F., Ng, P.-S., Wu, Y.-H., Lee, Y.-S., Cheong, M.-L., Chen, H. SFRP3 negatively regulates placental extravillous trophoblast cell migration mediated by the GCM1-WNT10B-FZD7 axis.

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V.angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V.angustifolium leaf extract (0 to 2×104 ng/ml) on trophoblast cell migration (n=4) and invasion (n=4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n=3) and trypan blue exclusion assays (n=3), respectively. At 20ng/ml, V.angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p<.01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n=3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted.

H19 long noncoding RNA alters trophoblast cell migration and invasion by regulating TβR3 in placentae with fetal growth restriction.

Fetal growth restriction (FGR) is a well-recognized risk factor for perinatal mortality and morbidity, as well as neurodevelopmental impairment and adulthood onset disorders. Here we report that the H19 long noncoding RNA (lncRNA) is significantly decreased in placentae from pregnancies with FGR. Downregulation of H19 leads to reduced migration and invasion of extravillous trophoblast (EVT) cells in vitro. This is consistent with reduced trophoblast invasion that has been observed in FGR. Genome-scale transcriptome profiling of EVT cells reveals significantly decreased expression of the type III TGF-β receptor (TβR3) following H19 knockdown. Decreased TβR3 expression is also seen in FGR placentae. TβR3 repression decreases EVT cell migration and invasion, owing to impaired TGF-β signaling through a non-canonical TGF-β signaling pathway. Further, we identify TβR3 as a novel regulatory target of microRNA let-7. We propose that dysregulation of this newly identified H19/TβR3-mediated regulatory pathway may contribute to the molecular mechanism of FGR. Our findings are the first to show a lncRNA-based mechanism of FGR, holding promise for the development of novel predictive, diagnostic, and therapeutic modalities for FGR.

Restraint of Trophoblast Invasion of the Uterus by Decorin: Role in Pre-eclampsia.

Decorin (DCN) is a leucine-rich, TGF-β binding proteoglycan produced by mesenchymal cells including chondrocytes, dermal fibroblasts, and uterine decidual cells. It exerts multiple physiological functions including collagen fibrillogenesis, myogenesis, angiostasis, and restraining placental invasiveness. We discovered that decidua-derived DCN restrains proliferation, migration, and invasion of extravillous trophoblast (EVT) cells of the human placenta in a TGF-β-independent manner. These functions were differentially mediated by binding of DCN to multiple tyrosine kinase receptors (TKR) including EGFR, IGFR1, and VEGFR2. DCN blocked VEGFR-2 dependent EVT cell migration and endovascular differentiation by inhibiting P38MAPK and ERK1/2 pathways.We identified the avid VEGFR2 binding site in DCN protein as a 12 amino acids (LGTNPLKSSGIE) span in the Leucine-rich-repeat (LRR) 5 region of domain III. A single amino acid mutation (substitution of K to A) of DCN at this site abrogated VEGFR-2- dependent DCN actions. Also, DCN mRNA expression, measured with in situ hybridization, was selectively upregulated in decidual cells in placentas from mothers suffering from pre-eclampsia (PE), whereas the expression levels remained unchanged in chorionic villus mesenchymal cells. This difference between PE and control placentas was present at all gestational ages, indicating the pathogenic role of DCN in PE. We hypothesize that increased blood DCN levels could be a candidate biomarker for PE.

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation.

ObjectiveExtravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells.MethodsWestern blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells.ResultsHypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells.DiscussionThe methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta.

CCN3 (NOV): A key player in trophoblast differentiation promoting cell cycle exit and migration of extravillous trophoblast cells and its role in preeclampsia

CCN3 (NOV): A key player in trophoblast differentiation promoting cell cycle exit and migration of extravillous trophoblast cells and its role in preeclampsia

The term basal plate of the human placenta as a source of functional extravillous trophoblast cells

BackgroundExtravillous trophoblast (EVT) cells are of pivotal importance in human embryo implantation and homeostasis of the maternal fetal interface. Invasion of the endometrium by EVT contributes to placental anchorage, spiral artery remodeling, immunological defense, tolerogenic responses, and several collaborative cross talks involved in establishing and maintaining a successful pregnancy. We report here an improved protocol for the isolation of fully differentiated EVT cells from the basal plate of the human term placenta.MethodsThe basal plate was carefully dissected from the villous tissue and the amniochorion membrane prior to enzymatic digestion. Term basal EVT cells were isolated using a 30 and 60% Percoll gradient. A panel of markers and characteristics of the isolated cells were used to confirm the specificity and efficiency of the method so that their potential as an investigative tool for placental research could be ascertained.ResultsIsolated cells were immunoreactive for cytokeratin-7 (CK-7), placental growth factor, placental alkaline phosphatase, human leukocyte antigen G1 (HLA-G1), and α1 and α5 integrins, similarly to the EVT markers from first trimester placental villi. Around 95% of the isolated cells labeled positively for CK-7 and 82% for HLA-G1. No significant change in viability was observed during 48 h of EVT culture as indicated by propidium iodide incorporation and trypan blue test exclusion. Genes for metalloproteinases MMP-2 and MMP9 (positive regulators of trophoblast invasiveness) were expressed up to 48 h of culturing, as also the gelatinolytic activity of the isolated cells. Transforming growth factor (TGF)-beta, which inhibits proliferation, migration, and invasiveness of first-trimester EVT cells, also reduced invasion of isolated term EVT cells in transwell assays, whereas epidermal growth factor was a positive modulator.ConclusionsTerm basal plate may be a viable source of functional EVT cells that is an alternative to villous explant-derived EVT cells and cell lines. Isolated term EVT cells may be particularly useful in investigation of the role of trophoblast cells in pathological gestations, in which the precise regulation and interactive ability of extravillous trophoblast has been impaired.

Matricellular CCN1 and CCN3 proteins are key regulators at the fetal-maternal interface by promoting cell cycle exit and migration of extravillous trophoblast cells

Matricellular CCN1 and CCN3 proteins are key regulators at the fetal-maternal interface by promoting cell cycle exit and migration of extravillous trophoblast cells

Improvement of insulin sensitivity promotes extravillous trophoblast cell migration stimulated by insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) has been shown to stimulate extravillous trophoblast (EVT) cell migration and invasion, and to play a crucial role in placental function, thereby, influencing placental development and fetal growth. Insufficient invasion of EVT cells into the uterine endometrium leads to pregnancy-related complications, including spontaneous abortion, fetal growth restriction (FGR), and pregnancy-induced hypertension (PIH). Insulin-resistant conditions such as polycystic ovary syndrome (PCOS) and gestational diabetes mellitus (GDM) have also been associated with abortion and PIH. However, the effects of IGF-I on EVT cells under insulin-resistant conditions have not been elucidated yet. The current study was undertaken to analyze the effects of IGF-I under insulin-resistant conditions and to determine whether improvement in insulin sensitivity alters IGF signaling and cell migration in the EVT. Incubation with pioglitazone, an insulin sensitizer, increased peroxisome proliferator-activated receptor-γ (PPARγ) expression after 48 h. A 48-h pre-incubation with insulin reduced the phosphorylation and concentration of the insulin receptors, which were increased by insulin treatment. Long-term exposure to insulin reduced phosphorylation of the IGF-I receptor, insulin receptor substrate-1 (IRS-1), and Akt, and also reduced EVT cell migration. However, when the cells were incubated with pioglitazone in addition to insulin for 48 h, the phosphorylation of these proteins was restored. This combination partially reversed the inhibitory effect of insulin on EVT cell migration. These results suggest that abnormalities in pregnancy that are induced by loss of insulin sensitivity can be treated by improving insulin sensitivity.