Trophoblast Phenotype Research Articles

Epithelioid trophoblastic tumor in male patients with germ cell tumor: A clinicopathologic analysis of five cases

Epithelioid trophoblastic tumor (ETT) is an extremely rare chorionic-type neoplasm in the testis, with only seven cases reported in the literature. Here, we report five cases of testicular ETT from a single institution, constituting the largest series of this rare tumor to date. The patients had a mean age of 44 years (range, 20–68 years). Four patients had a previous history of testicular germ cell tumor (GCT) treated with chemotherapy, and they developed ETT in metastatic sites in a mean of 11 years (range, 3–15 years) after the initial diagnosis of testicular GCT. Only one patient had ETT in the testis. Three patients had a normal serum beta–human chorionic gonadotropin (β-hCG) level, and two patients had a level that was slightly elevated, but far below that typically seen in patients with choriocarcinoma. ETT was characterized by a proliferation of intermediate trophoblastic cells with abundant eosinophilic cytoplasm, and the tumors frequently had coagulative necrosis with eosinophilic debris, mimicking keratinizing squamous cell carcinoma. The trophoblastic phenotype of ETT was supported by its immunoreactivity for trophoblastic markers, including GATA-3 (3 of 3 cases tested), α-inhibin (3/4), p63 (3/5), and β-hCG (3/4). ETT was also positive for cytokeratin (4/4) and GCT marker SALL4 (3/3). Despite surgery and chemotherapy, two patients died of disease 17 months after ETT diagnosis, and three patients were alive with metastatic disease at a mean of 20 months (range, 15–28 months). Our results demonstrate that ETT may be an aggressive disease associated with distinct pathologic features and poor clinical outcome.

Spatial proteomics and transcriptomics of the maternal-fetal interface in placenta accreta spectrum

In severe Placenta Accreta Spectrum (PAS), trophoblasts gain deep access in the myometrium (placenta increta). This study investigated alterations at the fetal-maternal interface in PAS cases using a systems biology approach consisting of immunohistochemistry, spatial transcriptomics and proteomics. We identified spatial variation in the distribution of CD4+, CD3+ and CD8+ T-cells at the maternal-interface in placenta increta cases. Spatial transcriptomics identified transcription factors involved in promotion of trophoblast invasion such as AP-1 subunits ATF-3 and JUN, and NFKB were upregulated in regions with deep myometrial invasion. Pathway analysis of differentially expressed genes demonstrated that degradation of extracellular matrix (ECM) and class 1 MHC protein were increased in increta regions, suggesting local tissue injury and immune suppression. Spatial proteomics demonstrated that increta regions were characterised by excessive trophoblastic proliferation in an immunosuppressive environment. Expression of inhibitors of apoptosis such as BCL-2 and fibronectin were increased, while CTLA-4 was decreased and increased expression of PD-L1, PD-L2 and CD14 macrophages. Additionally, CD44, which is a ligand of fibronectin that promotes trophoblast invasion and cell adhesion was also increased in increta regions. We subsequently examined ligand receptor interactions enriched in increta regions, with interactions with ITGβ1, including with fibronectin and ADAMS, emerging as central in increta. These ITGβ1 ligand interactions are involved in activation of epithelial–mesenchymal transition and remodelling of ECM suggesting a more invasive trophoblast phenotype. In PAS, we suggest this is driven by fibronectin via AP-1 signalling, likely as a secondary response to myometrial scarring.

Human placental organoids as a model to probe early gestation maternal immune dynamics.

During pregnancy, the human placenta establishes tolerance toward fetal allogeneic tissue, where specialized trophoblast subtypes play a complex role in local and peripheral immunomodulation. However, due to inadequate models to study the early gestation of the human placenta, each trophoblast subtype's role in modulating the maternal immune response has remained elusive. Here, we derived human placental organoids from early gestation trophoblast stem cells to (1) identify patterns of immunomodulatory protein expression by trophoblast subtype and (2) evaluate the effects of the placental organoid secretome on immune cell activation and regulation. We show that the three primary trophoblast phenotypes had distinct influences on immune cell phenotype and activation and that three-dimensional culture significantly alters trophoblast immunomodulation relative to traditional two-dimensional trophoblast culture.

USING A MULTI-OMICS LANDSCAPE OF THE MATERNAL-FETAL INTERFACE TO MODEL THE LONGITUDINAL PATHOGENESIS OF EARLY-ONSET PRE-ECLAMPSIA

Objective: Pre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications. We currently lack efficient screening tools and early therapies to address PE. Design and method: To identify candidate biomarkers and operative pathways in early onset PE (eoPE, severe PE with onset before week 34), we performed spatio-temporal multi-omics profiling of human eoPE placentae and healthy controls, and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-sequencing combined with spatial proteo- and transcriptomics and mechanistic in vitro signalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. Results: We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts in eoPE. We found a significant increase in the senescence markers in preeclamptic maternal serum in early gestation and late gestation, before the development of clinical symptoms, indicating that the placental syndrome drives systemic maternal syndrome, even before clinical manifestation of eoPE. Conclusions: Our work describes a new disease progression model, starting with dysregulated transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.

Downregulated PDIA3P1 lncRNA Impairs Trophoblast Phenotype by Regulating Snail and SFRP1 in PE.

Preeclampsia (PE) manifests as a pregnancy-specific complication arising from compromised placentation characterized by inadequate trophoblast invasion. A growing body of evidence underscores the pivotal involvement of pseudogenes, a subset of long noncoding RNAs, in the pathological processes of PE. This study presents a novel finding, demonstrating a significant downregulation of the pseudogene PDIA3P1 in PE placental tissues compared to normal tissues. In vitro functional assays revealed that suppressing PDIA3P1 hindered trophoblast proliferation, invasion, and migration, concurrently upregulating the expression of secreted frizzled-related protein 1 (SFRP1). Further exploration of the regulatory role of PDIA3P1 in PE, utilizing human trophoblasts, established that PDIA3P1 exerts its function by binding to HuR, thereby enhancing the stability of Snail expression in trophoblasts. Overall, our findings suggest a crucial role for PDIA3P1 in regulating trophoblast properties and contributing to the pathogenesis of PE, offering potential targets for prognosis and therapeutic intervention.

Linking genotype to trophoblast phenotype in preeclampsia and HELLP syndrome associated with STOX1 genetic variants

Preeclampsia is a major hypertensive pregnancy disorder with a 50% heritability. The first identified gene involved in the disease is STOX1, a transcription factor, whose variant Y153H predisposes to the disease. Two rare mutations were also identified in Colombian women affected by the hemolysis, elevated liver enzyme, low platelet syndrome, a complication of preeclampsia (T188N and R364X). Here, we explore the effects of these variants in trophoblast cell models (BeWo) where STOX1 was previously invalidated. We firstly showed that STOX1 knockout alters response to oxidative stress, cell proliferation, and fusion capacity. Then, we showed that mutant versions of STOX1 trigger alterations in gene profiles, growth, fusion, and oxidative stress management. The results also reveal alterations of the STOX interaction with DNA when the mutations affected the DNA-binding domain of STOX1 (Y153H and T188N). We also reveal here that a major contributor of these effects appears to be the E2F3 transcription factor.

EGR1 modulates EPHB4-induced trophoblast dysfunction in recurrent spontaneous abortion†.

Recurrent spontaneous abortion, defined as at least three unexplained abortions occurring before the 20-24 week of pregnancy, has a great impact on women's quality of life. Ephrin receptor B4 has been associated with trophoblast function in preeclampsia. The present study aimed to verify the hypothesis that ephrin receptor B4 regulates the biological functions of trophoblasts in recurrent spontaneous abortion and to explore the upstream mechanism. Ephrin receptor B4 was overexpressed in mice with recurrent spontaneous abortion. Moreover, ephrin receptor B4 inhibited trophoblast proliferation, migration, and invasion while promoting apoptosis. Downregulation of early growth response protein 1 expression in mice with recurrent spontaneous abortion led to ephrin receptor B4 overexpression. Poor expression of WT1-associated protein in mice with recurrent spontaneous abortion reduced the modification of early growth response protein 1 mRNA methylation, resulting in decreased early growth response protein 1 mRNA stability and expression. Overexpression of WT1-associated protein reduced the incidence of recurrent spontaneous abortion in mice by controlling the phenotype of trophoblasts, which was reversed by early growth response protein 1 knockdown. All in all, our findings demonstrate that dysregulation of WT1-associated protein contributes to the instability of early growth response protein 1, thereby activating ephrin receptor B4-induced trophoblast dysfunction in recurrent spontaneous abortion. Our study provides novel insights into understanding the molecular pathogenesis of recurrent spontaneous abortion.

LncRNA GHET1 regulates extravillous trophoblastic phenotype via EZH2/LSD1-mediated MT2A epigenetic suppression in pre-eclampsia.

Pre-eclampsia (PE) is usually defined as new-onset hypertension with albuminuria or other organ damage. Herein, the role and mechanism of long noncoding RNA (lncRNA) gastric carcinoma high expressed transcript 1 (GHET1) during PE are investigated. Expression of GHET1 in PE pregnancies was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Proliferation and cell cycle of extravillous trophoblasts were assessed by Cell Counting Kit-8 (CCK-8), colony formation, 5-Ethynyl-2'-deoxyuridine (EdU) assays, and flow cytometry, respectively. Migration, invasion, and network formation of trophoblasts were measured by wound healing, transwell system, and tube formation assays. RNA immunoprecipitation (RIP), RNA pull-down, and chromatin immunoprecipitation (ChIP) assays were used to confirm the molecular interaction. GHET1 was markedly decreased in the placenta of PE patients. GHET1 promoted the proliferation and cell cycle of extravillous trophoblasts, as well as migration, invasion, and network formation in vitro. Metallothionein 2A (MT2A) functioned as a downstream effector of GHET1, which was negatively correlated with GHET1 in PE. GHET1 directly bound with zeste 2 polycomb repressive complex 2/lysine-specific demethylase 1 (EZH2/LSD1). Knockdown of GHET1 reduced the occupancies of H3K27me3 and H3K4me2 in the MT2A promoter region by recruiting EZH2 and LSD1. MT2A knockdown reversed GHET1 inhibition mediated biological functions. GHET1 regulates extravillous trophoblastic phenotype via EZH2/LSD1-mediated MT2A epigenetic suppression in PE.

Chorionic villus-derived mesenchymal stem cell-mediated NRG1 upregulation promotes HTR-8/SVneo cells proliferation through the activation of the NF-κB signaling pathway

In a prior study, our group found that chorionic villus-derived mesenchymal stem cells (CV-MSCs) were capable of promoting trophoblast proliferative and invasive activity. The mechanistic basis for this activity, however, has yet to be clarified. As such, an RNA-Seq analysis was conducted using trophoblasts that were treated with or without CV-MSC-conditioned media. Of the differentially expressed genes identified when comparing these two groups of cells, 23 proliferation-associated genes were identified and knocked down to test their functional roles in trophoblasts. These analyses revealed that inhibiting neuregulin 1 (NRG1) expression was sufficient to suppress proliferation and induce cell cycle arrest in trophoblasts. Placental samples from patients with preeclampsia exhibited significantly increased NRG1 expression relative to samples from healthy pregnancies. Following treatment with CV-MSC-conditioned media, NRG1 was upregulated in trophoblasts at the mRNA and protein levels. Relative to control trophoblasts, those in which NRG1 had been knocked down exhibited significantly impaired proliferation and DNA replication with the inactivation of the NF-κB signaling pathway. In contrast, overexpressing NRG1 yielded the opposite trophoblast phenotypes. Even in cells overexpressing NRG1, inhibition of NF-κB signaling was sufficient to significantly suppress trophoblast proliferation (P < 0.05). These results indicate that elevated NRG1 expression may play a role in the ability of CV-MSCs to induce proliferative activity in trophoblasts through the NF-κB signaling axis.

MALAT1 modulates trophoblast phenotype via miR-101–3p/VEGFA axis

Preeclampsia is a potentially life-threatening condition that can arise due to poor placentation and consequent abnormal uterine spiral artery remodeling. Abnormal placentation, in turn, is associated with aberrant trophoblast cell proliferation and apoptosis. Here, we investigated the lncRNA MALAT1 in trophoblast proliferation during early-onset preeclampsia (ePE). MALAT1 levels were examined in placental tissue samples from ePE patients and control patients. The effects and underlying mechanism of MALAT1 on proliferation, migration, invasion and apoptosis were investigated in the first-trimester extravillous trophoblast HTR-8/SVneo cells and the human choriocarcinoma JAR cells. MALAT1 levels were decreased in the placental tissue samples of ePE patients compared with those of control patients, and the levels of MALAT1 were positively correlated with the neonate birth-weight. Knockdown of MALAT1 attenuated the cell viability, proliferation, migration, invasion and the cell cycle progression of trophoblasts, but promoted the apoptosis of trophoblasts. The MALAT1 knockdown promoted miR-101–3p upregulation and VEGFA downregulation. Inhibitor of miR-101–3p increased vascular endothelial growth factor A (VEGFA) expression, and miR-101–3p mimic inhibited VEGFA expression. Luciferase assays showed that miR-101–3p could bind to both MALAT1 and VEGFA. The MALAT1 knockdown-induced induction in the cell vitality and proliferation were attenuated by miR-101–3p inhibitor. We conclude that endogenous MALAT1 promotes proliferation, migration and invasion of trophoblasts by inhibiting the miR-101–3p expression and the subsequent VEGFAupregulation. The reduced MALAT1 level in placental tissue may be involved in the pathogenesis of the ePE.

P-523 3D Sw71 blastocyst-like model and its validation by primary trophoblasts

Abstract Study question Are the in vitro developed 3D-Sw71 blastocyst-like model and the primary trophoblast 3D model interchangeable? Summary answer We confirmed that 3D-Sw71 model can be successfully replaced by 3D primary trophoblast model. What is known already Overcoming the challenges and ethical considerations of manipulating human embryos, researchers use cell lines to resemble blastocyst morphology and behavior. We have constructed 3D-Sw71 blastocyst-like model from non-cancerous trophoblast cell line. Like human blastocyst, our Sw71 spheroids undergo compactization and cavitation, have similar shape and size and attach to endometrial epithelium, invade extracellular matrix and migrate between endometrial stromal cells. Models’Sw71 cells have the hybrid epithelial-mesenchymal phenotype of the extravillous trophoblasts (EVT), important for building functional placenta. 3D-Sw71 models have the unique invariant HLA-profile of EVTs (HLA-G+, HLA-C+) providing tolerogenic maternal-embryo immune recognition. Sw71-2D and 3D -cultures produced CD63-positive exosomes. Study design, size, duration To validate our models in vivo, we performed direct comparison between 3D Sw71 spheroids and analogous spheroids built from isolated primary chorion-derived trophoblasts (1st trimester of pregnancy). The comparison was done in context of the following criteria: generation/ differentiation, morphology (shape, size), function, invariant HLA profile, presence of hybrid phenotype and exosomes secretion. For each condition of validation at least 3 independent experiments were conducted. Participants/materials, setting, methods 3D-Sw71 spheroids were constructed from Sw71 cells and 3D primary trophoblasts spheroids – from trophoblasts from placenta of pregnant women, directed to elective early pregnancy termination. Trophoblast cells were isolated by enzymes treatment and Percoll gradient separation. The generation and migration of the spheroids was monitored by live cells imaging and EMT and HLA molecules were detected by FACS and immunocytochemistry. The exosomes isolated by ultrafiltration were characterized by TEM, dot-blot analysis and immunogold staining. Main results and the role of chance From pure trophoblast cultures (&amp;gt; 80%, assessed by FACS) were generated long-lived clones, cells from which were used for 3D primary trophoblasts spheroids construction. Our results showed that in both 3D culture settings we obtained stable, round shaped, multilayered and relatively symmetric spheroid structures followed by compaction (12–24 h) and formation of single differentiated spheroid (24-48 h) with an intact periphery and easy to manipulate. The primary trophoblasts 3D spheroids resemble 3D blastocyst-like structures in shape and size. Primary trophoblast spheroids migrated successfully in the same time frame as 3D in vitro blastocyst-like structure (BLS), (48 hours). The trophoblasts cells of the primary 3D spheroids have hybrid phenotype (Vim+Ck7+) as Sw71 cells of 3D-Sw71 BLS. These expressed the invariant HLA-G and -C molecules as well. The HLA molecules were proven also in situ in early human placenta by IHC. Sw71 and primary trophoblasts secrete exosomes, proven by TEM as 20-120 nm EV, positive for CD63 (exclusive exosome marker). In conclusion, 3D-Sw71 blastocyst-like structure represent a valuable model for in vitro trophoblast and implantation studies as well as trophoblasts-immune cells interactions. Limitations, reasons for caution Primary trophoblasts cultures have limited viability, which makes difficulty to perform more than three independent experiments for all conditions. In general, manually transferring 3D spheroids to new media conditions is very laborious, time-consuming and not amenable to high-throughput screenings. Wider implications of the findings Mimicking the implanting embryo has huge implications for a plethora of studies, such as toxicity and drug screens, development of implantation-promoting compounds. 3D-Sw71 model has a potential to integrate additional components such as immune cells, endothelial cells/vessels and bioengineered in vitro models of the uterine wall/glands to uncover human implantation. Trial registration number not applicable

CircSTAM inhibits migration and invasion of trophoblast cells by regulating miR-148a-5p/PTEN axis

BackgroundThe mechanisms underlying the pathogenesis of preeclampsia (PE) remains unclear. Exploring the molecular players in PE progression can provide insights into targeted therapy.MethodsThe expression levels of circSTAM in placental chorionic tissues of PE patients and normal pregnant women were compared by RT-qPCR. CircSTAM was knocked down by small interfering RNA to investigate its role in migration, invasion and epithelial-mesenchymal transformation (EMT) of trophoblast HTR-8/SVneo cells. The downstream target of circSTAM was predicted using online bioinformatics resources, and their molecular interaction was examined by luciferase reporter assay.ResultsCircSTAM was upregulated in PE placenta tissues in comparison to normal placental tissues. CircSTAM knockdown significantly enhanced cellular invasion, migration, as well as EMT. Mir-148a-5p was identified as a target of circSTAM to regulate cell migration and invasion. Mir-148a-5p negatively regulated PTEN expression in trophoblast HTR-8 /SVneo cells.ConclusionIn summary, circSTAM upregulation in PE trophoblasts promoted the invasion, migration and EMT. CircSTAM may modulate trophoblast phenotype by impinging on mir-148a-5p/PTEN axis. These data provided novel insights into the pathogenesis of PE.

Downregulated PDIA3P1 contributes to impair trophoblastic phenotype via inactivation of PDIA3P1/HuR/Snail pathway and regulation of SFRP1 in PE

Downregulated PDIA3P1 contributes to impair trophoblastic phenotype via inactivation of PDIA3P1/HuR/Snail pathway and regulation of SFRP1 in PE

MiR-218-5p Induces Interleukin-1β and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor β-SMAD2 Pathway.

The acquisition of an endovascular trophoblast (enEVT) phenotype is essential for normal placental development and healthy pregnancy. MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating gene expression. We have recently reported that miR-218-5p promotes enEVT differentiation and spiral artery remodeling in part by targeting transforming growth factor β2 (TGFβ2). We also identified IL1B, which encodes interleukin 1β (IL1β), as one of the most highly upregulated genes by miR-218-5p. In this study, we investigated how miR-218-5p regulates IL1B expression and IL1β secretion and the potential role of IL1β in enEVT differentiation. Using two cell lines derived from extravillous trophoblasts (EVTs), HTR-8/SVneo and Swan 71, we found that stable overexpression of miR-218-5p precursor, mir-218-1, or transient transfection of miR-218-5p mimic, significantly increased IL1B mRNA and IL1β protein levels in cells and conditioned media. We also showed that miR-218-5p directly interacted with SMAD2 3’UTR and reduced SMAD2 at mRNA and protein levels. Knockdown of SMAD2 induced IL1B expression and attenuated the inhibitory effect of TGFβ2 on IL1B expression. On the other hand, overexpression of SMAD2 reduced IL1β levels and blocked the stimulatory effects of miR-218-5p on IL1B expression, trophoblast migration and endothelial-like network formation. In addition, treatment of trophoblasts with IL1β induced the formation of endothelial-like networks and the expression of enEVT markers in a dose-dependent manner. These results suggest that miR-218-5p inhibits the TGFβ/SMAD2 pathway to induce IL1β and enEVT differentiation. Finally, low doses of IL1β also inhibited the expression of miR-218-5p, suggesting the existence of a negative feedback regulatory loop. Taken together, our findings suggest a novel interactive miR-218-5p/TGFβ/SMAD2/IL1β signaling nexus that regulates enEVT differentiation.

The role of integrins in the formation of the placental increment &lt;i&gt;(placenta accreta spectrum)&lt;/i&gt;

One of the most serious pregnancy complications is currently considered as a violation of cytotrophoblast invasion, particularly, a pathologically increased depth of invasion, leading to the formation of the placental increment (placenta accreta), ingrowth (placenta increta), and germination (placenta percreta). Cytotrophoblast invasion is regulated through subtle intercellular interactions, an important role among which is played by integrins, transmembrane glycoproteins that contribute to the immersion of cytotrophoblast in the endometrium and myometrium. The balance disturbance in the expression of 11, 51, and 64 integrins after normal implantation form a pathological extravillous trophoblast (EVT) phenotype by the blastocyst. This review examines the importance of integrins, collagens, and fibronectin in the formation of placental ingrowth (placenta accreta spectrum).

Generation of Trophoblast-Like Cells From Hypomethylated Porcine Adult Dermal Fibroblasts.

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial components of the placenta, and which supports the fetus during the intrauterine life. However, the epigenetic and paracrine controls at work in trophectoderm differentiation are still to be fully elucidated and the creation of dedicated in vitro models is desirable to increase our understanding. Here we propose a novel approach based on the epigenetic conversion of adult dermal fibroblasts into trophoblast-like cells. The method combines the use of epigenetic erasing with an ad hoc differentiation protocol. Dermal fibroblasts are erased with 5-azacytidine (5-aza-CR) that confers cells a transient high plasticity state. They are then readdressed toward the trophoblast (TR) phenotype, using MEF conditioned medium, supplemented with bone morphogenetic protein 4 (BMP4) and inhibitors of the Activin/Nodal and FGF2 signaling pathways in low O2 conditions. The method here described allows the generation of TR-like cells from easily accessible material, such as dermal fibroblasts, that are very simply propagated in vitro. Furthermore, the strategy proposed is free of genetic modifications that make cells prone to instability and transformation. The TR model obtained may also find useful application in order to better characterize embryo implantation mechanisms and developmental disorders based on TR defects.

Examination of FERMT1 expression in placental chorionic villi and its role in HTR8-SVneo cell invasion.

Transmembrane integrin receptors mediate cell-extracellular matrix as well as cell-cell adhesion. As placental trophoblast cells undergo differentiation they display changes in integrin expression or switching, but the mechanism(s) of integrin activation that supports this differentiation is still unknown. The Fermitin family of adapter proteins (FERMT 1-3) are integrin activators that mediate integrin-mediated signaling. In this study, we examined the spatiotemporal pattern of expression of FERMT1 in human chorionic villi throughout gestation and its role in HTR8-SVneo substrate adhesion and invasion. Placental villous tissue was obtained from patients undergoing elective terminations at weeks 8-14, as well as from term deliveries at weeks 37-40 and analyzed by immunofluorescence. Additionally, HTR8-SVneo trophoblast cells were transfected with FERMT1-specific siRNA or non-targeting siRNA (control) and used in cell-substrate adhesion as well as invasion assays. FERMT1 was primarily localized to membrane-associated regions at the base or around the periphery of the villous cytotrophoblast and proximal as well as distal cell column trophoblast. FERMT1 was also localized to endothelial cells of blood vessels in chorionic villi. siRNA-mediated depletion of FERMT1 in HTR8-SVneo cells did not markedly alter HTR8-SVneo cell-substrate adhesion but did significantly decrease invasion (P < 0.05) compared to control cells. These novel findings identify the presence of the integrin activator FERMT1 in trophoblast cells and that FERMT1 can regulate HTR8-SVneo cell invasion. FERMT1 may directly influence integrin activation and the subsequent integrin-mediated signaling and differentiation that underlies the acquisition of the invasive trophoblast phenotype in vivo.

Preeclampsia and phototherapy

Preeclampsia is a leading cause of maternal morbidity and mortality worldwide, complicating 2–8% of pregnancies, and is recognized as an independent risk factor for CVD by the ACC/AHA. The etiology of preeclampsia is poorly understood. However, the role of placental extracellular matrix (pECM) and extravillous trophoblast in placental blood vessel formation and preeclampsia is understood.The objective is to identify phenotypical changes in extravillous trophoblast (HTR8/SVneo) in a preeclampsia microenvironment using healthy and preeclamptic patient placental samples vs Dahl S rats (SS) as an animal model of superimposed preeclampsia and explore a novel therapeutic modality restoring trophoblastic phenotype by phototherapy using a 670nm light (NIR).Placentas from rats and patients were decellularized and characterized using pECM markers galectin‐3 and heparan sulfate proteoglycan 2 (HSP2) by western blot and immunofluorescence. pECM was used as substrate for HTR8/SVneo. Proliferation, migration, and apoptosis assays were used to determine cell behavior in the placental microenvironment. Immunofluorescence for placental growth factor (PLGF) and transforming growth factor (TGFβ) was performed on HTR8/SVneo cultured on both pECM. NIR was applied to HTR8/SVneo cultured on patient and rat pECM with 4J/cm2 intensity.In patient samples galectin‐3 expression was reduced by 34.21%±13.16 in preeclamptic PE compared to control patient pECM. HSP2 was reduced by 14.75%±6.11 in PE compared to control patient pECM. In rats galectin‐3 expression was reduced to 51.58%±3.78 in SS compared to control rat pECM. HSP2 was reduced to 45.15%± 0.63 in SS compared to control rat pECM.Migration of HTR8/SVneo on pECM (patient: control 11.08±0.82, PE 6.45±0.88; rats: control 3.87±0.96, SS 4.42±1.18) and proliferation of HTR8/SVneo on pECM were decreased in patients (# of cells X 1000) control: 59.125±6262.9, PE:67.125±7706.5 and rat control 5.16±0.79, SS 4.25±0.6 samples. Proliferation and migration were restored by NIR treatment: proliferation patient: control 101.375±2075.4; PE 106.875±4870.7, migration rat: control 12.58±7.06, SS 5.11±2.55. An increased number of apoptotic HTR8/SVneo was observed in preeclamptic pECM compared to healthy pECM: patient control 2.4±0.24, PE 3±0.4 and rat apoptotic cells control 5.2±0.89; SS 7.4±1.23). NIR decreased the number of apoptotic cells, patient:1.2 ±0.58, PE 1.4±0.5; rat control 3.9±0.43, SS 5.4±0.73.PLGF and TGFβ were downregulated in HTR8/SVneo when cultured on preeclamptic pECM from patients compared to healthy pECM and unchanged in rats. Fluorescence Intensity of PLGF in patient pECM; control 102.12±14.91, PE 68.77±9.73, TGFβ control 88.86±2.51, PE 63.59±4.83 and rats; PLGF control 65.18±0.79, SS 61.09±0.68; TGFβ control 60.78±0.48, SS 61.47 ± 0.45. The decrease was reversed by NIR treatment in patients; PLGF control 165.05±12.32, PE 175.14±12.48, TGFβ control 74.99±7.17, PE 91.03±2.27; rat PLGF control 66.58±0.99, SS 65.57±1.52, TGFβ control 66.67±1.58, SS 63.82±1.53).Extracellular matrix is altered in preeclampsia, and HTR8/SVneo trophoblasts respond similarly to altered matrix in preeclamptic placentas from patients and rats. 670nm light application restored the phenotype of HTR8/SVneo on preeclamptic pECM in patients and rats suggesting NIR as a therapeutic.

Down-regulated lncRNA AGAP2-AS1 contributes to pre-eclampsia as a competing endogenous RNA for JDP2 by impairing trophoblastic phenotype.

Recently, growing evidence has shown that aberrant long non‐coding RNA (lncRNA) expression in conjunction with an impaired trophoblastic phenotype could implicate the pathological process of pre‐eclampsia (PE). However, only a small portion of lncRNAs has been characterized with regard to the function and molecular mechanisms involved in PE. There are still gaps in the available knowledge; as a result, there are currently only a few applicable treatments for PE in the context of lncRNA. Here, we found that lncRNA AGAP2‐AS1 is abnormally down‐regulated in severe PE placenta tissues. Using human trophoblasts, we established that AGAP2‐AS1 knockdown could inhibit trophoblasts proliferation and invasion and promote cell apoptosis. Further, we showed that overexpression of AGAP2‐AS1 substantially stimulated the development of the trophoblastic phenotype. Through high‐throughput sequencing analysis, we demonstrated that silencing of AGAP2‐AS1 favourably regulated various genes which are relevant to trophoblastic growth and invasion. Mechanistically, AGAP2‐AS1 promoted the suppressor protein, Jun dimerization protein 2 (JDP2), by sponging miR‐574‐5p. Resultantly, further impairment of the trophoblastic phenotype was achieved by way of inhibiting cell growth, apoptosis and invasion. We also determined that the expression of AGAP2‐AS1 could be mediated by FOXP1. Our results showed that the down‐regulated expression of lncRNA AGAP2‐AS1 might serve as a key suppressor in PE via inhibition of JDP2 at the post‐transcriptional level by competing for miR‐574; thus, this presents a novel therapeutic strategy for PE.

Downregulated placental expression of linc00468 contributes to trophoblast dysfunction by inducing epithelial-mesenchymal transition.

BackgroundPreeclampsia (PE) is a widespread progressive condition that can occur pregnancy and is related to high maternal morbidity and fetal mortality in the perinatal period. However, the exact mechanism responsible has not been specific. Accumulating evidence has highlighted the prominent role of the epithelial‐mesenchymal transition (EMT) in the biological behaviors of PE.MethodsWe explored the role of a lincRNA in extravillous trophoblast (EVTs) cell viability, migration, invasion and apoptosis in vitro, along with the use of linc00468 knockdown or overexpression. Clinically, we discovered that the expression of linc00468 was frequently correlated with adverse clinical features and poor prognosis of PE patients.ResultsWe uncovered that linc00468 was downregulated in PE samples compared to in healthy tissues and in trophoblast cells. Functionally, gain and loss-of-function experiments demonstrated that linc00468 inhibited cell proliferation, migration, invasion and linc00468 accelerated apoptosis of the trophoblast phenotype in cell lines. Moreover, we demonstrated that downregulation of linc00468 promoted the expression of E-cadherin and β-catenin but reduced the expression of N-cadherin, Vimentin and Snail, resulting in progression of EMT.ConclusionsIn conclusion, linc00468 promoted EMT and a consequent increase in invasiveness in HTR-8/Svneo and JAR EVT cell lines. Our study provides the first evidence that linc00468 has a pivotal role in cell invasion and promotes intrinsic and extrinsic EMT ability of PE.