Preeclamptic Placental Tissue Research Articles

BMP5 promotes trophoblast functions upon N-glycosylation via the BMP5-SMAD1/5 signaling pathway in preeclampsia

IntroductionPreeclampsia (PE) is one of the most common pregnancy-related complications worldwide and currently lacks an effective treatment. While trophoblast cell dysfunction has been identified as the fundamental cause of PE, the underlying mechanisms remain unclear. Bone morphogenetic protein 5 (BMP5) is a secreted glycoprotein highly expressed in the placenta that is involved in cell proliferation, migration, and invasion. However, the role and mechanism of BMP5 glycosylation of trophoblast cell function remain unclear. MethodsThe expression of BMP5 and N-glycosylation in preeclamptic placental tissues was investigated. We predicted and validated the N-glycosylation sites of BMP5. Additionally, we evaluated the effect of BMP5 N-glycosylation on the proliferation, migration, invasion, and angiogenesis of human immortalized trophoblastic HTR-8/SVneo cells. Furthermore, the role of N-glycosylated BMP5 in activating the BMP5-SMAD1/5 signaling pathway and regulating trophoblastic cell functions was explored. ResultsOur study reveals that PHA-E + L (recognizing branching N-glycans) reactive N-glycans and BMP5 expression levels are lower in preeclamptic villous tissues compared to normal placental tissues. Additionally, we demonstrated that BMP5 is an N-glycosylation-modified protein. Furthermore, N-glycosylated BMP5 promoted the functional trophoblastic cells (HTR-8/SVneo). We also revealed that N-glycosylation of BMP5 regulates multiple cell functions through the BMP5-SMAD1/5 signaling pathway. ConclusionN-glycosylated BMP5 promotes trophoblast cell proliferation, migration, invasion, and angiogenesis. This study provides mechanistic insight as to how N-glycosylation of BMP5 in trophoblast cells can contribute to the pathogenesis of preeclampsia and provides a new basis for its diagnosis and treatment.

Impact of the Immunomodulatory Factor Soluble B7-H4 in the Progress of Preeclampsia by Inhibiting Essential Functions of Extravillous Trophoblast Cells.

A key aspect of preeclampsia pathophysiology is the reduced invasiveness of trophoblasts and the impairment of spiral artery remodelling. Understanding the causes of altered trophoblast function is critical to understand the development of preeclampsia. B7-H4, a checkpoint molecule, controls a wide range of processes, including T-cell activation, cytokine release, and tumour progression. Our previous findings indicated that B7-H4 levels are elevated in both maternal blood and placental villous tissue during the early stages of preeclampsia. Here, we investigated the function of B7-H4 in trophoblast physiology. Recombinant B7-H4 protein was used to treat human SGHPL-5 extravillous trophoblast cells. Biological functions were investigated using MTT, wound healing, and transwell assays. Signalling pathways were analysed by immunoblotting and immunofluorescence. The functionality of B7-H4 was further confirmed by immunoblotting and immunohistochemical analysis in placental tissues from control and preeclamptic patients following therapeutic plasma exchange (TPE) or standard of care treatment. This study showed that B7-H4 inhibited the proliferation, migration, and invasion capacities of SGHPL-5 extravillous cells while promoting apoptosis by downregulating the PI3K/Akt/STAT3 signalling pathway. These results were consistently confirmed in placental tissues from preterm controls compared to early-onset preeclamptic placental tissues from patients treated with standard of care or TPE treatment. B7-H4 may play a role in the development of preeclampsia by inhibiting essential functions of extravillous trophoblast cells during placental development. One possible mechanism by which TPE improves pregnancy outcomes in preeclampsia is through the elimination of B7-H4 amongst other factors.

Combined analysis of the effects of hypoxia and oxidative stress on DNA methylation and the transcriptome in HTR-8/SVneo trophoblast cells.

The alterations in DNA methylation and transcriptome in trophoblast cells under conditions of low oxygen and oxidative stress have major implications for pregnancy-related disorders. However, the exact mechanism is still not fully understood. In this study, we established models of hypoxia (H group) and oxidative stress (HR group) using HTR-8/SVneo trophoblast cells and performed combined analysis of genome-wide DNA methylation changes using reduced representation bisulphite sequencing and transcriptome expression changes using RNA sequencing. Our findings revealed that the H group exhibited a higher number of differentially methylated genes and differentially expressed genes than the HR group. In the H group, only 0.90% of all differentially expressed genes displayed simultaneous changes in DNA methylation and transcriptome expression. After the threshold was expanded, this number increased to 6.29% in the HR group. Notably, both the H group and HR group exhibited concurrent alterations in DNA methylation and transcriptome expression within Axon guidance and MAPK signalling pathway. Among the top 25 differentially methylated KEGG pathways in the promoter region, 11 pathways were commonly enriched in H group and HR group, accounting for 44.00%. Among the top 25 KEGG pathways in transcriptome with significant differences between the H group and HR group, 10 pathways were consistent, accounting for 40.00%. By integrating our previous data on DNA methylation from preeclamptic placental tissues, we identified that the ANKRD37 and PFKFB3 genes may contribute to the pathogenesis of preeclampsia through DNA methylation-mediated transcriptome expression under hypoxic conditions.

Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients.

The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells. Here, we optimized the protocol for studying BER in human placental tissue because the placenta is a non-invasive tissue for biomonitoring of early-life exposures, and it can be used to investigate molecular mechanisms associated with prenatal disorders. The optimal protein concentration of placental protein extracts for optimal damage recognition and incision was 2 mg protein/mL. The addition of aphidicolin did not lead to reduced non-specific incisions and was, therefore, not included in the optimized protocol. The interval between sample collection and analysis did not affect BER activity up to 70 min. Finally, this optimized protocol was tested on pre-eclamptic (PE) placental tissues (n = 11) and significantly lower BER activity in PE placentas compared to controls (n = 9) was observed. This was paralleled by a significant reduction in the expression of BER-related genes and increased DNA oxidation in PE placentas. Our study indicates that BER activity can be determined in placentas, and lower activity is present in PE compared with healthy. These findings should be followed up in prospective clinical investigations to examine BER's role in the advancement of PE.

Definition and Quantification of Three-Dimensional Imaging Targets to Phenotype Pre-Eclampsia Subtypes: An Exploratory Study

Pre-eclampsia is a severe placenta-related complication of pregnancy with limited early diagnostic and therapeutic options. Aetiological knowledge is controversial, and there is no universal consensus on what constitutes the early and late phenotypes of pre-eclampsia. Phenotyping of native placental three-dimensional (3D) morphology offers a novel approach to improve our understanding of the structural placental abnormalities in pre-eclampsia. Healthy and pre-eclamptic placental tissues were imaged with multiphoton microscopy (MPM). Imaging based on inherent signal (collagen, and cytoplasm) and fluorescent staining (nuclei, and blood vessels) enabled the visualization of placental villous tissue with subcellular resolution. Images were analysed with a combination of open source (FIJI, VMTK, Stardist, MATLAB, DBSCAN), and commercially (MATLAB) available software. Trophoblast organization, 3D-villous tree structure, syncytial knots, fibrosis, and 3D-vascular networks were identified as quantifiable imaging targets. Preliminary data indicate increased syncytial knot density with characteristic elongated shape, higher occurrence of paddle-like villous sprouts, abnormal villous volume-to-surface ratio, and decreased vascular density in pre-eclampsia compared to control placentas. The preliminary data presented indicate the potential of quantifying 3D microscopic images for identifying different morphological features and phenotyping pre-eclampsia in placental villous tissue.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles loaded with TFCP2 activate Wnt/β-catenin signaling to alleviate preeclampsia.

Failures in invasive extravillous trophoblasts (EVTs) migration into the maternal uterus have been noticed in preeclampsia (PE). Human umbilical cord mesenchymal stem cell (hUCMSC)-derived extracellular vesicles (EVs) have been highlighted for the role as a potential therapeutic method in PE. This study intends to investigate the mechanistic basis of hUCMSCs-derived EVs loaded with bioinformatically identified TFCP2 in the activities of EVTs of PE. Primary human EVTs were exposed to hypoxic/reoxygenation (H/R) to mimic the environment encountered in PE. The in vivo PE-like phenotypes were induced in mice by reduced uterine perfusion pressure (RUPP) surgery. CCK-8, Transwell and flow cytometry assays were performed to detect proliferation, migration, invasion and apoptosis of H/R-exposed EVTs. More importantly, EVs were extracted from hUCMSCs and transduced with ectopically expressed TFCP2, followed by co-culture with EVTs. TFCP2 was found to be down-regulated in the preeclamptic placental tissues and in H/R-exposed EVTs. hUCMSCs-EVs loaded with TFCP2 activated the Wnt/β-catenin pathway, thereby promoting the proliferative, migratory, and invasive potential of EVTs. Furthermore, overexpression of TFCP2 alleviated PE-like phenotypes in mice, which was associated with activated Wnt/β-catenin pathway. From our data we conclude that hUCMSCs-EVs overexpressing TFCP2 may be instrumental for the therapeutic targeting and clinical management of PE.

Elevated PGT promotes proliferation and inhibits cell apoptosis in preeclampsia by Erk signaling pathway

Prostaglandins participate in maternal recognition of pregnancy, implantation and maintenance of gestation. Prostaglandin transporter (PGT), as a candidate molecule of prostaglandin carriers, might be involved in the pathogenesis of preeclampsia. In preeclampsia (PE) patients’ placental tissue, we identified PGT by RNA sequencing, measured its expression pattern by quantitative real-time PCR and Western blot. PGT was found to be upregulated in preeclamptic placental tissue. The expression pattern of PGT in PE was double confirmed by eight Gene Expression Omnibus (GEO) databases. In abortion tissues at 6–8 weeks, we then observed the cellular location of PGT by Immunofluorescence technique (IF) and found PGT located in trophoblast cell of the placenta of early pregnancy. In vitro studies revealed that forced expression of PGT in HTR8/Sveno cell inhibited its apoptosis, but promoted its proliferation by activating Erk signaling. In vivo study, we used reduced uterine perfusion pressure (RUPP) rat model and L-NAME-induced preeclampsia-like rats to study the possible role of PGT in preeclampsia. And PGT was found to be upregulated in both preeclampsia rat models by Immunohistochemical (IHC) staining. Newly identified PGT plays an important role in trophoblast proliferation via Erk signaling, providing new insights for understanding the pathogenesis of PE.

RGS2 promotes estradiol biosynthesis by trophoblasts during human pregnancy

Production of estradiol (E2) by the placenta during human pregnancy ensures successful maintenance of placental development and fetal growth by stimulating trophoblast proliferation and the differentiation of cytotrophoblasts into syncytiotrophoblasts. Decreased levels of E2 are closely associated with obstetrical diseases such as preeclampsia (PE) in the clinic. However, the mechanisms underlying the inhibition of placental E2 biosynthesis remain poorly understood. Here, we report that regulator of G-protein signaling 2 (RGS2) affects E2 levels by regulating aromatase, a rate-limiting enzyme for E2 biosynthesis, by using human trophoblast-derived JEG-3 cells and human placental villus tissues. RGS2 enhanced the protein degradation of the transcription factor heart and neural crest derivatives expressed 1 (HAND1) by suppressing ubiquitin-specific protease 14 (USP14)-mediated deubiquitination of HAND1, resulting in the restoration of HAND1-induced trans-inactivation of the aromatase gene and subsequent increases in E2 levels. However, aromatase bound to RGS2 and repressed RGS2 GTPase activating protein (GAP) activity. Moreover, we observed a positive correlation between RGS2 and aromatase expression in clinical normal and preeclamptic placental tissues. Our results uncover a hitherto uncharacterized role of the RGS2-aromatase axis in the regulation of E2 production by human placental trophoblasts, which may pinpoint the molecular pathogenesis and highlight potential biomarkers for related obstetrical diseases.

Placental DAAM2 is unaltered in preeclampsia, but upregulated by treatment with proton pump inhibitors.

Dishevelled Associated Activator Of Morphogenesis 2 (DAAM2) levels are elevated in the maternal circulation and placenta in pregnancies complicated by fetal growth restriction. However, placental DAAM2 levels in cases of preeclampsia have not previously been explored. Here, we examined placental DAAM2 in pregnancies complicated by preterm preeclampsia, and whether candidate preeclampsia therapeutics altered its expression. DAAM2 mRNA and protein levels were assessed in placental tissue from cases of preterm preeclampsia and gestation-matched controls (delivering ≤ 34weeks; qPCR and western blot respectively). Short interfering RNAs were used to silence DAAM2 in isolated primary cytotrophoblast under normoxic (8% O2) and hypoxic (1% O2) conditions, and expression of anti-angiogenic sFLT-1, angiogenic PGF, antioxidant, fetal growth, and inflammatory genes assessed. DAAM2 expression was measured in placental explant tissue from pregnancies complicated by preeclampsia, treated with three proton pump inhibitors (100µM esomeprazole, lansoprazole, and rabeprazole). DAAM2 expression was significantly reduced in preeclamptic placental tissue compared to controls, but protein production was unchanged. Silencing DAAM2 in hypoxic cytotrophoblast increased sFLT-i13 isoform expression, but did not alter sFLT-e15a or PGF expression, or sFLT-1 secretion. DAAM2 knockdown did not alter expression of antioxidant (NQO-1, TXN, GCLC), fetal growth (SPINT1), or inflammasome (NLRP3) genes. Esomeprazole and lansoprazole, but not rabeprazole, increased DAAM2 expression in placental explant tissue from cases of preeclampsia. Placental DAAM2 protein is not significantly altered in placental tissue in cases of preeclampsia, and its suppression does not alter sFLT-1 secretion. Hence, placental DAAM2 is unlikely to drive the pathogenesis associated with preeclampsia.

WNT3 hypomethylation counteracts low activity of the Wnt signaling pathway in the placenta of preeclampsia

Preeclampsia is a hypertensive disorder of pregnancy. Many studies have shown that epigenetic mechanisms may play a role in preeclampsia. Moreover, our previous study indicated that the differentially methylated genes in preeclampsia were enriched in the Wnt/β-catenin signaling pathway. This study aimed to identify differentially methylated Wnt/β-catenin signaling pathway genes in the preeclamptic placenta and to study the roles of these genes in trophoblast cells in vitro. Using an Illumina Infinium HumanMethylation 850 K BeadChip, we found that the Wnt signaling pathway was globally hypermethylated in the preeclamptic group compared with the term birth group, but hypomethylated in the preeclamptic group compared with the preterm birth group. Among all Wnt/β-catenin signaling pathway factors, WNT3 was the most significantly differentially expressed gene and was hypomethylated in the preeclamptic group compared to the nonhypertensive groups, namely, the preterm birth group and term birth group. This result was confirmed by pyrosequencing. Through quantitative real-time PCR and western blot analysis, the WNT3 gene was found to be highly expressed in preeclamptic placental tissues, in contrast to other WNT factors, which were previously reported to be expressed at low levels in placental tissues. Additionally, in the HTR8/SVneo cell line, knockdown of WNT3 suppressed the Wnt/β-catenin signaling pathway, consistent with the findings for other WNT factors. These results prompted us to speculate that the WNT3 gene counteracts the low activation state of the Wnt signaling pathway in the preeclamptic placenta through methylation modification.

Expression of CD44 and IL-10 in normotensive and preeclamptic placental tissue.

We aimed to demonstrate cell-cell adhesion and apoptotic changes in preeclamptic placentas by examining the expression of CD44 and IL-10. Placenta samples of 15 preeclamptic and 15 healthy 35-38th week-pregnant women were involved in the study. Tissue samples were taken only from the maternal side of the placenta and fixed in 10% formaldehyde, then blocked in paraffin wax and 5 μm-thick sections were cut and stained with Masson Trichrome. Antigen retrieval was performed for sections, incubated with CD44 antibody and anti-IL-10 antibody. After the application of streptavidin peroxidase followed by AEC chromogen solution, sections were counterstained with Mayer hematoxylin. In the preeclampsia group, increased CD44 positive expression was observed in maternal decidua cells and fibroblast cells close to root villi. CD44 was positively expressed in muscle cells around the blood vessels, mucosal connective tissue areas, syncytial nodes, and syncytial bridges. In the preeclampsia group, significant increased IL-10 expression was seen in subendothelial layers of the medium-sized vessels in the maternal region. IL-10 was also positively expressed in decidua cells outside the vessels, and inflamed connective tissue areas, chorionic villus cells with intense inflammation in intervillous spaces. CD44 was found to be an essential molecule in the regulation of vascular permeability, inflammatory response, activation of the cells, cell-to-cell interaction, and the signaling pathways to which they are associated. Since IL-10 regulates appropriate pregnancy outcomes and contributes to the balance of anti-inflammatory signals via both paracrine and autocrine regulators of trophoblast activity, we proposed that it might be a key to elucidate the etiology of preeclampsia with CD44 receptor.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Novel Mechanisms of Preeclampsia Prevention via SGK1

Preeclampsia (PE), a hypertensive disorder of pregnancy, involves poor trophoblast invasion, poor spiral artery remodeling, and an early systemic inflammatory immune response. In the chronic infusion of vasopressin (AVP) mouse model of PE, the mice showed similar obstetric and immune alterations observed in humans. We have shown that a single dose of betamethasone (BMTZ) early in pregnancy in AVP infused dams will reverse pregnancy‐induced hypertension and proteinuria. However, the mechanism of this reversal is still unclear. SGK1 is a serine/threonine kinase that is stimulated by BMTZ. SGK1 dysregulation, as well as human genetic variants in SGK1, have been implicated in the development of hypertension through a decrease in the anti‐inflammatory TH2 renal milieu. Further SGK1−/− mice exhibit fetal growth restriction and demise via this pro‐inflammatory shift leading to decidual apoptosis and poor trophoblast invasion, a feature well demonstrated in PE. Further, miR365 has been shown to decrease SGK‐1 expression in human deciduas from pregnancies with poor placentation. Together, these data suggest BMTZ reverses AVP‐induced preeclampsia via upregulation of SGK1 in mice, and that human preeclamptic women have a reduced expression of SGK1 via regulation by miR365. Thus, we hypothesized that AVP and SGK1 regulate the expression of miR365 in placental cells, playing a role in the development of preeclampsia.To address our hypothesis, we utilized primary placental tissues and HTR8/SVneo trophoblast cells to examine the expression of miR365 and SGK1. Placental tissues were used to determine if miR‐365a‐3p is differentially expressed in human preeclampsia. Human preeclamptic and non‐preeclamptic samples were obtained from the Maternal Fetal Tissue Bank (MFTB) (IRB# 200910784) at the University of Iowa. HTR8/SVneo cells were treated for 24 hours with either saline, AVP (300ng/mL), AVP+BMTZ (10−4 mM), AVP+SGK1 inhibitor (1mM), or AVP+BMTZ+SGK1 inhibitor. Quantitative real‐time PCR was used to measure miR365 and SGK1. In primary placental tissues, there is a 1.5‐fold lower expression of miR365 in preeclamptic compared to non‐preeclamptic samples (p=0.016, n=20). Although, there was no difference in expression of SGK1 in tissues from preeclamptic versus non‐preeclamptic (p>0.05, n=20) placentas. HTR8/SVneo cells showed no difference in the expression of miR365 (p>0.05, n=4) between different treatments. No difference was observed in the expression of SGK1 in HTR8/SVneo treated cells (p>0.05, n=4). These findings support the conclusion that miR365 is differentially expressed in preeclamptic placental tissues versus non‐preeclamptic placental tissues. Additionally, treatments with AVP, BMTZ, and/or SGK1 inhibitor does not change the expression of SGK1 and miR365. Our data suggest the reduced expression of miR365 observed in preeclamptic pregnancies is mediated via an SGK1 independent mechanism.Support or Funding InformationFunding provided by The American Physiological Society STRIDE fellowship (CL), American Heart Association Strategically Focused Research Network in Hypertension (MS, JG, SS, DS), American Heart Association Postdoctoral Fellowship (SS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

EGFR (Epidermal Growth Factor Receptor) Signaling and the Mitochondria Regulate sFlt-1 (Soluble FMS-Like Tyrosine Kinase-1) Secretion.

Preeclampsia is a hypertensive disorder of pregnancy characterized by maternal endothelial dysfunction and end-organ damage. The antiangiogenic factor, sFlt-1 (soluble FMS-like tyrosine kinase-1) has been strongly implicated in the pathogenesis of preeclampsia. sFlt-1 is secreted into the maternal circulation where it antagonizes VEGF (vascular endothelial growth factor) and ultimately disrupts vascular homeostasis. However, the upstream mechanisms regulating release of sFlt-1 are poorly characterized. We investigated the roles of key prosurvival pathways, EGFR (epidermal growth factor receptor) signaling, and the mitochondria, in regulating sFlt-1 production. We initially found that the mRNA and protein of EGFR and downstream adaptor molecules were significantly increased in preeclamptic placental tissue relative to normotensive controls. Inhibiting the EGFR signaling cascade using siRNA (small interfering ribonucleic acid) or small molecule inhibitors significantly reduced sFlt-1 release from primary cytotrophoblast (placental cells). Additionally, inhibiting the mitochondrial electron transport chain or activating downstream energy-sensing molecules (AMPK [AMP-activated kinase], SIRT1 [sirtuin 1 ], and PGC1α [PPAR-γ co-activator 1]) also significantly reduced sFlt-1 secretion from cytotrophoblast cells, without affecting EGFR signaling. In vivo, treating pregnant mice with gefitinib (an EGFR inhibitor) or resveratrol (perturbs mitochondrial function) significantly reduced circulating murine sFlt-1 compared with vehicle control. Furthermore, treating primary cytotrophoblasts with therapeutics which have been previously found to reduce sFlt-1 secretion, either reduced EGFR signaling (esomeprazole and statins) or perturbed mitochondrial function (esomeprazole, resveratrol, and metformin). Additionally, targeting both pathways simultaneously produced additive reductions in sFlt-1 secretion. Thus, we have identified 2 key survival pathways that seem to be overactive in preeclampsia and involved in the regulation of placental sFlt-1 secretion.

MiR-616-3p modulates cell proliferation and migration through targeting tissue factor pathway inhibitor 2 in preeclampsia.

Despite improvements in diagnosis and treatment, preeclampsia (PE) continues to pose a significant risk of maternal and foetal morbidity and mortality if not addressed promptly. An increasing number of studies have suggested that tissue factor pathway inhibitor 2 (TFPI2) acts as a suppressor gene, possibly inhibiting multiple serine proteases affecting cell proliferation and migration. It plays an essential role in the occurrence and development of PE, but the pathogenesis remains unclear. In our research, we performed western blotting, immunohistochemistry and qPCR assays to investigate TFPI2 and miR-616-3p expression in preeclamptic placental tissues. Cell assays were performed in HTR-8/SVneo and JEG3 cell lines. Cell proliferation and migration events were investigated by MTT, EdU and transwell assays. In conjunction with bioinformatics analysis, luciferase reporter assays were performed to elucidate the mechanism by which miR-616-3p binds to TFPI2 mRNA. We established that TFPI2 protein levels were significantly upregulated in PE placental tissues. In addition, we found that miR-616-3p binds specifically to the 3'-UTR region of TFPI2 mRNA. Furthermore, miR-616-3p knockdown or TFPI2 overexpression substantially impaired cell growth and migration, whereas miR-616-3p upregulation or TFPI2 knockdown stimulated cell proliferation and migration. This miR-616-3p/TFPI2 axis was also found to affect the epithelial-mesenchymal transition process in PE. Our results demonstrated that TFPI2 plays a vital role in the progression of PE and might provide a prospective therapeutic strategy to mitigate the severity of the disorder.

Downregulated lncRNA HOXA11-AS Affects Trophoblast Cell Proliferation and Migration by Regulating RND3 and HOXA7 Expression in PE

The long noncoding RNA HOXA11-AS displays abnormal expression in numerous human diseases. However, its function and biological mechanisms remain unclear in preeclampsia (PE). In this study, we report that HOXA11-AS is significantly downregulated in preeclamptic placental tissues and could contribute to the occurrence and development of PE. Silencing of HOXA11-AS expression could significantly suppress trophoblast cell growth and migration, whereas HOXA11-AS overexpression facilitated cell growth in the HTR-8/SVneo, JEG3, and JAR cell lines. RNA-seq analysis also indicated that HOXA11-AS silencing preferentially regulated numerous genes associated with cell proliferation and cell migration. Mechanistic analyses showed that HOXA11-AS could recruit Ezh2 and Lsd1 protein and regulate RND3 mRNA expression in the nucleus. In the cytoplasm, HOXA11-AS modulates HOXA7 expression by sponged miR-15b-5p, affecting trophoblast cell proliferation. Together, these data confirm that aberrant expression of HOXA11-AS is involved in the occurrence and development of PE and may act as a prospective diagnosis and therapeutic target in PE.

FN1, FOS, and ITGA5 induce preeclampsia: Abnormal expression and methylation

ABSTRACTObjective: As a frequent hypertensive disorder in pregnancy, preeclampsia causes 15% of all preterm deliveries. This study aims to understand its mechanism via analyzing gene expression and methylation data.Methods: Human gene expression microarray data (GSE73374) and corresponding DNA methylation microarray data (GSE73375) of preeclamptic placental tissues (n = 19) and normotensive placental tissues (n = 17) were obtained from the Gene Expression Omnibus. Differentially expressed genes (DEGs), differentially methylated regions (DMRs) and CpG islands (DMIs) were identified and compared. Pathway enrichment analysis was performed, and the functional interactions were investigated.Results: A total of 392 DEGs, 2667 DMRs, and 464 DMIs were found between the two groups, and a functional interaction network of DEGs was established. Then, 28 potential key genes were identified, which were oppositely regulated in preeclampsia regarding expression and methylation. Eight of the potential key genes had DMIs, among which ITGA5 interacted with FN1 and FOS. The 3 DEGs were up-regulated, and their DMIs were hypomethylated.Conclusion: FN1, FOS, and ITGA5 might promote the development of preeclampsia via modulating differentiation/apoptosis/invasion of human extravillous trophoblasts, as well as focal adhesion, VEGFR3 signaling, β1 integrin cell surface interactions, α9β1/α4β1 integrin signaling, AP-1/ATF-2/HIF-1α transcription factor networks, and Fc-epsilon receptor I signaling in mast cells.

Fatty acid-binding protein3 expression in BeWo cells, a human placental choriocarcinoma cell line

Cellular uptake of long chain fatty acids in human placental trophoblasts is thought to be mediated by several membrane- and cytoplasmic fatty acid-binding proteins (FABP). FABP3 was shown to be involved in long chain polyunsaturated fatty acids (LCPUFA) uptake in human trophoblastic choriocarcinoma cells, BeWo as the uptake of arachidonic acid,20:4n-6 (ARA) was decreased in FABP3-knockdown BeWo cells. However, the regulation of expression of FABP3 in these cells is not yet well known. The aim of the present study was to examine the FABP3 expression by LCPUFAs, insulin and LXR agonists in BeWo cells. Among all these fatty acids tested, only ARA dose-dependently stimulated the expression of FABP3 protein in these cells after 24h incubation while other fatty acids had no such effect. In addition, LXR agonist and insulin dose-dependently increased FABP3 protein expression in these cells after 24h incubation. Insulin-stimulated FABP3 protein expression was accompanied with an increased arachidonic acid uptake. Differentiated BeWo cells had lesser expression of FABP3 protein than in the undifferentiated cells as the cellular differentiation state was measured by hCG production. In preeclamptic placental tissue, lowered expression of FABP3 protein was observed compared with those in normal pregnancy. All these data indicate that FABP3 may in be part involved in ARA uptake in these cells and its expression may be regulated by ARA, insulin, LXR and the state of cellular differentiation.

Long Non-Coding RNA Uc.187 Is Upregulated in Preeclampsia and Modulates Proliferation, Apoptosis, and Invasion of HTR-8/SVneo Trophoblast Cells.

Among the preeclampsia-related long non-cording RNAs (lncRNAs) screened with a gene chip in our preliminary study, uc.187 attracted our attention because of its high conservation across different species and significant positive correlation with preeclampsia (PE). The literature and bioinformatics analysis suggested that lncRNA uc.187 might be associated with cell growth, invasion, and apoptosis. The expression of uc.187 in severe preeclamptic placentas (n = 31) and normal placentas (n = 18) was evaluated by real-time reverse transcription polymerase chain reaction (qRT-PCR). We constructed a silencing lentivirus vector (uc.187 siRNA) to explore the biological function of uc.187 in the development and progression of HTR-8/SVneo trophoblast cells in vitro. Furthermore, we utilized CCK8 analysis, a transwell invasion assay, and flow cytometry to determine the role of uc.187 in the proliferation, invasion, and apoptosis of HTR-8/SVneo trophoblast cells. The proteins related to proliferation (PCNA, Ki67), invasion (MMP-2/-9 and TIMP-1), and apoptosis (caspase-3, Bcl-2) were evaluated with a Western blot assay. The results showed that there was an obvious upregulation of uc.187 expression in preeclamptic placental tissues. In addition, uc.187 silencing enhanced cell proliferation and invasion and reduced the cellular apoptotic response. Taken together, our findings suggest for the first time that abnormal expression of lncRNA uc.187 may lead to the aberrant biological behavior of HTR-8/SVneo cells. Therefore, we propose uc.187 as a novel lncRNA molecule that might contribute to the development of PE and might represent a potential diagnostic and therapeutic target for this disease. J. Cell. Biochem. 118: 1462-1470, 2017. © 2016 Wiley Periodicals, Inc.

Overexpressed HO-1 is associated with reduced STAT3 activation in preeclampsia placenta and inhibits STAT3 phosphorylation in placental JEG-3 cells under hypoxia.

IntroductionInadequate trophoblast invasion and placentation are widely believed to contribute to preeclampsia, and multiple lines of evidence indicate the involvement of hypoxia in preeclampsia. However, the molecular mechanisms underlying the association of placental hypoxia with preeclampsia are not clear.Material and methodsThe present study focused on the role in preeclampsia of heme oxygenase 1 (HO-1), which is an inducible isoform of HO in response to hypoxia, via examining the expression of HO-1 and the expression and phosphorylation (Tyr705) of Signal transducer and activator of transcription (STAT) 3 in preeclamptic placentas via the immunohistochemical method, western blotting assay and RT-qPCR method. Then we investigated the regulation by HO-1 of the expression and phosphorylation of STAT3 in human placental choriocarcinoma JEG-3 cells under hypoxia.ResultsThere was upregulation of HO-1 at both mRNA (1.506 ±0.08347 (N = 37) vs. 1.000 ±0.08854 (N = 31), p < 0.0001) and protein (0.630 ±0.155 (N = 35) vs. 0.310 ±0.052, 0.630 ±0.155 (N = 35), p < 0.001) levels and a reduced level of STAT3 phosphorylation (Tyr 705) in the preeclamptic placental tissues, compared to normal placental tissues (0.143 ±0.027 (N = 35) vs. 0.194 ±0.028 (N = 35), p < 0.01). Also, in vitro experiments demonstrated that HO-1 was markedly promoted by hypoxia in human placental choriocarcinoma JEG-3 cells, 6 or 12 h post treatment (p < 0.05 or p < 0.01). However, the STAT3 phosphorylation (Tyr 705) was attenuated by sustained hypoxia (p < 0.01). Moreover, it was demonstrated that HO-1 overexpression significantly inhibited the hypoxia-promoted STAT3 phosphorylation (Tyr 705).ConclusionsHO-1 was overexpressed in PE placenta, in association with reduced STAT3 phosphorylation (Tyr 705). HO-1 inhibits the STAT3 phosphorylation in placental JEG-3 cells under hypoxia. Thus, we speculate that overexpressed HO-1 might contribute to the reduced STAT3 phosphorylation (Tyr 705) and the pathogenesis of preeclampsia.