Placenta Of Preeclampsia Patients Research Articles

MicroRNA-203 Targets Insulin-Like Growth Factor Receptor 1 to Inhibit Trophoblast Vascular Remodeling to Augment Preeclampsia.

Preeclampsia (PE) is a pregnancy-specific condition featured by high blood pressure, edema, and proteinuria. Research about the role of microRNA (miR)-203 in PE remains insufficient. This experiment is designed to investigate the specific role of miR-203 in trophoblasts in PE. miR-203 expression in placenta tissues of normal pregnant women and PE patients was examined to analyze the relevance between miR-203 and PE diagnostic efficiency and between miR-203 and blood pressure (systolic pressure and diastolic pressure) and proteinuria of PE patients. miR-203 expression was downregulated in hypoxia-cultured trophoblasts using miR-203 inhibitor to assess matrix metalloproteinase-9 (MMP-9) level. Then, the angiogenesis of trophoblasts with different treatments was determined. Subsequently, the target relation between miR-203 and insulin-like growth factor receptor 1 (IGF-1R) was predicted and verified. Additionally, the effect of IGF-1R in the mechanism of miR-203 modulating trophoblast vascular remodeling was detected. miR-203 was overexpressed in the placenta of PE patients and it acted as a promising diagnostic indicator for PE. Moreover, miR-203 was positively associated with blood pressure (systolic pressure and diastolic pressure) and proteinuria of PE patients. miR-203 silencing in hypoxia-cultured trophoblasts enhanced trophoblast vascular remodeling. Mechanically, miR-203 bound to IGF-1R to suppress its transcription. IGF-1R downregulation counteracted the promotive effect of miR-203 silencing on trophoblast vascular remodeling. miR-203 was overexpressed in PE, and it targeted IGF-1R to limit trophoblast vascular remodeling. · miR-203 is overexpressed in the placenta of PE patients.. · miR-203 acts as a potential diagnostic marker for PE.. · miR-203 targets IGF-1R to reduce trophoblast vascular remodeling in PE..

Identification of Key Molecules and lncRNA-miRNA-mRNA ceRNA Network in Preeclampsia.

BackgroundPreeclampsia (PE) is an idiopathic hypertensive disorder of pregnancy and is the leading cause of maternal death, fetal malformation, and premature birth. The purpose of this study is to identify the key molecules and lncRNA-related competitive endogenous (ceRNA) regulatory network in PE.MethodsThe differentially expressed mRNAs (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified between PE and control using the Deseq R package. In addition, we performed Geno ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) on DEGs and DELs-target genes to explore their function. The ceRNA network was established based on lncRNA-miRNA and miRNA-mRNA interactions and visualized using Cytoscape software. LINCO2532, SLCO4A1-AS1, miR23a-5p, and DYNLRB1 were selected for qRT-PCR assay.ResultsUsing microarray analysis, we screened 726 DELs (456 upregulated and 370 downregulated), 49 DEMs (37 upregulated and 12 downregulated), and 318 DEGs (230 upregulated and 88 downregulated) between PE patients and control. Based on lncRNA-miRNA pairs and miRNA-mRNA pairs, the ceRNA network was constructed, which contained 16 lncRNA, 1 miRNA (miR-23a-5p), and 1 mRNA (DYNLRB1). LncRNA (LINCO2532 and SLCO4A1-AS1) and DYNLRB1 were downregulated and the expression of miR23a-5p was upregulated in PE patients compared with healthy controls.ConclusionIn this study, the novel ceRNA network was established in the placentas of PE patients. It elucidated the regulatory mechanism of PE, and identified novel PE biomarkers, which have important guiding significance for clinical treatment and further scientific research of PE.

The abnormal expression of Tim-3 is involved in the regulation of myeloid-derived suppressor cells and its correlation with preeclampsia.

Maternal immune system tolerance to the semi-allogeneic fetus is critical to a successful pregnancy. We previously reported that myeloid-derived suppressor cells (MDSC) was associated with maternal immune imbalance. T cell immunoglobulin and mucin-containing protein 3 (Tim-3)/Galectin-9 (Gal-9) pathway modulates function of various immune cells in maternal-fetal interface. However, the regulatory effects of Tim-3/Gal-9 signaling on MDSCs and its role in preeclampsia (PE) remain unclear. In the current study we investigated the expression of Tim-3 on MDSC in preeclampsia (PE) patients to further explore the pathogenesis of PE. The proportion of Tim-3+ M-MDSC (monocytic MDSC) cells was higher in PE patients than in healthy control. Meanwhile, the protein expression of Gal-9, as the ligand of Tim-3, was increased in placenta of PE patients. M-MDSC also expressed a higher level of interferon-γ (IFN-γ) and a lower level of transforming growth factor-β (TGF-β) in PE. Furthermore, our study suggested that blocking Tim-3 could attenuate the inhibitory function of MDSC. The abnormal expression of Tim-3 on MDSC might be involved in the pathogenesis of PE, and could be a marker to evaluate the immune function in PE.

TRIB3-Regulated Akt Signal Pathway Affects Trophoblast Invasion in the Development of Preeclampsia.

The aim of the study is to explore the mechanism of tribbles pseudokinase 3 (TRIB3)-regulated Akt pathway in the development of preeclampsia (PE). TRIB3 expression in the placenta of PE patient was determined by quantitative reverse transcriptase polymerase chain reaction and western blotting. Then HTR-8/SVneo or JEG-3 cells were transfected and divided into Mock, Control siRNA, TRIB3 siRNA-1, and TRIB3 siRNA-2 groups. Cell proliferation, invasion, and migration were determined by MTT assay, Transwell assay, and wound healing test, while the expression of TRIB3 and Akt pathway was measured by western blotting. PE rats were treated with TRIB3 siRNA, and blood pressure, 24-hour urinary protein, as well as serum levels of sFlt-1 and vascular endothelial growth factor (VEGF) were measured. The placenta of PE patients presented with increased TRIB3 expression. In comparison with Mock group, the proliferation, invasion, and migration of HTR-8/SVneo and JEG-3 cells in TRIB3 siRNA-1 group and TRIB3 siRNA-2 group increased, with decreased TRIB3 expression but enhanced expression of p-Akt/Akt, MMP-2, and MMP-9. Rats in PE group showed increases in mean arterial pressure, SBP, 24-hour urinary protein, and serum sFlt-1 levels, but decreases in serum VEGF levels, fetal weight, and placental efficiency. Moreover, TRIB3 expression was upregulated, while p-Akt/Akt was downregulated in the placenta of rats in PE group. However, indicators above were significantly improved in rats treated with TRIB3 siRNA. TRIB3 was upregulated in the PE placenta, while silencing TRIB3 activated the Akt signaling pathway to promote the invasion and migration of trophoblast both in vitro and in vivo and ameliorated the development of PE symptoms in the PE rat model. · The TRIB3 expression was increased in the placenta of PE patient. · Silencing TRIB3 activates Akt signal pathway.. · Silencing TRIB3 improves the pathological process of preeclampsia rat..

The Role of IL-37 and IL-38 in Obstetrics Abnormalities.

There are two fairly common complications during pregnancy, i.e., gestational diabetes mellitus (GDM) and pre-eclampsia, which are independent, but are also closely linked in prevalence in pregnant women, with potential serious adverse consequences. IL-37 and IL-38, which belong to the IL-1 superfamily, participate in anti-inflammatory responses. Dysregulation of IL-37 and IL-38 has been observed in many auto-immune diseases. IL-37 is substantially reduced in the umbilical cords and placentas of GDM subjects, but IL-37 is significantly induced in the placentas of pre-eclampsia patients, suggesting there are differential regulatory roles of IL-37 in obstetrics, despite IL-37 being an anti-inflammatory mediator. Furthermore, IL-38 is substantially increased in the umbilical cords and placentas of GDM subjects, but minimal difference is observed in the placentas from pre-eclampsia patients. These data imply that IL-38 is also regulated independently within the diseased placentas. This review provides some insight for both basic scientists and medical practitioners to manage these patients effectively.

Elevated S100A9 in preeclampsia induces soluble endoglin and IL-1β secretion and hypertension via the NLRP3 inflammasome.

Maternal systemic and placental inflammatory responses participate in the pathogenesis of hypertensive disorders of pregnancy including preeclampsia, a pregnancy-specific syndrome, although the role of inflammation remains unclear. The NLRP3 inflammasome has been implicated in the control of sterile inflammation involved in preeclampsia. In the present study, we hypothesized that S100A9, as major alarmin, are associated with the pathogenesis of preeclampsia and induction of a preeclampsia-like phenotype in pregnant mice. Plasma were taken from normal pregnant women and preeclampsia patients. Human placental tissues, trophoblast cell line Sw.71 cells, and human umbilical vein endothelial cells (HUVEC) were treated with S100A9 with or without inhibitors associated with NLRP3 inflammasome. Pregnant mice were administered S100A9. S100A9 was elevated in plasma and released from placentas of preeclampsia patients. S100A9 activated the NLRP3 inflammasome, resulting in IL-1β secretion, by human placental tissues and trophoblasts. In addition, secretion of soluble endoglin, a main contributor to the pathogenesis of preeclampsia, is regulated via S100A9-stimulated NLRP3 inflammasome activation in the human placenta and HUVECs. S100A9 administration significantly elevated maternal blood pressure and neutrophil accumulation within the placentas of pregnant mice, and both were significantly decreased in Nlrp3-knock out pregnant mice. The results of this study demonstrated that S100A9 acts as a danger signal to activate the NLRP3 inflammasome in the placenta, associating with hypertension during pregnancy.

Vitamin D-Binding Protein is Involved in the Pathogenesis of Preeclampsia by Inhibiting the Tyrosine Phosphorylation of Vascular Endothelial Growth Factor Receptor-2 in Endothelial Cells

Abstract Objective: The role of Vitamin D-binding protein (DBP) in preeclampsia (PE) pathogenesis is unknown. In this study, we compared the expression of DBP in the placentas of PE patients with the placentas of normotensive pregnant women with placenta previa controls, and aimed to explore the effect of DBP on endothelial cells (ECs) and the underlying mechanism. Methods: DBP expression in placental tissues collected from PE patients and controls was evaluated by immunohistochemistry. The downregulation and upregulation of DBP expression in HTR-8/SVneo cells were examined using DBP-targeting small interfering RNA (siRNA) and DBP-expression vector, respectively. The conditioned media of these DBP-overexpressing and DBP-siRNA HTR-8/SVneo cells were collected and added to human umbilical vein EC (HUVEC) cultures. Angiogenic effects on HUVECs were assessed by tube formation assays, and the proliferation and migration of HUVECs were examined using the Real-Time Cell Analyzer. The expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2, as well as the phosphorylation of different residues of VEGFR-2 in HUVECs, were determined by western blotting. Results: DBP expression was significantly increased in the placental tissues collected from PE patients. The conditioned medium of DBP-overexpressing HTR-8/SVneo cells potently inhibited tube formation by HUVECs, in addition to their proliferation and migration. Furthermore, treatment of HUVECs with the conditioned medium of DBP-overexpressing HTR-8/SVneo cells decreased the phosphorylation of VEGFR-2 at tyrosine 996, whereas the treatment of these cells with the conditioned medium of DBP-siRNA HTR-8/SVneo cells increased the phosphorylation of VEGFR-2 at tyrosine 951, 996, and 1,175. Conclusions: The expression of DBP is increased in the placentas of PE patients. DBP plays potential roles in endothelial dysfunction, which contributes to PE development, by inhibiting tyrosine phosphorylation of VEGFR-2 in ECs.

Long intergenic noncoding RNA 00473 promoting migration and invasion of trophoblastic cell line HTR-8/SVneo via regulating miR-424-5p-mediated wnt3a/β-catenin signaling pathway.

Preeclampsia (PE) is a serious obstetric complication. Recent studies point out that the functions of long intergenic noncoding RNA 00473 (linc00473), miR-424-5p, and Wnt/β-catenin signaling pathway were involved in the invasion and migration of extravillous trophoblast. Here, we investigated the role and mechanism of linc00473 in HTR-8/SVneo trophoblastic cell line and its role in PE. The expression levels of linc00473 and miR-424-5p in placental tissues and the transfection efficiency of miR-424-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). HTR-8/SVneo cell invasion and proliferation were determined by transwell and Cell Counting Kit-8 (CCK-8) assays. The protein expressions of wnt3a, p-GSK3β, GSK3β, active β-catenin, and total β-catenin were detected by Western blot. The apoptosis and migration of HTR-8/SVneo cells were detected by flow cytometry and wound healing assays. The targeting relationships between linc00473, miR-424-5p, and wnt3a were predicted by ENCORI database and TargetScan V7.2 and were determined using dual-luciferase reporter assay. The expression level of linc00473 was low and miR-424-5p was high in placenta of PE patients. Linc00473 can target miR-424-5p, while miR-424-5p target wnt3a. High expression of linc00473 and wnt3a promoted cell proliferation, migration, invasion, and inhibited cell apoptosis. However, miR-424-5p mimic inhibited HTR-8/SVneo cells proliferation, migration, invasion, while promoted cell apoptosis, partially reversed the effect of linc00473, while wnt3a overexpression partially counteracted the effect of miR-424-5p mimic. Linc00473 mediates the regulation of Wnt/β-catenin signaling pathway by miR-424-5p to affect the invasion and migration ability of trophoblastic cell line HTR-8/SVneo. It indicated that linc00473 is involved in PE and could be a therapeutic target.

Ubiquitin-Specific Peptidase 5 is Involved in the Proliferation of Trophoblast Cells by Regulating Wnt/β-Catenin Signaling.

Preeclampsia (PE) is a pathologic condition in pregnant women which accounts for the inhibition of proliferation, migration and invasion of trophoblast cells. This study aimed to investigate the regulation of ubiquitin-specific peptidase 5 (USP5) on the trophoblast cells in PE. Expressions of USP5 in the placentas of PE patients and healthy donors were examined by qRT-PCR and Western blot. Hypoxia/reoxygenation (H/R) model in trophoblast cells was further established. Cell viability was examined using CCK-8 assay. Finally, the effect of overexpression and silence of USP5 using lentivirus transduction was studied. Our results showed that USP5 was lowly expressed in the placentas of PE patients as well as in H/R-induced trophoblast cells. In the experiments of overexpression, USP5 promoted the proliferation of trophoblast cells, and up-regulated the expressions of β-catenin and the downstream signals c-Myc and Cyclin D1 in trophoblast cells. On the other hand, silence of USP5 elicited the opposite results. The overexpression of USP5 in the H/R model greatly released the H/R-induced inhibition in the trophoblast cells, and moderated the down-regulation of β-catenin and c-Myc induced by H/R. We concluded that USP5 promoted the proliferation of trophoblast cells via the up-regulation of the Wnt/β-catenin signaling pathway.

Effect of lncRNA-ATB/miR-651-3p/Yin Yang 1 pathway on trophoblast-endothelial cell interaction networks.

Our previous studies have confirmed that lncRNA‐ATB may be involved in the pathogenesis of preeclampsia, however, it is uncertain whether lncRNA‐ATB influence the interaction between trophoblast and endothelial cells, which is crucial to the uterine spiral artery remodelling. Scratch wound healing and transwell invasion assay were conducted to test the migration and invasion of trophoblast cells. Co‐culture model was used to simulate the physiological environment in vivo. The expression levels of lncRNA‐ATB were analyzed in placenta tissues from healthy pregnant women and preeclampsia patients. Subsequently, the binding site of lncRNA‐ATB and miR‐651‐3p was verified using dual‐luciferase reporter assay, and the rescue experiment was used to study the effects of these two on the biological function. The direct effects of miR‐651‐3p and Yin Yang 1 (YY1) were verified using similar methods. LncRNA‐ATB was found to be down‐regulated in the placenta of preeclampsia patients. LncRNA‐ATB knockdown decreased trophoblast migration, invasion and colocalisation with human umbilical vein endothelial cells. MiR‐651‐3p was a direct target of lncRNA‐ATB and they had opposite effects. Moreover, the expression of lncRNA‐ATB and miR‐651‐3p in placental tissues was negatively correlated. MiR‐651‐3p has been confirmed to directly target the 3′ untranslated region of YY1. The inhibitory effects of YY1 low expression on biological function was rescued by miR‐651‐3p depletion. Western blot analysis showed that lncRNA‐ATB could regulate YY1 expression by sponging miR‐651‐3p. LncRNA‐ATB functioned as a competitive endogenous RNA of miR‐651‐3p to regulate YY1 on progress of spiral artery remodelling.

Downregulation of Cullin 3 Ligase Signaling Pathways Contributes to Hypertension in Preeclampsia.

Background: Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality; however, its etiology and pathophysiology remain obscure. PE is initiated by inadequate spiral artery remodeling and subsequent placental ischemia/hypoxia, which stimulates release of bioactive factors into maternal circulation, leading to hypertension and renal damage.Methods and Results: Abundance of key components of cullin 3-ring ubiquitin ligase (CRL3), including cullin 3 (CUL3) and its neddylated modification, and adaptors including Kelch-like 2 (KLHL2) and Rho-related BTB domain containing protein 1 was all decreased in spiral arteries and placentas of PE patients. Similar changes were found in aortic tissues and renal distal tubules of pregnant mice treated with Nω-nitro-l-arginine methyl ester hydrochloride. The downregulation of CRL3 function led to accumulation of with-no-lysine kinases, phosphodiesterase 5, and RhoA in vessels and renal distal tubules, which promoted vasoconstriction and Na–Cl cotransporter activation in the distal convoluted tubule (DCT), as well as vascular and DCT structure remodeling. Proton pump inhibitor esomeprazole partially restored CRL3 function. In vitro studies have shown that increased abundance of JAB1, a component of the COP9 signalosome, inhibited CUL3 neddylation and promoted the expression of hypoxia-inducible factor 1α, which downregulated peroxisome proliferator–activated receptor γ and further promoted CUL3 inactivation. KLHL3/2 was degraded by increased autophagy.Conclusion: These findings support that the downregulation of CRL3 function disrupts the balance of vasoconstriction and vasodilation and aggravates excess reabsorption of sodium in PE.

Vasohibin 1, a clinically relevant biomarker, contributes to pre-eclampsia.

Pre-eclampsia is a leading health threat for pregnant women which is characterised by hypertension and proteinuria. The detailed mechanism is elusive and no effective therapy is available. Predictive biomarkers are needed for accurate diagnosis. Vasohibin-1 (VASH1) is an intrinsic inhibitor of angiogenesis induced by angiogenic factors in endothelial cells. This study aimed to evaluate the role of VASH1 as a useful biomarker for pre-eclampsia. VASH1 level was examined by ELISA and immunoblotting assay in the serum and placental samples from healthy pregnant women and pre-eclampsia patients. Cellular assay was performed to assess cell migration and invasion with different levels of VASH1. The level of VASH1 was measured under different oxygen conditions by qPCR. VASH1 was highly expressed in the serum and placenta of pre-eclampsia patients. Overexpression of VASH1 led to attenuated cell migration and invasion ability and reduced levels of matrix metallopeptidase 2 and 9. VASH1 was significantly induced in primary human trophoblast cells and placental explants under hypoxic condition in hypoxia-inducible factor 1 alpha-dependent manner. Our study suggested that VASH1 could be used as a potentially novel biomarker for pre-eclampsia and its level may positively correlate with the disease status.

Identification of downstream targets and signaling pathways of long non-coding RNA NR_002794 in human trophoblast cells

ABSTRACT Preeclampsia (PE) is a huge threat to pregnant women. Our previous study demonstrated that long non-coding RNA (lncRNA) NR_002794 was highly expressed in placentas of PE patients and could regulate the phenotypes of trophoblast cells. However, the downstream regulatory mechanisms of NR_002794 remain unknown. In this text, some potential downstream targets or signaling pathways of NR_002794 were identified through RNA sequencing (RNA-seq) and bioinformatics analysis in SWAN71 trophoblast cells. Western blot assay demonstrated that NR_002794 inactivated protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways and activated cell apoptotic signaling in SWAN71 cells. Both RNA-seq and reverse transcription-quantitative PCR (RT-qPCR) outcomes showed that NR_002794 up-regulation could notably inhibit the expression of C-C motif chemokine ligand 4 like 2 (CCL4L2), interleukin 15 receptor subunit alpha (IL15RA), interleukin 32 (IL32), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), while NR_002794 knockdown induced these gene expressions in SWAN71 cells. CCK-8, BrdU, Transwell, wound healing, and flow cytometry analyses showed that NR_002794 inhibited cell proliferation and migration and induced cell apoptosis through down-regulating TIE1 in SWAN71 cells. In conclusion, lncRNA NR_002794 could exert its functions by regulating AKT and ERK1/2 pathways and TIE1 expression in human trophoblast cells.

Expression of urotensin II is positively correlated with pyroptosis-related molecules in patients with severe preeclampsia

ABSTRACT Purpose: We studied the expression of urotensin II (UII) and its relationships with markers of pyroptosis in preeclampsia. Methods: 48 pregnant subjects were recruited consisting of 28 severe preeclampsia pregnancies (SPE) and 20 healthy pregnancies. We detected expressions of UII and markers of pyroptosis such as NLR-family pyrin domain (PYD)-containing 3 (NLRP-3), caspase-1/4/5, interleukin-1β (IL-1β), and gasdermin D (GSDMD) in placentas of patients with SPE and healthy pregnancies. Results: SPE group have higher expression of UII and NLRP-3, caspase-1, interleukin-1β (IL-1β), and GSDMD than that normal controls by IHC, real-time PCR, and western blot. IHC analysis manifests that the expressions of UII and pyroptosis-related molecules are mainly located in the placental cytotrophoblasts. Expressions of UII mRNA and protein are significantly positively correlated with pyroptosis marker such as NLRP3, caspase-1, GSDMD mRNA and protein by Pearson correlation analysis. Moreover, UII, NLRP-3, caspase-1, interleukin-1β (IL-1β), and GSDMD are positively related with systolic blood pressure, meanwhile caspase-1 and GSDMD are positively correlated with urine protein in SPE patients. We firstly verify that UII has a positive correlation with pyroptosis markers in placentas of preeclampsia patients; besides, pyroptosis-related proteins are positively correlated with systolic blood pressure and urine protein in patients with severe preeclampsia.

Overexpression of LVRN impedes the invasion of trophoblasts by inhibiting epithelial-mesenchymal transition.

Laeverin (LVRN) was first detected on the outer layer of the chorion laeve and migrating extravillous trophoblasts (EVTs). It is an enzyme that plays an important role in the placentation and pathophysiology of preeclampsia (PE). Previous studies have indicated that LVRN may be required for the invasion of human trophoblast cells. Paradoxically, LVRN was found to be highly expressed in the trophoblasts of PE patients with impaired invasive capacities. In this study, we detected the expression of LVRN in the placentas of PE patients (n=5) and normal term pregnancy women (n=5) as a control group by immunohistochemistry. LVRN was elevated in decidua (P=0.0083) and villi (P=0.0079) of PE patients. Next, LVRN was overexpressed via adeno-associated virus-mediated gene transfer in trophoblastic cell lines HTR8, Swan71, and JAR. Matrigel transwell assay and wound healing assay showed that overexpression of LVRN impeded the invasion of these three cell lines. Western blot analysis showed that LVRN overexpression caused downregulation of N-cadherin and vimentin and upregulation of E-cadherin, suggesting the inhibitory role of LVRN in epithelial-mesenchymal transition (EMT). Moreover, our data indicated that long noncoding RNA NONSTAT103348 (lnc10-7) was elevated in PE patients. Silencing lnc10-7 led to decreased LVRN expression. Taken together, although the basal level of LVRN may be crucial for cell invasion, overexpression of LVRN may abrogate the cell invasiveness, suggesting a multifaceted role of LVRN in the pathogenesis of PE.

EGR1 Overexpression Inhibits the Occurrence of Preeclampsia by Binding to MicroRNA-574 Promoter and Upregulating GAB1.

Early growth response protein 1 (EGR1) is potent in modulating placental trophoblast cell growth and shows a differential expression in preeclampsia (PE). We aimed to identify the downstream mechanism of EGR1 in PE. RT-qPCR showed EGR1 was significantly decreased in PE placenta. Overexpression of EGR1 facilitated the proliferation and invasion of HTR-8/Svneo cells, and reduced the concentration of human chorionic gonadotrophin (HCG) in the supernatant. Bioinformatics prediction, ChIP, and luciferase reporter experiments revealed that EGR1 inhibited miR-574 expression by binding to miR-574 promoter and that miR-574 targeted GAB1. Furthermore, overexpression of miR-574 inhibited the proliferation and invasion of HTR-8/Svneo cells. GAB1 was downregulated in placenta of PE patients, which was positively correlated with EGR1 and negatively correlated with miR-574. Inhibition of GAB1 attenuated the effect of EGR1 overexpression on the proliferation and invasion of HTR-8/Svneo cells. All in all, EGR1 upregulated GAB1 by inhibiting miR-574, thus contributing to trophoblast cell proliferation and invasion.

Overexpression of tissue factor pathway inhibitor 2 attenuates trophoblast proliferation and invasion in preeclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.

Complement Component C4d Deposition in the Placenta of Preeclampsia Patients and Renal Glomeruli in 1 Postpartum Renal Biopsy.

The classic disorder of placental malperfusion is preeclampsia (PE), in which the kidney is also a target organ, leading to renal dysfunction. Although the precise pathogenesis of PE is unknown, increasing evidence suggests that PE is associated with complement dysregulation. The maternal immune response to an allogenic fetus and excessive activation of the complement system may both be involved in the pathogenesis of PE. C4d deposition is considered to be evidence of antibody-mediated rejection in an allograft. This study investigated a correlation between C4d expression in the placenta and clinicopathologic features of PE patients. Immunohistochemical staining for C4d was performed on placental tissue of PE patients (n=70) and normal pregnancy patients (n=30). Clinicopathologic features, such as maternal age and parity, placental weight, proteinuria, and histologic features of the placenta were evaluated. One PE patient who suffered from proteinuria after delivery received a renal biopsy. C4d expression was demonstrated in syncytiotrophoblast of chorionic villi. The expression of C4d was significantly more frequent in the placenta with PE (50%) than in the placenta lacking complications (14.3%) (P=0.001). C4d expression was significantly accompanied by increased syncytial knots in PE (P=0.045). Among PE patients, C4d expression was significantly correlated with low placental weight (P=0.001) and high proteinuria (P=0.018, Mann-Whitney U test). Renal biopsy of a PE patient after delivery also showed deposition of C4d along the glomerular capillary walls. C4d may play an important role in placental tissue injury and in renal complications in PE.

Evidence for lysosomal biogenesis proteome defect and impaired autophagy in preeclampsia

ABSTRACT The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation: atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1

Identification of unexplored substrates of the serine protease, thrombin, using N-terminomics strategy

The function and regulation of thrombin is a complex as well as an intriguing aspect of evolution and has captured the interest of many investigators over the years. The reported substrates of thrombin are coagulation factors V, VIII, XI, XIII, protein C and fibrinogen. However, these may not be all the substrate of thrombin and therefore its functional role(s), may not have been completely comprehended. The purpose of our study was to identify hitherto unreported substrates of thrombin from human plasma using a N-terminomics protease substrate identification method. We identified 54 putative substrates of thrombin of which 12 are already known and 42 are being reported for the first time. Amongst the proteins identified, recombinant siglec-6 and purified serum alpha-1-acid glycoprotein were validated by cleavage with thrombin. We have discussed the probable relevance of siglec-6 cleavage by thrombin in human placenta mostly because an upregulation in the expression of siglec-6 and thrombin has been reported in the placenta of preeclampsia patients. We also speculate the role of alpha-1-acid glycoprotein cleavage by thrombin in the acute phase as alpha-1-acid glycoprotein is known to be an inhibitor of platelet aggregation whereas thrombin is known to trigger platelet aggregation.