Early Onset Preeclamptic Placentas Research Articles

The Impact of Hydroxychloroquine on Primary Feto-Placental Endothelial Cells from Healthy and Early-Onset Preeclamptic Placentas.

Hydroxychloroquine (HCQ), an anti-malarial drug, is suggested as a promising candidate for the treatment of pregnancy-related disorders associated with endothelial activation, among which there is preeclampsia (PE). Arterial feto-placental endothelial cells (fpECAs) were isolated from control (CTR) and early-onset preeclamptic (EO-PE) placentas. The aim of this study was to test potential protective effects of HCQ in an in vitro model of endothelial activation as well as in cells isolated from EO-PE placentas. To mimic PE conditions, CTR fpECAs were exposed to a pro-inflammatory environment consisting of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β (furtherly referred as MIX) with or without varying concentrations of HCQ (1 µg/mL and 10 µg/mL). Their effect on wound healing and endothelial barrier integrity was analyzed. Variations in the expression of IL-8 and leukocyte adhesion molecules (LAM) on both mRNA and protein levels were determined between CTR and PE fpECAs in the presence or absence of HCQ. MIX decreased wound healing and stability of the endothelial barrier, but HCQ did not affect it. Significant differences between CTR and EO-PE fpECAs were observed in IL-8 mRNA, protein secretion, and vascular cell adhesion protein 1 (VCAM-1) mRNA expression levels. After challenging CTR fpECAs with MIX, upregulation of both mRNA and protein levels was observed in all molecules. Combined treatment of HCQ and MIX slightly lowered VCAM-1 total protein amount. In CTR fpECAs, treatment with low concentrations of HCQ alone (1 µg/mL) reduced basal levels of IL-8 and VCAM-1 mRNA and secretion of IL-8, while in EO-PE fpECAs, a higher (10µg/mL) HCQ concentration slightly reduced the gene expression of IL-8. Conclusion: These results provide additional support for the safety of HCQ, as it did not adversely affect endothelial functionality in control fpECAs at the tested concentration. Furthermore, the observed limited effects on IL-8 secretion in EO-PE fpECAs warrant further investigation, highlighting the need for clinical trials to assess the potential therapeutic effects of HCQ in preeclampsia. Conducting clinical trials would offer a more comprehensive understanding of HCQ's efficacy and safety, allowing us to explore its potential benefits and limitations in a real-world clinical setting.

Transfer and Vascular Effect of Endothelin Receptor Antagonists in the Human Placenta.

Increasing evidence suggests a role for the ET (endothelin) system in preeclampsia. Hence, blocking this system with endothelin receptor antagonists (ERAs) could be a therapeutic strategy. Yet, clinical studies are lacking due to possible teratogenic effects of ERAs. In this study, we investigated the placental transfer of ERAs and their effect on ET-1-mediated vasoconstriction. Term placentas were dually perfused with the selective ETAR (ET type A receptor) antagonists sitaxentan and ambrisentan or the nonselective ETAR/ETBR antagonist macitentan and subsequently exposed to ET-1 in the fetal circulation. ET-1 concentration-response curves after incubation with sitaxentan, ambrisentan, macitentan, or the selective ETBR antagonist BQ-788 were also constructed in isolated chorionic plate arteries using wire-myography, and gene expression of the ET-system was quantified in healthy and early onset preeclamptic placentas. At steady state, the mean fetal-to-maternal transfer ratios were 0.32±0.05 for sitaxentan, 0.21±0.02 for ambrisentan, and 0.05±0.01 for macitentan. Except for BQ-788, all ERAs lowered the response to ET-1, both in the perfused cotyledon and isolated chorionic plate arteries. Placental gene expression of ECE-1, ETAR, and ETBR were comparable in healthy and preeclamptic placentas, while ET-1 expression was higher in preeclampsia. Our study is the first to show direct transfer of ERAs across the term human placenta. Furthermore, ETAR exclusively mediates ET-1-induced constriction in the fetoplacental vasculature. Given its limited transfer, macitentan could be considered as potential preeclampsia therapy. Extending knowledge on placental transfer to placentas of preeclamptic pregnancies is required to determine whether ERAs might be applied safely in preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Altered DNA methylation and transcription of WNT2 and DKK1 genes in placentas associated with early-onset preeclampsia

PurposeThe purpose of this study was to characterize the changes in DNA methylation and transcription of WNT2 and DKK1 genes in placentas associated with early-onset preeclampsia. MethodsThe study includes three groups: patients with early-onset preeclampsia, normotensive preterm and term births. Placental tissues were collected and pyrosequencing was performed on DKK1 and WNT2 proximal promoters. Transcriptional levels of DKK1 and WNT2 genes were determined with real-time PCR. ResultsDKK1 gene methylation levels were lower in placentas associated with early-onset preeclampsia compared to those associated with preterm birth (P<0.05). DKK1 mRNA expression was higher in early-onset preeclampsia placentas than those in preterm placentas (P < 0.05). WNT2 mRNA expression in early-onset preeclamptic placentas was lower than that in other two groups (P < 0.05). In the preterm and early-onset preeclampsia groups, the mRNA levels for WNT2 and DKK1 were negatively correlated (P < 0.05). In all the subjects, the levels of DKK1 mRNA and methyaltion were negatively correlated (P < 0.05). ConclusionDecreased methylation of DKK1 promoter in early-onset preeclamptic placenta tissues may up-regulate the expression of DKK1. The increased expression of DKK1 and decreased expression of WNT2 may be involved in the pathogenesis of early-onset preeclampsia.

Long non-coding RNA RPAIN regulates the invasion and apoptosis of trophoblast cell lines via complement protein C1q.

Long non-coding RNAs (lncRNAs) are key regulatory molecules that are involved in a variety of biological processes and human diseases. Their impact on early onset preeclampsia remains unclear. In this study, we tested the expression of RPAIN (transcript variant 12 of RPA interacting protein, a non-coding RNA, NR_027683.1) in placenta tissues derived from 25 pregnant women with PE and 15 healthy pregnant women using quantitative real-time PCR. The effect of RPAIN on trophoblast proliferation, invasion, and apoptosis and the underlying mechanisms were examined in trophoblast cell lines (HTR-8/SVneo). The results showed that RPAIN expression levels were significantly increased in early onset preeclamptic placentas compared to normal controls. The proliferation and invasive abilities of the trophoblast cells were significantly inhibited, and the apoptosis abilities of the trophoblast cells were significantly promoted when RPAIN was overexpressed. In addition, the overexpression of RPAIN inhibited the expression of complement protein C1q. Furthermore, C1q overexpression rescued the decreased cell invasion and enhanced cell apoptosis in RPAIN-overexpressing trophoblast cells. Our results suggest that increased RPAIN levels may contribute to the development of preeclampsia through regulating trophoblast invasion and apoptosis via C1q. Therefore, we proposed RPAIN as a novel lncRNA molecule, which might contribute to the development of PE (preeclampsia) and might compose a potential diagnostic and therapeutic target for this disease.

P87. Alteration of Delta-like ligand 1 and Notch 1 receptor in various placental disorders with special reference to early-onset preeclampsia

Introduction Notch signaling pathway has been shown to be dysregulated in placentas with preeclampsia, but there has been a lack of studies on methylation of Notch family genes in this disorder. Objectives: To investigate the methylation status of DLL 1 and Notch 1 and expression of those proteins in placenta with preeclampsia. Methods We therefore executed methylation-specific polymerase chain reaction and immunostaining for Notch 1 receptor and the activating ligand, Delta-like (DLL) 1, with placental tissues from cases of preeclampsia (early-onset, n =18; late-onset, n =19) and other placental disorders, including maternal complications such as diabetes mellitus and collagen disease (n = 10), fetal growth restriction ( n =17), fetal anomaly ( n =23), preterm birth ( n =15), miscarriage ( n =25), and hydatidiform moles ( n =9) as well as term births ( n =12). Results The frequency of DLL1 methylation was higher in early-onset preeclamptic placentas (61%) than the other subjects (0–36%; P ⩽0.016). Appreciable samples (36%) of miscarriage also represented DLL1 methylation. None of the samples studied showed Notch 1 methylation. On gestational period-matched analysis, the rate of DLL1 methylation was higher in early-onset preeclampsia (83.3%) than preterm birth (13.3%; P Conclusion Altered Notch signaling via methylation of DLL1 is likely involved in possible disease-related mechanisms of early-onset preeclampsia. Alternatively, DLL1 methylation in early-onset preeclampsia could be a manifestation of a lack of placental maturation, similar to miscarriage.

Alteration of Delta-like ligand 1 and Notch 1 receptor in various placental disorders with special reference to early onset preeclampsia.

Notch signaling pathway has been shown to be dysregulated in placentas with preeclampsia, but there has been a lack of studies on methylation of Notch family genes in this disorder. We therefore executed methylation-specific polymerase chain reaction and immunostaining for Notch 1 receptor and the activating ligand, Delta-like (DLL) 1, with placental tissues from cases of preeclampsia (early onset, n = 18; late-onset, n = 19) and other placental disorders, including maternal complications such as diabetes mellitus and collagen disease (n = 10), fetal growth restriction (n = 17), fetal anomaly (n = 23), preterm birth (n = 15), miscarriage (n = 25), and hydatidiform moles (n = 9) as well as term births (n = 12). The frequency of DLL1 methylation was higher in early onset preeclamptic placentas (61%) than the other subjects (0%-36%; P ≤ .016). Appreciable samples (36%) of miscarriage also represented DLL1 methylation. None of the samples studied showed Notch 1 methylation. On gestational period-matched analysis, the rate of DLL1 methylation was higher in early onset preeclampsia (83.3%) than preterm birth (13.3%; P < .001), with no significant differences in clinical backgrounds between the two. In this analysis, increase of syncytial knots and accelerated villous maturation were most prominent in DLL1-methylated placentas with early onset preeclampsia. Notch 1 and DLL1 expressions in villous trophoblasts and endothelial cells were significantly lower in early onset preeclamptic placentas as compared with preterm birth controls. In conclusion, altered Notch signaling via methylation of DLL1 is likely involved in possible disease-related mechanisms of early onset preeclampsia. Alternatively, DLL1 methylation in early onset preeclampsia could be a manifestation of a lack of placental maturation, similar to miscarriage.

Placental SEMA3B expression is not altered in severe early onset preeclampsia

Recent evidence suggests that Semaphorin 3B (SEMA3B) is upregulated in severe preeclampsia, and a major driver of cytotrophoblast aberrations in this disease. Here we independently assess whether SEMA3B expression is altered in a large cohort of severe early onset preeclamptic placentas. We demonstrate that SEMA3B relative mRNA expression and copy number are not changed in PE placentas. We confirm this at the protein level by western blot. Interestingly, exposure of term trophoblasts or explants to hypoxia induced a significant down regulation of SEMA3B mRNA, but a trend towards increased SEMA3B protein expression. We conclude that SEMA3B mRNA and protein is not altered in severe early onset preeclamptic placentas.

TREM-1 Expression Is Increased in Human Placentas From Severe Early-Onset Preeclamptic Pregnancies Where It May Be Involved in Syncytialization

Preeclampsia, a major cause of maternal and perinatal morbidity and mortality, is thought to be attributable to dysregulation of trophoblast invasion and differentiation. Microarray studies have shown that triggering receptor expressed on myeloid cells (TREM) 1, a cell surface molecule involved in the inflammatory response, is increased in preeclamptic placentas. The aim of this study was to determine the level of TREM-1 expression in severe early-onset preeclamptic placentas and its functional role in trophoblast differentiation. Placenta was obtained from women with severe early-onset preeclampsia (n = 19) and gestationally matched preterm controls placentas (n = 8). The TREM-1 expression was determined by quantitative reverse transcriptase polymerase chain reaction and Western blotting. The effect of TREM-1 small interfering RNA on cell fusion and differentiation was assessed in BeWo cells. The effect of oxygen tension on TREM-1 levels, in basal or forskolin-treated BeWo cells, was also assessed. The TREM-1 was localized to the syncytiotrophoblast layer, and TREM-1 messenger RNA and protein expression was significantly increased in preeclamptic placentas. The BeWo cells treated with forskolin were associated with increased TREM-1 expression. The TREM-1 knockdown inhibited forskolin-induced expression of the differentiation marker β-human chorionic gonadotropin but had no effect on the cell-fusion marker E-cadherin. The increase in TREM-1 expression in BeWo cells treated with forskolin during normoxic conditions was reduced in forskolin-treated cells under hypoxic conditions. In conclusion, TREM-1 is increased in preeclamptic placentas and by forskolin treatment. Knockdown of TREM-1 by RNA interference inhibits cell differentiation but has no effect on cell-cell fusion. Finally, we show that TREM-1 upregulation is attenuated under hypoxic conditions in which cell differentiation is impaired.

PAPPA2 is increased in severe early onset pre-eclampsia and upregulated with hypoxia

Severe early onset pre-eclampsia is a serious pregnancy complication, believed to arise as a result of persistent placental hypoxia due to impaired placentation. Pregnancy-associated plasma protein A2 (PAPPA2) is very highly expressed in the placenta relative to all other tissues. There is some evidence that PAPPA2 mRNA and protein are increased in association with pre-eclampsia. The aim of the present study was to characterise the mRNA and protein expression, as well as localisation, of PAPPA2 in an independent cohort of severe early onset pre-eclamptic placentas. We also examined whether exposing placental explants to hypoxia (1% oxygen) changed the expression of PAPPA2. Expression of PAPPA2 mRNA and protein was upregulated in severe early onset pre-eclamptic placentas compared with preterm controls and localised to the syncytiotrophoblast. Interestingly, protein localisation was markedly reduced in term placenta. Syncytialisation of BeWo cells did not change PAPPA2 expression. However, hypoxia upregulated PAPPA2 mRNA and protein expression in primary placental explants. Together, our data suggest that PAPPA2 may be upregulated in severe pre-eclampsia and, functionally, this may be mediated via increased placental hypoxia known to occur with this pregnancy disorder.

Corin, an enzyme with a putative role in spiral artery remodeling, is up-regulated in late secretory endometrium and first trimester decidua

What is the nature of cellular Corin expression in human gestational tissues? CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT-PCR, and levels of CORIN within human sera were measured by ELISA. CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare that no competing interests exist.

PLAC4 is upregulated in severe early onset preeclampsia and upregulated with syncytialisation but not hypoxia

IntroductionSevere early onset preeclampsia is one of the most serious complications of pregnancy. Placental specific 4 (PLAC4) is very highly expressed in placenta relative to all other tissues. Recently in a biomarker screening study, we found PLAC4 mRNA was significantly upregulated in maternal whole blood and placenta obtained from cases of severe preeclampsia. Intriguingly however, very little is known about its expression or functional role in either normal pregnancy or pregnancies complicated by preeclampsia. MethodsThe objective of this study was to characterize the protein expression and localization of PLAC4 in severe early onset preeclamptic placenta. Given so little of the biology of PLAC4 is known, we also examined whether the expression of PLAC4 alters with syncytialisation or placental hypoxia. ResultsWe found PLAC4 protein expression was significantly (p < 0.05) upregulated in severe early onset preeclamptic placentas (n = 24) compared to gestationally matched preterm controls (n = 12). PLAC4 protein was specifically localized to the syncytiotrophoblast of preterm, preeclamptic and term placentas. Functional analysis of PLAC4 mRNA and protein expression revealed a significant (p < 0.05) increase with syncytialisation of BeWo cells. However, exposure of either syncytialised BeWo cells or primary term placental explants to hypoxia (1% oxygen) did not alter the expression of either PLAC4 mRNA or protein. ConclusionIn conclusion, we have found PLAC4 is significantly upregulated in association with severe preterm preeclampsia. Furthermore, it is upregulated with syncytialisation, but not hypoxia. It is possible PLAC4 may have a role in the pathogenesis of preeclampsia, and its biology merits further investigation.

The imprinted H19 gene regulates human placental trophoblast cell proliferation via encoding miR-675 that targets Nodal Modulator 1 (NOMO1)

Preeclampsia is a pregnancy-specific syndrome mainly characterized by hypertensive disorder and proteinuria after gestational weeks 20. So far the etiology of preeclampsia remains unclear. We previously reported that preeclamptic placentas exhibited decreased mRNA expression and hypermethylation in promoter region of the paternally imprinted H19 gene compared with normal placentas. H19 has recently been identified to encode the precursor of miR-675, indicating a possible novel functional pathway of the imprinting gene. The aim of the present study was to identify the roles of H19 gene via miR-675 pathway in human trophoblast cells, and to figure out the involvement of this pathway in pathogenesis of preeclampsia. Knockdown of H19 gene or inhibition of miR-675 exhibited similar proliferation-promoting effect in human trophoblastic JEG-3 cells. Target gene prediction in combination with luciferase assay revealed that miR-675 could directly downregulate Nodal Modulator 1 (NOMO1) protein expression by binding to 3′-UTR sequence of NOMO1. Overexpression of NOMO1 in JEG-3 cells could rescue miR-675-surppressed cell proliferation and phosphorylation of Smad2, while Nodal had additive effect with miR-675 in suppression cell proliferation and activation of Smad2. In early-onset preeclamptic placentas, expression levels of H19 gene and miR-675 were appreciably lower, while NOMO1 protein level was higher than those in normal placentas. Taken together, our data suggested that H19 gene could inhibit human trophoblast cell proliferation via encoding miR-675 that targeted NOMO1, and aberrantly lowered expression of H19 in placenta may participate in the excessive proliferation of trophoblast cells observed in early-onset severe preeclampsia by downregulating miR-675 which targets NOMO1 and interferes with nodal signaling.

Detection of global DNA methylation and paternally imprinted H19 gene methylation in preeclamptic placentas

Preeclampsia (PE) is a severe hypertensive disorder associated with pregnancy; despite substantial research effort in the past several years, the etiology of PE is still unclear. The role of epigenetic factors in the etiology of PE, including DNA methylation, has been poorly characterized. In the present study, we investigated global DNA methylation as well as DNA methylation of the paternally imprinted H19 gene in preeclamptic placentas. Using 5-methylcytosine immunohistochemistry and Alu and LINE-1 repeat pyrosequencing, we found that the global DNA methylation level and the DNA (cytosine-5) methyltransferase 1 mRNA level were significantly higher in the early-onset preeclamptic placentas when compared with the normal controls. Data from methylation-sensitive high resolution melting demonstrated hypermethylation of the promoter region of the H19 gene, and results of real-time PCR showed decreased mRNA expression of H19 gene in the early-onset preeclamptic placentas as compared with the normal controls. Our results suggest that abnormal DNA methylation during placentation might be involved in the pathophysiology of PE, especially early-onset preeclampsia.

DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate <10% and methylation difference >10%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P=0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.