MicroRNA analysis of medium/large placenta extracellular vesicles in normal and preeclampsia pregnancies.

Background: Preeclampsia (PE) is a hypertensive disorder of pregnancy, affecting 2%-8% of pregnancies worldwide, and is the leading cause of adverse maternal and fetal outcomes. The disease is characterized by oxidative and cellular stress and widespread endothelial dysfunction. While the precise mechanisms are not entirely understood, the pathogenesis of PE is closely linked to placental dysfunction and, to some extent, syncytiotrophoblast extracellular vesicle release (STB-EVs). These vesicles can be divided into the less well-studied medium/large EVs (220-1,000 nm) released in response to stress and small EVs (<220 nm) released as a component of intercellular communication. The previously described production of m/lSTB-EVs in response to cellular stress combined with the overwhelming occurrence of cellular and oxidative stress in PE prompted us to evaluate the microRNAome of PE m/lSTB-EVs. We hypothesized that the microRNAome profile of m/lSTB-EVs is different in PE compared to normal pregnancy (NP), which might permit the identification of potential circulating biomarkers not previously described in PE. Methods/study design: We performed small RNA sequencing on medium/large STB-EVs isolated from PE and NP placentae using dual-lobe ex vivo perfusion. The sequencing data was bioinformatically analyzed to identify differentially regulated microRNAs. Identified microRNAs were validated with quantitative PCR analysis. We completed our analysis by performing an in-silico prediction of STB-EV mechanistic pathways. Results: We identified significant differences between PE and NP in the STB-EVs micro ribonucleic acid (microRNA) profiles. We verified the differential expression of hsa-miR-193b-5p, hsa-miR-324-5p, hsa-miR-652-3p, hsa-miR-3196, hsa-miR-9-5p, hsa-miR-421, and hsa-miR-210-3p in the medium/large STB-EVs. We also confirmed the differential abundance of hsa-miR-9-5p in maternal serum extracellular vesicles (S EVs). In addition, we integrated the results of these microRNAs into the previously published messenger RNA (mRNA) data to better understand the relationship between these biomolecules. Conclusions: We identified a differentially regulated micro-RNA, hsa-miR-9-5p, that may have biomarker potential and uncovered mechanistic pathways that may be important in the pathophysiology of PE.

A cross-sectional analysis of syncytiotrophoblast membrane extracellular vesicles-derived transcriptomic biomarkers in early-onset preeclampsia.

Background: Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. Objective: The objective of this study was to identify the difference in the transcriptome of placenta and STB-EVs between preeclampsia and normal pregnancy (NP) and mechanistic pathways. Methods/study design: We performed RNA-sequencing on placental tissue, medium/large and small STB-EVs from PE (n = 6) and NP (n = 6), followed by bioinformatic analysis to identify targets that could be used in the future for EV-based diagnostic tests for preeclampsia. Some of the identified biomarkers were validated with real-time polymerase chain reactions. Results: Our analysis identified a difference in the transcriptomic STB-EV cargo between PE and NP. We then identified and verified the differential expression of FLNB, COL17A1, SLC45A4, LEP, HTRA4, PAPP-A2, EBI3, HSD17B1, FSTL3, INHBA, SIGLEC6, and CGB3. Our analysis also identified interesting mechanistic processes via an in silico prediction of STB-EV-based mechanistic pathways. Conclusions: In this study, using comprehensive profiling of differentially expressed/carried genes of three linked sample subtypes in PE, we identified potential biomarkers and mechanistic gene pathways that may be important in the pathophysiology of PE and could be further explored in future studies.

In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium.

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules.

Disturbances in endoplasmic reticulum (ER) function lead to ER stress which, when severe or prolonged, may result in apoptosis. Severe ER stress has been implicated in several pathological conditions including pre-eclampsia, a multisystem disorder of pregnancy associated with the release of pro-inflammatory factors from the placenta into the maternal circulation. Here, we show that severe ER stress induced by two distinct mechanisms in BeWo choriocarcinoma cells leads to the release of extracellular vesicles (EVs) carrying pro-inflammatory damage-associated molecular pattern (DAMP) molecules. Co-treatment with the antioxidant pyrrolidine dithiocarbamate results in a reduction in ER stress-induced EV-associated DAMP release. We further demonstrate that severe ER stress is associated with changes in the expression of several stress-related proteins, notably Cited-2 and phosphorylated JNK. Together, these data indicate that severe ER stress-mediated release of EV-associated DAMPs may contribute to the heightened systemic maternal inflammatory response characteristic of pre-eclampsia and may also be relevant to other chronic inflammatory diseases which display elevated ER stress.

Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development.

Some neuropsychiatric disease, including schizophrenia, may originate during prenatal development, following periods of gestational hypoxia and placental oxidative stress. Here we investigated if gestational hypoxia promotes damaging secretions from the placenta that affect fetal development and whether a mitochondria-targeted antioxidant MitoQ might prevent this. Gestational hypoxia caused low birth-weight and changes in young adult offspring brain, mimicking those in human neuropsychiatric disease. Exposure of cultured neurons to fetal plasma or to secretions from the placenta or from model trophoblast barriers that had been exposed to altered oxygenation caused similar morphological changes. The secretions and plasma contained altered microRNAs whose targets were linked with changes in gene expression in the fetal brain and with human schizophrenia loci. Molecular and morphological changes in vivo and in vitro were prevented by a single dose of MitoQ bound to nanoparticles, which were shown to localise and prevent oxidative stress in the placenta but not in the fetus. We suggest the possibility of developing preventative treatments that target the placenta and not the fetus to reduce risk of psychiatric disease in later life.

Syncytiotrophoblast extracellular vesicles - Circulating biopsies reflecting placental health.

The ability to directly monitor the status of the placenta throughout pregnancy would be a major advance in both general and personalized obstetric care, allowing treatments to be tailored to the dynamic changes that can occur in gestation. Syncytiotrophoblast extracellular vesicles (STBEV) are membrane bound vesicles, released from the surface of the placenta directly into the maternal circulation, in the form of exosomes, microvesicles and apoptotic bodies. They carry many syncytiotrophoblast derived factors such as proteins, lipids, glycans and nucleic acids, which together could dynamically signal to the mother the status of the placenta. We review STBEV research and discuss the potential for STBEV to be used as circulating syncytiotrophoblast biopsies, accessible via a simple blood sample throughout pregnancy, giving a real-time readout of syncytiotrophoblast health. We also highlight advances in the use of extracellular vesicles as circulating tumour derived biopsies in the field of cancer research, which could prove beneficial to obstetric care.

Secretions from placenta, after hypoxia/reoxygenation, can damage developing neurones of brain under experimental conditions.

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.

RhoE is regulated by cyclic AMP and promotes fusion of human BeWo choriocarcinoma cells.

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N(6)-phenyl-cAMP, and a specific inhibitor of PKA (14-22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.

Downregulation of caveolin-1 enhances fusion of human BeWo choriocarcinoma cells.

BACKGROUND: Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. Caveolin-1 has been shown to be expressed in human villous cytotrophoblast and to be downregulated during fusion into syncytiotrophoblast but it is unclear whether it plays a role in this process. METHODOLOGY/PRINCIPAL FINDINGS: We used RNA interference to determine whether caveolin-1 plays a role in differentiation and fusion in the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control. Furthermore, caveolin-1 knockdown alone was sufficient to promote spontaneous fusion. In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment. Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr(308). CONCLUSIONS/SIGNIFICANCE: Taken together, these results suggest that caveolin-1 regulates BeWo cell differentiation and fusion, possibly through a mechanism involving modulation of Akt activity.

Overexpression of p65/RelA potentiates curcumin-induced apoptosis in HCT116 human colon cancer cells.

Curcumin, the yellow pigment in the spice turmeric, has potent chemopreventive activities that involve diverse molecular pathways. It is widely believed that curcumin pro-apoptotic properties are mediated by downregulation of NF kappa B (NFkappaB). The p65/RelA subunit of NFkappaB may influence cell death, in part by activation of NFkappaB anti-apoptotic target genes including X-linked inhibitor of apoptosis (XIAP), A20, bcl-xL and inhibition of sustained activation of c-Jun N-terminal kinase (JNK). We have shown previously that curcumin inhibits NFkappaB, activates JNK and promotes apoptosis in HCT116 colorectal cancer cells. Here, we show that forced overexpression of p65 does not affect curcumin-induced JNK activation. Indeed, overexpression of p65 enhanced curcumin-mediated apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and poly(ADP-ribose) polymerase (PARP) cleavage. This potentiating effect of p65 upon curcumin-mediated apoptosis was reversed by transfection of cells with an IkappaB super-repressor (DeltaNIkappaB). Curcumin treatment inhibited expression of NFkappaB anti-apoptotic target genes in mock-transfected and in p65-overexpressing HCT116 cells, although expression levels remained higher in the latter. Taken together, these results show that curcumin-mediated activation of JNK or induction of apoptosis does not require inhibition of p65. Furthermore, curcumin/p65 synergy in promotion of apoptosis cannot be attributed to active repression of NFkappaB anti-apoptotic genes.

Chemopreventive properties of curcumin.

Inhibition of defined molecular steps of tumorigenesis by natural nontoxic compounds may be an efficient means to tackle the population cancer burden. Extensive research has addressed the chemotherapeutic potential of curcumin (diferuloylmethane), a relatively nontoxic plant-derived polyphenol. This review considers the following properties of curcumin: anticancer effects in animal model systems; metabolism; biological structure and pharmacokinetics; biological properties implicated in chemoprevention; antioxidant properties; influences upon Phase I and II carcinogen-metabolizing enzymes; signal transduction properties and the neoplastic phenotype; apoptosis evasion, cell proliferation, de-differentiation, migration and invasion and clinical studies. This review will summarize the unique properties of curcumin that may be exploited for successful clinical cancer prevention.

Curcumin induces c-jun N-terminal kinase-dependent apoptosis in HCT116 human colon cancer cells.

Curcumin, the major pigment of the dietary spice turmeric has the potential for chemoprevention by promotion of apoptosis. Mitogen-activated protein kinase (MAPK) and NF-kappa B (NFkappaB) signalling cascades are thought to regulate apoptosis and cell survival. While curcumin inhibits NFkappaB, its effects upon the MAPK pathways are unclear. This study investigates curcumin effects upon MAPK signalling and apoptosis in HCT116 cells. Here we report that curcumin time- and dose-dependent induction of apoptosis were accompanied by sustained phosphorylation and activation of c-jun N-terminal kinase (JNK) and p38 MAPK as well as inhibition of constitutive NFkappaB transcriptional activity. Curcumin treatment also induced JNK-dependent sustained phosphorylation of c-jun and stimulation of AP-1 transcriptional activity. Curcumin-mediated c-jun phosphorylation and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125. Conversely, the p38-specific inhibitor SB203580 had no effect upon curcumin-induced apoptosis. Curcumin treatment had no effect on the activity of extracellular signal-regulated protein kinase (ERK). Taken together, our data show for the first time that JNK, but not p38 or ERK signalling, plays an important role in curcumin-mediated apoptosis in human colon cancer cells that may underlie its chemopreventive effects.