DNA methylation landscape in pregnancy-induced hypertension: progress and challenges.

Gestational hypertension (PIH), especially pre-eclampsia (PE), is a common complication of pregnancy. This condition poses significant risks to the health of both the mother and the fetus. Emerging evidence suggests that epigenetic modifications, particularly DNA methylation, may play a role in initiating the earliest pathophysiology of PIH. This article describes the relationship between DNA methylation and placental trophoblast function, genes associated with the placental microenvironment, the placental vascular system, and maternal blood and vascular function, abnormalities of umbilical cord blood and vascular function in the onset and progression of PIH, as well as changes in DNA methylation in the progeny of PIH, in terms of maternal, fetal, and offspring. We also explore the latest research on DNA methylation-based early detection, diagnosis and potential therapeutic strategies for PIH. This will enable the field of DNA methylation research to continue to enhance our understanding of the epigenetic regulation of PIH genes and identify potential therapeutic targets.

Prenatal dexamethasone exposure impaired vascular reactivity in adult male offspring cerebral arteries.

BACKGROUND: Cerebrovascular disease is one of the leading causes of death worldwide. Middle cerebral artery (MCA) is the largest and most complex of cerebral arteries. The prenatal period is a critical time for development, which largely determines lifelong health. Clinically, glucocorticoids (GCs) administration to accelerate preterm fetal lung maturation has become standard practice. Prenatal GCs administration increases cardiovascular risks in offspring, but little is known regarding the side effects on offspring MCA function. OBJECTIVE: We investigated the alterations of MCA reactivity following prenatal GCs administration in postnatal offspring. METHOD AND RESULTS: Pregnant Sprague-Dawley rats received synthetic GCs (dexamethasone, DEX) during the last week of pregnancy, and we examined vascular reactivity, cellular electrophysiology, and gene promoter epigenetic modifications in the male offspring MCA. Our results showed that prenatal DEX exposure increased the sensitivity of offspring MCA to Angiotensin II, which was resulted from the increased Cav1.2 (L-type Ca2+ channels subunit alpha1 C). Mechanistically, prenatal DEX exposure resulted in a transcriptionally active chromatin structure at the Cav1.2 gene promoter by altering histone modifications. This activation led to increased expression of vascular Cav1.2 gene, ultimately resulting in increased MCA contractility in offspring. CONCLUSION: The present study is the first to demonstrate that the adverse effects of prenatal GCs administration on cerebrovascular tone persist into adulthood, providing new insights into developmental origins of cerebrovascular disease.

Preeclampsia impedes foetal kidney development by delivering placenta-derived exosomes to glomerular endothelial cells.

BACKGROUND: Foetal renal dysplasia is still the main cause of adult renal disease. Placenta-derived exosomes are an important communication tool, and they may play an important role in placental (both foetal and maternal) function. We hypothesize that in women with preeclampsia, foetal renal dysplasia is impeded by delivering placenta-derived exosomes to glomerular endothelial cells. METHODS: In the present study, we established a PE trophoblast oxidative stress model to isolate exosomes from supernatants by ultracentrifugation (NO-exo and H/R-exo) and collected normal and PE umbilical cord blood plasma to isolate exosomes by ultracentrifugation combined with sucrose density gradient centrifugation (N-exo and PE-exo), then we investigated their effects on foetal kidney development by in vitro, ex vivo and in vivo models. RESULTS: The PE trophoblast oxidative stress model was established successfully. After that, in in vitro studies, we found that H/R-exo and PE-exo could adversely affect glomerular endothelial cell proliferation, tubular formation, migration, and barrier functions. In ex vivo studies, H/R-exo and PE-exo both inhibited the growth and branch formation of kidney explants, along with the decrease of VE-cadherin and Occludin. In in vivo studies, we also found that H/R-exo and PE-exo could result in renal dysplasia, reduced glomerular number, and reduced barrier function in foetal mice. CONCLUSIONS: In conclusion, we demonstrated that PE placenta-derived exosomes could lead to foetal renal dysplasia by delivering placenta-derived exosomes to foetal glomerular endothelial cells, which provides a novel understanding of the pathogenesis of foetal renal dysplasia. Video Abstract.

Exosomal miR-146a-5p derived from human umbilical cord mesenchymal stem cells can alleviate antiphospholipid antibody-induced trophoblast injury and placental dysfunction by regulating the TRAF6/NF-κB axis.

Exosomes originating from human umbilical cord mesenchymal stem cells (hucMSC-exos) have become a novel strategy for treating various diseases owing to their ability to regulate intercellular signal communication. However, the potential of hucMSC-exos to improve placental injury in obstetric antiphospholipid syndrome and its underlying mechanism remain unclear. Our objective was to explore the potential application of hucMSC-exos in the treatment of obstetric antiphospholipid syndrome and elucidate its underlying mechanism. In our study, hucMSC-exos ameliorated the functional impairment of trophoblasts caused by antiphospholipid antibodies in vitro and attenuated placental dysfunction in mice with obstetric antiphospholipid syndrome by delivering miR-146a-5p. Exosomal miR-146a-5p suppressed the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibited the activation of NF-κB signaling, leading to the down-regulation of IL-1ß and IL-18 to rescue inflammation and modulation of Cleaved-CASP3, BAX, and BCL2 to inhibit apoptosis in HTR8/SVneo cells and mice placenta. This study identified the potential molecular basis of how hucMSC-exos improved antiphospholipid antibody-induced placental injury and highlighted the functional importance of the miR-146a-5p/TRAF6 axis in the progression of obstetric antiphospholipid syndrome. More importantly, this study provided a fresh outlook on the promising use of hucMSC-exos as a novel and effective treatment approach in obstetric antiphospholipid syndrome.

Effect of peroxiredoxin 1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia.

PURPOSE: PE is a pregnancy-specific syndrome and one of the main causes of maternal, fetal, and neonatal mortality. PRDX1 is an antioxidant that regulates cell proliferation, differentiation, and apoptosis. The aim of this study is to investigate the effect of PRDX1 on the regulation of trophoblast function by affecting autophagy and oxidative stress in preeclampsia. METHODS: Western blotting, RT-qPCR, and immunofluorescence were used to examine the expression of PRDX1 in placentas. PRDX1-siRNA was transfected to knockdown PRDX1 in HTR-8/SVneo cells. The biological function of HTR-8/SVneo cells was detected by wound healing, invasion, tube formation, CCK-8, EdU, flow cytometry, and TUNEL assays. Western blotting was used to detect the protein expression of cleaved-Caspase3, Bax, LC3II, Beclin1, PTEN, and p-AKT. DCFH-DA staining was used to detect ROS levels by flow cytometry. RESULTS: PRDX1 was significantly decreased in placental trophoblasts in PE patients. Following the exposure of HTR-8/SVneo cells to H2O2, PRDX1 expression was significantly decreased, LC3II and Beclin1 expression was notably increased, and ROS level was also markedly increased. PRDX1 knockdown impaired migration, invasion, and tube-formation abilities and promoted apoptosis, which was accompanied by an increased expression of cleaved-Caspase3 and Bax. PRDX1 knockdown induced a significant decrease in LC3II and Beclin1 expression, along with an elevated p-AKT expression and a decreased PTEN expression. PRDX1 knockdown increased intracellular ROS levels, and NAC attenuated PRDX1 knockdown-induced apoptosis. CONCLUSION: PRDX1 regulated trophoblast function through the PTEN/AKT signaling pathway to affect cell autophagy and ROS level, which provided a potential target for the treatment of PE.

Protection by hydroxychloroquine prevents placental injury in obstetric antiphospholipid syndrome.

Obstetric antiphospholipid syndrome (OAPS) is mediated by antiphospholipid antibodies (aPLs, and anti-ß2 glycoprotein I antibody is the main pathogenic antibody), and recurrent abortion, preeclampsia, foetal growth restriction and other placental diseases are the main clinical characteristics of placental pathological pregnancy. It is a disease that seriously threatens the health of pregnant women. Hydroxychloroquine (HCQ) was originally used as an anti-malaria drug and has now shown benefit in refractory OAPS where conventional treatment has failed, with the expectation of providing protective clinical benefits for both the mother and foetus. However, its efficacy and mechanism of action are still unclear. After clinical data were collected to determine the therapeutic effect, human trophoblast cells in early pregnancy were prepared and treated with aPL. After the addition of HCQ, the proliferation, invasion, migration and tubule formation of the trophoblast cells were observed so that the therapeutic mechanism of HCQ on trophoblast cells could be determined. By establishing an obstetric APS mouse model similar to the clinical situation, we were able to detect the therapeutic effect of HCQ on pathological pregnancy. The normal function of trophoblast cells is affected by aPL. Antibodies reduce the ability of trophoblast cells to invade and migrate and can impair tubule formation, which are closely related to placental insufficiency. HCQ can partially reverse these side effects. In the OAPS mouse model, we found that HCQ prevented foetal death and reduced the incidence of pathological pregnancy. Therefore, HCQ can improve pregnancy outcomes and reverse the aPL inhibition of trophoblast disease. In OAPS, the use of HCQ needs to be seriously considered.

A Critical Role of Formyl Peptide Receptors in Host Defense against Escherichia coli.

Formyl peptide receptors (FPRs, mouse Fprs) belong to the G protein-coupled receptor superfamily and mediate phagocyte migration in response to bacteria- and host-derived chemoattractants; however, knowledge about their in vivo roles in bacterial pathogenesis is limited. In this study, we investigated the role of Fpr1 and Fpr2 in host defense against Escherichia coli infection. In vitro, we found that supernatants from E. coli cultures induced chemotaxis of wild-type (WT) mouse bone marrow-derived neutrophils and that the activity was significantly reduced in cells genetically deficient in either Fpr1 or Fpr2 and was almost absent in cells lacking both receptors. Consistent with this, E. coli supernatants induced chemotaxis and MAPK phosphorylation in HEK293 cells expressing either recombinant Fpr1 or Fpr2 but not untransfected parental cells. WT bone marrow -derived neutrophils could actively phagocytose and kill E. coli, whereas both activities were diminished in cells lacking Fpr1 or Fpr2; again, an additive effect was observed in cells lacking both receptors. In vivo, Fpr1 and Fpr2 deficiency resulted in reduced recruitment of neutrophils in the liver and peritoneal cavity of mice infected with inactivated E. coli Moreover, Fpr1-/- and Fpr2-/- mice had significantly increased mortality compared with WT mice after i.p. challenge with a virulent E. coli clinical isolate. These results indicate a critical role of Fprs in host defense against E. coli infection.

Identification and ultrasensitive photoelectrochemical detection of LncNR_040117: a biomarker of recurrent miscarriage and antiphospholipid antibody syndrome in platelet-derived microparticles.

The abnormal expression of long non-coding RNAs (LncRNAs) in platelet-derived microparticles (PMPs) is closely related to immune disorders and may lead to antiphospholipid antibody syndrome and recurrent miscarriage. To understand the association between the LncRNAs in PMPs and RM/APS, the differences in the expression of LncRNAs in RM/APS patients and healthy controls were analyzed. Microarray analysis and RT-qPCR detection proved that RM/APS patient exhibited high levels of LncNR_040117 expression. The lentiviral silent expression transfection of HTR-8/SVneo cells indicated that LncNR_040117 downregulation decreased the activity of HTR-8/SVneo cells and inhibited the MAPK signaling pathway, further confirming the biomarker proficiency of LncNR_040117 for RM/APS. After that, we proposed a ß-In2S3@g-C3N4 nanoheterojunction-based photoelectrochemical (PEC) biosensor to achieve the ultrasensitive detection of LncNR_040117. The nanoheterojunction aids in the effective separation of photogenerated carriers and significantly improve the photocurrent response of the biosensor. The conjugation of LncNR_040117 onto the PEC biosensing platform increased the steric hindrance between electrolyte and electrode, subsequently decreasing the photocurrent signal. The PEC biosensor showed a wide detection range of 0.1-106 fM and a low limit of detection of 0.025 fM. For clinical sample testing, the results of the PEC and RT-qPCR were highly consistent. Overall, LncNR_040117 in PMPs was identified as an effective biomarker for RM/APS and could be accurately detected by the proposed PEC biosensor, which is expected to provide a reliable diagnostic platform for RM/APS.

Suppression of FPR2 expression inhibits inflammation in preeclampsia by improving the biological functions of trophoblast via NF-κB pathway.

PURPOSE: The dysfunction of trophoblast during inflammation plays an important role in PE. Formyl peptide receptor 2 (FPR2) plays crucial roles in the development of inflammation-associated disease. This present study aimed to explore the effect of FPR2 on a trophoblast cellular model of preeclampsia. METHODS: The expression of FPR2 in placenta was detected by immunohistochemical staining and western blotting. Transfection of siRNA was used to knockdown FPR2 in HTR-8/SVneo cells. Inflammatory cytokines were detected by ELISA. CCK8, Transwell, wound healing, FACS and tube formation assays were performed to observe the abilities of cell proliferation, migration, invasion, apoptosis and angiogenesis. Western blotting was implemented to clarify that NF-κB signaling pathway was downstream of FPR2. RESULTS: The expression levels of FPR2 were higher in placental tissues of patients with PE. Knockdown of FPR2 expression by siFPR2 or inhibition of its activity by WRW4 decreased the release of proinflammatory cytokines in HTR8/SVneo cells treated with LPS. Knockdown of FPR2 expression or inhibition of its activity further reversed the LPS-induced attenuation of the proliferation, migration, invasion and angiogenesis and increase in apoptosis in HTR8/SVneo cells. Moreover, the NF-κB signaling pathway was activated in both placental tissues of patients with PE and LPS-treated HTR8/SVneo cells. However, the activation was attenuated when FPR2 was knocked down or inhibited. CONCLUSION: Suppression of FPR2 expression alleviated the effects of inflammation induced by LPS on trophoblasts via the NF-κB signaling pathway, which provided a novel and potential strategy for the treatment of PE.

Influence of indications on perinatal outcomes after radio frequency ablation in complicated monochorionic pregnancies: a retrospective cohort study.

BACKGROUND: Radiofrequency ablation (RFA) is recommended to prevent potential neurological injury or intrauterine foetal death (IUFD) of the co-twin(s) in complicated monochorionic (MC) pregnancies. However, the impacts of various indications on the pregnancy outcome following RFA remain unclear. This study aimed to determine how the indications influence the perinatal outcomes in complicated MC pregnancies undergoing radiofrequency ablation. METHODS: This was a retrospective cohort study performed in a single centre. All consecutive MC pregnancies treated with RFA between July 2011 and July 2019 were included. The adverse perinatal outcomes and the survival rate were analysed based on various indications. The continuous variables with and without normal distribution were compared between the groups using Student's t-test and Mann-Whitney U test, respectively, and for categorical variables, Chi-square and Fisher's exact tests were used. P < 0.05 indicated a significant difference. RESULTS: We performed 272 RFA procedures in 268 complicated MC pregnancies, including 60 selective intrauterine growth restriction (sIUGR), 64 twin-twin transfusion syndrome (TTTS), 12 twin reversed arterial perfusion sequence (TRAPs), 66 foetal anomaly and 66 elective foetal reduction (EFR) cases. The overall survival rate of the co-twin was 201/272 (73.9%). The overall technical successful rate was determined at 201/263 (76.7%). The IUFD rate in the co-twin was 20/272 (7.4%). The TTTS group had recorded the lowest survival rate (37/64, 57. 8%), and the survival rate was significantly correlated with Quintero stages (P = 0.029). Moreover, the sIUGR III subgroup had a lower survival rate compared with sIUGR II (55.6%, versus 84.3%). The subgroup of foetal anomaly of gastroschisis or exomphalos had the highest IUFD rate (4/10, 40%), followed by sIUGR III (2/9, 22.2%) and dichorionic triamniotic (DCTA) subgroup (8/46, 17.9%). In EFR group, eight IUFD cases were all coming from the DCTA subgroup and received RFA before 17 weeks. CONCLUSIONS: The perinatal outcome of RFA was correlated with the indications, with the lowest survival rate in TTTS IV and the highest IUFD incidence in abdominal wall defect followed by sIUGR III. Elective RFA after 17 weeks may prevent IUFD in DCTA pregnancies.

Uterine metabolic disorder induced by silica nanoparticles: biodistribution and bioactivity revealed by labeling with FITC.

Extensive application of nanomaterials has dramatically increased the risk of silica nanoparticle (SiNP, SiO2) exposure, yet their biological effect on reproduction has not been fully elucidated. By tracking the uterine biodistribution of SiNP in pregnant mice, this study was conducted to evaluate the biological effect of SiNP on reproduction. First, SiNP was conjugated with FITC, and then the FITC-SiNP was administrated to trophoblast (100 µg/mL, 24 h) in vitro and pregnant mice (0.25 mg/mouse, 2-24 h) in vivo. It was found that the FITC-SiNP was internalized by trophoblast and deposited in the uterus. The internalization of SiNP caused trophoblast dysfunction and apoptosis, while SiNP accumulation in the uterus induced diffuse inflammatory infiltration. The genome-wide alteration of gene expression was studied by high throughput sequencing analysis, where 75 genes were found to be dysregulated after SiNP exposure, among which ACOT2, SCD1, and CPT1A were demonstrated to regulate the biosynthesis of unsaturated fatty acids. Moreover, the suppression of unsaturated fatty acids caused mitochondrial overload of long-chain fatty acyl-CoA (LACoA), which further induced both trophoblast apoptosis and endometrial inflammation. In conclusion, the successful conjugation of FITC onto SiNP facilitated the tracking of SiNP in vitro and in vivo, while exposure to FITC-SiNP induced uterine metabolic disorder, which was regulated by the ACOT/CPT1A/SCD1 axis through the biosynthesis of unsaturated fatty acids signaling pathway.

MicroRNA-513c-5p is involved in the pathogenesis of preeclampsia by regulating of low-density lipoprotein receptor-associated protein 6.

BACKGROUND: Preeclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality. Studies on the role of microRNAs (miRNAs), in the pathogenesis of PE through their effects on trophoblast function have been reported, but roles for some miRNAs including miR-513c-5p, have not been identified. We aimed to evaluate potential miRNA candidates that regulate the LRP6 mRNAand to elucidate the possible mechanism in PE. Potential miRNAs were selected by bioinformatics analysis, PCR of placenta tissues and dual luciferase reporter assay of HTR-8/SVneo cells. METHODS: A bioinformatics analysis (Gene Expression Omnibus, GEO; miRWalk) was performed to screen the possible miRNAs that participate in the pathology of PE. Placentas from patients with PE and women with a normal pregnancy were collected to detect the expression of predicted miRNAs by RT-qPCR. A dual luciferase reporter assay was used to test the binding of the potential miRNAs to LRP6. The effects of miR-513c-5p on the biological functions of HTR-8/SVneo cells were further evaluated by performing EdU staining, flow cytometry, wound healing assays and Transwell assays. RESULTS: GEO and miRWalk predicted 16 miRNAs that might target LRP6. Hsa-miR-371a-5p, hsa-miR-513c-5p, hsa-miR-126-3p, hsa-miR-145-5p, hsa-miR-193b-5p and hsa-miR-296-5p were 6 miRNAs upregulated in the PE placenta. LRP6 was downregulated in patients with PE compared to normal women. miR-513c-5p mimics inhibited LRP6 expression in HTR-8/SVneo cells, and LRP6 is the target gene of miR-513c-5p. miR-513c-5p mimics also inhibited invasion, migration and proliferation of HTR-8/SVneo cells but promoted their apoptosis. CONCLUSIONS: Our study reveals that overexpression of placenta miR-513c-5p is involved in PE by regulating the biological functions of trophoblasts through the inhibition of LRP6.

Obstetric outcomes for twins from different conception methods - A multicenter cross-sectional study from China.

INTRODUCTION: The effects of assisted reproductive technology on the outcomes of twin pregnancies are controversial. Therefore, the purpose of this study was to compare the maternal and perinatal outcomes of twin pregnancies conceived spontaneously and those conceived by assisted reproductive technology. MATERIAL AND METHODS: This was a cross-sectional study performed at Peking Union Medical College Hospital (PUMCH). Data on twin pregnancies (conceived spontaneously and by in vitro fertilization [IVF]/intracytoplasmic sperm injection [ICSI]) were obtained from the National Birth Registry of China for the period between 1 October 2016, and 30 September 2017. The primary obstetric outcomes were compared between twin pregnancies conceived by different methods. Logistic regression analysis with 95% confidence intervals (95% CI) was used for the multivariate analysis. RESULTS: A total of 3270 twin pregnancies (2003 and 1209 conceived spontaneously and by IVF/ICSI, respectively) were identified. The proportion of twin pregnancies among all pregnancies was 3.4% (3332/97 278). Multiple regression analysis indicated that the incidences of gestational diabetes mellitus (adjusted odds ratio [AOR] = 1.42, 95% CI 1.10-1.83, p = 0.007), preterm premature rupture of membranes (AOR = 1.65, 95% CI 1.21-2.25, p = 0.002), placenta accreta spectrum (AOR = 2.12, 95% CI 1.42-3.17, p < 0.001) and postpartum hemorrhage (AOR = 1.38, 95% CI 1.02-1.86, p = 0.037) were significantly higher in the IVF/ICSI group than in the natural pregnancy group. Multivariate analysis also revealed that conception mode was not an independent risk factor for neonate outcomes. CONCLUSIONS: In twin pregnancies, IVF/ICSI is independently associated with multiple maternal complications, including gestational diabetes mellitus, preterm premature rupture of membranes and placenta accreta spectrum compared with spontaneous conception, although potential residual confounders due to indications for assisted reproductive technology exist.

Effect of RvD1/FPR2 on inflammatory response in chorioamnionitis.

Chorioamnionitis (CAM), as a common intrauterine infectious disease, is the leading cause of premature birth, stillbirth, neonatal infection and sepsis. The formyl peptide receptor 2 (FPR2) is a member of GPCRs widely distributed in a variety of tissues and is associated with many inflammatory diseases. With the discovery of FPR2 in human placenta, the possibility of exploring the function of FPR2 in obstetrics is evolving. The Resolvin D1 (RvD1) plays an important role in the resolution of inflammation by combining with FPR2. In this study, we evaluated the role of FPR2 and RvD1 in CAM, not only in the human placenta but also in mouse models. The expression of FPR2 increased in the placenta of CAM patients and the downstream PPARγ/NF-κB signalling changed accordingly. Moreover, Fpr2-/- mice were highly susceptible to LPS, displaying a worse CAM symptom, compared with WT mice. By establishing a model of trophoblast inflammation in vitro, it was confirmed that RvD1 rescued the effect of LPS on inflammation by combining with FPR2 and its downstream PPARγ/NF-κB pathway. Otherwise, RvD1 improved the preterm labour in a mouse model of CAM induced by LPS. Altogether, these findings show that RvD1 alleviated the inflammation of trophoblast in vivo and in vitro through FPR2/PPARγ/NF-κB pathway, suggesting RvD1/FPR2 might be a novel therapeutic strategy to alleviate CAM.

Antiphospholipid antibody-activated NETs exacerbate trophoblast and endothelial cell injury in obstetric antiphospholipid syndrome.

Despite the widespread use of antiplatelets and anticoagulants, women with antiphospholipid syndrome (APS) may face pregnancy complications associated with placental dysplasia. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of many autoimmune diseases, including vascular APS; however, their role in obstetric APS is unclear. Herein, we investigated the role of NETs by quantifying cell-free DNA and NET marker levels. Live-cell imaging was used to visualize NET formation, and MAPK signalling pathway proteins were analysed. Cell migration, invasion and tube formation assays were performed to observe the effects of NETs on trophoblasts and human umbilical vein endothelial cells (HUVECs). The concentrations of cell-free DNA and NETs in sera of pregnant patients with APS were elevated compared with that of healthy controls (HCs) matched to gestational week. APS neutrophils were predisposed to spontaneous NET release and IgG purified from the patients (APS-IgG) induced neutrophils from HCs to release NETs. Additionally, APS-IgG NET induction was abolished with inhibitors of reactive oxygen species, AKT, p38 MAPK and ERK1/2. Moreover, NETs were detrimental to trophoblasts and HUVECs. In summary, APS-IgG-induced NET formation deserves further investigation as a potential novel therapeutic target in obstetrical APS.

LRP6 regulates Rab7-mediated autophagy through the Wnt/ß-catenin pathway to modulate trophoblast cell migration and invasion.

Pre-eclampsia is a common complication during pregnancy; however, the underlying mechanisms of the crosstalk between low-density lipoprotein receptor-related protein 6 (LRP6) and autophagy in trophoblast cells are still not fully explored. Messenger RNA (mRNA) and protein levels of LRP6, beclin 1, Unc-51-like autophagy activating kinase 1 (ULK1), p62, vimentin, matrix metallopeptidase-9 (MMP-9), ß-catenin, c-Myc, and Rab7, as well as the ratio of LC3-II/LC3-I, were analysed by quantitative real-time polymerase chain reaction or Western blot analysis, respectively. An MTT assay was used to measure cell growth, and transwell and wound healing assays were carried out to evaluate the invasion and migration abilities of the trophoblasts used. An immunofluorescence assay was used to measure LC3. The mRFP-GFP-LC3 tandem fluorescence assay was applied to detect autophagic flow. LRP6 overexpression was achieved by constructing pcDNA3.1-LRP6 vectors. LRP6 was expressed at low levels in HTR-8/SVneo cells under hypoxia/reoxygenation (H/R) conditions. H/R inhibited the activation of autophagy. LRP6 overexpression promoted cell proliferation and activated autophagy, which led to the upregulation of beclin 1 and ULK1, as well as the ratio of LC3-II/LC3-I and the downregulation of p62. Furthermore, LRP6 overexpression elevated the migration and invasion abilities of the indicated cells and increased vimentin and MMP-9 expression levels. Furthermore, LRP6 upregulated Rab7 and activated autophagy through the Wnt/ß-catenin pathway. The late autophagy inhibitor bafilomycin A1 (Baf-A1) and the Wnt/ß-catenin pathway inhibitor PKF115-584 reversed the effects of LRP6 on trophoblast autophagy, migration and invasion. LRP6 promotes Rab7-mediated autophagy by activating the Wnt/ß-catenin pathway, which leads to increasing migration and invasion of trophoblast cells. Our study paves a new avenue for clinical treatment, and LRP6 may serve as an essential target in pre-eclampsia.

Implication of nuclear factor-erythroid 2-like 2/heme oxygenase 1 pathway in the protective effects of coenzyme Q10 against preeclampsia-like in a rat model.

BACKGROUND: Preeclampsia has ranked as one of the leading causes of both maternal and prenatal morbidity and mortality around the world. The hypotensive effect of coenzyme Q10 has been widely reported in preeclampsia rat model. However, the detailed mechanism remains unclear. METHODS: L-NAME was utilized to establish the preeclampsia rat model. Biomarker assessments were performed to identify the levels of vascular factors including soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PlGF), the circulating cytokines including interleukin 6, tumor necrosis factor α and interleukin 1ß, and oxidative stress factors including malondialdehyde, H2 O2 , glutathione, superoxide dismutase, glutathione peroxidase, and Catalase. QRT-PCR was used to demonstrate the levels of cytokines in placenta tissues, and Western blot was performed to estimate the nuclear factor-erythroid 2-like 2 (Nrf2) and heme oxygenase 1 (HO-1) protein levels. RESULTS: Coenzyme Q10 treatment decreased the blood pressure in rat model with preeclampsia by regulating the circulating levels of sFlt-1 and PlGF. Coenzyme Q10 attenuated serum and placental inflammation and oxidative stress in L-NAME-induced preeclampsia rats. Coenzyme Q10 activated the placental Nrf2/HO-1 pathway in L-NAME-induced preeclampsia rats. CONCLUSION: Coenzyme Q10 attenuated placental inflammatory and oxidative stress, thereby protecting the rats against preeclampsia by activating the Nrf2/HO-1 pathway.

Decorin promotes apoptosis and autophagy via suppressing c-Met in HTR-8 trophoblasts.

Decorin (DCN) regulates a vast array of cellular processes including proliferation, migration, apoptosis, and autophagy, and its aberrant expression has been associated with poor extravillous trophoblasts (EVT) invasion of the uterus, which underlies the occurrence of preeclampsia (PE) and intrauterine growth restriction (IUGR). In this study, we aim to elucidate the molecular mechanism of how the DCN regulates the cell functions through the use of trophoblast cell line, HTR-8. Using a series of cell function assays, including CCK8, RTCA, transwell, scratch-wound assay, and Annexin V staining, we found that DCN suppressed proliferation and invasion, while promoted autophagy and apoptosis of HTR-8 in a dose-dependent manner. Transient stimulation of DCN have increased the activity of c-Met and its downstream effectors - Akt, FAK and m-TOR. However, a prolonged exposure to DCN have significantly downregulated the expression of c-Met, leading to suppression of its downstream effectors. Lentivirus that overexpressed c-Met targeting shRNA was used to knockdown c-Met expression and crizotinib was used to selectively inhibit the kinase activity of c-Met in HTR-8 cells. A combination of DCN and c-Met knockdown/inhibition have reduced the proliferation and invasion in HTR-8 cells; however, DCN-induced autophagy and apoptosis were not synergistically enhanced by c-Met inhibition. In conclusion, DCN promotes autophagy and apoptosis predominantly through downregulating c-Met/Akt/mTOR activity in human trophoblasts.

Platelet-derived microparticles from recurrent miscarriage associated with antiphospholipid antibody syndrome influence behaviours of trophoblast and endothelial cells.

Platelet-derived microparticles (PMPs) are a type of microparticle budding from platelets undergoing activation or apoptosis in many autoimmune diseases, including antiphospholipid antibody syndrome (APS). PMPs may also contribute to recurrent miscarriage, although the exact mechanism is unclear. The aim of this study was to determine the potential biological mechanism by which abnormal PMP activation may affect recurrent miscarriage. PMPs were counted by fluorescence-activated cell sorting (FACS) and compared between the healthy control (HC) and recurrent miscarriage/APS groups. Different effects of PMPs isolated by FACS from patients with recurrent miscarriage/APS and HCs were explored. Capillary electrophoresis immunoquantification, RT-qPCR, Luminex xMAP and immunofluorescence staining were performed to investigate all these different effects of PMPs. We found that the difference in the counts of PMP was not significant. However the expression of the inflammatory cytokine tumour necrosis factor-α (TNF-α) and the adhesion molecules intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were increased by PMPs derived from the recurrent miscarriage/APS group. PMPs isolated from patients with recurrent miscarriage/APS also more potently stimulated monocyte recruitment, inhibited angiogenesis and promoted human umbilical vein endothelial cell (HUVEC) apoptosis, in comparison to PMPs from HCs matched for gestational week. Moreover, PMPs could be ternalized by HTR-8/SVneo cells and could increase apoptosis of these cells and decrease trophoblastic invasion and migration. To supplement our work, the limited sample size needs to be increased, and further in-vivo work is necessary. Findings from this study indicate that abnormal activation of PMPs contributes to recurrent miscarriage/APS progression and provides potential therapeutic targets.