Hyperglycemia-induced endothelial exosomes trigger trophoblast dysregulation and abnormal placentation through PUM2-mediated repression of SOX2.

BACKGROUND: Hyperglycemia is closely related to adverse pregnancy outcomes including pre-eclampsia (PE), a life-threatening complication with a substantial morbidity and mortality. However, the pathogenesis of abnormal placentation in gestational diabetes mellitus (GDM)-associated PE remains elusive. METHOD: Here we isolated exosomes from the human umbilical vein endothelial cells (HUVECs) treated with normal level of glucose (NG) and high levels of glucose (HG). The exosomes were added to HTR-8a/SVneo cells, a trophoblast cell line. High-throughput RNA-sequencing was performed to analyzed the changed RNAs in the exosomes and exosome-treated HTR-8a/SVneo cells. HTR-8a/SVneo cell phenotypes were evaluated from the aspects of cell proliferation, cell invasion and DNA damage. RESULTS: After treatment with HG, the changed RNAs in exosomes was enriched in RNA stabilization and oxidative stress. The altered RNAs in the HTR-8a/SVneo cells treated with exosomes from HG-induced HUVECs were enriched in pathways related to cell adhesion, migration, DNA damage response and angiogenesis. The HG-induced exosomes impaired the proliferation and invasion of HTR-8a cells and caused the DNA damage. HG up-regulated PUM2 in the exosomes and exosome-treated HTR-8a/SVneo cells. PUM2 interacted with SOX2 mRNA, resulting in the mRNA degradation. Overexpression of SOX2 prevented the damage to HTR-8a/SVneo cells caused by the exosomes from HG-induced HUVECs. CONCLUSIONS: We demonstrate that high glucose-induced endothelial exosomes mediate abnormal phenotypes of trophoblasts through PUM2-mediated repression of SOX2. Our results reveal a novel regulatory mechanism of hyperglycemia in development of abnormal placentation and provide potential targets for preventing adverse pregnancy outcomes.

Circular RNA expression profiles in umbilical cord blood exosomes from normal and gestational diabetes mellitus patients.

Circular RNA (circRNA) is a novel member of endogenous noncoding RNAs with widespread distribution and diverse cellular functions. Recently, circRNAs have been identified for their enrichment and stability in exosomes. However, the roles of circRNAs from umbilical cord blood exosomes in gestational diabetes mellitus (GDM) occurrence and fetus growth remains poorly understood. In the present study, we used microarray technology to construct a comparative circRNA profiling of umbilical cord blood exosomes between GDM patients and controls. We found the exosome particle size was larger, and the exosome concentration was higher in the GDM patients. A total of 88,371 circRNAs in umbilical cord blood exosomes from two groups were evaluated. Of these, 229 circRNAs were significantly up-regulated and 278 circRNAs were significantly down-regulated in the GDM patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses demonstrated that circRNA parental genes involved in the regulation of metabolic process, growth and development were significantly enriched, which are important in GDM development and fetus growth. Further circRNA/miRNA interactions analysis showed that most of the exosomal circRNAs harbored miRNA binding sites, and some miRNAs were associated with GDM. Collectively, these results lay a foundation for extensive studies on the role of exosomal circRNAs in GDM development and fetus growth.

The restoration of Wnt/ß-catenin signalling activity by a tuna backbone-derived peptide ameliorates hypoxia-induced cardiomyocyte injury.

Myocardial infarction (MI) is a serious disease with high morbidity and mortality worldwide. Reducing myocardial reperfusion injury in MI patients remains a challenge. The generation of excessive reactive oxygen species (ROS) during reperfusion is known to be responsible for injury. A peptide from tuna backbone protein (APTBP) captured our attention due to its strong antioxidant activity. Here, we aimed to assess the function of APTBP in protecting against myocardial ischaemia-reperfusion (I/R) injury and to clarify the associated mechanism. Two in vitro models generated by hypoxia and cobalt chloride treatment were used to determine the effect of APTBP on cardiomyocytes under hypoxic stress. In vivo, a rat model of I/R was generated to evaluate APTBP functions. As a result, APTBP attenuated hypoxia- or cobalt chloride-induced injury to H9C2 cells and primary cardiomyocytes. Moreover, hypoxia-induced apoptosis, ROS generation and impaired mitochondrial function were also suppressed by APTBP administration. In vivo, tail vein injection of APTBP ameliorated pathological damage and mildly restored cardiac function. To clarify the mechanism, RNA-seq was performed and revealed that the Wnt signalling pathway may be associated with this mechanism. Rescue analysis showed that ß-catenin knockdown diminished the protective effect of APTBP and that the expression of an ROS generator abolished the restoration of Wnt/ß-catenin signalling induced by APTBP. Collectively, our findings suggest that APTBP reduces cardiomyocyte apoptosis and protects against myocardial ischaemia-reperfusion injury by scavenging ROS and subsequently restoring Wnt/ß-catenin signalling.

Differential mRNA and Long Noncoding RNA Expression Profiles in Umbilical Cord Blood Exosomes from Gestational Diabetes Mellitus Patients.

Background and Aims: Exosomes contain numerous RNAs and transfer them between cells or organs, thereby establishing intercellular or interorgan communication. The roles of mRNAs and long noncoding RNAs (lncRNAs) from umbilical cord blood exosomes in gestational diabetes mellitus (GDM) occurrence and fetus growth remain poorly understood. We aimed to establish the differential mRNA and lncRNA expression profiles in umbilical cord blood exosomes from GDM patients compared with normal controls. Results: Using microarray technology, we identified 84 mRNAs and 256 lncRNAs as differentially expressed in umbilical cord blood exosomes of GDM patients compared with controls. The protein-protein interaction network revealed that the differentially expressed mRNAs were associated with glucagon signaling pathway, an important GDM-related pathway. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses were performed for mRNAs associated with differentially expressed lncRNAs. The results indicated that metabolic process, growth, and development were significantly enriched, which are important in GDM development and fetus growth. Moreover, pathway network was constructed to reveal the key pathways in GDM, such as metabolic pathways and insulin signaling pathway. Further lncRNA/miRNA interaction analysis showed that most of the exosomal lncRNAs harbored miRNA binding sites, and some were associated with GDM. Conclusion: These results showed that exosomal mRNAs and lncRNAs are aberrantly expressed in the umbilical cord blood of GDM patients and play potential roles in GDM development and fetus growth.

Identification of Differentially Expressed Proteins in Serum of Obese Patients by Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Coupled 2D LC-MS.

BACKGROUND The aim of this study was to identify the differentially expressed proteins of obese patients compared with normal participants and to provide a potential target for future investigation of obesity. MATERIAL AND METHODS We enrolled 10 obese male adults and 10 matched normal subjects. Serum samples were collected to get total protein extraction, denaturation, deoxidation, and enzymatic hydrolysis. Differentially expressed proteins were distinguished with mass spectrometry after samples were labeled with iTRAQ. RESULTS A total of 9622 differentially expressed peptides were identified, corresponding to 733 proteins; 118 proteins of these showed significant differential expression, with 15 upregulated and 103 downregulated. CONCLUSIONS iTRAQ is an effective technique to identify differentially expressed proteins in obese patients. The development of obesity is correlated with a series of complex elements and mutual effects. The proteins identified in this study may provide novel directions and targets for future pathological studies of obesity.

Genetic Susceptibility to Gestational Diabetes Mellitus in a Chinese Population.

Introduction: New genetic variants associated with susceptibility to obesity and metabolic diseases have been discovered in recent genome-wide association (GWA) studies. The aim of this study was to investigate the association of theses risk variants with gestational diabetes mellitus (GDM). Methods: We performed a case-control study including 964 unrelated pregnant women with GDM and 1,021 pregnant women with normal glucose tolerance (as controls). A total of 33 genetic variants confirmed by GWA studies for obesity and metabolic diseases were selected and measured. Results: We observed that FTO rs1121980 and KCNQ1 rs163182 conferred a decreased GDM risk in the dominant and additive model [additive model: OR (95% CI) = 0.79 (0.67-0.94), P = 0.007 for rs1121980; OR(95%CI) = 0.84 (0.73-0.96), P = 0.009 for rs163182], whereas MC4R rs12970134 and PROX1 rs340841 conferred an increased GDM risk in the dominant, recessive, and additive model [additive model: OR(95%CI) = 1.25 (1.07-1.46), P = 0.006 for rs12970134; OR(95%CI) = 1.22 (1.07-1.39), P = 0.002 for rs340841). With the increasing number of risk alleles of the four significant SNPs, GDM risk was significantly increased in a dose-dependent manner (Ptrend < 0.001). And the significant positive associations between the weighted genetic risk score and risk of GDM persisted. Further function annotation indicated that these four SNPs may fall on the functional elements of human pancreatic islets. The genotype-phenotype associations indicated that these SNPs may contribute to GDM by affecting the expression levels of their nearby or distant genes. Conclusion: Our study suggests that FTO rs1121980, KCNQ1 rs163182, MC4R rs12970134, and PROX1 rs340841 may be markers for susceptibility to GDM in a Chinese population.

miR-30-5p-mediated ferroptosis of trophoblasts is implicated in the pathogenesis of preeclampsia.

Oxidative stress is a major cause of adverse outcomes in preeclampsia (PE). Ferroptosis, i.e. programmed cell death from iron-dependent lipid peroxidation, likely mediates PE pathogenesis. We evaluated specific markers for ferroptosis in normal and PE placental tissues, using in vitro (trophoblasts) and in vivo (rat) models. Increase in malondialdehyde content and total Fe2+ along with reduced the glutathione content and glutathione peroxidase activity was observed in PE placenta. While the trophoblasts experienced death under hypoxia, inhibitors of ferroptosis, apoptosis, autophagy, and necrosis increased the cell viability. Microarrays, bioinformatic analysis, and luciferase reporter assay revealed that upregulation of miR-30b-5p in PE models plays a pivotal role in ferroptosis, by downregulating Cys2/glutamate antiporter and PAX3 and decreasing ferroportin 1 (an iron exporter) expression, resulting in decreased GSH and increased labile Fe2+. Inhibition of miR-30b-5p expression and supplementation with ferroptosis inhibitors attenuated the PE symptoms in rat models, making miR-30b-5p a potential therapeutic target for PE.

Application of iTRAQ proteomics in identification of the differentially expressed proteins of placenta of pregnancy with preeclampsia.

OBJECTIVE: To explore the differential protein profile of preeclampsia and identify its potential biomarker. METHODS: Around 20 pregnant women with preeclampsia (preeclampsia group) and 20 normal-term pregnancy (normal group) were collected from 2017 to 2018 in the study. Total protein of placenta tissues was extracted, denaturized, deoxidized, and enzymolyzed. The sample was labeled with isobaric tags for relative and absolute quantitation (iTRAQ) and analyzed with mass spectrum to identify differentially expressed proteins. RESULTS: There were 37 proteins, which were differentially expressed with significance (P < 0.05). Among them, 17 proteins were upregulated and 20 proteins were downregulated with significance in the placenta of preeclampsia group compared with control group, those proteins may have an induction or protection function during the development of preeclampsia. CONCLUSION: iTRAQ technology can effectively screen the differentially expressed proteins in the placenta, which can effectively diagnose the preeclampsia during pregnancy.

miR-362-3p regulates cell proliferation, migration and invasion of trophoblastic cells under hypoxia through targeting Pax3.

Preeclampsia (PE), a common obstetrical disorder, is one of the leading causes of pregnancy associated death. PE is closely linked with impaired migration and invasion ability of trophoblastic cells. miR-362-3p recently received our particular attention due not only to its aberrant expression in the placentas of patients with PE, but also to its important roles in regulating migration and invasion of various cells. This study was thus conducted to investigate the roles of miR-362-3p in PE and the related mechanism. The expression of miR-362-3p and Pax3 was examined in placentas of patients with PE and in normal placentas. HTR8/SVneo cells were cultured under hypoxia and transfected with miR-362-3p mimics, miR-362-3p inhibitors or Pax3 over-expression vectors. Results showed up-regulation of miR-362-3p but down-regulation of Pax3 in placentas of preeclamptic pregnancies. Luciferase report assay confirmed that Pax3 is a direct target of miR-362-3p. Although Pax3 was predicted to be targeted by miR-30a-3p and miR-181a-5p as well, their expression either had no difference between placentas of PE patients and normal placentas or showed less increment in placentas of PE patients than miR-362-3p. Exposure to hypoxia inhibited cell viability, migration and invasion of HTR8/SVneo cells. Increasing miR-362-3p by the mimics conferred improved effects on the inhibition. However, deletion of miR-362-3p or overexpression of Pax3 abolished the inhibiton. These results suggest that miR-362-3p/Pax3 axis regulates cell viability, migration and invasion of HTR8/SVneo cells under hypoxia. The present study adds to the further understanding of the pathogenesis of PE.

Fasudil attenuates soluble fms-like tyrosine kinase-1 (sFlt-1)-induced hypertension in pregnant mice through RhoA/ROCK pathway.

Preeclampsia (PE) has become the leading cause of maternal and fetal morbidity and mortality in the world, which is characterized by a systemic maternal inflammatory response associated with endothelial dysfunction, hypertension, and proteinuria. The development of PE is still barely predictable and thus challenging to prevent and manage clinically. Fasudil (FSD), the first-generation Rho/ROCK inhibitor, has been studied widely and applied in clinical practice with high safety and efficacy in treating hypertension and other cardiovascular diseases. However, few studies have focused on the effect of fasudil on preeclampsia in vivo and in vitro. Therefore, the aim of this study is to investigate the effects of fasudil on hypoxia/reoxygenation injury in vitro and its role on preeclamptic animal model. Here, we found that RhoA/ROCK pathway was significantly activated in H/R-challenged endothelial cells and in placenta and umbilical vessel of PE mice. And fasudil pre-treatment can protect vascular endothelial cells from H/R-induced apoptosis. In addition, inhibition of RhoA/ROCK pathway with fasudil can reduce the high blood pressure and urine protein levels as well as the concentration of s-Flt in peripheral and umbilical blood in a dose-dependent manner, thus resulting in prevention of the development of PE. Thus, Fasudil attenuates soluble fms-like tyrosine kinase-1 (sFlt-1)-induced hypertension in pregnant mice through RhoA/ROCK pathway, which would become a potential strategy for PE therapy.

Alpha-1-antitrypsin suppresses oxidative stress in preeclampsia by inhibiting the p38MAPK signaling pathway: An in vivo and in vitro study.

This present study was designed to investigate the effects of alpha-1-antitrypsin (AAT) on oxidative stress in preeclampsia (PE) by regulating p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. HTR8/SVneo cells were randomly assigned into normal, hypoxia/reoxygenation (H/R), HR + AAT and HR + siRNA-AAT groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expressions of p-p38MAPK, AAT, signal transducer and activator of transcription 1 (STAT1) and activating transcription factor2 (ATF2). Flow cytometry, scratch test, cell counting kit-8 (CCK-8) assay and the 3-(4,5)-dimethylthiazol (-z-y1)-3,5-di- phenyltetrazolium bromide (MTT) assay were conducted to detect reactive oxygen species (ROS) and cell apoptosis, cell migration, proliferation and cytotoxicity, respectively. Mouse models in PE were established, which were divided into normal pregnancy (NP), PE and PE + AAT groups with blood pressure and urine protein measured. Chromatin immunoprecipitation (ChIP) and enzyme-linked immunosorbent assay (ELISA) were conducted to detect the activity of oxidative stress-related kinases and expressions of inflammatory cytokines and coagulation-related factors in cells and mice placenta. Immunohistochemistry, Western blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect AAT and p38MAPK expressions, apoptosis-related protein expressions, and apoptosis rate in mice placenta. Compared with the normal group, the H/R group had decreased expression of AAT, activity of superoxide dismutase (SOD) and GSH-Px, cell proliferation and migration, but increased p38MAPK, STAT1, ATF2, MDA, H2O2, inflammatory cytokines, coagulation-related factors, cell cytotoxicity, ROS, apoptotic factors and apoptosis rate. Compared with the H/R group, the HR + ATT group had increased expressions of AAT, activity of SOD and GSH-Px, cell proliferation and migration but decreased p38MAPK, STAT1, ATF2, malonyldialdehyde (MDA), H2O2, inflammatory cytokines and coagulation-related factors, cell cytotoxicity, ROS, apoptotic factors and apoptosis rate, while opposite results were observed in the HR + siRNA-ATT group. Compared with the NP group, the PE group had decreased activity of SOD and GSH-Px but increased MDA, H2O2, AAT, p38MAPK, inflammatory cytokines, coagulation-related factors and apoptosis rate. The indexes in the PE + AAT group were between the NP and PE groups. Thus, we concluded that AAT suppressed oxidative stress in PE by inhibiting p38MAPK signaling pathway.

SIRT1 inhibits releases of HMGB1 and HSP70 from human umbilical vein endothelial cells caused by IL-6 and the serum from a preeclampsia patient and protects the cells from death.

Preeclampsia (PE), a pregnancy-specific disorder, is associated with inappropriate maternal inflammatory response, oxidative stress, and vascular endothelial cell dysfunction and damage. Releases of high mobility group box-1 (HMGB1) and heat-shock protein 70 (HSP70) into serum are considered to participate in the pathogenesis of PE. The deacetylase, sirtuin 1 (SIRT1), has protective effects against inflammation, apoptosis, and oxidative stress in various pathological conditions. We established a PE mouse model by injection of phosphatidylserine/dioleoyl-phosphatidycholine compounds, followed by measurement of the SIRT1 protein level in the placenta via Western blotting and the serum HMGB1 and HSP70 concentrations via enzyme-linked immunosorbent assay (ELISA). SIRT1 was down-regulated in the placenta of PE mice, in accompany with increased serum HMGB1 and HSP70 concentrations. We incubated human umbilical vein endothelial cells (HUVECs) with IL-6 and the serum from a PE patient individually to mimic status of vein endothelial cells in PE. Western blot and Immunofluorescent assays showed that HMGB1 and HSP70 protein levels were decreased in the cells, but they were increased in the cell medium based on ELISA. These suggested that HMGB1 and HSP70 were forced to be released from the cells. SIRT1 knockdown promoted the releases of HMGB1 and HSP70, whereas its over-expression inhibited the releases. Moreover, SIRT1 protected the cells from death. Collectively, SIRT1 inhibits the releases of HMGB1 and HSP70 from HUVECs caused by IL-6 and the serum from PE patient and protects the cells from death, thus SIRT1 is probably a potentially protective factor in placenta against PE.

Alpha-1-antitrypsin functions as a protective factor in preeclampsia through activating Smad2 and inhibitor of DNA binding 4.

Pre-eclampsia (PE) is one of the most common reason for high morbidity and mortality of maternal and prenatal infants. Production from oxidative stress results in maternal ROS system and anti-oxidation defense system imbalance to promote tissue ischemia and hypoxia, and ultimately impairs the maternal organs and placenta. Our previous study showed that exogenous Alpha-1-antitrypsin (AAT) and overexpression of AAT in umbilical vein cell (HUVEC) hypoxia-reoxygenation model could increase the activity of antioxidant enzymes, and played a protective role in preeclampsia animal model. In this study, we aim to investigate the underlying mechanism by which AAT prevents PE progress. Whole-exome sequencing was performed to screen the genes altered by AAT. We found that AAT knockdown altered the expression of Smad family and Id family genes, and further demonstrated that AAT positively regulated Id4 expression through activating Smad2. Reduced Id4 expression and Smad2 phosphorylation were observed in preeclampsia animal model, which was also confirmed in human placenta tissues. In addition, AAT protected HUVEC cells from hypoxia/reoxygenation injury and relieved preeclampsia symptoms through Smad2/Id4 axis. Our data illustrate AAT/Smad2/Id4 axis is an important mediator of placenta and vascular function during pregnancy. These findings provide insights into events governing pregnancy-associated disorders, such as preeclampsia.

Alpha-1 Antitrypsin Prevents the Development of Preeclampsia Through Suppression of Oxidative Stress.

Preeclampsia (PE) and its complications have become the leading cause of maternal and fetal morbidity and mortality in the world. And the development of PE is still barely predictable and thus challenging to prevent and manage clinically. Oxidative stress contributes to the development of the disease. Our previous study demonstrated that exogenous Alpha-1 antitrypsin (AAT) played a cytoprotective role in vascular endothelial cell by suppressing oxidative stress. In this study, we aim to investigate whether AAT contributes to the development of PE, and to identify the mechanism behind these effects. We found that AAT levels were significantly decreased in placenta tissues from women with PE compared that of healthy women. Notably, we demonstrate that AAT injection is able to relieve the high blood pressure and reduce urine protein levels in a dose-dependent manner in PE mice. In addition, our results showed that AAT injection exhibited an anti-oxidative stress role by significantly reducing PE mediated-upregulation of ROS, MMP9 and MDA, and increasing the levels of SOD, eNOS, and GPx with increased dosage of AAT. Furthermore, we found that AAT injection inactivated PE mediated activation of PAK/STAT1/p38 signaling. These findings were confirmed in human samples. In conclusion, our study suggests that exogenous AAT injection increases the antioxidants and suppresses oxidative stress, and subsequent prevention of PE development through inactivation of STAT1/p38 signaling. Thus, AAT would become a potential strategy for PE therapy.