MiRNA-26a-5p inhibits preterm labor initiation by targeting and regulating TRPC3 ion channel protein expression.

The incidence of preterm birth (PTB) is increasing annually worldwide, leading to various health problems or even fetal deaths. Our previous work demonstrated the activation of transient receptor potential cation channel subfamily C 3 (TRPC3) in mice with PTB, and its activation could promote inward flow of calcium ions and uterine smooth muscle (USM) contraction via regulation of Cav3.2, Cav3.1, and Cav1.2. However, the upstream regulators of TRPC3 and its mechanisms remain unknown. In the present study, the binding of miR-26a-5p to the 3' untranslated region of TRPC3 was predicted by bioinformatics databases (TargetScanHuman and starBase v3.0) and confirmed by a dual-luciferase assay. MiR-26a-5p was downregulated, while TRPC3 was upregulated in the USM tissues of patients with PTB compared to people without PTB. The results showed that miR-26a-5p mimic transfection markedly reduced TRPC3 expression in LPS-stimulated USM cells. Additionally, miR-26a-5p regulated intracellular Ca2+ levels in USM cells by targeting TRPC3. Furthermore, miR-26a-5p inhibited the CPI17/PKC/PLCγ signaling pathway and reduced the expression of Cav3.2, Cav3.1, and Cav1.2. In conclusion, miR-26a-5p regulated the initiation of PTB by targeting TRPC3 and regulating intracellular Ca2+ levels. This study provides a promising diagnostic biomarker and therapeutic target for PTB.

Functionalized TMC and ε-CL elastomers with shape memory and self-healing properties.

Introduction: Smart elastomers, which possess self-healing and shape memory capabilities, have immense potential in the field of biomedical applications. Polycarbonates and polyesters have gained widespread interest due to their remarkable biocompatibility over the last century. Nevertheless, the lack of functional versatility in conventional polyesters and polycarbonates means that they fall short of meeting the ever-evolving demands of the future. Methods: This paper introduced a new smart elastomer, named mPEG43-b-(PMBC-co-PCL)n, developed from polyester and polycarbonate blends, that possessed shape memory and self-heal capabilities via a physical crosslinking system. Results: The material demonstrated a significant tensile strength of 0.38 MPa and a tensile ratio of 1155.6%, highlighting its favorable mechanical properties. In addition, a conspicuous shape retrieval rate of 93% was showcased within 32.5 seconds at 37°C. Remarkably, the affected area could be repaired proficiently with no irritation experienced during 6h at room temperature, which was indicative of an admirable repair percentage of 87.6%. Furthermore, these features could be precisely modified by altering the proportion of MBC and ε-CL to suit individual constraints. Discussion: This innovative elastomer with exceptional shape memory and self-heal capabilities provides a solid basis and promising potential for the development of self-contracting intelligent surgical sutures in the biomedical field.

Prevention of Preterm Birth by Cervical Pessary Combined with Vaginal Progesterone: a Systematic Review and Meta-analysis with Trial Sequential Analysis.

This study was to assess the effectiveness of cervical pessary combined with vaginal progesterone for the prevention of preterm birth (PTB). Ten studies about singleton [five randomized controlled trials (RCTs), vs vaginal progesterone; four cohorts, vs vaginal progesterone; two cohorts, vs cervical cerclage + vaginal progesterone] and two cohort studies about multiple pregnancies (vs vaginal progesterone) were included after searching electronic databases. For singleton pregnancies, the meta-analysis of three non-RCTs [relative risk (RR) = 0.41, p = 0.001] or total trials in non-Asian country (RR = 0.56, p = 0.03) revealed that compared with vaginal progesterone alone, cervical pessary + vaginal progesterone treatment had significant effectiveness on preventing PTB < 34 weeks, but not for five RCTs; meta-analysis of two trials showed that cervical pessary + vaginal progesterone had no significant prevention effects of PTB compared with cervical cerclage + vaginal progesterone. For multiple pregnancies, meta-analysis of two trials showed that compared with vaginal progesterone, cervical pessary + vaginal progesterone treatment increased neonatal birth weight (standardized mean difference = 0.50, p = 0.01). Trial sequential analysis implied additional studies were required. Four studies vs other controls (pessary, three-combined, tocolysis, conservative or no treatment; one study, each) were selected for systematic review. In conclusion, cervical pessary combined with vaginal progesterone may be safe and effective to prevent PTB in singleton pregnancies and increase neonatal birth weight in the multiple pregnancies compared with vaginal progesterone alone.

Umbilical Cord Blood-Derived Exosomes in Maternal-Fetal Disease: a Review.

The nutrients and other factors transported by umbilical cord blood, which is vital for fetal survival, play crucial roles in fetal development. There are various communication modes between the fetal-placental system and the maternal-placental system, and these communication modes are all mediated by umbilical cord blood. During the process of umbilical cord blood transportation, the changes of some nutrients and factors may play a key role in fetal development. Exosomes, which are members of the extracellular vesicle family, are present in the umbilical cord blood and play roles in information transmission as a result of their efficient cellular communication activity. The study of umbilical cord blood-derived exosomes provides a new approach for research on the etiology of maternal-fetal diseases and they may be useful for the development of intrauterine treatments. This review summarizes specific functions and research directions regarding umbilical cord blood-derived exosomes, and their potential associations with pregnancy complications.

Dendrimer-Based N-Acetyl Cysteine Maternal Therapy Ameliorates Placental Inflammation via Maintenance of M1/M2 Macrophage Recruitment.

Intrauterine inflammation (IUI) is the primary cause of spontaneous preterm birth and predisposes neonates to long-term sequelae, including adverse neurological outcomes. N-acetyl-L-cysteine (NAC) is the amino acid L-cysteine derivative and a precursor to the antioxidant glutathione (GSH). NAC is commonly used clinically as an antioxidant with anti-inflammatory properties. Poor bioavailability and high protein binding of NAC necessitates the use of high doses resulting in side effects including nausea, vomiting, and gastric disruptions. Therefore, dendrimer-based therapy can specifically target the drug to the cells involved in inflammation, reducing side effects with efficacy at much lower doses than the free drug. Towards development of the new therapies for the treatment of maternal inflammation, we successfully administered dendrimer-based N-Acetyl Cysteine (DNAC) in an animal model of IUI to reduce preterm birth and perinatal inflammatory response. This study explored the associated immune mechanisms of DNAC treatment on placental macrophages following IUI, especially on M1/M2 type macrophage polarization. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the pro-inflammatory cytokine mRNA of Il1ß and Nos2, and decreased CD45+ leukocyte infiltration in the placenta following IUI. Furthermore, we found that DNAC altered placental immune profile by stimulating macrophages to change to the M2 phenotype while decreasing the M1 phenotype, thus suppressing the inflammatory responses in the placenta. Our study provides evidence for DNAC therapy to alleviate IUI via the maintenance of macrophage M1/M2 imbalance in the placenta.

Placental Macrophages Demonstrate Sex-Specific Response to Intrauterine Inflammation and May Serve as a Marker of Perinatal Neuroinflammation.

Preterm birth (PTB) is considered to be one of the most frequent causes of neonatal death. Prompt and effective measures to predict adverse fetal outcome following PTB are urgently needed. Placenta macrophages are a critical immune cell population during pregnancy, phenotypically divided into M1 and M2 subsets. An established mouse model of intrauterine inflammation (IUI) was applied. Placenta (labyrinth) and corresponding fetal brain were harvested within 24 hours post injection (hpi). Flow cytometry, Western blot, real-time qPCR, and regular histology were utilized to examine the cytokines, macrophage polarization, and sex-specificity. Placental exposure to LPS led to significantly reduced labyrinth thickness compared to PBS-exposed controls as early as 3 hpi, accompanied by apoptosis and necrosis. Pro-inflammatory M1 markers, Il-1ß, and iNOS, and anti-inflammatory M2 marker Il-10 increased significantly in placentas exposed to IUI. Analysis of flow cytometry revealed that fetal macrophages (Hofbauer cell, HBCs) were mostly M1-like and that maternal inter-labyrinth macrophages (MIM) were M2-like in their features in IUI. Male fetuses displayed significantly decreased M2-like features in HBCs at 3 and 6 hpi, while female fetuses showed significant increase in M2-like features in MIM at 3 and 6 hpi. Furthermore, there was a significant correlation between the frequency of HBCs and corresponding microglial marker expression at 3 and 6 hpi. Placental macrophages demonstrated sex-specific features in response to IUI. Specifically, HBCs may be a potential biomarker for fetal brain injury at preterm birth.

Case Report: Compound Heterozygous Phosphatidylinositol-Glycan Biosynthesis Class N (PIGN) Mutations in a Chinese Fetus With Hypotonia-Seizures Syndrome 1.

Multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1) caused by phosphatidylinositol-glycan biosynthesis class N (PIGN) mutations is an autosomal recessive disease involving many systems of the body, such as the urogenital, cardiovascular, gastrointestinal, and central nervous systems. Here, compound heterozygous variants NM_012327.6:c.2427-2A > G and c.963G > A in PIGN were identified in a Chinese proband with MCAHS1. The features of the MCAHS1 family proband were evaluated to understand the mechanism of the PIGN mutation leading to the occurrence of MCAHS1. Ultrasound was conducted to examine the fetus, and his clinical manifestations were evaluated. Genetic testing was performed by whole-exome sequencing and the results were verified by Sanger sequencing of the proband and his parents. Reverse transcription-polymerase chain reaction was performed, and the products were subjected to Sanger sequencing. Quantitative PCR (Q-PCR) was conducted to compare gene expression between the patient and wild-type subjects. The compound heterozygous mutation NM_012327.6:c.2427-2A > G and c.963G > A was identified by whole-exome sequencing and was confirmed by Sanger sequencing. The NM_012327.6:c.2427-2A > G mutation led to skipping of exon 26, which resulted in a low expression level of the gene, as measured by Q-PCR. These findings provided a basis for genetic counseling and reproduction guidance in this family. Phenotype-genotype correlations may be defined by an expanded array of mutations.

Melatonin for prevention of fetal lung injury associated with intrauterine inflammation and for improvement of lung maturation.

Inflammation is associated with injury to immature lungs, and melatonin administration to preterm newborns with acute respiratory distress improves pulmonary outcomes. We hypothesized that maternally administered melatonin may reduce inflammation, oxidative stress, and structural injury in fetal lung and help fetal lung maturation in a mouse model of intrauterine inflammation (IUI). Mice were randomized to the following groups: control (C), melatonin (M), lipopolysaccharide (LPS; a model of IUI) (L), and LPS with melatonin (ML). Pro-inflammatory cytokines, components of the Hippo pathway, and Yap1/Taz were analyzed in the fetal lung at E18 by real-time RT-qPCR. Confirmatory histochemistry and immunohistochemical analyses (surfactant protein B, vimentin, HIF-1ß, and CXCR2) were performed. The gene expression of IL1ß in the fetal lung was significantly increased in L compared to C, M, and ML. Taz expression was significantly decreased in L compared to C and M. Taz gene expression in L was significantly decreased compared with those in ML. Immunohistochemical analyses showed that the expression of HIF-1ß and CXCR2 was significantly increased in L compared to C, M, and ML. The area of surfactant protein B and vimentin were significantly decreased in L than C, M, or ML in the fetal and neonatal lung. Antenatal maternally administered melatonin appears to prevent fetal lung injury induced by IUI and to help lung maturation. The results from this study results suggest that melatonin could serve as a novel safe preventive and/or therapeutic medicine for preventing fetal lung injury from IUI and for improving lung maturation in prematurity.

The Role of circRNA-SETD2/miR-519a/PTEN Axis in Fetal Birth Weight through Regulating Trophoblast Proliferation.

Abnormal birth weight is the one of the major causes of adulthood diseases such as obesity, metabolic syndrome, cardiovascular disease, type 2 diabetes, and hypertension. Accumulating evidence has suggested that the placental trophoblast is one of the most important reasons that influence birth weight. Our previous study showed that miR-519a are correlated with low fetal birth weight through regulating trophoblast proliferation. To further clarify the detailed mechanisms on how it is regulated, we screened the placental-specific circular RNAs (circRNAs) via microarray assay. The result identified that circ-SETD2 was highly expressed in the placenta of the patients with fetal macrosomia compared with healthy donors. Furthermore, bioinformatic analyses and the luciferase reporter assay revealed that miR-519a possessing the binding sites for both circ-SETD2 and phosphate and tensin homolog was deleted on chromosome 10 (PTEN). Interestingly, upregulation of circ-SETD2 enhanced the proliferation and invasion of the human trophoblast-like cell line HTR8/SVneo cell. A parallel study performed by Western blotting showed that overexpression of circ-SETD2 reduced miR-519a levels and increased PTEN levels in HTR8/SVneo cells. Importantly, the enhancement of HTR8/SVneo cell activity by circ-SETD2 overexpression was nullified when the cells were cotransfected by circ-SETD2 and miR-519a, suggesting the involvement of the circ-SETD2/miR-519a/PTEN axis in trophoblast activity. Taken together, we illustrate the role of circ-SETD2, as an upstream signaling of miR-519a/PTEN, in placenta development via regulating trophoblast proliferation and invasion. These findings improve our understanding of the mechanisms of progression of fetal macrosomia and will guide future development of therapeutic strategies against the disease by targeting the circ-SETD2/miR-519a/PTEN axis.

Human umbilical cord mesenchymal stem cell-derived exosome-mediated transfer of microRNA-133b boosts trophoblast cell proliferation, migration and invasion in preeclampsia by restricting SGK1.

OBJECTIVE: Exosomes have been documented to function in human diseases, yet their transfer of microRNA (miRNA) in preeclampsia (PE) has seldom been reported. This study intends to discuss the role of miR-133b derived from exosomes in human umbilical cord mesenchymal stem cells (hUC-MSCs) in trophoblast cell development in PE. METHODS: Placentas from PE patients and normal pregnant women were collected. The hUC-MSCs and their exosomes were obtained and identified. Trophoblast cell HPT-8 and HTR8-S/Vneo were obtained and co-cultured with hUC-MSCs-derived exosomes that had been transfected with different miR-133b plasmids. MiR-133b and glucocorticoid-regulated kinase 1 (SGK1) expression in placental tissues and HPT-8 and HTR8-S/Vneo cells was determined. HTR8-S/Vneo and HPT-8 cell proliferation, cell cycle distribution, apoptosis rate, migration and invasion were detected. RESULTS: MiR-133b was down-regulated and SGK1 was up-regulated in placental tissues of PE patients. MiR-133b expression was inversely related to SGK1 expression in HTR8-S/Vneo and HPT-8 cells co-cultured with hUC-MSC-derived exosomes. Exosomes promoted HTR8-S/Vneo and HPT-8 cell proliferation, migration and invasion abilities, cell cycle entry and inhibited apoptosis. Elevated exosome-derived miR-133b from hUC-MSCs boosted HTR8-S/Vneo and HPT-8 cell proliferation, cell cycle progression, migration and invasion and limited cell apoptosis. MiR-133b targeted SGK1. CONCLUSION: Collectively, we demonstrate that miR-133b is down-regulated and SGK1 is up-regulated in PE, and miR-133b derived from exosomes in hUM-MSCs facilitates trophoblast cell proliferation, migration and invasion in PE via constraining SGK1.

Dose-dependent structural and immunological changes in the placenta and fetal brain in response to systemic inflammation during pregnancy.

PROBLEM: Systemic maternal inflammation is associated with adverse neonatal sequelae. We tested the hypothesis that IL-1ß is a key inflammatory regulator of adverse pregnancy outcomes. METHOD OF STUDY: Pregnant mice were treated with intraperitoneal injections of IL-1ß (0, 0.1, 0.5, or 1 µg) from embryonic day (E)14 to E17. Placenta and fetal brains were harvested and analyzed for morphologic changes and IL-1ß signaling markers. RESULTS: As compared with non-treated dams, maternal injections with IL-1ß resulted in increased p-NF-κB and caspase-1 in placentas and fetal brains, but not consistently in spleens, suggesting induction of intrinsic IL-1ß production. These findings were confirmed by increased levels of IL-1ß in the placentas of the IL-1ß-treated dams. Systemic treatment of dams with IL-1ß suppressed Stat1 signaling. Maternal inflammation caused by IL-1ß treatment reduced fetal viability to 80.6% and 58.9%, in dams treated with either 0.5 or 1 µg of IL-1ß, respectively. In the placentas, there was an IL-1ß dose-dependent distortion of the labyrinth structure, decreased numbers of mononuclear trophoblast giant cells, and reduced proportions of endothelial cells as compared to placentas from control dams. In fetal brains collected at E17, there was an IL-1ß dose-dependent reduction in cortical neuronal morphology. CONCLUSION: This work demonstrates that systemic IL-1ß injection causes dose-dependent structural and functional changes in the placenta and fetal brain.

The effect of intrauterine inflammation on mTOR signaling in mouse fetal brain.

Fetuses exposed to an inflammatory environment are predisposed to long-term adverse neurological outcomes. However, the mechanism by which intrauterine inflammation (IUI) is responsible for abnormal fetal brain development is not fully understood. The mechanistic target of rapamycin (mTOR) signaling pathway is closely associated with fetal brain development. We hypothesized that mTOR signaling might be involved in fetal brain injury and malformation when fetuses are exposed to the IUI environment. A well-established IUI model was utilized by intrauterine injection of lipopolysaccharide (LPS) to explore the effect of IUI on mTOR signaling in mouse fetal brains. We found that microglia activation in LPS fetal brains was increased, as demonstrated by elevated Iba-1 protein level and immunofluorescence density. LPS fetal brains also showed reduced neuronal cell counts, decreased cell proliferation demonstrated by low Ki67-positive density, and elevated neuron apoptosis evidenced by high expression of cleaved Caspase 3. Furthermore, we found that mTOR signaling in LPS fetal brains was elevated at 2 hr after LPS treatment, declined at 6 hr and showed overall inhibition at 24 hr. In summary, our study revealed that LPS-induced IUI leads to increased activation of microglia cells, neuronal damage, and dynamic alterations in mTOR signaling in the mouse fetal brain. Our findings indicate that abnormal changes in mTOR signaling may underlie the development of future neurological complications in offspring exposed to prenatal IUI.

Edge stabilization in reduced-dimensional perovskites.

Reduced-dimensional perovskites are attractive light-emitting materials due to their efficient luminescence, color purity, tunable bandgap, and structural diversity. A major limitation in perovskite light-emitting diodes is their limited operational stability. Here we demonstrate that rapid photodegradation arises from edge-initiated photooxidation, wherein oxidative attack is powered by photogenerated and electrically-injected carriers that diffuse to the nanoplatelet edges and produce superoxide. We report an edge-stabilization strategy wherein phosphine oxides passivate unsaturated lead sites during perovskite crystallization. With this approach, we synthesize reduced-dimensional perovskites that exhibit 97 ± 3% photoluminescence quantum yields and stabilities that exceed 300 h upon continuous illumination in an air ambient. We achieve green-emitting devices with a peak external quantum efficiency (EQE) of 14% at 1000 cd m-2; their maximum luminance is 4.5 × 104 cd m-2 (corresponding to an EQE of 5%); and, at 4000 cd m-2, they achieve an operational half-lifetime of 3.5 h.

Maternal inflammation leads to different mTORC1 activity varied by anatomic locations in mouse placenta†.

Maternal inflammation (MI) is associated with many adverse perinatal outcomes. The placenta plays a vital role in mediating maternal-fetal resource allocation. Studies have shown that MI contributes to placental dysfunction, which then leads to adverse birth outcomes and high health risks throughout childhood. Placental mammalian target of rapamycin complex 1 (mTORC1) signaling pathway links maternal nutrient availability to fetal growth; however, the impact of MI on mTORC1 signaling in the placenta remains unclear. In this study, we sought to explore the changes of mTORC1 signaling in the mouse placenta at late gestation by using two models of MI employing lipopolysaccharide (LPS) and interleukin-1ß (IL-1ß) to mimic acute (aMI) and sub-chronic (cMI) inflammatory states, respectively. We determined placental mTORC1 activity by measuring the activity of mTORC1 downstream molecules, including S6k, 4Ebp1, and rpS6. In the aMI model, we found that mTORC1 activity was significantly decreased in the placental decidual and junctional zone at 2 and 6 h after LPS surgery, respectively; however, mTORC1 activity was significantly increased in the placental labyrinth zone at 2, 6, and 24 h after LPS treatment, respectively. In the cMI model, we observed that mTORC1 activity was increased only in the placental labyrinth zone after consecutive IL-1ß exposure. Our study reveals that different parts of the mouse placenta react differently to MI, leading to variable mTORC1 activity throughout the placenta. This suggests that different downstream molecules of mTORC1 from different parts of the mouse placenta may be used in clinical research to monitor the fetal well-being during MI.

Exposure to systemic and intrauterine inflammation leads to decreased pup survival via different placental mechanisms.

PROBLEM: Exposure to systemic maternal inflammation (i.e., maternal sepsis, influenza, human immunodeficiency virus, or pyelonephritis) and intrauterine (IU) inflammation (i.e., chorioamnionitis or preterm labor) have been associated with adverse perinatal sequelae. Whether systemic and localized inflammation leading to adverse outcomes have similar placental mechanisms remain unclear. METHOD OF STUDY: We conducted a study by murine modeling systemic and localized IU inflammation with injections of either intraperitoneal (IP) or IU interleukin-1ß (IL-1ß) and compared fetoplacental hemodynamic changes, cytokine/chemokine expression, and fetal loss. RESULTS: IU IL-1ß exposure reduced offspring survival by 31.1% and IP IL-1ß exposure by 34.5% when compared with control pups. Despite this similar outcome in offspring survival, Doppler analysis revealed a stark difference: IU group displayed worsened fetoplacental hemodynamic changes while no differences were found between IP and control groups. While both IU and IP groups had increases in pro-inflammatory cytokines and chemokines, specific gene expression trends differed between the two groups, once again highlighting their mechanistic differences. CONCLUSION: While both IP and IU IL-1ß exposure similarly affected pup survival, only IU inflammation resulted in fetoplacental hemodynamic changes. We speculate that exposure to maternal systemic and IU inflammation plays a key role in fetal injury by utilizing different placental inflammatory pathways and mechanisms.

Melatonin for prevention of placental malperfusion and fetal compromise associated with intrauterine inflammation-induced oxidative stress in a mouse model.

Melatonin has been shown to reduce oxidative stress and mitigate hypercoagulability. We hypothesized that maternally administered melatonin may reduce placental oxidative stress and hypercoagulability associated with exposure to intrauterine inflammation (IUI) and consequently improve fetoplacental blood flow and fetal sequelae. Mice were randomized to the following groups: control (C), melatonin (M), lipopolysaccharide (LPS; a model of IUI) (L), and LPS with melatonin (ML). The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. The systolic/diastolic ratio, resistance index, and pulsatility index in uterine artery (UtA) and umbilical artery (UA) were significantly increased in L compared with other groups when analyzed by Doppler ultrasonography. The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. Vascular endothelial damage and thrombi formation, as evidenced by fibrin deposits, were similarly increased in L compared to other groups. Maternal pretreatment with melatonin appears to modulate maternal placental malperfusion, fetal cardiovascular compromise, and fetal neuroinflammation induced by IUI through its antioxidant properties.

Maternal Supplementation of Low Dose Fluoride Alleviates Adverse Perinatal Outcomes Following Exposure to Intrauterine Inflammation.

Maternal periodontal disease has been linked to adverse pregnancy sequelae, including preterm birth (PTB); yet, root planing and scaling in pregnancy has not been associated with improved perinatal outcomes. Fluoride, a cariostatic agent, has been added to drinking water and dental products to prevent caries and improve dental health. The objective of this study was to explore the effects of fluoride supplementation using a mouse model of preterm birth and perinatal sequalae. Pregnant mice were fed low dose fluoride (LF-) or high dose fluoride (HF-) and given intrauterine injections of lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). We found that LPS + LF- significantly increased livebirths, pup survival, and litter size compared to LPS alone. Moreover, offspring from the LPS + LF- group exhibited significantly improved neuromotor performance and more neurons compared to those from the LPS group. Additionally, LF- treatment on human umbilical vein endothelial cells (HUVECs) increased cell viability and decreased oxidative stress after treatment with LPS. Collectively, our data demonstrates that maternal LF- supplementation during pregnancy postpones the onset of PTB, acts to increase the liveborn rate and survival time of newborns, and reduces perinatal brain injury in cases of intrauterine inflammation.

YAP Is Decreased in Preeclampsia and Regulates Invasion and Apoptosis of HTR-8/SVneo.

Preeclampsia (PE) is a gestational disorder with hypertension and proteinuria leading to maternal and fetal morbidity and mortality. Yes-associated protein (YAP), a transcription coactivator of Hippo pathway, was identified as an oncoprotein participated in tumorigenesis. However, the effect of YAP on trophoblast has not been investigated. In our study, YAP expression levels in first-trimester, full-term, and PE placentas were detected using quantitative real-time polymerase chain reaction (PCR), Western blot assays, and immunohistochemistry. Yes-associated protein expression was also detected in BeWo and HTR-8/SVneo. Overexpression plasmid and YAP small interfering RNA were introduced into trophoblast cells. Furthermore, we utilized a Transwell invasion assay, flow cytometry, and Cell Counting Kit-8 analysis to examine the role of YAP in the invasion, apoptosis, and proliferation of HTR-8/SVneo trophoblast cells. The result showed that both YAP messenger RNA (mRNA) and protein expression levels were less in preeclamptic placentas. Yes-associated protein mRNA and protein expression levels were more highly expressed in BeWo. Yes-associated protein enhanced cell invasion, reduced the cellular apoptotic response, and had no effect on proliferation. In addition, the overexpression of YAP activated the expression of caudal-related homeobox transcription factor 2 (CDX2), whereas reduced expression of YAP inhibited the expression of CDX2. Our results demonstrate that decreased YAP levels may contribute to the development of PE by regulating trophoblast invasion and apoptosis involving regulation of CDX2. Collectively, we proposed decreased YAP may contribute to trophoblast dysfunction, which suggests it might represent a prognostic biomarker and therapeutic target for PE.

Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked ß-N-acetylglucosamine signaling.

AIM: Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. METHODS: HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. RESULTS: Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. CONCLUSION: mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling.

Altered expression of mTOR and autophagy in term normal human placentas.

Mammalian target of rapamycin (mTOR) and autophagy have been implied in trophoblast cells proliferation and invasion. This study investigated whether mTOR and autophagy were involved in placental development and fetal programming. A total of 90 term normal placentas (37-42 gestational weeks) were collected from women who underwent elective cesarean section, with large for gestational age (LGA), fetal growth restriction (FGR), and appropriate for gestational age (AGA) infants (n=30, respectively). Capillary volume density within peripheral villi significantly decreased in the FGR placentas compared with the AGA group (p<0.01). Autophagic vacuoles were more prominent in the FGR placentas than in the AGA pregnancies (p<0.001). LC3B-II and Beclin1 in placentas of FGR were increased by 99% and 83%, respectively, while p62 was descended by 39% (p<0.001), compared with that in the AGA group. Whereas, there was no significant differences in these autophagy-related proteins between the LGA and AGA pregnancies. Compared to the AGA pregnancies, placental mTOR was reduced by 30% in the FGR group and raised by 26% in the LGA group (p<0.05). Pearson's correlation analysis showed a significantly inverse correlation between mTOR level and LC3B-II expression (r=-0.456, p<0.01), as well as between mTOR and Beclin1 expression in term human placentas (r=-0.468, p<0.01). These results demonstrate that altered expression of mTOR and autophagy may be associated with the development of placentas and pathophysiology of FGR, and that there may be a reciprocal regulation between mTOR and autophagy in human placentas.