Upregulation of GPR133 expression impaired the phagocytosis of macrophages in recurrent spontaneous miscarriage.

Decidual macrophages are the second-largest immune cell group at the maternal-foetal interface. They participate in apoptotic cell removal, and protect the foetus from microorganisms or pathogens. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia and recurrent spontaneous miscarriage (RSM). However, the mechanisms by which decidual macrophages are involved in the occurrence of adverse pregnancy outcomes have not been elucidated. Here we integrated DNA methylation and gene expression data from decidua macrophages to identify potential risk factors related to RSM. GPR133 was significantly hypomethylated and upregulated in decidual macrophages from RSM patients. Further demethylation analysis demonstrated that GPR133 expression in decidual macrophages was significantly increased by 5-Aza-dC treatment. In addition, the influence of GPR133 on the phagocytic ability of macrophages was explored. Phagocytosis was impaired in the decidual macrophages of RSM patients with increased GPR133 expression. Increased GPR133 expression induced by demethylation treatment in the decidual macrophages of healthy control patients led to a significant decrease in phagocytic function. Importantly, knockdown of GPR133 resulted in a significant improvement in the phagocytic function of THP-1 macrophages. In conclusion, the existing studies have shown the influence of GPR133 on the phagocytic function of decidual macrophages and pregnancy outcomes, providing new data and ideas for future research on the role of decidual macrophages in RSM.

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. In vivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

Effects of Metabolism on Macrophage Polarization Under Different Disease Backgrounds.

Macrophages are versatile immune cells associated with various diseases, and their phenotypes and functions change on the basis of the surrounding environments. Reprogramming of metabolism is required for the proper polarization of macrophages. This review will focus on basic metabolic pathways, the effects of key enzymes and specific products, relationships between cellular metabolism and macrophage polarization in different diseases and the potential prospect of therapy targeted key metabolic enzymes. In particular, the types and characteristics of macrophages at the maternal-fetal interface and their effects on a successful conception will be discussed.

Decreased nitric oxide content mediated by asymmetrical dimethylarginine and protein l-arginine methyltransferase 3 in macrophages induces trophoblast apoptosis: a potential cause of recurrent miscarriage.

STUDY QUESTION: Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? SUMMARY ANSWER: Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal-foetal interface. WHAT IS KNOWN ALREADY: Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. STUDY DESIGN, SIZE, DURATION: A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. PARTICIPANTS/MATERIALS, SETTING, METHODS: NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. MAIN RESULTS AND THE ROLE OF CHANCE: Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. LIMITATIONS, REASONS FOR CAUTION: All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Downregulation of HDAC8 expression decreases CD163 levels and promotes the apoptosis of macrophages by activating the ERK signaling pathway in recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) is a systemic disorder that has been defined as two or more pregnancies lost before the 20th week of gestation. Although the impaired function of macrophages at the maternal-fetal interface has been reported to be associated with RSM, the underlying mechanisms have not been fully elucidated. Here, we revealed that HDAC8 plays a critical role in RSM. Our results show that the mRNA and protein expression of HDAC8 was decreased in decidual macrophages from RSM patients. Moreover, the knockdown of HDAC8 resulted in a significant decrease in CD163 expression and an increase in apoptosis in dTHP-1 macrophages. Mechanistically, the ERK signaling pathway was activated in HDAC8-knockdown macrophages. When HDAC8-knockdown cells were pretreated with the ERK inhibitor U0126, expression levels of CD163, activated caspases 3, 7 and 9, and the apoptosis rate, were rescued. Taken together, our current results suggest that HDAC8 plays an important role in macrophage activation and apoptosis and may contribute to maintaining normal pregnancy by increasing the expression of M2 marker genes and inhibiting the apoptosis of macrophages at the maternal-fetal interface.

Macrophage Polarization in Physiological and Pathological Pregnancy.

The immunology of pregnancy is complex and poorly defined. During the complex process of pregnancy, macrophages secrete many cytokines/chemokines and play pivotal roles in the maintenance of maternal-fetal tolerance. Here, we summarized the current knowledge of macrophage polarization and the mechanisms involved in physiological or pathological pregnancy processes, including miscarriage, preeclampsia, and preterm birth. Although current evidence provides a compelling argument that macrophages are important in pregnancy, our understanding of the roles and mechanisms of macrophages in pregnancy is still rudimentary. Since macrophages exhibit functional plasticity, they may be ideal targets for therapeutic manipulation during pathological pregnancy. Additional studies are needed to better define the functions and mechanisms of various macrophage subsets in both normal and pathological pregnancy.

Hypoxic-stabilized EPAS1 proteins transactivate DNMT1 and cause promoter hypermethylation and transcription inhibition of EPAS1 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality globally. Although cigarette smoking is by far the most important risk factor for lung cancer, the aberrant expression of oncogenes and tumor suppressor genes contributes a great deal to tumorigenesis. Here, we reveal that aberrant expression of endothelial PAS domain-containing protein 1 ( EPAS1) gene, which encodes hypoxia inducible factor 2α, has a critical role in NSCLC. Our results showed EPAS1 mRNA was down-regulated in 82.5% of NSCLC tissues, and a new region of EPAS1 promoter was found to be highly methylated in lung cancer cell lines and NSCLC tissues. Moreover, the methylation rates were negatively correlated to EPAS1 mRNA expression in lung tissues. Further, demethylation analysis demonstrated EPAS1 was regulated by DNA methyltransferases (DNMTs) in NSCLC. In contrast, DNMT1 was verified as an EPAS1 target gene by chromatin immunoprecipitation assay and could be transactivated by stabilized EPAS1 proteins in hypoxic lung cells, thereby decreasing EPAS1 mRNA expression by methylation regulation. Collectively, our study suggests there might be a mechanism of negative-feedback regulation for EPAS1 in NSCLC. That is, hypoxic-stabilized EPAS1 proteins transactivated DNMT1, which further promoted the hypermethylation of EPAS1 promoter and decreased EPAS1 mRNA expression levels in NSCLC.-Xu, X.-H., Bao, Y., Wang, X., Yan, F., Guo, S., Ma, Y., Xu, D., Jin, L., Xu, J., Wang, J. Hypoxic-stabilized EPAS1 proteins transactivate DNMT1 and cause promoter hypermethylation and transcription inhibition of EPAS1 in non-small cell lung cancer.

Fluorescent Chemosensor for Selective Detection of Phosgene in Solutions and in Gas Phase.

The detection of highly toxic chemicals in a convenient, fast, and reliable manner is essential for coping with serious threats to humankind and public security caused by unexpected terrorist attacks and industrial accidents. In this paper, a highly selective fluorescent probe has been constructed through o-phenylenediamine covalently linking to coumarin (o-Pac), which can respond to phosgene in turn-on fluorescence mode. The response time is less than 0.5 min and the detection limit is as low as 3 nM in solutions. More importantly, the sensor exhibits good selectivity to phosgene over triphosgene and various acyl chlorides. Furthermore, a portable test paper has been fabricated with polystyrene membrane containing o-Pac for real-time selective monitoring of phosgene in gas phase.

BODIPY-Based Fluorescent Sensor for the Recognization of Phosgene in Solutions and in Gas Phase.

As a highly toxic and widely used chemical, phosgene has become a serious threat to humankind and public security because of its potential use by terrorists and unexpected release during industrial accidents. For this reason, it is an urgent need to develop facile, fast, and selective detection methods of phosgene. In this Article, we have constructed a highly selective fluorescent sensor o-Pab for phosgene with a BODIPY unit as a fluorophore and o-phenylenediamine as a reactive site. The sensor o-Pab exhibits rapid response (∼15 s) in both colorimetric and turn-on fluorescence modes, high selectivity for phosgene over nerve agent mimics and various acyl chlorides and a low detection limit (2.7 nM) in solutions. In contrast to most undistinguishable sensors reported, o-Pab can react with phosgene but not with its substitutes, triphosgene and biphosgene. The excellent discrimination of o-Pab has been demonstrated to be due to the difference in highly reactive and bifunctional phosgene relative to its substitutes. Furthermore, a facile testing paper has been fabricated with poly(ethylene oxide) immobilizing o-Pab on a filter paper for real-time selective monitoring of phosgene in gaseous phase.

Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis and autophagy in human hepatic L-02 cells.

Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb of Aster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo, and a toxicity-guided isolation method led to the identification of a cyclopeptide astin B. Astin B is structurally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivo and that hepatic injury was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked oxidative stress-associated inflammation in hepatocytes as evidenced by increased levels of reactive oxygen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol, the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle fluorescence, increased LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increased LC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely together with other members of astins, is the substance that is primarily responsible for the hepatotoxicity of A.tataricus.

Pathway-based association analyses identified TRAIL pathway for osteoporotic fractures.

INTRODUCTION: Hip OF carries the highest morbidity and mortality. Previous studies revealed that individual genes/loci in the Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) pathway were associated with bone metabolism. This study aims to verify the potential association between hip OF and TRAIL pathway. METHODS: Using genome-wide genotype data from Affymetrix 500 K SNP arrays, we performed novel pathway-based association analyses for hip OF in 700 elderly Chinese Han subjects (350 with hip OF and 350 healthy matched controls). RESULTS: The TRAIL pathway achieved a significant p value (p = 0.01) for association with hip OF. Among the 38 genes in the TRAIL pathway, seven genes achieved nominally significant association with hip OF (p<0.05); the TNFSF10 (TRAIL) gene obtained the most significant p value (p = 1.70×10(-4)). SNPs (rs719126, rs6533015, rs9594738, rs1805034, rs11160706) from five genes (CFLAR, NFKB1, TNFSF11, TNFRSF11A, TRAF3) of the pathway had minor alleles that appear to be protective to hip OF. SNPs (rs6445063 and rs4259415) from two genes (TNFSF10 and TNFRSF10B) of the pathway had minor alleles (A) that are associated with an increased risk of hip OF, with the ORs (odds ratios) of 16.51 (95%CI:3.83-71.24) and 1.37 (95%CI:1.08-1.74), respectively. CONCLUSIONS: Our study supports the potential role of the TRAIL pathway in the pathogenesis of hip OF in Chinese Han population. Further functional study of this pathway will be pursued to determine the mechanism by which it confers risk to hip OF.

Molecular genetic studies of gene identification for osteoporosis: the 2009 update.

Osteoporosis is a complex human disease that results in increased susceptibility to fragility fractures. It can be phenotypically characterized using several traits, including bone mineral density, bone size, bone strength, and bone turnover markers. The identification of gene variants that contribute to osteoporosis phenotypes, or responses to therapy, can eventually help individualize the prognosis, treatment, and prevention of fractures and their adverse outcomes. Our previously published reviews have comprehensively summarized the progress of molecular genetic studies of gene identification for osteoporosis and have covered the data available to the end of September 2007. This review represents our continuing efforts to summarize the important and representative findings published between October 2007 and November 2009. The topics covered include genetic association and linkage studies in humans, transgenic and knockout mouse models, as well as gene-expression microarray and proteomics studies. Major results are tabulated for comparison and ease of reference. Comments are made on the notable findings and representative studies for their potential influence and implications on our present understanding of the genetics of osteoporosis.

Association analyses of RANKL/RANK/OPG gene polymorphisms with femoral neck compression strength index variation in Caucasians.

Femoral neck compression strength index (fCSI), a novel phenotypic parameter that integrates bone density, bone size, and body size, has significant potential to improve hip fracture risk assessment. The genetic factors underlying variations in fCSI, however, remain largely unknown. Given the important roles of the receptor activator of the nuclear factor-kappaB ligand/receptor activator of the nuclear factor-kappaB/osteoprotegerin (RANKL/RANK/OPG) pathway in the regulation of bone remodeling, we tested the associations between RANKL/RANK/OPG polymorphisms and variations in fCSI as well as its components (femoral neck bone mineral density [fBMD], femoral neck width [FNW], and weight). This was accomplished with a sample comprising 1873 subjects from 405 Caucasian nuclear families. Of the 37 total SNPs studied in these three genes, 3 SNPs, namely, rs12585014, rs7988338, and rs2148073, of RANKL were significantly associated with fCSI (P = 0.0007, 0.0007, and 0.0005, respectively) after conservative Bonferroni correction. Moreover, the three SNPs were approximately in complete linkage disequilibrium. Haplotype-based association tests corroborated the single-SNP results since haplotype 1 of block 1 of the RANKL gene achieved an even more significant association with fCSI (P = 0.0003) than any of the individual SNPs. However, we did not detect any significant associations of these genes with fBMD, FNW, or weight. In summary, our findings suggest that the RANKL gene may play an important role in variation in fCSI, independent of fBMD and non-fBMD components.

Genome-wide association study suggested copy number variation may be associated with body mass index in the Chinese population.

Obesity is a major public health problem characterized with high body mass index (BMI). Copy number variations (CNVs) have been identified to be associated with complex human diseases. The effect of CNVs on obesity is unknown. In this study, we explored the association of CNVs with BMI in 597 Chinese Han subjects using Affymetrix GeneChip Human Mapping 500K Array Set. We found that one CNV at 10q11.22 (from 46.36 Mb to 46.56 Mb) was associated with BMI (the raw P=0.011). The CNV contributed 1.6% of BMI variation, and it covered one important obesity gene-pancreatic polypeptide receptor 1(PPYR1). It was reported that PPYR1 was a key regulator of energy homeostasis. Our findings suggested that CNV might be potentially important for the BMI variation. In addition, our study suggested that CNV might be used as a genetic marker to locate genes associated with BMI in Chinese population.

Genome-wide copy-number-variation study identified a susceptibility gene, UGT2B17, for osteoporosis.

Osteoporosis, a highly heritable disease, is characterized mainly by low bone-mineral density (BMD), poor bone geometry, and/or osteoporotic fractures (OF). Copy-number variation (CNV) has been shown to be associated with complex human diseases. The contribution of CNV to osteoporosis has not been determined yet. We conducted case-control genome-wide CNV analyses, using the Affymetrix 500K Array Set, in 700 elderly Chinese individuals comprising 350 cases with homogeneous hip OF and 350 matched controls. We constructed a genomic map containing 727 CNV regions in Chinese individuals. We found that CNV 4q13.2 was strongly associated with OF (p = 2.0 x 10(-4), Bonferroni-corrected p = 0.02, odds ratio = 1.73). Validation experiments using PCR and electrophoresis, as well as real-time PCR, further identified a deletion variant of UGT2B17 in CNV 4q13.2. Importantly, the association between CNV of UGT2B17 and OF was successfully replicated in an independent Chinese sample containing 399 cases with hip OF and 400 controls. We further examined this CNV's relevance to major risk factors for OF (i.e., hip BMD and femoral-neck bone geometry) in both Chinese (689 subjects) and white (1000 subjects) samples and found consistently significant results (p = 5.0 x 10(-4) -0.021). Because UGT2B17 encodes an enzyme catabolizing steroid hormones, we measured the concentrations of serum testosterone and estradiol for 236 young Chinese males and assessed their UGT2B17 copy number. Subjects without UGT2B17 had significantly higher concentrations of testosterone and estradiol. Our findings suggest the important contribution of CNV of UGT2B17 to the pathogenesis of osteoporosis.

Polymorphisms in the estrogen receptor genes are associated with hip fractures in Chinese.

INTRODUCTION: Hip fractures (HF) are a major cause of public health burden with strong genetic determination. However, the true causal genes remain largely unknown. MATERIALS AND METHODS: Based on the important biological role of estrogens in bone homeostasis, this study aimed to investigate whether the estrogen receptor genes, ESR1 and ESR2, affect the onset of HF in 700 elderly Chinese subjects (350 with osteoporotic HF and 350 healthy controls). We genotyped 32 SNPs in total and examined their associations both by the single-SNP and haplotype tests. RESULTS: We identified two novel SNPs of ESR1, rs3020314 and rs1884051, were significantly associated with HF (rs3020314: P=0.0004, OR=1.66, 95%CI: 1.25-2.18; rs1884051: P=0.0004, OR=1.46, 95%CI: 1.19-1.81). We firstly detected significant association of ESR2 with HF (rs960070: P=0.0070, OR=1.43, 95%CI: 1.10-1.86). Haplotype analyses corroborated our single-SNP results. CONCLUSION: Our findings have important implications for understanding the pathology of osteoporotic fractures. Independent replication studies are needed to validate our results and explore the most possible functional variants for molecular studies.