Liraglutide ameliorates high glucose-induced vascular endothelial injury through TRIB3/NF-κB signaling pathway.

As one of the most commonly used antidiabetic medications clinically, liraglutide is involved in the protection of vascular endothelium, and whether it can relieve high glucose-induced vascular endothelial damage was unknown. This study aims to address the response of liraglutide (LIRA) on human umbilical vein endothelial cells, as well as to elucidate its possible underlying mechanism. We established a vascular endothelial cell injury model by exposing human umbilical vein endothelial cells (HUVECs) to high glucose, and used LIRA pretreatment before HG treatment to address the endothelial protective effect of LIRA. Our results suggest that LIRA prevented HG-induced HUVEC apoptosis, oxidative stress, inflammasome activation, and pyroptosis. Furthermore, silencing of tribbles homolog 3 (TRIB3) could markedly reduce HG-induced HUVEC apoptosis, ROS level, the expressions of TXNIP, cleaved caspase3, NLRP3, and caspase1, indicating TRIB3 inhibition protected HUVECs against HG-induced vascular endothelial injury. In addition, LIRA restrained NF-κB/IκB-α signaling pathway activation in HUVECs. Thus, LIRA appears to mitigate HG-induced apoptosis, oxidative stress, inflammasome activation, and pyroptosis in HUVECs via regulating the TRIB3/NF-κB/IκB-α signaling pathway. Our study provides new insight into the mechanisms underlying the protective activity of LIRA against the vascular endothelial injury in diabetic vascular complication.

The targeting of DAPK1 with miR-190a-3p promotes autophagy in trophoblast cells.

Pre-eclampsia (PE) is a dangerous pathological status that occurs during pregnancy and is a leading reason for both maternal and fetal death. Autophagy is necessary for cellular survival in the face of environmental stress as well as cellular homeostasis and energy management. Aberrant microRNA (miRNA) expression is crucial in the pathophysiology of PE. Although studies have shown that miRNA (miR)-190a-3p function is tissue-specific, the precise involvement of miR-190a-3p in PE has yet to be determined. We discovered that miR-190a-3p was significantly lower and death-associated protein kinase 1 (DAPK1) was significantly higher in PE placental tissues compared to normal tissues, which is consistent with the results in cells. The luciferase analyses demonstrated the target-regulatory relationship between miR-190a-3p and DAPK1. The inhibitory effect of miR-190a-3p on autophagy was reversed by co-transfection of si-DAPK1 and miR-190a-3p inhibitors. Thus, our data indicate that the hypoxia-dependent miR-190a-3p/DAPK1 regulatory pathway is implicated in the development and progression of PE by promoting autophagy in trophoblast cells.

Identification and validation of feature genes associated with M1 macrophages in preeclampsia.

Preeclampsia (PE) is a pregnancy-specific cardiovascular complication that is the leading cause of maternal and neonatal morbidity and mortality. Previous studies have indicated the importance of immune cells, such as M1 and M2 macrophages, in the pathogenesis of PE. However, the mechanisms leading to immune dysregulation are unclear. Data-independent acquisition proteomic analysis was performed on placental tissues collected from patients with PE and healthy controls. Transcriptome data for placenta samples from patients with PE and their corresponding controls were obtained from the Gene Expression Omnibus database. Differential analysis of transcriptome and proteome data between PE and control groups was performed using R software. Immunocytic infiltration scoring was performed using the quantiseq algorithm. Weighted gene co-expression network analysis (WGCNA) screened for feature genes associated with M1 cell infiltration. Protein-protein interaction (PPI) analysis identified hub genes. We confirm that the infiltration score of M1 macrophages was significantly increased in the placental tissues of patients with PE. Differential analysis, WGCNA, and PPI analysis identified four hub molecules associated with M1 cell infiltration (HTRA4, POGK, MFAP5, and INHBA). The hub molecules displayed dysregulated expression in PE tissues. The qPCR, Western blots, and immunohistochemistry analyses confirmed that Inhibin, beta A (INHBA) was highly expressed in placental tissues of patients with PE. Immunofluorescence revealed the extensive infiltration of M1 macrophages in the placental tissues of patients with PE and their co-localization with INHBA. The collective results identified hub genes associated with M1 macrophage infiltration, providing potential targets for the pathogenesis and treatment of PE.

MiR-141-3p promotes hypoxia-induced autophagy in human placental trophoblast cells.

Preeclampsia (PE) is a pregnancy-specific disorder and a significant contributor to maternal, fetal and neonatal morbidity and mortality worldwide. Its pathogenesis is generally accepted as insufficient trophoblast invasion of the maternal endometrium and inadequate remodeling of the maternal spiral arteries. These impairments lead to elevated levels of hypoxia and oxidative stress. Autophagy has become a highly researched field in obstetrics, and this process may be essential for preimplantation development beyond the four- and eight-cell stages, and for blastocyst survival, extra-villous trophoblast functions, invasion and vascular remodeling. Several studies have shown that autophagy activation, shown by an increase in autophagy vacuoles or microtubule-associated protein 1 A/1B-light chain 3 (LC3) dots, was more common in PE than in normal pregnancy. Thus, changes in autophagic status are seen in preeclamptic placentas. MicroRNA-141-3p (miR-141-3p), a multifunctional miRNA, is involved in a variety of physiological and pathological processes, including PE and autophagy. However, the influence of miR-141-3p on autophagy regulation in trophoblast cells has yet to be described. Therefore, the objective of our study was to investigate the role of miR-141-3p in autophagy induced by hypoxia in human placental trophoblast cells. Our results found that hypoxia induced autophagy in trophoblast cells and dramatically elevated the expression of miR-141-3p. Overexpression of miR-141-3p improved autophagic activity, whereas low expression of miR-141-3p inhibited autophagic activity. Therefore, our data demonstrated that miR-141-3p promoted hypoxia-induced autophagy in placental trophoblast cells, which may be related to the development of preeclampsia.

Functional mechanism and clinical implications of miR-141 in human cancers.

Cancer is caused by the abnormal proliferation of local tissue cells under the control of many oncogenic factors. MicroRNAs (miRNAs) are a class of evolutionarily conserved, approximately 22-nucleotide noncoding small RNAs that influence transcriptional regulationby binding to the 3'-untranslated region of target messenger RNA. As a member of the miRNA family, miR-141 acts as a suppressor or an oncomiR in various cancers and regulates cancer cell proliferation, apoptosis, invasion, and metastasis through a variety of signaling pathways, such as phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and constitutive activation of nuclear factor-κB (NF-κB). Target gene validation and pathway analysis have provided mechanistic insight into the role of this miRNA in different tissues. This review also outlines novel findings that suggest miR-141 may be useful as a noninvasive biomarker and as a therapeutic target in several cancers.

Gut microbiota dysbiosis promotes age-related atrial fibrillation by lipopolysaccharide and glucose-induced activation of NLRP3-inflammasome.

AIMS: Ageing is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). The gut microbiota dysbiosis is involved in age-related diseases. However, whether the aged-associated dysbiosis contributes to age-related AF is still unknown. Direct demonstration that the aged gut microbiota is sufficient to transmit the enhanced AF susceptibility in a young host via microbiota-intestinal barrier-atria axis has not yet been reported. This study aimed to determine whether gut microbiota dysbiosis affects age-related AF. METHODS AND RESULTS: Herein, by using a faecal microbiota transplantation (FMT) rat model, we demonstrated that the high AF susceptibility of aged rats could be transmitted to a young host. Specially, we found the dramatically increased levels of circulating lipopolysaccharide (LPS) and glucose led to the up-regulated expression of NOD-like receptor protein (NLRP)-3 inflammasome, promoting the development of AF, which depended on the enhanced atrial fibrosis in recipient host. Inhibition of inflammasome by a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, resulted in a lower atrial fibrosis and AF susceptibility. Then, we conducted cross-sectional clinical studies to explore the effect of ageing on the altering trends with glucose levels and circulating LPS among clinical individuals in two China hospitals. We found that both of serum LPS and glucose levels were progressively increased in elderly patients as compared with those young. Furthermore, the ageing phenotype of circulating LPS and glucose levels, intestinal structure and atrial NLRP3-inflammasome of rats were also confirmed in clinical AF patients. Finally, aged rats colonized with youthful microbiota restored intestinal structure and atrial NLRP3-inflammasome activity, which suppressed the development of aged-related AF. CONCLUSIONS: Collectively, these studies described a novel causal role of aberrant gut microbiota in the pathogenesis of age-related AF, which indicates that the microbiota-intestinal barrier-atrial NLRP3 inflammasome axis may be a rational molecular target for the treatment of aged-related arrhythmia disease.

Genistein alleviates H2O2-induced senescence of human umbilical vein endothelial cells via regulating the TXNIP/NLRP3 axis.

CONTEXT: Genistein (Gen) has shown protective effects against ageing process. OBJECTIVE: To explore the role of Gen on the senescence of H2O2-induced human umbilical vein endothelial cells (HUVECs) and investigate the possible mechanism. MATERIALS AND METHODS: HUVECs were treated with different concentrations of H2O2 (50, 100, 200 and 400 µmol/L) for 1 h or Gen administration (20, 40, 80 and 160 µg/mL) for 24 h. Functional experiments (cell counting kit-8, ß-galactosidase staining and flow cytometry) were used to detect the effect of Gen on H2O2-induced HUVECs. After HUVECs were transfected with TXNIP overexpression plasmids, the expression of p16, p21, thioredoxin-interacting protein (TXNIP), nucleotide-binding and oligomerization domain-like receptor 3 (NLRP3), cleaved caspase-3 and cleaved caspase-1 in HUVECs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. RESULTS: H2O2 (200 and 400 µmol/L) inhibited the proliferation of HUVECs. At concentrations of >50 µmol/L, H2O2 induced the cell cycle progression arrests in G1 phase and promoted cell senescence of HUVECs. Gen had no obvious cytotoxicity to HUVECs below 160 µg/mL. H2O2-induced HUVEC senescence and the expression of TXNIP and NLRP3 in HUVECs were down-regulated by Gen (40 and 80 µg/mL). Expressions of TXNIP and NLRP3 in HUVECs were up-regulated by H2O2 but down-regulated by Gen. Overexpressed TXNIP partially reversed the suppressive effect of Gen on H2O2-induced senescence and apoptosis of HUVECs. Expressions of p16, p21, TXNIP, NLRP3, cleaved caspase-3 and cleaved caspase-1 in H2O2-treated HUVECs were inhibited by Gen, while the inhibition as such was partially reversed by overexpressed TXNIP. DISCUSSION AND CONCLUSIONS: H2O2-induced HUVEC senescence was alleviated by Gen via suppressing the TXNIP/NLRP3 axis, which may offer a potential therapeutic approach for improving HUVEC senescence and provide a new direction for the treatment of cardiovascular disease.

Construction of a comprehensive observer-based scale assessing aging-related health and functioning in captive rhesus macaques.

Aging-related health and functioning are difficult to quantify in humans and nonhuman primates. We constructed an observer-based scale for daily application in assessing the aging-related health and functioning of rhesus macaques. Ten items referring to an aging appearance, musculoskeletal aging and aging-related eating behavior were selected through a panel consensus. The Aging-related Health and Functioning Scale (AHFS) was constructed based on these scored items form 57 healthy rhesus macaques. High reliability of the AHFS was shown based on Cronbach's alpha coefficient (0.877). The structure of the AHFS was validated by three exploratory factors. The largest factor, whose four components were dietary uptake, iliac muscle mass, hair condition and fragility, and sex, explained 50.5% of the variation in aging-related health and functioning scores. The second factor, involving age, tooth loss and tooth wear, explained 15.5% of the variation. The lowest-ranking factor comprised only facial redness and accounted for 10% of the variation. A hierarchical cluster analysis validated the good applicability of the scale in distinct samples. From these scale-scored results, complicated aging phenomena observed in humans, including the sex-survival paradox and the calorie-related health-survival paradox, were both demonstrated in rhesus macaques. Therefore, the AHFS provides a valuable approach for aging-related research.

Reduced expression of the lncRNA NRON is a potential hallmark of the CMV-amplified CD8+ T cell accumulations commonly seen in older humans.

The characteristic accumulation of late-stage differentiated CD8+ T cells is enhanced by lifelong latent cytomegalovirus (CMV) persistence, which makes it challenging to screen for subclinical biomarkers of immune aging in the elderly. We systematically identified predominantly preformed, long, noncoding RNAs (lncRNAs) as integrative biomarkers of CD8+ T cell aging in 14 elderly CMV carriers over 80 years of age. After sorting the CD28nullCD8+ T cell subset and its CD28bearingCD8+ counterpart in five nonagenarians, we profiled the differential expression of lncRNAs and genes in CD28nullCD8+ T cells via array detection. We focused on 11 differentially expressed antisense lncRNAs and cross-referenced them with previously identified age-accumulated lncRNAs to create a set of candidates in CD28nullCD8+T cells. We performed intracellular validation on the age-accumulated candidate lncRNAs paired with their antisense target genes using quantitative polymerase chain reaction (qPCR). Simultaneously, we sorted the CMVpp65-specific CD8+ T cell subset and its counterpart from participant cells with the HLA-A-*0201 genotype. The validated age-accumulated lncRNAs in CD28nullCD8+ T cells were intracellularly cross-validated in CMVpp65CD8+ T cells. Finally, we identified the immunity-related gene(s) that acted as potential target(s) to the cross-validated age-accumulated lncRNA(s), using bioinformatics techniques. The potential regulation of the final identified lncRNA-gene pair(s) was simultaneously predicted in two pathway-integrated networks. We concluded that expression of an age-accumulated lncRNA (NRON) was decreased, whereas that of its immunity-related target gene (NFAT) was increased, in both CD28nullCD8+ T cells and CMVpp65CD8+ T cells of elderly individuals with persistent CMV infection. The identification of NRON as a potential biomarker suggests that NRON contributes to CMV-enhanced CD28nullCD8+ T cell aging by modulating phosphorylation and/or IL-4-dependent NFAT signaling.

Magnetic nanosphere-guided site-specific delivery of vascular endothelial growth factor gene attenuates restenosis in rabbit balloon-injured artery.

OBJECTIVE: New and efficient strategies to protect endothelium or to enhance endothelial regrowth are important for treatment of restenosis after percutaneous transluminal angioplasty. Magnetic DNA microspheres are used to accelerate vascular endothelial growth factor (VEGF) re-endothelialization and to attenuate intimal hyperplasia in balloon-injured artery. This study aimed to assess DNA-gelatin magnetic nanospheres containing VEGF expression plasmids in vascular restenosis attenuation. METHODS: Ninety-six rabbits underwent balloon injury and were randomly divided for gene transfer with naked VEGF plasmids (NAK group), magnetic VEGF microspheres (MIC group), and LacZ (CON group: naked LacZ plasmid and LacZ nanosphere subgroups). Serum and tissue VEGF levels were measured. Also, the ratios of intima area to media area were determined to assess neointima formation. RESULTS: Microsphere gene delivery through the artery by a magnet resulted in VEGF overexpression in transfected arterial segments. Tissue VEGF integral optical densities were significantly increased in MIC rabbits compared with NAK animals. Serum VEGF was below detection in all animals. X-Gal staining showed higher transfection efficiency in the CON group. The impact of neointimal thickening was evaluated by light microscopy as the ratio of intima area to media area in cross sections. Significant differences in the ratio of intima area to media area were obtained between the NAK group (0.12 ± 0.02, 0.41 ± 0.03, 0.61 ± 0.05, and 0.72 ± 0.04 at 1, 2, 3, and 4 weeks, respectively) and the MIC group (0.06 ± 0.03, 0.20 ± 0.05, 0.25 ± 0.04, and 0.26 ± 0.03 at 1, 2, 3, and 4 weeks, respectively) at 2, 3, and 4 weeks (P < .05). CONCLUSIONS: Intra-arterial VEGF gene delivery by magnetic microspheres significantly increased DNA stability, transfection efficiency, and targeting specificity, resulting in exogenous VEGF overexpression and attenuated intimal hyperplasia in balloon-injured artery.

MiR-34a, miR-21 and miR-23a as potential biomarkers for coronary artery disease: a pilot microarray study and confirmation in a 32 patient cohort.

The aim of this study was to investigate the expression of circulating microRNAs (miRNAs) in apolipoprotein E (apoE) knockout mice (apoE(-/-)) and to validate the role of these miRNAs in human coronary artery disease (CAD). Pooled plasma from 10 apoE(-/-) mice and 10 healthy C57BL/6 (B6) mice was used to perform the microarray analysis. The results showed that miR-34a, miR-21, miR-23a, miR-30a and miR-106b were differentially expressed in apoE(-/-) mice, and these expression changes were confirmed by real-time quantitative reverse-transcription PCR. Then, miR-34a, miR-21, miR-23a, miR-30a and miR-106b were detected in the plasma of 32 patients with CAD and of 20 healthy controls. Only miR-34a, miR-21 and miR-23a were significantly differentially expressed in the plasma of CAD patients (all P<0.01). In conclusion, miR-34a, miR-21 and miR-23a were elevated in CAD patients, which means that these miRNAs might serve as biomarkers of CAD development and progression.

Differentiated miRNA expression and validation of signaling pathways in apoE gene knockout mice by cross-verification microarray platform.

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE-/-) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA-miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA-miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE-/- mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT-PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT-PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression.