This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.
Previous studies have shown that the expression of claudin-4 is upregulated in breast cancer. The aim of the present study was to investigate the role and the regulation of claudin-4 in MCF-7 breast cancer cells. For the in vitro experiments, MCF-7 cells were treated with recombinant vectors carrying cDNA for claudin-4 overexpression or short hairpin RNAs (shRNAs) for claudin-4 silencing. Cell proliferation was determined by an MTT assay and cell migration ability was measured by a wound-healing assay. The cell cycle profile and apoptotic rate were analyzed using flow cytometry. The effect of methylation status on claudin-4 expression was determined by PCR and western blotting. For the in vivo tumorigenesis analysis, MCF-7 cells with or without claudin-4 silencing were transplanted into nude mice. In vivo cell growth was evaluated 14 days after transplantation. We found that claudin-4 overexpression increased MCF-7 cell proliferation and migration, and reduced the rate of cell apoptosis. Silencing of claudin-4 induced the opposite effects in MCF-7 cells. In addition, claudin-4 expression was upregulated by demethylation. Moreover, the size of tumor formation was reduced in nude mice transplanted with claudin-4 silenced MCF-7 cells. These observations suggested that claudin-4, which was regulated by methylation status, plays an important role in breast cancer growth and malignancy via the control of cell proliferation, migration and apoptosis.
Endothelial microvesicles (EMVs) could reflect the status of endothelial cells (ECs) which are involved in the pathogenesis of ischemic stroke (IS). MiR-155 could regulate EC functions. However, their roles in IS remain unclear. This study aimed to investigate the levels of plasma EMVs and EMVs carrying miRNA-155 (EMVs-miR-155) in IS patients to explore their potential roles as biomarkers. Ninety-three IS patients and 70 controls were recruited in this study. The levels of circulating EMVs and EMVs-miR-155 were detected by fluorescence nanoparticle tracking analysis and quantitative real-time PCR, respectively. The correlations between level of EMVs/ EMVs-miR-155 and the onset time, severity, infarct volume, and subtypes of IS were analyzed. The severity and infarct volume were assessed by NIHSS and magnetic resonance imaging, respectively. Multivariate logistic regression analysis was used to investigate the risk factors of IS. The ROC curve and area under ROC curve (AUC) of EMVs and EMVs-miR-155 were determined. The levels of plasma EMVs and EMVs-miR-155 were increased significantly in acute and subacute stages of IS and remained unchanged in chronic stage, and were positively related to the infarct volume and NIHSS scores and were associated with large artery atherosclerosis and cardioembolism subtypes defined by Trial of Org 10 172 in acute stroke treatment (TOAST) classification. Multivariate logistic regression analysis demonstrated that plasma EMVs and EMVs-miR-155 were significant and independent risk factors of IS and their AUC were 0.778 and 0.851, respectively, and increased to 0.892 after combination. Our study suggests that plasma EMVs and EMVs-miR-155 are promising biomarkers for IS. The diagnostic value of EMVs-miR-155 is higher and their combination is the best.
Pompe disease is an autosomal recessive hereditary lysosomal disorder and correlated with acid α-glucosidase enzyme (GAA) deficiencies, which lead to accumulation of glycogen in all tissues, most notably in skeletal muscles. Adult late-onset Pompe disease (LOPD) is a slowly progressive disease of proximal myopathy with later involvement of the respiratory muscles, resulting in respiratory failure. In this study, we reported a 22-year-old Chinese woman with inability to withstand heavy physical activity since childhood, who presented with respiratory and ambulation weakness in 2 months. On admission, her bilateral upper limbs strength was 4/5 and lower limbs strength was 3/5 according to Medical Research Council (MRC) score. The patient had compound heterozygotes containing a newly identified 4 nt deletion of coding sequence (deletion nt 1411_1414) in one of the acid α-glucosidase alleles and a c.2238G>C (p.Trp746Cys) missense mutation. This deletion has been reported in infant-onset Pompe disease (IOPD) but not LOPD. Intriguingly, this deletion mutation was not found in the patient's family and was considered as pathogenic. Muscle biopsy showed scattered vacuoles with basophilic granules inside the subsarcolemmal area, which were strongly stained by periodic acid-Schiff (PAS). Laboratory tests revealed a significant increase of creatine kinase MB isoenzyme (CK-MB) and lactate dehydrogenase (LDH). GAA level was 9.77 nmol/1 h/mg and was not sufficient for the diagnosis of GAA activity deficiency (0–3.78 nmol/1 h/mg). In summary, mutational analysis of GAA and muscle biopsy are crucial in the diagnosis of Pompe disease.
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