Abstract
Background and Aims: Increased O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins by O-GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation.Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining.Results: HG upregulated OGT mRNA and protein expression and protein O-GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3′-untranslated region (3′-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O-GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3′-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O-GlcNAcylation levels; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Additionally, siRNA-mediated OGT depletion reduced HG-induced protein O-GlcNAcylation; ICAM-1, VCAM-1, and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion, confirming that HG-induced endothelial inflammation is partially mediated via OGT-induced protein O-GlcNAcylation. These results were validated in vivo: tail-vein injection of miR-200a/200b mimics downregulated endothelial OGT and ICAM-1 expression in db/db mice.Conclusion: miR-200a/200b are involved in modulating HG-induced endothelial inflammation by regulating OGT-mediated protein O-GlcNAcylation, suggesting the therapeutic role of miR-200a/200b on vascular complications in diabetes.
Highlights
Diabetic vascular disease—a low-grade, chronic inflammatory disease—is a major cause of death in developed countries
We investigated the role of miR200 members in regulating endothelial inflammation mediated by O-GlcNAc transferase (OGT)-induced O-GlcNAcylation in HG-treated human aortic endothelial cells (HAECs) and examined the aortic endothelial tissues in the miR-200a/200b mimics-treated db/db type 2 diabetic mice used as an in vivo model
The expression of intercellular adhesion molecule 1 (ICAM-1) was significantly upregulated after 24 h HG stimulation (Figure 1F) and was associated with a 1.95-fold increase in the adhesion of the THP-1 cells to the HAECs (Figure 1G)
Summary
Diabetic vascular disease—a low-grade, chronic inflammatory disease—is a major cause of death in developed countries. Increased levels of reactive oxygen species (ROS) in endothelial cells are involved in the pathophysiology of diabetic vascular complications by activating downstream pathways, leading to increases in polyol pathway flux, advanced glycation end product formation, advanced glycation end product receptor formation, protein kinase C isoform activation, and hexosamine biosynthetic pathway (HBP) flux (Giacco and Brownlee, 2010). A small amount of fructose-6-phosphate (2–5%) is converted to glucosamine-6phosphate by glutamine:fructose 6-phosphate amidotransferase. This conversion commits fructose-6-phosphate to entering the HBP, which produces UDP-N-acetylglucosamine (UDP-GlcNAc) as an important sensor of nutrition flux by integrating the metabolic processes of glucose, fatty acids, amino acids, and nucleotides (Bond and Hanover, 2015). Oxidative stress promotes endothelial inflammation during diabetes. We examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation
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