title: Lysosome-membrane fusion mediated superoxide production in hyperglycaemia-induced endothelial dysfunction.
abstract: Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91(phox), a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O₂·⁻) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91(phox) colocalization to LRs clusters which were proved to mediate the HG induced O₂·⁻ formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91(phox) colocalization to LRs clusters, O₂·⁻ formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O₂·⁻ production.
authors.affiliation: Department of Aerospace Physiology, Fourth Military Medical University, Xi'an, People's Republic of China.
authors.forename: Jun-Xiang
authors.initials: JX
authors.lastname: Bao
authors.forename: Hui
authors.initials: H
authors.lastname: Chang
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Lv
authors.forename: Jin-Wen
authors.initials: JW
authors.lastname: Yu
authors.forename: Yun-Gang
authors.initials: YG
authors.lastname: Bai
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.forename: Yue
authors.initials: Y
authors.lastname: Cai
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Jin
authors.initials: J
authors.lastname: Ma
authors.forename: Yao-Ming
authors.initials: YM
authors.lastname: Chang
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D020789:Cathepsin C / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology; D003470:Culture Media; D004727:Endothelium / Q000187:drug effects / Q000503:physiopathology; D005453:Fluorescence; D020868:Gene Silencing / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D066298:In Vitro Techniques; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology; D019255:NADPH Oxidases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002518:Ceramides; D003470:Culture Media; D034741:RNA, Small Interfering; D013481:Superoxides; D009569:Nitric Oxide; D019255:NADPH Oxidases; D013108:Sphingomyelin Phosphodiesterase; D001619:beta-N-Acetylhexosaminidases; D020789:Cathepsin C; D005947:Glucose