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 gp91phox, 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 gp91phox 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, gp91phox 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.
Highlights
Cardiovascular complications such as atherosclerosis, myocardial infarction, and stroke etc are quite common in people with hyperglycaemia or diabetes
High glucose incubation induced the movement of lysosomal marker to cellular membrane To investigate the effects of high glucose incubation on lysosomal fusion to cellular membrane, the cells were cultured in the medium with normal glucose (5.6 mmol/l, Ctrl) or high glucose (22 mmol/l, HG) for 6 h, 12 h or 24 h
To clarify HG-induced lysosome-membrane fusion further, we examined the activity of b-hexosaminidase, cathepsin C or lactic dehydrogenase (LDH) in cultural medium. b-hexosaminidase and cathepsin C are the symbolized enzymes of lysosome which could be transported into medium after lysosomal fusing to cellular membrane while LDH is located in cytoplasm and the increase of its concentration in medium indicates ruptured or dead cell
Summary
Cardiovascular complications such as atherosclerosis, myocardial infarction, and stroke etc are quite common in people with hyperglycaemia or diabetes. To our knowledge, elevated glucose levels could generate ROS by several pathways including nonenzymatic oxidation of glucose, increased mitochondrial oxidative phosphorylation, and increased activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase etc [2,3]. As far as the NADPH oxidase is concerned, the activation of it depends largely on the assembling and aggregation of its subunits, including membrane-associated subunits gp91phox or its isoforms (NOX) and p22phox, as well as cytosolic subunits p47phox, p40phox, p67phox, and Rac [4,5,6]. The clustering of lipid rafts (LRs) has shown great promise to act as a driving force for the NOX assembling and aggregation [5,6,7]. The LRs clustering has been proved to be promoted remarkably by the lysosomal exocytosis and fusion to cellular membrane [5,6,7]
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