The Mechanisms Of Reactive Oxygen Species Generation In High Concentration Glucose-treated Human Vascular Endothetial Cells

High concentration of glucose in circulation is the main character of diabetes. Oxidative stress plays a key role in atherosclerosis accompanyed with diabetes. Reactive oxygen species (ROS) are second messengers in regulating vascular function and cell growth. The potential sources of endothelial ROS include nicotinamide vadenine dinucleotede phosphate oxidase (NADPH oxidase, NOX), cytochrome p-450, xanthine oxidase, uncoupled NO synthase and lipoxygenase. However, ROS in vascular endothelial cells are mostly derived from NOX. gp91phox, the complex enzyme NOX composed of five subunits including cytosolic components (p47phox, P67phox and Rac1) and cytomembrane components (p22phox and NOX2), was firstly found in phagocyte cells. In recent years, the novel gp91phox homologues of NOX families such as NOX1, NOX3, NOX4 and NOX5 have been found in different organs and vascular cells. Latest studies indicated that NOX4 is the one of the most important enzymes in the ROS generation in vascular endothelial cells. Our investigations show that high concentration of glucose could upregulate the expression of NOX4 and stimulate the production of ROS in HUVECs. Preincubation with DPI, the specific inhibitor of NOX could reverse those effects. Furthermore, the regulation of NOX in those processes was investigated. Objectives: To approach the main access of the generation of ROS in HUVECs, to detect the expression of NOX subunits in HUVECs treated with high concentration of glucose and investigate the mechanism of ROS generation.Method: Mophology of HUVECs was observed by inverted microscope. Expression of factorâ…§related antigen in HUVECs was investigated by immunofluorescence, and expression of NOX subunits was detected with PCR and western blot. Intracellular ROS production was measured by flow cytometry, location of NOX subunits in HUVECs was observed by immunofluorescence.Results: The HUVECs were detached successfully. High concentration of glucose induced ROS generation in HUVECs mainly through NOX. NOX4 but not any other subunit was upregulated, however, p47phox was translocated from cytoplasm to nuclear membrane in this process, and the specific inhibitor of NOX could reverse these effects. PKC participated in regulating ROS generation.Conclusions: High concentration of glucose induces ROS generation in HUVECs mainly through NOX. p47phox was translocated to nuclear membrane from cytoplasm to mediate the upregulation of NOX4 and the generation of ROS. Moreover, PKC can activate p47phox translocation.