Mechanisms Of NADPH Oxidase4Involved In High Glucose-induced Injury Of Vascular Endothelial Cell

Diabetes mellitus is caused by absolute or relative insufficient insulin and hyperglycemia is one of the main characteristics of diabetes mellitus. Diabetes is an important risk factor of cardiovascular diseases and dysglycemia is one of major causes resulted in vascular complications of diabetes. High glucose-induced morphological changes and dysfunction of vascular endothelium are the early symbols of vascular complications of diabetes. Hyperglycemia-induced oxidative stress plays a key role in atherosclerosis accompanied with diabetes. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. The potential sources of ROS in vascular system include nicotinamide vadenine dinucleotede phosphate oxidase (NADPH oxidase, NOX), xanthine oxidase, cytochrome p-450, uncoupled endothelial nitric oxide synthase and lipoxygenase. NOX is considered as the main source of ROS in vascular endothelium. NOX2, the complex enzyme NOX composed of five subunits including cytomembrane components (p22PHOX and NOX2) and cytosolic components (p47PHOX, p67PHOX and Rac), was firstly found in phagocyte cells. NOX2, which has potential binding sites of NADPH, heme and FAD, and p22PHOX are regulated by p47PHOX and Rac. Seven members of NOX family including NOX1, NOX2(formerly named gp91PHOX), NOX3, NOX4, NOX5, Duox1(Dual oxidase1) and Duox2have been identified in different cells. It is reported the both NOX2and NOX4are expressed in endothelial cells, and the level of NOX4mRNA in endothelial cells is20fold higher than NOX2. Further studies indicated that NOX4is the most important enzyme in the ROS generation in vascular endothelial cells. The enzyme activity of NOX is regulated through phosphorylation and translocation in membrane of p47PHOX and Rac-1.In order to investigate the mechanisms of high glucose-induced oxidative stress in vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) were treated with20mmol/L glucose, and ROS level was detected by flow cytometry. Comparing with the control group, treatment of20mmol/L glucose could lead to excessive production of ROS in HUVECs. However, preincubation of HUVECs with DPI, the NOX inhibitor, but not Rotenone, Oxypurinol or L-NAME could inhibit the generation of ROS, indicating that NOX is the main source of high glucose-induced ROS generation. Further, the expression of NOX subunits was investigated by RT-PCR, Real-time PCR and Western blot. The results showed that NOX4and sub-units p22PHOX, p47PHOX, p67PHOX, Rac-1were expressed in HUVECs, but only the expression of NOX4was significantly upregulated after20mmol/L glucose treatment in HUVECs. The gene profile screened through SuperArray’s PCR array indicated that the level of Dual Oxidase2mRNA could be increased by nearly13fold, while the level of Superoxide dismutase3mRNA could be significantly decreased in HUVECs treated with20mmol/L glucose. p47PHOx and Rac-1were translocated to cell membrane from cytoplasm to mediate the activation of NOX4and the generation of ROS, and PKC was also participated in regulating NOX4-mediated ROS generation. It was found that high concentration of glucose could stimulate the production of ROS and induce apoptosis through upregulating the expression of NOX4in HUVECs. Moreover, two strategies, lost of function and gain of function, were carried out to observe effects of NOX4on ROS generation and injury of HUVECs. The NOX4was knocked down by transfection with NOX4-siRNA, and overexpressed by transfection with NOX4-expressing vector in HUVECs. The results showed that overexpression of NOX4could induce the excessive production of ROS and apoptosis of HUVECs. Contrast, down-regulation of NOX4significantly reduced the level of ROS and prevented HUVECs from apoptosis. Furthermore, to study the mechanisms of apoptosis induced by glucose, nuclear factor-κ B (NF-κB) and p38MAPK pathways were analyzed by Western blot and immunofluorescence staining. After glucose treatment, phosphorylated IkB level was elevated and IκB was degraded, and NF-κB was also phosphorylated and translocated into nucleus in where NF-κB upregulated the expression of inflammatory factors such as ICAM-1and VCAM-1. p38MAPK pathway is considered as an important signaling pathway participated in regulation of endothelial apoptosis. The high glucose resulted in phosphorylation of p38MAPK and the translocation of p38MAPK into nucleus. SB203580, a specific inhibitor of p38MAPK, could inhibit high glucose-induced upregulation of P-p38MAPK and suppress high glucose-induced apoptosis, as measured by Hoechst/PI, TUNEL and PI/Annexin V-FITC staining. These pointed out that p38MAPK played a key role in the glucose-induced apoptosis. Recent results have revealed that beyond lipid-lowering activity, statins exhibit functions such as modulating inflammatory responses, improving antioxidant effects and maintaining plaque stability. Our previous results provided that Simvastatin could downregulate the expression of NOX4but not NOX2and impair the excessive production of ROS induced by treatment of0.8g/L LDL. These results indicated that Simvastatin could lower ROS level by suppression of NOX4expression. To further investgate Simvastin’s antioxidant effects in vivo, oxidative stress was analyzed in atherosclerotic Apo E-/-mice treated with Simvastatin. The results showed that Simvastatin could reduce the levels of TG and MDA in serum and the expression of NOX in vascular system, in term impaired the oxidative stress of atherosclerotic mice. In addition, the average area of atherosclerotic lesion was decreased, and the thickness and integrity of fibrous cap were improved in Simvastatin-treated mice, suggesting that Simvastatin has the ability to stabilize the atherosclerotic plaque.In summary, our results provide the molecular evidence that NOX4-derived ROS could play an important role in oxidative stress of vascular endothelial cells induced by high concentration of glucose. Specifically inhibiting NOX activity may be a novel target preventing vascular complications of diabetes.