The Expression Of NOX4 Gene In Human Vascular Cells And Its Role In The Apoptosis Of Endothelial Cells

Reactive oxygen species (ROS) are important regulators of vascular function. It has been demonstrated that ROS play an important role in the generation and progression of atherosclerosis through oxidative damage of vascular endothelial cells. Potential sources of endothelial ROS generation that are implicated in disease progress include mitochondria, xanthine oxidase (XO), uncoupled NO synthase, cytochrome p-450 enzymes, and NADPH oxidase (NOX) . ROS of human vascular system mostly derive from NADPH oxidase. Gp91phox was firstly found in phagocyte cells, and the novel gp91phox homologues of NOX families such as N0X1, N0X3, N0X4, N0X5 have been found in different organs (colon, kidney, spleen) and vascular smooth muscle cells in recent years. Latest studies indicate that N0X4 gene may be important in the ROS generation in endothelial cells. Firstly, mRNA expression of NOX gene was detected in human umbilical vein endothelial cells (HUVECs), ECV304, HU-LT and human umbilical artery smooth muscle cells (HUASMCs) by reverse transcription PCR, it was found that N0X4 is the major NOX gene in these cells. To further clarify the role of N0X4 gene in the apoptosis and ROS generation of HUVECs, we observed the change of N0X4 mRNA and ROS level as well as apoptosis in HUVECs stimulated by high concentration of glucose, low density lipoprotein(LDL), angiotensin II(AngII) and serum-free medium.Part I Effects of high concentration of glucose, LDL, Ang II and serum-free medium on N0X4 mRNA expression , ROS generation andapoptosis of HUVECsObjectives: To investigate the mRNA expression of NOX gene in HUVECs, ECV304, HU-LT and HUASMCs, then to elucidate the change of N0X4 mRNA expression, intracellular ROS level ,and apoptosis in HUVECs stimulated by high concentration ofglucose, low density lipoprotein(LDL), angiotensin II (Ang II) and serum-free medium. Methods: Morphology of HUVECs and HUASMCs was observed by inverted microscope, expression of factor Vffl related antigen in HUVECs was investigated by immunohistochemistry and immunofluorescence, expression of a-actin in HUASMCs was measured by immunohistochemistry. PcDNA3.1-N0Xl plasmid, U937 cell, HepG2 cell and PcDNA3.1-NOX4 plasmid were used as the positive control of mRNA expression of N0X1, N0X2, N0X3 and N0X4 respectively. NOX expression of HUVECs, HUASMCs, ECV304 and HU-LT was measured by RT-PCR. mRNA expression of N0X4, intracellular ROS level, and apoptosis in HUVECs were detected by RT-PCR, flow cytometry, Hoechst33342 staining respectively. Results: The results showed that there was expression of factor VBI related antigen in HUVECs and a-actin in HUASMCs. N0X4 was highly expressed in HUVECs, HUASMCs and HU-LT, while only a weak band of N0X2 was detected in HUVECs. However, all members of NOX were absent in ECV304. mRNA expression of N0X4, intracellular ROS level, and apoptosis were all increased in HUVECs stimulation with high glucose, LDL, Ang II and serum-free medium. Conclusions: (1) N0X4 is the major NOX families in HUVECs and HUASMCs.(2) The mRNA expression of NOX4 increases significantly along with the up-regulation of apoptosis, intracellular ROS level in HUVECs stimulated by high glucose, LDL, Ang II and serum-free medium.Part II Effects of high level expression of N0X4on HUVECs apoptosisObjectives: To elucidate the change of intracellular ROS level and apoptosis in HUVECs after transfection with PcDNA3.1-NOX4 plasmid.Methods: N0X4 mRNA expression in HUVECs and ECV304 transfection with PcDNA3.1-NOX4 plasmid or PcDNA3.1-GFP plasmid was measured by RT-PCR. Intracellular ROS level and apoptosis were detected with flow cytometry. The apoptosis of HUVECs and ECV304 was observed by Hoechst staining and TUNEL staining. Results: N0X4 mRNA expression and ROS level were increased significantly in...