High Glucose Induced Rat Vascular Smooth Muscle Cell And Human Umbilical Vein Endothelial Cell Injury: Involvement Of Protein Tyrosine Nitration

The hazards of diabetes to body mostly come from complications induced by hyperglycemia. Oxidative stress plays an important role in the development and progression of diabetic complications. When oxidative stress induced by high glucose happens, overproduction of superoxide anion (O2.-) would decrease the bioactivity of nitric oxide (NO), thus leading to the generation of more potent oxidant, such as peroxynitrite (ONOO-), which will damage protein and produce 3-NO2Tyr (3-NT). Though 3-NT has been found in many diabetic complications, the mechanism has been not studied systematically. In this study, human umbilical vein endothelial cells (ECV304) and rat vascular smooth muscle cell (VSMC) were cultured in high glucose medium or incubated with exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1), then the cell oxidative and nitrative injury was studied and the role of protein tyrosine nitration in cell injury, as well as the difference between protein tyrosine nitration induced by high glucose and exogenous nitrating agents were discussed. The main results in this work are as follows:1. Protein tyrosine nitration presents in high glucose induced human umbilical vein endothelial cell injuryThe dysfunction and further damage of endothelium play an important role in the development and progression of diabetic vascular complications. Protein tyrosine nitration is involved in endothelial cell injury induced by high glucose. Little is known about protein nitration in human umbilical vein endothelial cells (ECV304) induced by high glucose. In the present article, ECV304 was cultured in 30 mM glucose and 40 mM glucose for 72h, then the cell viability, lipid peroxidation, intracellular glutathione, intracellular reactive oxygen species, nitrite and nitrate contents and protein tyrosine nitration were assayed. After 72h treatment, it was found that high glucose stimulated ECV304 injury in a dose-dependent manner, including reducing cell viability, increasing malondialdehyde (MDA) content, decreasing glutathione (GSH) content, increasing intracellular reactive oxygen species (ROS), increasing the production of nitric oxygen (NO) (increased nitrite content in cell and nitrate content in medium) and generating protein tyrosine nitration ( protein nitration with molecular masses ~ 66, 50, 43, 34, 30 and 23 kDa), which demonstrated that high glucose could induce cell oxidative injury to ECV304.2. Protein tyrosine nitration presents in high glucose induced rat vascular smooth muscle cell injuryThe dysfunction of vascular smooth muscle cell involves in the development and progression of diabetic vascular complications. When oxidative stress induced by high glucose happens, reactive oxygen species involve in vascular smooth muscle cell, then generates protein tyrosine nitration. No investigation on protein tyrosine nitration in vascular smooth muscle cell has been found. In the present study, exposure of rat vascular smooth muscle cell (VSMC) to 30 mM glucose and 40 mM glucose for 3d and 7d, then the cell viability, intracellular glutathione, intracellular reactive oxygen species, nitrite content, protein tyrosine nitration were assayed. After 3d and 7d treatment, it was found that high glucose stimulated VSMC injury in a dose-dependent and time-dependent manner, including increasing cell viability, inducing cell oxidative injury (decreased GSH content and increased intracellular ROS content), increasing the production of NO (increased nitrite content in cell). It was demonstrated that protein tyrosine nitration with molecular masses ~ 55, 30, 26 and 20 kDa was generated in cell. The cell oxidative injury induced by high glucose would aggravate with the dose and time.3. Protein tyrosine nitration induced by high glucose and exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1) are differentThe development and progression of diabetic vascular complications is complicated and there are multiple pathways leading to protein tyrosine nitration. It is well accepted that heme or heme containing proteins could catalyze NO2--H2O2 to nitrate tyrosine residue of protein. This is a more possible mechanism under physiological and pathological conditions to produce tyrosine nitration than peroxynitrite. There is no comparison between protein tyrosine nitration induced by high glucose and exogenous nitrating agents. In the present study, exposure of ECV304 and VSMC to high glucose and exogenous nitrating agents (hemin-nitrite-H2O2 and SIN-1) for 72h, the cell oxidative injury in ECV304 and VSMC induced by high glucose and exogenous nitrating agents was studied by cell viability and protein tyrosine nitration. After 72h treatment, it was found that with the addition of nitrite, the cytotoxicities of hemin-H2O2 on ECV304 and VSMC were significantly augmented, at the same time, protein nitration in cellular protein of both cell lines was also significantly increased. Secondly, with regard to some proteins, protein nitration induced by high glucose was similar to those induced by extrinsic factors (hemin-nitrite-H2O2 system and SIN-1). Furthermore, the difference between protein tyrosine nitration induced by high glucose condition and extrinsic factors (hemin-nitrite-H2O2 system and SIN-1) was that the later could also generate protein nitration with low molecular masses (~11, 17, and 20 kDa). By comparison protein tyrosine nitration induced by high glucose condition with those induced by extrinsic factors (hemin-nitrite-H2O2 system and SIN-1), it may be speculated that protein tyrosine nitration is selective in diabetic vascular complications.