Dynamic Change And Mechanism Of Anion Exchange 2 (AE2) Expression Induced By High Glucose In Vascular Endothelial Cells

Objective To observe the dynamic change of AE2 expression level in model of human umbilical vein endothelial cells (HUVECs) injury induced by high glucose (HG) and explore the expression mechanism from advanced glycation endproducts (AGEs) and reactive oxygen species (ROS) pathway. Methods In the experiment of concentration-effect relation, HUVECs were exposed to DMEM with 5.5, 17.8, 35.6, 71.2 and 142.4 mM of glucose or 142.4 mM of mannitol for 48 hours; in the study of time course, HUVECs were exposed to DMEM with 35.6 mM of glucose for indicated times (0, 12, 24, 48, 72, 120 and 168 h, respectively). In the other batch of experiment, HUVECs were exposed to DMEM containing 35.6 mM of glucose with or without AG (final concentration 5mM) or MTZ (1μM) for 48 h, respectively. The cell viability was determined by MTT. The HUVECs were harvested and total RNA and protein were isolated. AE2 mRNA and protein levels were analyzed by RT-PCR and western blots, respectively. Additionally, nucleus protein was isolated from cell extracts and binding activity of AP-1 was assayed by electrophoresis mobility shift assay (EMSA). Results (1) Cell viability by MTT: With the glucose concentration increased, cell viability was gradually decreased,while mannitol(MNT, 142.4 mM) appeared to have no effec(tP >0.05, vs 5.5 mM GLU); similarly, with the incubation hours elongated, cell viability was also gradually declined. (2) Concentration-effect relation of AE2 expression: AE2 mRNA levels increased with the increment of glucose concentrations, peaking at 35.6 or 71.2 mM, (P <0.01,or P <0.05,vs 5.5 mM GLU)and so did AE2 polypeptide levels. (3) Time course of AE2 expression: AE2 mRNA levels increased with elongation of the exposure time, peaking at 48 hours and sustaining to120 hours(P <0.01,or P <0.05,vs 0 h), and so did AE2 polypeptide levels. (4) Inhibition of AGEs pathway and ROS pathway downregulated the expression of AE2 mRNA and its protein. Exposure of HUVECs to 35.6 mM GLU for 48 h significantly upregulated the expression of AE2 mRNA and its protein, whereas pretreatment with aminoguanidine (AG,5 mM of final concentration) and Myxothiazole (MTZ, 1μM of final concentration) can normalize the expression level of AE2 mRNA and its protein.(5) HG increased AP-1 binding activity to AE2 promoter: Exposure of HUVECs to 35.6 mM GLU for 48 h considerably increased the AP-1 binding activity to AE2 promoter, whereas pretreatment with aminoguanidine (AG , 5 mM of final concentration) and Myxothiazole (MTZ, 1μM of final concentration) can normalize the the AP-1 binding activity.Conclusion HG upregulated the expression levels of AE2 mRNA and its protein in a time-and concentration-dependent manner in HUVECs; its mechanism is likely to increase AP-1 binding activity and resultantly upregulate the expression levels of AE2 mRNA and its protein through AGEs and ROS dependent pathway.