Role Of Anion Exchanger- 2(AE2 ) In Apoptosis Of Endothelial Cells Induced By Hyperglucose

Objective: Dysregulation of endothelial cells (ECs) is an initial and crucial step of angiopathy resulted in by diabetes mellitus (DM). Anion exchanger 2(AE2) is likely to play an important role in angiopathy. The purpose of this study is to examine the expression of AE2 in human umbilical vein cells (HUVECs) and investigate whether AE2 participates in HUVECs apoptosis induced by high glucose.Methods: HUVECs were treated with DMEM containing glucose (5.5, 17.8, 35.6 mmol/L, respectively) for 48 h. Cultured HUVECs were randomly divided into six groups: Control group, Low glucose group, High glucose group, Control+SITS group, High glucose+ SITS group and Mannitol group. Apoptosis was detected by Annexin-V/PI assays and AE2 mRNA as well as protein levels were examined by RT-PCR and Western blotting, respectively. Intracellular ROS generation was detected by flow cytometry using Peroxide-sensitive fluorescent probe DCFH-DA. Cell viability was determined using MTT assay. The activity of SOD in cells was measured using SOD kit.Results: 1. Cell viability: The cell viability of low glucose (55.1±10.1%) and high glucose (38.3±3.6%) group significantly decreased compared with that of control group (90.2±9.3%) (P<0.01); the viability of high glucose+SITS group (86.4±7.2%) increased 47.9% compared with that of high glucose (P<0.01); There was no significant difference in viability of the other three groups, high glucose +SITS group, Control+SITS group (88.6±6.2%) and Mannitol group (85.1±10.1% ), respectively, compared with that of control group (P>0.05). 2. Expression of AE2 mRNA: The expression of AE2 mRNA in the high glucose group (0.79±0.03) was significantly higher than that in Control group (0.33±0.03) (P<0.01); there was no significant difference between Mannitol group (0.31±0.06) and Control group (P>0.05). 3. Expression of AE2 Protein: AE2 protein is a basic expression in Control group. The expression elevels of AE2 Protein in high glucose group (0.36±0.05) was considerably increased compared with that of control group (0.25±0.04) (P<0.01). However, there was no significant difference between Mannitol group and Control group (P>0.05). 4. Cell apoptosis levels: In high glucose group, the apoptosis levels of HUVECs noticeably increased compared that of control group, while treatment with SITS, the rate of apoptosis was significantly decreased. 5. Activity of intracellular SOD: The activity of intracellular SOD in high glucose group (35.5±3.2 U/mL) significantly decreased compared with that of control group (60.2±8.5 U/mL) (P<0.01); and high glucose+SITS (50.6±4.2U/ml) also had a significant difference compared with high glucose (P<0.01), other groups had no significant difference (P>0.05) compared with control group except low glucose group. 6. ROS production: Mean fluorescence intensity(MFI) in high glucose group was more stronger, to reach 110.4 , compared with that of control group (22.6±2.5) (P<0.01), and high glucose +SITS also have a significant difference compared with high glucose (P<0.01). Other groups had no significant difference except low glucose group compared with control group (P>0.05). 7. Mitochondrial membrane potential: The mitochondrial membrane potential was demonstrated by the apoptosis rate after stained with rhodamine123 and PI. The LG group and HG group were significantly higher than that in Control group (P<0.01), while the high glucose +SITS group was significantly lower than that of HG group (P<0.01). There was no significant difference among Control+SITS group, Mannitol group and control group (P>0.05).Conclusion: High glucose upregulated expression of AE2 protein and induced HUVECs apoptosis. High glucose-induced apoptosis of HUVECs is AE2/ROS-dependent.