Tl1a Mediates High Glucose-induced Apoptosis By Upregulation Ros Generation Inthe Endothelial Cell

Background Accelerated endothelial cell apoptosis is a critical event in the process of diabetes-associated vascular disease. However, the underlying mechanisms by which high glucose induces apoptosis in human vascular endothelium need to be elucidated. High glucose exposure is associated with increased endothelial cell production of reactive oxygen spicies (ROS), and oxidative stress has been the mechanism most often implicated in the increase in the associated endothelial cell apoptosis. We have showed that high glucose up-regulated TL1A expression in human umbilical vein endothelial cell (HUVEC). TL1A, a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family, has been demonstrated to induce apoptosis in endothelial cells. However, it has not been reported whether TL1A involves in the process of diabetes-associated vascular disease, and whether TL1A-Ab can prevents high glucose-induced human vascular endothelial cell apoptosis through ROS inhibition.Objective To observe the effect of TL1A on ROS generation in human umbilical vein endothelial cells(HUVEC) and to investigate the role and mechanism of TL1A in the high glucose-mediated endothelial cell apoptosis.Methods 1. HUVEC were exposed to different concentrations of glucose (5.6, 11.2, 22.4, 33.6mM)for 24 hours or to 22.4 mM glucose for different time (0h, 6h, 12h, 24h, 48h). 5.6 mM glucose plus 16.8mM mannitol as an osmotic control. Expression of TL1A protein was evaluated by western blotting. 2. Cultured HUVECs were divided eight groups:①5.6mM glucose(NG) group;②NG and TL1A group;③22.4mM glucose (HG) control group;④HG with SOD and CAT group;⑤HG and TL1A-Ab1μg/ml group;⑥HG and TL1A-Ab3μg/ml group;⑦HG and TL1A-Ab9μg/ml group;⑧5.6 mM glucose and 16.8mM mannitol group. ROS generation was evaluated by DCFH-DA staining and flow cytometry. The apoptotic rate of HUVEC was determined by flow cytometry with annexinⅤ-FITC/PI double staining. Caspase-3 activity was detected by enzyme linked immunosorbent assay. Results Exposure of HUVEC to high glucose(11.2, 22.4 or 33.6mM) for 24h resulted in a significant increase in TL1A expression, compared with normal glucose(5.6mM, P<0.01), in which the highest expression in HUVEC TL1A was induced by 22.4mM glucose. High glucuse raised TL1A protein expression (P<0.01) in a time- and dose-dependent manner. Exposure of HUVEC to 22.4mM glocose for 24h resulted in a significant increase in ROS generation and apoptosis, compared with normal control group(76.96±6.61vs 46.80±4.10,23.70%±3.20% vs 4.09%±0.39%, P<0.01). ROS formation of HUVEC was increased to 93.75±9.12 after adding exogenous TL1A (50ng/ml) to media containing 5.6 mM of glucose for 24h, which just doubled the number of the NG group,and is significantly higher than HG group (P<0.01). TL1A-Ab reduced high glucose-induced ROS formation and apoptosis rate in HUVEC in a dose-dependent manner. HUVEC's ROS formation and apoptosis rate in HG+ TL1A-Ab9μg/ml group was not significantly different from those in HG+SOD+CAT group and NG group. Hyperosmolarity(22.4mM)failed to induce HUVEC apoptosis. The trend of caspase-3 activity was the same as that of apoptosis.Conclusion High glucose up-regulated TL1A expression in HUVEC, and TL1A mediate high-glucose-induced apoptosis by increasing ROS formation in the endothelial cell.