The Effects Of Endogenous Tgfβ1 And Tl1a On High Glucose-induced Apoptosis In Endothelial Cell

Background Excessive(Accelerated) endothelial cell apoptosis is a critical event in the process of diabetes-associated vascular disease. It's mechanism has not yet been clarified. The changes of cytokines synthesis and biological activity have been shown to play a potentially important role in high glucose-induced cell apoptosis. Both TGF 1 and TL1A were shown to mediate apoptosis. TGF 1 is considered a key modulator of endothelial cell apoptosis, there is, however, little evidence establishing a direct link between the effects of high glucose on apoptosis and autocrine action action of TGF 1 in endothelial cells. It remains contentious whether exposure to high glucose does induce TGF 1 production by endothelial cells. TL1A, as a recently newly discovered membre of tumaor necrosis factor (TNF)cytokine family, has been repored to play an important role in endothelial cell apoptosis. However, no study explore wether TL1Ainvolves in the process of diabetes-associated vascular disease.Objective To observe the TGF 1 and TL1A expression in human umbilical vein endothelial cell (HUVEC) under the high glucose(HG) condition, and to investigate the effects of endogenous TGF 1 and TL1A on high glucose- induced 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. TGF 1 and TL1A expression was evaluated by RT-PCR and Western Blotting. 2. Cultured HUVECs were divided six groups: 5.6mM glucose(NG) group NG and TGF 1group NG and TL1A group 22.4mM glucose (HG) control group HG and TGF 1-Ab group HG and TL1A-Ab group. The apoptotic rate of HUVEC was determined by flow cytometry with annexin -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 TGF 1 and TL1A expression, compared with normal glucose(5.6mM, P 0.01), glucose at 22.4mM induced the highest expression in HUVEC TGF 1 and TL1A.( the highest expression in HUVEC TGF 1 and TL1A were induced by 22.4mM glucose). Under exposure of HUVEC to 22.4mM glucose for 6h, 12h, 24h and 48h, HUVEC TGF 1 and TL1A expression showed significant increase compared with 0h (P 0.01). The peak expression of TGF- 1 in HUVEC was at the 12th hour, while TL1A expression peaks at the 48th hour after treatment with high glucose. In addition, HG raised TL1A mRNA and protein expression (P 0.01) in a time- and dose-dependent manner. Compared with normal glucose group, the apoptosis rate of HUVEC was greatly raised in HG control group (21.06% vs 4.09%, P0.01). HUVEC apoptosis rate significantly increased after adding exogenous TGF- 1 or TL1A to media containing 5.6 mM of glucose for 24h 15.26 %±2 .93% 35.45%±3.91% vs 4. 09%±0.32% P 0.01). 9 g/ml TL1A antibody prevent high glucose-induced endothelial apoptosis, but 30 g/ml TGF- 1 antibody does not. Hyperosmolarity does not cause HUVEC apoptosis. The trend of caspase-3 activity was the same as that of apoptosis.Conclusion 1. High glucose up-regulated TGF 1 and TL1A expression in HUVEC;2. TL1A is a mediator of high glucose-induced endothelial apoptosis; High glucose-induced endothelial apoptosis is independent of endogenous TGF 1.