Effect of high glucose on function of cultured rabbit vascular endothelial cells

Son, Seok Man; Kim, In Ju; Kim, Yong Ki; Kim, Chi Dae; Hong, Ki Whan

Abstract

BACKGROUND: Vascular disease accounts for the majority of the clinical complications of diabetes mellitus. Changes in local control of vascular tone such as imbalanced production of relaxing and contracting factors by endothelium may be related to the initiation and maintenance of abnormal vascular reactivity characteristically seen in diabetic vascular complications. Cytokines and growth factors released from injured endothelial cells, T-cells, and macro-phages enhance atherogenesis. In this study, we examined NO and TNF-a released from cultured rabbit aortic endothelial cells(RAECs) under different glucose concentration to investigate the relationship between high glucose and endothelial cell dysfunction.
METHODS
The thoracic and abdominal aortae of rabbit(23kg) were isolated and periadventitial connective tissue was carefully removed. Rabbit aortic endothelial cells in primary culture were prepared by the m.ethod of Schwartz with modification. RAECs were grown to confluence in 25 cm2 flask in DMEM supplemented with 20% FBS, 150pg/mL endothelial cell growth supplernent, 90pg/mL heparin, 100 U/mL penicillin and 100pg/mL streptomycin at 37'C in humidified 5% carbon dioxide in air. For experiments, confluent cells were replaced in 1 1 mm, 48 well plate containing same medium composition. Cells were then incubated in the presence or absence of FBS for various times up to 48 hours(time course) to eveluate the NO and TNF-a response to different glucose concentrations(0, 5.5, 11, 22, and 44 mmol/ L). Cells were also incubated with various concentration of ACH and ADP(10, 10', 10 and 10' mol/L) and 10' mol/L of ACH or ADP with different glucose concentrations for 24 hours to evaluate stimulated effect of ACH and ADP on NO release.
RESULTS
1) Total NO release from RAECs was significantly in a time-dependent. After 48 hours incubation, the total secretion of NO was significantly higher in culture medium with FRS than without FBS. 2) Glucose concentration resembling severe hyper-glycemic conditions(22 and 44 mmol/L) significantly inhibited NO release from RAECs, 3) Acetylcholine and ADP induced a clear dose-dependent NO release in RAECs. 4) Stimulation of acetylcholine and ADP on NO release according to different glucose concentration was not significantly higher than NO release in culture medium with glucose alone. 5) The increment in TNF-a levels was associated with a significant increase at higher glucose concentration, 6) There was a negative correlation between NO and TNF-a release in culture medium with FBS but not in culture medium without FBS.
CONCLUSION
Our data show that decreased NO release and increased TNF-a release from RAECs were noted under high glucose concentration. Such interaction could play a significant role in the development of diabetic vascular complication in hyperglycernic conditions.