[Effects of vascular endothelial growth factor on apoptosis of sinusoidal endothelial cells caused by ethanol: experiment with rats]

To investigate the cell death pattern of sinusoidal endothelial cells (SECs) caused by ethanol and the effects of vascular endothelial growth factor (VEGF) on this cell death, as well as the underlying mechanism involving Ets-1 and Caspase-8.

SECs were isolated from male Wistar rats and cultured in medium containing ethanol (25 - 100 mmol/L). VEGF (20 - 30 ng/ml) was added into the medium to be co-incubated for up to 6 h. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) technique. The protein expression of Ets-1, prototype of anti-apoptotic Ets family, was determined by Western blotting and the Caspase-8 was measured by FLICE/caspase-8 colorimetric protease assay kit.

Three days after culture, the SECs showed spindle-like shapes and nearly confluent, however, the cells tended to shrink and die during the time course of ethanol incubation under phase contrast microscope. The control SECs contained only a few percent of TUNEL-positive cells; however, the TUNEL-positive cells started to increase 2 hours after the addition of ethanol (100 mmol/L), and about 75% of the cells were TUNEL-positive 6 hours after ethanol incubation (P < 0.05) under fluorescent microscope. Again, TUNEL-positive cells increased 6 hours after ethanol (25 - 100 mmol/L) incubation in a dose dependent manner (P < 0.05). Six hours after VEGF (20 - 30 ng/ml) was added into the medium with ethanol (100 mmol/L) the percentage of TUNEL positive SECs decreased in a dose dependent manner (P < 0.05), the level of ethanol-induced apoptotic cells in the presence of VEGF (30 ng/ml) being around 71% 6 hours after ethanol incubation alone. The Ets-1 protein level of the SECs decreased 6 hours after ethanol (100 mmol/L) incubation, which was prevented almost completely by VEGF (30 ng/ml). The Caspase-8 activity level was significantly increased to 44.9 +/- 14.3 2 hours after ethanol (100 mmol/L) incubation, and decrease to 30.4 +/- 2.0 and 25.2 +/- 2.2 respectively after the addition of VEGF (20 - 30 ng/ml) (both P < 0.05).

VEGF prevents the apoptosis of primary cultured SECs induced by ethanol, through at least in part, inhibition of ethanol-induced down-regulation of Ets-1 protein expression and ethanol-induced up-regulation of Casepase-8 activity in SECs.