Inhibition of alphastatin on angiogenesis in human endothelial cells and the mechanism thereof: an experimental study

Abstract

To explore the effect of alphastatin on angiogenesis activity of human umbilical vein endothelial cells and mechanism thereof. Human umbilical vein endothelial cells of the line ECV304 was cultured. Hepatic growth factor (HGF) and alphastatin of the concentrations of 10, 100, or 1000 nmol/L were use in migration test ECV304 cells at logarithmic stage were inoculated in a 96-well plate. Alphastatin of different concentrations was added, 24, 48, 72, and 96 hours later MTT was added to detect the A(490) nm values with and draw a growth curve. Phorbol 12-myristate 13-acetate (PMA) was used as positive group. The formation of capillary-like structure was examined with fluorescent microscopy. RT-PCR was used to examine the expression of sphingosine kinase (SPK) mRNA in the ECV304 cells. ECV304 cells were treated with alphastatin of 10 nmol/L, 100 nmol/L, or 1000 nmol/L respectively. SPK activity was examined by using [gamma-(32)P] ATP. The number of migrated cells treated with alphastatin of the concentrations of 10, 100, and 1000 nmol/L were (103 +/- 4), (75 +/- 3), and (13 +/- 1) respectively, all significantly lower than that of the HGF group (131 +/- 4, all P < 0.05). The values at different time points of the ECV304 cells treated with alphastatin of different concentrations were not significantly different from that of the HGF group. The area of capillary-like structure of the PMA group was 2996 +/- 31 microm(2)/field, and those of the groups of alphastatin of the concentrations of 100 and 1000 nmol/L were 1509 +/- 30 microm(2)/field and 1301 +/- 20 microm(2)/field respectively, all significantly less than that of the PMA group (2996 +/- 31 microm(2)/field, all P < 0.05). However, there was not a dose-dependent manner in the inhibitive activity of alphastatin on the formation of capillary-like structure. RT-PCR showed that quantity of SPK mRNA product was amplified from the ECV304 cells. The S1P activity of the ECV304 treated with alphastatin was significantly lower than that of the control group, especially when the concentration of alphastatin was 100 nmol/L and 12 hours after treatment. Alphastatin shows a potent antiangiogenic property, which may be closely associated with the downregulation of endothelial cells SPK activity.