Figure 5

Amifostine inhibits human umbilcal vein endothelial cells (HUVEC) proliferation and vascular endothelial growth factor A (VEGF-A)-induced migration and differentiation. (A) Amifostine inhibits HUVEC proliferation. HUVEC growing in endothelial growth medium-2 supplemented with 0, 0.25, 0.5, 1 or 2 mM of WR-2721 were counted for 3 days. The insert shows the inhibition of cell proliferation after 2 days of treatment, in percentage of untreated cells. (B) Amifostine inhibits VEGF-A dependant HUVEC migration. A Transwell migration assay was used. HUVEC were seeded in upper compartments and incubated in Dulbecco's modified eagle medium containing (DMEM) 0.5% fetal bovine serum (FBS), 0.1% bovine serum albumine and increasing concentrations of WR-1065. 10 ng/mL VEGF-A were, or were not, added in the lower compartment and cells were allowed to migrate for 7 h. Cells that migrated were fixed, stained and quantified. Left panel: quantification of VEGF-A-dependent HUVEC migration (% of the migration versus control untreated cells). Significant changes were determined by Student paired tests (*P < 0.05; **P < 0.01). Right panel: corresponding photomicrographs of HUVEC. (C) Amifostine inhibits VEGF-A induced capillary-like structures formation. HUVEC were seeded on GFR-Matrigel. After a 1 h incubation in 0.5% FBS-containing DMEM to allow cell adhesion, cells were treated with WR-1065 for 3 h, in the presence or absence of 10 ng/mL VEGF-A. Total tubule length was determined using NIS image analysis software. Left panel: quantification of total tubule length in the different experimental conditions. Results are expressed as percentages of the average tubule length in the control condition, set to 100%. The control condition corresponds to absence of both VEGF-A and WR-1065. Significant changes, were determined by Student paired tests (*P < 0.05; **P < 0.01). Right panel: phase-contrast microphotographs of tubule networks formed by HUVEC, at a 200-fold magnification.