Effect of anti-VEGF on the transmigration of human CD4+ and CD8+ T cells across ECs. Endothelial cells (ECs) were cultured to confluency on FluoroBlok 3μM pore membranes in the upper chambers of transwells. Subsequently, CFSE-labeled T cells (5 × 105 cells) were added into the upper chamber, and migration into the lower chamber was monitored real-time by the assessment of increasing fluorescence. The number of transmigrated cells in each lower chamber was determined using a standard curve, and the percentage of transmigrated cells at each time point was calculated. Transmigration assays were performed in the presence of control IgG (black dots) or anti–human VEGF (Genentech; 1μg/mL, open squares). (A-B) Representative experiment illustrating transmigration across unactivated ECs. No difference between anti-VEGF versus control IgG treatment is evident. (C-D) Representative experiment illustrating transmigration across 6-hour TNFα–activated ECs. (E-F) The mean percentage inhibition of transmigration by anti-VEGF across unactivated (□) or TNFα–activated ECs (■, n = 5 per experimental group). Anti-VEGF significantly inhibits transmigration across activated ECs at times > 2 hours. *P < .01 vs control IgG.
