Effect of BV14 and BV13 on VE-cadherin organization at junctions and on permeability of confluent endothelial cells.

(A) VE-cadherin+/+ (i) and VE-cadherin−/− (ii) cells were incubated with BV13 mAb (50 μg/mL) after fixation. Only VE-cadherin–positive cells expressed the antigen at junctions, while VE-cadherin null cells were negative to staining. VE-cadherin+/+ cells were incubated with 50 μg/mL BV14 (iii) or BV13 (iv) for 7 hours before fixation. After this time, cells were fixed, and VE-cadherin staining was evidenced by a rhodhamine-conjugated secondary antibody. (Magnification × 1000.) (B) Permeability across cell monolayers was measured by seeding the cells on Transwell filters and measuring the passage of FITC-dextran from the upper to the lower compartment. VE-cadherin−/− cells presented higher permeability values as compared to VE-cadherin+/+ cells. Addition of BV13 (50 μg/mL) to VE-cadherin–positive cells induced an increase in permeability at 3 hours, similar to that observed in VE-cadherin−/− cells. BV14 at the same concentration was ineffective. Increasing the concentration of the mAbs up to 100 μg/mL did not further increase permeability. Data are expressed as percent increase in comparison to VE-cadherin+/+ cells and are means ± SEM of quadruplicates from a typical experiment out of 4 performed.