Induction of EC dispersion by an antagonistic MoAb for VEGFR-3 (AFL4).

Sorted ECs were cultured on OP9 in the presence or absence of MoAbs, and the cultures were immunostained by Mec13.3. Addition of 100 μg/mL AFL4 induced dispersion of ECs (panels C-D) compared with the matched 100 μg/mL control MoAb (A7R34; panel A). AFL4-induced EC dispersion was not observed in the culture in which endogenous VEGF-A was sequestered by mFlt-1-hIgG (panel B). Dispersion was induced by addition of anti–VE-cadherin MoAb VECD-1 (panels E-F); no alteration in shape was observed. Scale bar, 200 μm. (G) Quantitative analysis of the number of round EC clusters. Reduction in the number of packed colonies by EC dispersion by Ab treatment is expressed as a percentage against untreated cultures. Experiments were performed using different ES cell lines: EB5 (open bars) and CCE (black bars). Each error bar represents mean ± SD of 5 independent experiments. *P < .05. P values were calculated using the Mann-Whitney U test.