Fig. 7. Inhibition of HUVEC in vitro angiogenesis by anti-VEGFR-3 and anti-VEGFR-2 antibodies. (A) HUVECs were analyzed by FACS for expression of surface VEGFR-2 and VEGFR-3 using monoclonal antibodies IMC-1121 and hF4-3C5. Both receptors were detected, with approximately equal staining intensity. (B) HUVECs were grown for 24 hours on a solid gel of basement matrix proteins (Chemicon In Vitro Angiogenesis Kit) in the presence or absence of monoclonal antibodies. The images were captured using a 4x objective of a Nikon TE2000 inverted microscope. Blocking of tube formation is evident with either mAb IMC-1C11 or hF4-3C5. Simultaneous addition of both antibodies increases the inhibitory effect. Bar, 100 µM. (C) Total sprouting response was measured in replicate wells using digital imaging (see Materials and Methods). Branching was counted manually as the number of tube branch points per field. The results are shown as percent inhibition relative to the average of untreated controls. The extent of tube-forming inhibition is the same whether total sprouting or branching is measured.