J., Rosen L. show that these small molecule inhibitors of endocytosis block angiogenesis and test (values of less than 0.05 were considered to be statistically significant). RESULTS Small Molecule Inhibitors of Endocytosis Suppress the Internalization of VEGFR2 in Endothelial Cells To address the role of receptor internalization in the activation of ERK1/2, we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis (46, 47). To confirm that pitstop and dynasore can inhibit the internalization of VEGFR2 in endothelial cells, we used an antibody feeding assay similar to that used to monitor the fate of internalized VEGF receptors in other studies (33, 40, 41, 48). Plasma membrane VEGFR2 molecules were labeled on ice with a VEGFR2 extracellular domain-specific antibody. Examination of cells fixed directly after this labeling period demonstrated the retention of the VEGFR2 antibody at the cell surface and no colocalization with endosomes (Fig. 1and 0.05; **, 0.001. = 5 m. indicate VEGFR2-positive endosomes. indicate VEGFR2 membrane staining. We also performed surface biotinylation assays to SPDB-DM4 confirm that pitstop and dynasore treatment resulted in retention of VEGFR2 at the cell surface. Cells were either incubated with vehicle alone, or they were incubated with VEGF in the presence of vehicle, pitstop, or dynasore. Cells were then subjected to cell surface biotinylation with a membrane-impermeant biotinylation reagent. The biotinylated fraction was immunoprecipitated and probed for VEGFR2. The amount of cell surface VEGFR2 was significantly reduced in cells incubated with VEGF and vehicle compared with cells incubated with vehicle alone (Fig. 1and and and represent vehicle, represent pitstop. = 3 independent experiments. *, 0.05; **, 0.01; ***, 0.001, = no significant difference. and and and represent vehicle, represent SPDB-DM4 dynasore. = 3 independent experiments. *, 0.05; **, 0.01; ***, 0.001; = no significant difference. Vwf and and = 3 independent experiments. and = 3 independent experiments. *, 0.05. Internalization Is Required for ERK1/2 Activation in Stimulated Endothelial Cells but Not in Non-endothelial Cells We then examined whether internalization is required for ERK1/2 activation in endothelial cells stimulated with other pro-angiogenic growth factors, namely FGF2 and HGF. Although receptors for FGF2 and HGF are well known to undergo internalization in cells (51, 52), it is not known whether internalization of these receptors is important for signal transduction in endothelial cells. Stimulation of endothelial cells with FGF2 or HGF resulted in activation of ERK1/2, but this was abrogated by pitstop and dynasore (Fig. 5, and and and assay of endothelial tubule formation (19, 43). Latex beads coated with endothelial cells were embedded in a three-dimensional fibrinogen matrix and then incubated with VEGF and FGF2 in the presence of vehicle, pitstop, or dynasore. Tubule formation was inhibited by dynasore and pitstop in a dose-dependent fashion (Fig. 7, using the subcutaneous sponge assay (44). Inert sponges implanted subcutaneously under the back skin of mice SPDB-DM4 were injected three times a week with control solution (vehicle in PBS), dynasore (dynasore in PBS), growth factors (VEGF, FGF2, and vehicle in PBS), or growth factors plus dynasore (VEGF, FGF2, and dynasore in PBS). Microvessel density in the group receiving growth factor treatment was significantly enhanced compared with the.