FIGURE 3.

S156C TIMP-3 is an inefficient inhibitor of VEGF-mediated EC proliferation, migration, and actin reorganization. Serum-starved PAE_KDR cells expressing WT (W) or S156C TIMP-3 (M) or vector alone (V) were incubated with or without VEGF and analyzed for proliferation, migration, actin reorganization, and tube formation. A, proliferation of endothelial cells in response to VEGF (15 ng/ml) for 40 h (right panel). [³H]Thymidine incorporation was measured during the last 4 h of incubation. Optimal concentration of 15 ng/ml was determined from a dose-response curve generated using PAE_KDR vector-transfected cells (left panel). p < 0.01 (**) and p < 0.05 (*) versus unstimulated controls. B, migration of endothelial cells toward VEGF (50 ng/ml) (right panel). Optimal concentration of 50 ng/ml was determined from a dose-response curve generated using PAE_KDR vector-transfected cells (left panel). Migrating cell number is expressed as the means ± S.E. of quadruplicate samples. C, actin reorganization in PAE_KDR cells stimulated with or without 50 ng/ml VEGF for 15 min at 37 °C. The arrows indicate membrane edge ruffling. Magnification ×1000. D, PAE_KDR cells expressing WT (W) or S156C TIMP-3 (M) or vector alone (V) were cultured between two layers of type 1 collagen gel in the presence or absence of 50 ng/ml VEGF as indicated. Pictures were taken at the indicated times after inoculation. Images are of representative microscope fields (×200).