VEGF₁₆₅ effects on pro-uPA and MMP-2 activation are PI3-kinase dependent. (A) Surface pro-uPA levels of endothelial cells analyzed in fluorocytometric ELISA. VEGF effects on cell surface pro-uPA are abolished by the PI3-kinase inhibitors wortmannin or Ly294002. Serum-deprived human endothelial cells, preincubated for 30 minutes with different inhibitors (100 nM wortmannin, 10 μM Ly294002, 20 μM H-89, 10 μM PD98059, and 10 μM SB203580), were stimulated with 50 ng/mL VEGF₁₆₅ for 60 minutes, or were left unstimulated. VEGF₁₆₅ induced a PI3-kinase-dependent (wortmannin- or Ly294002-treated cells) decrease of cell surface pro-uPA level. c indicates control; V, VEGF; wort, wortmannin; Ly, Ly294002; PD, PD98059; and SB, SB203580; mean ± SDs; n = 3; ^*P < .05; ^**P < .01. (B) VEGF-induced increase in d-dimer concentration in cell supernatants (measured by an ELISA) was partially blocked by the PI3-kinase inhibitor wortmannin. Serum-deprived human endothelial cells were incubated with or without wortmannin (100 nM) and with or without VEGF (50 ng/mL). Mean ± SDs; n = 2. (C) Western blots for MMP-2 from endothelial cells lysates. The increase in active MMP-2 (62 kDa) in response to VEGF₁₆₅ is wortmannin (100 nM) dependent.