FIGURE 2.

Stimulation of mitogenesis in endothelial cells by VEGF-A requires uPA. A, DNA synthesis in response to VEGF in LMVECs isolated from WT or uPA^−/− mice. Endothelial cells were plated in 96-well plates, cultured for 24 h in complete medium, starved in 1% FBS/EBM for 24 h, and then stimulated with VEGF (25 ng/ml) for an additional 24 h. DNA synthesis was assessed by measuring incorporation or 5-ethynyl-2′-deoxyuridine. y axes denote -fold stimulation of DNA synthesis in response to VEGF relative to untreated cells (1-fold). The results, representative of one of three independent experiments performed in triplicate, are shown as the mean ± S.E. B, re-expression of uPA in uPA^−/− endothelial cells restored DNA synthesis in response to VEGF. uPA^−/− LMVEC transfected either with “empty” (con) LV or uPA-bearing LVs were plated in 96-well plates, cultured, and serum-deprived as in Panel A. VEGF was added for 24 h at the indicated concentrations, and DNA synthesis was measured as in panel A. x axes denote concentrations of VEGF in ng/ml. y axes denote -fold stimulation of DNA synthesis in response to VEGF relative to untreated cells (1-fold). Data from three independent experiments performed in triplicate are shown as the mean ± S.E. * denotes p < 0.05, ** denotes p < 0. 001. C, re-expression of uPA in uPA^−/− endothelial cells amplified VEGF-induced signaling. uPA^−/− LMVEC were transfected with empty (con) LV or uPA-bearing LV, starved overnight, and lysed at the indicated times after stimulation with VEGF (10 ng/ml). Western blot analysis shows the time course of ERK1/2 and ribosomal S6 unit phosphorylation, which reflects activation of the PI3k/Akt/mTOR/S6 Kinase (S6K) pathway (60). Anti-GAPDH was used to ensure equal protein loading. The illustrated blots are representative of three independent experiments. D, uPA potentiates DNA synthesis in response to VEGF in hLMVECs. hLMVEC were plated in 48-well plates, cultured for 24 h in complete medium, starved in 1% FBS/EBM for 24 h in the absence or presence of 20 nm uPA, and stimulated with VEGF at the indicated concentrations for an additional 24 h. DNA synthesis was assessed by measuring [³H]thymidine incorporation into DNA. Data are representative of three experiments performed in triplicate are shown as mean ± S.E.