Figure 7
Survivin expression augmented VEGF-dependent angiogenesis in the chick-CAM assay: A,B: HEK293T cells (2×106) were seeded in 60 mm plates, transfected with pEGFP-C1 or pEGFP-survivin (5μg) and after 48 h supernatants were collected and centrifuged. Also, cells in suspension were obtained (106/mL). The solutions, fresh medium alone, or media obtained from non-transfected cells or cells in suspension, were pipetted onto chick chorioallantoic membranes (CAM) as described in Materials and Methods. Per condition 3 eggs were employed. A week later CAMs were fixed and stained with hematoxilin-eosin. Three sections per egg were analyzed. A: Vessels (arrows) were photographed and counted (10 photographs per section). B: The number of vessels per mm2 optic field (mean ± s.e.m., n = 3, *: p < 0.05) are depicted. C: Supernatants of pEGFP-C1 and pEGFP-survivin obtained as described (A,B), were treated with increasing concentrations of anti-VEGF neutralizing antibodies (0.1, 1, 10 μg/mL). As an IgG control, anti-β3 integrin was used at the highest concentration (10 μg/mL). Numerical data shown are the means ± s.e.m. of results obtained in three independent experiments. Statistically significant differences compared to mock controls are indicated (* = p < 0.05). D: Model: β-catenin translocates to the nucleus where it binds Tcf/Lef and induces the expression of target genes (survivin, vegf, cox-2 are depicted). Survivin (by unknown mechanisms) favors PI3K/Akt activation, which increases β-catenin-Tcf/Lef transcriptional activity, thereby promoting its own expression (feeding this positive feedback loop), as well as that of cox-2 and vegf. The protein VEGF is secreted to the extracellular compartment where it induces angiogenesis.