Figure 6. Endothelial VEGF Mediates Survival in a Cell-Autonomous Manner.

(A–B) Endothelial cells from control (blue) and VEGF^ECKO (green) mice were cultured in the presence (A) or absence (B) of serum and viable cells were counted at indicated times. Endothelial cells were stained for x-gal and nuclear fast red (right panels, B). Note the difference in cell number between control and VEGF^ECKO cells.

(C) Endothelial cells from control (blue) and VEGF^ECKO (green) mice were cultured in the presence of CoCl₂, active caspases were fluorescence-labeled and quantified by fluorometer.

(D) Endothelial cells from control (blue) and VEGF^ECKO (green) mice were cultured in the presence of recombinant hVEGF₁₆₅ or CoCl₂ and viable cells assessed after 48hs.

(E) HUVECs were cultured under hypoxia for 24 h and analyzed for VEGFR2 phosphorylation (pVEGFR2, top) and VEGFR2 total levels (bottom). Lanes: 1, VEGF (100 ng/ml) for 5 min; 2–3, normoxia in the presence (2) and absence (3) of sodium orthovanadate (Na₃VO₄) for 24 h; 4–5, CoCl₂ (hypoxia mimetic) in the presence of Na₃VO₄ for 24 h.

(F–G) Endothelial cells from control (blue) and VEGF^ECKO (green) mice were cultured in the presence of CoCl₂ (100 µM) for 24 h and transcript levels of VEGF (F) and GAPDH (G) were determined by real-time RT-PCR. Relative RNA units (RRU) were normalized to β-actin. Data shown are means ± SD.

(H) Endothelial cells differentially labeled (Cy3 for wild type and BODIFY green for VEGF^ECKO) were cultured alone (left) or combined (right). Photographs were taken after 2 days and 5 days. Arrows indicate control cells (red). Arrowhead points to VEGF^ECKO cells (green).

(I) Quantification of cell survival following co-culture. Fluorescence labeled cells were counted at indicated times. Data are presented as percentage to control.