Under hypoxic conditions VEGF-mediated up-regulation of HLX and UNC5B mRNA is strongly reduced whereas sprouting activity is increased. (A) Comparison of VEGF-A effects on HLX, UNC5B, and VEGF-A mRNA under hypoxic and normoxic conditions: Starved HUVECs were treated with VEGF-A and were kept under normoxic or hypoxic conditions for 4, 8, 16, and 32 hours. The sample for the 1-hour value was kept under hypoxic conditions already for 4 hours before addition of VEGF. Cells were harvested and the RNA was isolated and subjected to real-time RT-PCR analysis. One representative experiment of 3 performed in triplicates is displayed. The values depict the mean of triplicates ± SEM. (B) Comparison of sprouting activity under hypoxic and normoxic conditions: HUVECs infected with Ad.con or Ad.HLX (20 MOI each) for 1 day were used in the spheroid sprouting assay. Normoxic or hypoxic conditions were used immediately after embedding into the collagen gel concomitant with VEGF addition. Results are displayed as fold induction ± SEM of total sprout length in comparison to spheroids incubated at normoxic conditions without VEGF. **P < .005, ***P < .001, t test.