Up-regulation of HIF-1 angiogenic factor transcriptional targets, persistent VEGFR2 signaling, and intrinsic down-regulation of endothelial proliferation in TetON-HIF-1 transgenic mice. (A) Ear tissue ELISA for VEGF (left), PlGF (middle), and SDF-1 (right). Immediate day 1 increase of VEGF and PlGF contrasts the gradual SDF-1 induction between day 3 and day 30. (B) TetON-HIF-1 endothelial cell proliferation kinetics determined by counting double-labeled 5-bromo-2′-deoxyuridine/MECA32-positive endothelial cells in four to six 10× fields per thin section normalized to the microvascular density. (C) Representative Western blot analysis of total ear tissue lysates for VE-cadherin, total and phosphorylated: VEGFR2^(Y1173), PLCγ^(Y783), ERK1/2^(T202/Y204), AKT^(S473), and cyclin D1. (D) Representative immunofluorescent images (top) and quantification (bottom) of cyclin D1. TetON-HIF-1 data at each DOX day were compared with NTG or TetON-HIF-1 day 0 data (data not shown), using unpaired Student t test: *P < .05; **P < .01; ***P < .001. Bar represents 100 μm.