Figure 5.
Hypoxia Drives Angiogenesis by a Macrophage-Generated Gradient of VEGF-A
(A) Representative images of sections of a rat sciatic nerve bridge, Day 2 and 3 after transection and 30 min after injection of hypoxyprobe-1, immunolabeled to detect hypoxyprobe-1 (green). Scale bar, 25 μm.
(B) As in (A) but immunolabeled to detect macrophages (Iba1+, red) and hypoxic cells (hypoxyprobe-1+, green). Scale bar, 25 μm.
(C) Graph showing percentage of hypoxic cells (hypoxyprobe-1+) in macrophage (Iba1+) and non-macrophage (Iba1−) populations from rat sciatic nerve bridges cultured at indicated oxygen conditions (n = 3).
(D) HUVECs or SCs were placed in the upper compartment of Boyden chambers and allowed to migrate into the lower chamber containing media with no factors (SATO), VEGF-A165, serum, or conditioned medium from bridge cells cultured at 1.5% O2 (n = 5). For (C) and (D) one-way ANOVA test was used for statistical analysis.
(E–H) Confocal images of longitudinal cryosections of injured sciatic nerves from PLP-EGFP mice, Day 5 or Day 7 after transection, following gavage of cabozantinib or control solvent on Day 4 (pre-vascularization), immunostained to detect ECs (CD31+, red) and axons (NF+, blue) Scale bar, 50 μm, quantified in (G) and (H) (n = 3).
(I) As for (F) but cabozantinib was administered on Day 5 (post-vascularization) and harvested on Day 6, quantified in (J) (n = 3). For reconstruction of longitudinal sections shown in (E), (F), and (I), multiple images from the same sample were acquired using the same microscope settings.
Graphs show mean value ± SEM. See also Figure S5.