VEGFR is required for neoangiogenesis during DO. Eight-week-old ΔVHL mice were injected i.p. with monoclonal antibodies against VEGFR-1 and -2 every 3 days after surgery for a total of five injections. Nonimmune IgG injection served as a negative control. At day 17 after surgery, mice were perfused with Microfil and analyzed for vessel formation in the distraction gap. (A) Representative μCT images of vasculature in Microfil-perfused distraction area are shown. (B) Quantitative measurements of vessel volume per total volume (VV/TV), vessel number, vessel separation, and vessel surface are shown. Data represent mean ± SD. *, P < 0.05, **, P < 0.01.

Pharmacological activation of the HIF-1α pathway increases angiogenesis in vitro. (A) U2OS cells expressing an HRE reporter gene were exposed to hypoxia (Hyp) or treated with colbalt chloride (CoCl₂, 125 μM), DFO (200 μM), ethyl 3,4-dihydroxybenzoate (DHB, 700 μM), or l-mim (700 μM) under normoxia. Cells were harvested 24 h after treatment and analyzed for luciferase activity. (B) MSCs were collected from bone marrow of WT mice by using standard methods and cultured until confluent. Cells were untreated (control), or treated with DFO (10 and 50 μM) or l-mim (300 and 500 μM) under normoxia for 24 h. Nuclear protein was extracted from the cells and HIF-1α protein level was examined by immunoblotting analysis by using an anti-HIF-1α monoclonal antibody. Immunoblot for TBP (TATA box-binding protein) was used as loading control. (C) Total RNA was also extracted from cells, and VEGF mRNA expression was determined by using quantitative real-time PCR. Data represent mean ± SD. *, P < 0.05. (D) Matrigel tube-formation assay. HUVEC were cultured on Matrigel chambers with the addition of DFO (50 and 200 μM) or l-mim (300 and 500 μM) with VEGF (10 ng/ml) as positive control. Tube formation was photographed 12 h after treatment. (Magnification, ×100.) (E) Quantification of tube-formation assay by counting tube-like structure numbers. Data represent mean ± SD. *, P < 0.05. (F) In vitro metatarsal endothelial sprouting assay. Metatarsals were dissected from C57BL/6 E17.5 fetuses and cultured for 3 days for attachment. The explants were then cultured for another 6 days and then treated with DFO (50 μM) or l-mim (300 μM) for 24 h with rhVEGF (10 ng/ml) as positive control, followed by the detection of endothelial sprouting by immunostaining with anti-CD31 monoclonal antibody. Representative images are shown. (Magnification, ×25.)

Genetic activation of the HIF-1α pathway increases neoangiogenesis and promotes bone regeneration. (A) Eight-week-old ΔVHL mice and control littermates were subjected to DO. Tissues were harvested at day 31 after surgery, and histological sections of the distraction gap were prepared. Representative sections from the ΔVHL mice and controls are shown after staining with antibodies against pVHL and HIF-1α. VEGF mRNA expression in bone-lining cells is shown by in situ hybridization and immunostaining; CD31 immunostaining is also shown. Arrows show positive cells. (B) Representative μCT images of vasculature in Microfil-perfused distraction area from control and the ΔVHL mice at day 17 after surgery. Quantitative measurements of vessel volume per total volume (VV/TV) and vessel number are shown. Data represent mean ± SD. *, P < 0.05. (C) Representative μCT images of distraction area from control and the ΔVHL mice at day 38 after surgery. Quantitative measurements of BV and BV/TV are shown. Data represent mean ± SD. *, P < 0.05. (D) Three-point bending tests (peak load and stiffness) and nanoindentation (elastic modulus and hardness) were performed on tibiae from the ΔVHL mice and controls at day 38 after surgery. Data shown represent mean ± SD. *, P < 0.05.

Disruption of HIF-1α in osteoblasts impairs angiogenesis and bone regeneration during DO. (A) Representative μCT images of vasculature in Microfil-perfused distraction area from control and the ΔHIF-1α mice at day 17 after surgery. Quantitative measurements of vessel volume per total volume (VV/TV) and vessel number are shown. (B) Representative μCT images of distraction area from control and ΔHIF-1α mice at day 31 after surgery. Quantitative measurements of BV and BV/TV are shown. Data represent mean ± SD. *, P < 0.05.

Pharmacological activation of the HIF-1α pathway by DFO increases angiogenesis and promotes bone regeneration. Eight-week-old wild-type (C57BL/6) mice were subjected to DO. Mice were injected with DFO (200 μM) or saline as control in the distraction gap every other day from days 7 to 17 after surgery. (A) Validation of local injection approach using methylene blue following the same protocol with DFO treatment. X-ray images of control and DFO-treated mice at day 31 after surgery show bone regeneration in the distraction gap. (B) Representative μCT images of vasculature in Microfil-perfused distraction area from DFO-treated and control mice at day 17 after surgery. (C) Representative μCT images of distraction area from DFO-treated and control mice at day 31 after surgery. (D) Quantitative measurements of vessel number and connectivity are shown. Data represent mean ± SD. *, P < 0.05. (E) Quantitative measurements of BV and BV/TV are shown. Data represent mean ± SD. *, P < 0.05.