Moreover, local application of VEGF has been used to enhance healing and angiogenesis in mouse, rat, and rabbit fracture and bone defect models (21C24). hybridization and immunohistochemistry (SI Fig. 6mice subsequently generated more dense woven bone in the distraction gap compared with controls (Fig. 1and SI Igf1 Fig. 7). CT measurements showed significantly increased bone volume (BV) and bone volume per total volume (BV/TV) at days 31 and 38 in the mice compared with controls (Fig. 1and SI Fig. 8). Thus, the increased vascularity observed in the mice at the conclusion of DO was followed by increased bone formation. Biomechanical testing of the bones by three-point bending showed that peak Ned 19 load and stiffness were significantly increased in mice compared with controls (Fig. 1mice led to an increase in structural integrity by increased bone Ned 19 volume with no difference in the material properties of the newly formed bone. Collectively, these results show that genetic activation of the HIF pathway in the mice increases angiogenesis and bone regeneration. Open in a separate window Fig. 1. Genetic activation of the HIF-1 pathway increases neoangiogenesis and promotes bone regeneration. (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 mice and controls are shown after staining with antibodies against pVHL and HIF-1. VEGF mRNA expression in bone-lining cells is usually shown by hybridization and immunostaining; CD31 immunostaining is also shown. Arrows show positive cells. (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. *, 0.05. (mice at day 38 after surgery. Quantitative measurements of BV and BV/TV are shown. Data represent mean SD. *, 0.05. (mice and controls at day 38 after surgery. Data shown represent mean SD. *, 0.05. VEGF Receptor Antibodies Inhibit Angiogenesis During DO. To determine the importance of VEGF signaling in the enhanced angiogenic response during bone regeneration in the mice, we administered VEGFR1 and VEGFR2 antibodies or nonimmune IgG i.p. every 3 days after surgery until day 17. CT angiography showed that mice given VEGF receptor antibodies had significantly decreased VV/TV, vessel number, and vessel surface with significantly increased vessel separation (Fig. 2 and mice requires VEGF. Open in a separate window Fig. 2. VEGFR is required for neoangiogenesis during DO. Eight-week-old Ned 19 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. ( 0.05, **, 0.01. Inactivation of HIF-1 Impairs Angiogenesis and Bone Regeneration. We next examined Ned 19 whether inhibiting HIF-1 would impair angiogenesis and bone healing. We developed a second mouse strain with a targeted deletion of HIF-1 in osteoblasts and subjected them to DO. CT angiography at day 17 showed decreased vascularity in the 0.05. Pharmacological Activation of the HIF-1 Pathway Stimulates Angiogenesis and Accelerates Bone Regeneration. A family of oxygen-sensitive prolyl hydroxylases (PHD1,2,3) hydroxylate HIFs under normoxia, which promotes their subsequent E-3 ligation and proteosomal destruction. To identify HIF activators, we tested several agents known to inhibit prolyl hydroxylases for their ability to activate a expression in primary mouse bone marrow mesenchymal stromal cells (MSCs). Treatment with DFO, and to a Ned 19 lesser extent, l-mim, increased HIF-1 nuclear accumulation and VEGF mRNA expression in MSCs maintained under normoxic conditions (Fig. 4 and and and SI Fig. 9). Continuous (14 days) exposure to DFO or l-mim was associated with detachment of the bone rudiments from the tissue culture plate, possibly because of the known effect of PHD inhibitors on collagen processing (17). However, exposure to DFO and, to a lesser degree, l-mim for a shorter period increased endothelial sprouting without obvious toxicity (Fig. 4as measured by alkaline phosphatase (ALP) staining (SI Fig. 10 and 0.05. ( 0.05. (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 M) or l-mim (300 M) for 24 h with rhVEGF (10 ng/ml) as positive control, followed by the detection of endothelial sprouting by immunostaining with anti-CD31.