Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair
Kai Hu, Bjorn R. Olsen
Kai Hu, Bjorn R. Olsen
Published January 5, 2016
Citation Information: J Clin Invest. 2016;126(2):509-526. https://doi.org/10.1172/JCI82585.
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Research Article Bone biology

Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair

Abstract

Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of Vegfa, we demonstrated that VEGF promoted macrophage recruitment and angiogenic responses in the inflammation phase, and optimal levels of VEGF were required for coupling of angiogenesis and osteogenesis in areas where repair occurs by intramembranous ossification. VEGF likely functions as a paracrine factor in this process because deletion of Vegfr2 in osteoblastic lineage cells enhanced osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte–derived VEGF stimulated recruitment of blood vessels and osteoclasts and promoted cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulated osteoclast formation in the final remodeling phase of the repair process. These findings provide a basis for clinical strategies to improve bone regeneration and treat defects in bone healing.

Authors

Kai Hu, Bjorn R. Olsen

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Figure 6

Osteoblast-derived VEGF stimulates macrophage-related angiogenesis and migration of BM cells.

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Osteoblast-derived VEGF stimulates macrophage-related angiogenesis and m...
(A) Increased anti-VEGF staining in hole (yellow rectangles) (17.6% ± 4.8%) and BM (blue rectangles) (20.3% ± 4.9%) of Vegfafl/fl compared with Vegfa CKO mice (4.2% ± 1.2% and 6.6% ± 2.0%) at PSD3; n= 5–6, P < 0.05. No significant differences outside hole (10.2% ± 2.9% vs. 9.4% ± 2.0%) (B) High anti-CD31 staining in hole (163/mm2 ± 5/mm2), adjacent BM (263/mm2 ± 18/mm2), and outside area (164/mm2 ± 26/mm2) of Vegfafl/fl compared with Vegfa CKO mice (58/mm2 ± 12/mm2, 65/mm2 ± 21/mm2, and 66/mm2 ± 17/mm2) at PSD3; n = 4–7, P < 0.05. (C) High density of F4/80+ macrophages in hole (321/mm2 ± 46/mm2) and BM (366/mm2 ± 69/mm2) of Vegfafl/fl compared with Vegfa CKO mice (82/mm2 ± 19/mm2 and 143/mm2 ± 26/mm2) at PSD3; n = 5–6, P < 0.01 (hole); P < 0.05 (BM); no difference outside (217/mm2 ± 38/mm2 vs. 166/mm2 ± 41/mm2). (D) High correlation between blood vessel and macrophage densities in hole. (E) Treating Vegfa CKO mice with 0.1 μg VEGF increases blood vessel density in hole (151/mm2 ± 7/mm2) and BM (115/mm2 ± 9/mm2) and macrophage density in hole (205/mm2 ± 34/mm2) at PSD5, compared with PBS (91/mm2 ± 12/mm2, 50/mm2 ± 6/mm2, and 87/mm2 ± 9/mm2); P < 0.05. Macrophage density in BM not affected (140/mm2 ± 25/mm2 vs. 94/mm2 ± 13/mm2). (F) In vitro wound closure; stippled lines indicate wound edges at time 0. Higher wound closure rate (μm/h) in BMSCs of Vegfafl/fl (8.1 ± 0.7 μm/h) than Vegfa CKO mice (5.1 ± 0.3 μm/h); n = 3, P < 0.05. (G) Transwell migration assay. After 14 hours, more migrated BMSCs from Vegfafl/fl than Vegfa CKO mice; n= 3, *P < 0.01. Scale bars: 50 μm (G), 100 μm (A, B, and E), 200 μm (C and F). CB, cortical bone; outside, area outside cortical bone. Spearman’s correlation coefficient test (D) and unpaired 2-tailed Student’s t test were used.