The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development
Ying Wang, … , Ernestina Schipani, Thomas L. Clemens
Ying Wang, … , Ernestina Schipani, Thomas L. Clemens
Published June 1, 2007
Citation Information: J Clin Invest. 2007;117(6):1616-1626. https://doi.org/10.1172/JCI31581.
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Category: Research Article

The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development

Abstract

Skeletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxia-inducible factor α (HIFα) pathway. Here we provide evidence that HIFα promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIFα in osteoblasts through selective deletion of the von Hippel–Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIFα pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone.

Authors

Ying Wang, Chao Wan, Lianfu Deng, Ximeng Liu, Xuemei Cao, Shawn R. Gilbert, Mary L. Bouxsein, Marie-Claude Faugere, Robert E. Guldberg, Louis C. Gerstenfeld, Volker H. Haase, Randall S. Johnson, Ernestina Schipani, Thomas L. Clemens

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Figure 7

Mice lacking Hif1a in osteoblasts have narrow, poorly vascularized long bones.

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Mice lacking Hif1a in osteoblasts have narrow, poorly vascularized long ...
(A) PCR analysis of Cre-mediated recombination in selected tissues from a ΔHif1a mouse. The recombined allele (Δflox) was present exclusively in bone tissue. (B) Representative histological sections of distal femurs from 6-week-old control and ΔHif1a mice after staining with antibodies against HIF-1α (left) or HIF-2α (right) as described in Methods. Sections were counterstained with hematoxylin. Red arrows indicate positive and black arrows negative staining in osteoblasts. Original magnification, ×400. (C) Representative images of femoral cross sections from control and ΔHif1a mice. Scale bars: 1.0 mm. (D) Representative μCT images of vasculature in Microfil-perfused femurs from 3-week-old ΔHif1a and control mice. Scale bar: 1.0 mm. (E and F) Confluent monolayers of Hif1a floxed primary osteoblasts were infected with either Ad-GFP or Ad-CreM1 (100 MOI). (E) Proteins in the cytoplasm and nucleus were extracted separately 48 hours after infection. Immunoblotting analysis was performed with antibodies against HIF-1α and HIF-2α. Immunoblots for TBP and α-tubulin were used as loading controls for nuclear and cytoplasmic proteins, respectively. (F) Total mRNA was extracted from confluent monolayers of osteoblasts 48 hours after infection. Hif1a, Hif2a, and Vegf mRNA expression was determined by quantitative real-time PCR. **P < 0.01.