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 hypoxiainducible 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. IntroductionThe development of the mammalian skeleton takes place in distinct phases involving the initial migration of cells to the site of future bone, condensation of mesenchymal cells, and finally the differentiation of progenitors into chondrocytes and osteoblasts. During intramembranous bone formation, which gives rise to the flat bones of the skull, mesenchymal cells differentiate directly into bone-forming osteoblasts. By contrast, in endochondral bone formation, bones are formed through a 2-stage mechanism that begins with the formation of a chondrocyte anlage, onto which osteoblasts then differentiate and deposit bone. Endochondral bone formation occurs in close spatial and temporal association and proximity to capillary invasion, suggesting that angiogenesis and osteogenesis are coupled.The initial signals for blood vessel invasion into bone are unknown, but tissue hypoxia is believed to be critical for commencement of the angiogenic cascade (1). Hypoxia triggers the changes in oxygen-regulated gene expression via the activation of the Per/Arnt/Sim (PAS)

The hypoxia-inducible factor-1alpha (HIF-1alpha) pathway is the central regulator of adaptive responses to low oxygen availability and is required for normal skeletal development. Here, we demonstrate that the HIF-1alpha pathway is activated during bone repair and can be manipulated genetically and pharmacologically to improve skeletal healing. Mice lacking pVHL in osteoblasts with constitutive HIF-1alpha activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1alpha in osteoblasts had impaired angiogenesis and bone healing. The increased vascularity and bone regeneration in the pVHL mutants were VEGF dependent and eliminated by concomitant administration of VEGF receptor antibodies. Small-molecule inhibitors of HIF prolyl hydroxylation stabilized HIF/VEGF production and increased angiogenesis in vitro. One of these molecules (DFO) administered in vivo into the distraction gap increased angiogenesis and markedly improved bone regeneration. These results identify the HIF-1alpha pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.

Skeletal trauma and impaired skeletal healing is commonly associated with diminished vascularity. Hypoxia inducible factor alpha (HIF-1) is a key transcription factor responsible for activating angiogenic factors during development and tissue repair. Small molecule inhibitors of the prolyl hydroxylase enzyme (PHD), the key enzyme responsible for degrading HIF-1, have been shown to activate HIF-1, and are effective in inducing angiogenesis. Here we examined the effects of several commercially available PHD inhibitors on bone marrow mesenchymal stromal cells (MSCs) in vitro and in a stabilized fracture model in vivo. Three PHD inhibitors [Desferrioxamine (DFO), L-mimosine (L-mim), and Dimethyloxalylglycine (DMOG)] effectively activated a HIF-1 target reporter, induced expression of vascular endothelial growth factor (VEGF) mRNA in vitro, and increased capillary sprouting in a functional angiogenesis assay. DFO and DMOG were applied by direct injection at the fracture site in a stabilized murine femur fracture model. PHD inhibition increased the vascularity at 14 days and increased callus size as assessed by microCT at 28 days. These results suggest that HIF activation is a viable approach to increase vascularity and bone formation following skeletal trauma.

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to recruit new blood vessels into bone. Reciprocal signals, likely emanating from the incoming vascular endothelium, stimulate mesenchymal cell specification through additional interactions with cells within the vascular stem cell niche. The hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process. We demonstrated that overexpression of HIF-1 in mature osteoblasts through disruption of the von Hippel-Lindau protein profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell nonautonomous mechanisms involving the action of vascular endothelial growth factor (VEGF) on the endothelial cells. The same occurred in the model of injury-mediated bone regeneration (distraction osteogenesis). Surprisingly, manipulation of HIF-1 does not influence angiogenesis of the skull bones, where earlier activation of HIF-1 in the condensing mesenchyme upregulates osterix during cranial bone formation.

ABSTRACT:Introduction: Distraction osteogenesis (DO) is characterized by the induction of highly vascularized new bone formation through an intramembranous process largely devoid of the formation of cartilage. Materials and Methods: To test the hypothesis that DO is strictly dependent on vascualrization, we inhibited vascular endothelial growth factor (VEGF) activity by antibody blockade of both receptors VEGFR1 (Flt-1) and VEGFR2 (Flk-1) or only VEGFR2 (Flk-1) in a previously developed murine tibia DO model. During normal DO, VEGFR1 (Flt-1), VEGFR2 (Flk-1), VEGFR3 (Flt4) and all four VEGF ligand (A, B, C, and D) mRNAs are induced. Results: The expression of mRNA for the receptors generally paralleled those of the ligands during the period of active distraction. Bone formation, as assessed by CT, showed a significant decrease with the double antibody treatment and a smaller decrease with single antibody treatment. Vessel volume, number, and connectivity showed progressive and significant inhibition in all of these of parameters between the single and double antibody blockade. Molecular analysis showed significant inhibition in skeletal cell development with the single and double antibody blockade of both VEGFR1 and 2. Interestingly, the single antibody treatment led to selective early development of chondrogenesis, whereas the double antibody treatment led to a failure of both osteogenesis and chondrogenesis. Conclusions: Both VEGFR1 and VEGFR2 are functionally essential in blood vessel and bone formation during DO and are needed to promote osteogenic over chondrogenic lineage progression.

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Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprised of self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e. RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration.

Osteogenesis and angiogenesis are tightly coupled during bone formation and repair. Blood vessels not only carry oxygen and nutrients to the developing bone, but also play an active role in bone formation and remodeling by mediating the interaction between osteoblasts, osteocytes, osteoclasts, and vascular cells at a variety of levels. Tissue hypoxia is believed to be a major stimulus for angiogenesis by activating hypoxia-inducible factor alpha (HIFalpha) pathway, which is a central regulator of hypoxia adaptation in vertebrates. HIFalpha remains inactive under normoxic conditions through pVHL-mediated polyubiquitination and proteasomal degradation. Activation of the HIFalpha pathway by hypoxia triggers hypoxia-responsive gene expression, such as vascular endothelial growth factor (Vegf), which plays a critical role in angiogenesis, endochondral bone formation, and bone repair following fracture. Recent work from our laboratory has shown that osteoblasts use the HIFalpha pathway to sense reduced oxygen tension and transmit signals that impinge on angiogenic and osteogenic gene programs during bone formation. Using a genetic approach, we have demonstrated that overexpression of HIFalpha in mouse osteoblasts through disruption of Vhl results in profound increases in angiogenesis and osteogenesis, which appear to be mediated by cell nonautonomous mechanisms involving VEGF. These studies suggest that VEGF exerts many of its actions on bone indirectly by stimulation of angiogenesis. Whether or to what extent this angiogenic factor functions independent of endothelial cells remains to be determined.