Shawn Gilbert

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

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 alpha (HIF alpha) pathway. Here we provide evidence that HIF alpha promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIF alpha 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 HIF alpha 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.

Prolyl hydroxylase inhibitors increase neoangiogenesis and callus formation following femur fracture in mice

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.

Bone Formation During Distraction Osteogenesis Is Dependent on Both VEGFR1 and VEGFR2 Signaling

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.

Role of HIF-1α in skeletal development

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.

Oxygen Sensing and Osteogenesis

Abstract:  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 α (HIFα) pathway, which is a central regulator of hypoxia adaptation in vertebrates. HIFα remains inactive under normoxic conditions through pVHL‐mediated polyubiquitination and proteasomal degradation. Activation of the HIFα 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 HIFα 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 HIFα 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.

Activation of the hypoxia-inducible factor-1α pathway accelerates bone regeneration

The hypoxia-inducible factor-1α (HIF-1α) 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-1α 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-1α activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1α 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-1α pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.

Biphasic peptide amphiphile nanomatrix embedded with hydroxyapatite nanoparticles for stimulated osteoinductive response.

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 comprising 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.

Hypoxia and HIF-1α expression in the epiphyseal cartilage following ischemic injury to the immature femoral head

UNLABELLED HIF-1alpha has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1alpha activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1alpha activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process. MATERIALS AND METHODS Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1alpha western blot, RT-qPCR of HIF-1alpha, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed. RESULTS Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1alpha protein level and HIF-1alpha expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p<0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks. CONCLUSIONS Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1alpha activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.

Increasing Vascularity to Improve Healing of a Segmental Defect of the Rat Femur

Background: Segmental bone loss remains a challenging clinical problem.A frequent mitigating factor is inadequate blood supply. Small molecules that activate the hypoxia-inducible factor pathway can be used to stimulate angiogenesis. We investigated an approach to promote healing using angiogenic and osteogenic compounds in combination with a biodegradable, weightbearing scaffold. Methods: Adult rats underwent removal of a 5-mm segment of femur stabilized by a cylindrical biodegradable implant and intramedullary fixation. Treatment groups included 1) saline (negative control); 2) desferrioxamine (DFO, a hypoxia-inducible factor activator; 3) low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 μg); 4) DFO and low-dose rhBMP-2 (5 μg); or 5) rh-BMP-2 (10 μg). Angiography was used to evaluate vascularity. Bone healing was assessed by radiographs, microcomputed tomography, histology, and biomechanical testing. Results: Increased vascularity was seen at 6 weeks in the DFO treatment group. There appeared to be increased bone bridging as assessed by radiographic scores and microcomputed tomography in the BMP groups, although the quantification of bone volume did not show statistically significant differences. Biomechanical testing revealed improved stiffness in the treatment groups. Conclusions: DFO improved angiogenesis and stiffness of bone healing in segmental defects. BMP improved radiographic scores and stiffness. Use of angiogenic compounds in segmental bone loss is promising. Clinical Relevance: Activation of the hypoxia-inducible factor pathway may prove useful for bone defects, particularly where impaired blood supply exists.The low-cost approach could be useful in segmental bone defects clinically.

Activation of the hypoxia-inducible factor-1 pathway accelerates bone regeneration

The hypoxia-inducible factor-1α (HIF-1α) 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-1α 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-1α activation in osteoblasts had markedly increased vascularity and produced more bone in response to distraction osteogenesis, whereas mice lacking HIF-1α 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-1α pathway as a critical mediator of neoangiogenesis required for skeletal regeneration and suggest the application of HIF activators as therapies to improve bone healing.