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)

Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of exogenous undercarboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion to control glucose homeostasis.

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.

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Synopsis Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mitochondrial DNA (mtDNA) sequence variation contributes to disease susceptibility. In this study we present a novel animal model of mtDNA polymorphisms, the mitochondrial nuclear exchange mouse (MNX), in which the mtDNA from C3H/HeN mouse has been inserted onto the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harboring the C57/BL6J mtDNA generate more reactive oxygen species (ROS) and have a higher mitochondrial membrane potential relative to those having the C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the “mitochondrial paradigm” for the development of disease susceptibility, and show that the mtDNA modulates, cellular bioenergetics, mitochondrial reactive oxygen species generation and susceptibility to cardiac stress.