Chao Wan

Insulin Receptor Signaling in Osteoblasts Regulates Postnatal Bone Acquisition and Body Composition

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 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.

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.

Concise review : Multipotent mesenchymal stromal cells in blood

Peripheral blood‐derived multipotent mesenchymal stromal cells circulate in low number. They share, most although not all, of the surface markers with bone marrow‐derived multipotent mesenchymal stromal cells, possess diverse and complicated gene expression characteristics, and are capable of differentiating along and even beyond mesenchymal lineages. Although their origin and physio‐pathological function are still unclear, their presence in the adult peripheral blood might relate to some interesting but controversial subjects in the field of adult stem cell biology, such as systemic migration of bone marrow‐derived multipotent mesenchymal stromal cells and the existence of common hematopoietic‐mesenchymal precursors. In this review, current studies/knowledge about peripheral blood‐derived multipotent mesenchymal stromal cells is summarized, and the above‐mentioned topics are discussed.

Activation of the hypoxia-inducible factor-1alpha 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.

Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice

Aging or glucocorticoid excess decrease bone strength more than bone mass in humans and mice, but an explanation for this mismatch remains elusive. We report that aging in C57BL/6 mice was associated with an increase in adrenal production of glucocorticoids as well as bone expression of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1, the enzyme that activates glucocorticoids. Aging also decreased the volume of the bone vasculature and solute transport from the peripheral circulation to the lacunar‐canalicular system. The same changes were reproduced by pharmacologic hyperglucocorticoidism. Furthermore, mice in which osteoblasts and osteocytes were shielded from glucocorticoids via cell‐specific transgenic expression of 11β‐HSD type 2, the enzyme that inactivates glucocorticoids, were protected from the adverse effects of aging on osteoblast and osteocyte apoptosis, bone formation rate and microarchitecture, crystallinity, vasculature volume, interstitial fluid, and strength. In addition, glucocorticoids suppressed angiogenesis in fetal metatarsals and hypoxia inducible factor‐1α transcription and vascular endothelial growth factor production in osteoblasts and osteocytes. These results, together with the evidence that dehydration of bone decreases strength, reveal that endogenous glucocorticoids increase skeletal fragility in old age as a result of cell autonomous effects on osteoblasts and osteocytes leading to interconnected decrements in bone angiogenesis, vasculature volume, and osteocyte‐lacunar‐canalicular fluid.

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.

Hedgehog signaling in mature osteoblasts regulates bone formation and resorption by controlling PTHrP and RANKL expression.

Hedgehog (Hh) signaling is required for osteoblast differentiation from mesenchymal progenitors during endochondral bone formation. However, the role of Hh signaling in differentiated osteoblasts during adult bone homeostasis remains to be elucidated. We found that in the postnatal bone, Hh signaling activity was progressively reduced as osteoblasts mature. Upregulating Hh signaling selectively in mature osteoblasts led to increased bone formation and excessive bone resorption. As a consequence, these mutant mice showed severe osteopenia. Conversely, inhibition of Hh signaling in mature osteoblasts resulted in increased bone mass and protection from bone loss in older mice. Cellular and molecular studies showed that Hh signaling indirectly induced osteoclast differentiation by upregulating osteoblast expression of PTHrP, which promoted RANKL expression via PKA and its target transcription factor CREB. Our results demonstrate that Hh signaling in mature osteoblasts regulates both bone formation and resorption and that inhibition of Hh signaling reduces bone loss in aged mice.

Bioreactor Expansion of Human Adult Bone Marrow‐Derived Mesenchymal Stem Cells

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft‐versus‐host disease, leading to a speedy recovery of hematopoiesis. To meet this clinical demand, a fast MSC expansion method is required. In the present study, we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors, including stem cell factor and interleukin‐3 and ‐6. After 8 days of culture, total cell numbers, Stro‐1+CD44+CD34− MSCs, and CD34+CD44+Stro‐1− HSCs were increased 9‐, 29‐, and 8‐fold, respectively. Colony‐forming efficiency‐fibroblast per day of the bioreactor‐treated cells was 1.44‐fold higher than that of the cells without bioreactor treatment. The bioreactor‐expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation, including osteocalcin (osteogenesis), type II collagen (chondrogenesis), and C/EBP‐α (CCAAT/enhancer‐binding protein‐α) (adipogenesis), were not detected. Upon induction, the bioreactor‐expanded MSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs.

Mesenchymal Stem Cells Expressing Osteogenic and Angiogenic Factors Synergistically Enhance Bone Formation in a Mouse Model of Segmental Bone Defect

The potential of mesenchymal stem cells (MSC) in tissue regeneration is increasingly gaining attention. There is now accumulating evidence that MSC make an important contribution to postnatal vasculogenesis. During bone development and fracture healing, vascularization is observed before bone formation. The present study determined the potential of MSC, transduced ex vivo with a recombinant adeno-associated virus 6 (rAAV6) encoding bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) in a mouse model of segmental bone defect created in the tibiae of athymic nude mice. Mouse MSC that were mock-transduced or transduced with rAAV6-BMP2:VEGF were systemically transplanted following radiographic confirmation of the osteotomy. Effects of the therapy were determined by enzyme-linked immunosorbent assay measurements for BMP2 and VEGF, dual-energy X-ray absorptiometry (DXA) for bone density, three-dimensional microcomputed tomography (microCT) for bone and capillary architecture, and histomorphometry for bone remodeling. Results of these analyses indicated enhanced bone formation in the group that received BMP2+VEGF-expressing MSC compared to other groups. The therapeutic effects were accompanied by increased vascularity and osteoblastogenesis, indicating its potential for effective use while treating difficult nonunion bone defects in humans.