Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro

Numerous studies have demonstrated the critical role of angiogenesis for su ccessful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-speci fic cytokine, has been shown to be mitogenic and chemotactic for endothelia l cells in vitro and angiogenic in many in vivo models. Based on previous w ork that (1) VEGF is up-regulated during membranous fracture healing, (2) t he fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isol ated osteoblasts in vitro stimulated by other fracture cytokines, the hypot hesis that hypoxia may regulate the expression of VEGF by osteoblasts was f ormulated. This hypothesis was tested in a series of in vitro studies in wh ich VEGF mRNA and protein expression was assessed after exposure of osteobl ast-like cells to hypoxic stimuli. Tn addition, the effects of a hypoxic mi croenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate ex pression of VEGF in osteoblast-like cells in a dose-dependent fashion. In a ddition, it is demonstrated that hypoxia results in decreased cellular prol iferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Ta ken together, these data suggest that osteoblasts, through the expression o f VEGF, may be in part responsible for angiogenesis and the resultant incre ased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decrease d oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.