- Paper Report
- Open Access
VEGF and bone resorption by osteoclasts
- Ewa Paleolog
© Current Science Ltd 1999
- Published: 20 August 1999
- Bone resorption
- macrophage colony-stimulating factor
Osteopetrotic (op/op) mice have a deficiency for osteoclasts that is caused by a mutation in the gene for macrophage colony-stimulating factor (M-CSF). A single injection of M-CSF is sufficient to induce osteoclast recruitment and survival in op/op mice. The action of M-CSF is mediated by the receptor c-Fms, a member of the platelet-derived growth factor (PDGF) receptor family. Vascular endothelial growth factor (VEGF), a potent stimulator of blood vessel formation, also acts through members of the PDGF receptor family, including Flt-1, Flk-1 and neuropilin. In contrast to endothelial cells, which express all of the VEGF receptor subtypes, cells of the monocyte/macrophage lineage express the Flt-1 receptor only. Because of the close lineage relationship between macrophages and osteoclasts, VEGF could compensate for the deficiency of M-CSF in op/op mice in terms of osteoclastic bone resorption.To investigate whether VEGF can substitute for M-CSF in stimulating osteoclast recruitment in op/op mice.
A single injection of either M-CSF or VEGF was sufficient to induce osteoclast recruitment in op/op mice. Osteoclasts were strongly stained with anti-Flt-1 antibody only, whereas endothelial cells were positive for both Flt-1 and Flk-1 receptors. Neutralisation of endogenously produced VEGF in op/op mice with injections of Flt-1/Fc chimeric protein decreased the number of M-CSF-recruited osteoclasts to ~25%. In contrast, when M-CSF was injected together with Flt-1/Fc chimeric protein, osteoclast number increased to the levels observed in mice consecutively injected with M-CSF alone. Resorption of bone trabeculae and replacement with bone marrow in femurs were observed in the femurs of op/op mice that had received either a single M-CSF injection only or consecutive injections of Flt-1/Fc and M-CSF in addition to the single M-CSF injection. A combination of VEGF and ODF supported the generation of TRAP-positive cells, and formed resorption lacunae, although lacunae were smaller than those supported by a combination of M-CSF and ODF. Progressive correction of osteopetrosis with age was accompanied by an increase in osteoclast numbers in op/op mice, due to endogenously produced VEGF. Injections of anti-VEGF polyclonal antibody significantly decreased osteoclast numbers.
Mice of the op/op genotype were injected with recombinant cytokines (VEGF, M-CSF), then killed; the femurs removed and decalcified. Longitudinal sections of whole femurs were stained for tartrate-resistant acid phosphatase (TRAP) activity and counter-stained with hematoxylin. TRAP-positive cells with two or more nuclei were counted as osteoclasts. To measure VEGF receptor expression, femurs of op/op mice were fixed, paraffin-embedded and stained with anti-Flt-1 or anti-Flk-1 antibodies. In another series of experiments, op/op mice were pretreated with a single injection of M-CSF, followed four days later by injection of Flt-1/Fc chimeric protein and/or M-CSF. Finally, osteoclasts were generated in vitro from bone marrow cells by culturing them with osteoclast differentiation factor (ODF) in the absence or presence of VEGF or M-CSF.