To examine whether VEGF can compensate for the absence of functional M-CSF in op/op
mice in the support of osteoclast recruitment, we first injected either rhM-CSF, rhVEGF165, rhVEGF121, or rhPlGF-1 into 12-d-old op/op
mice. As shown in , a single 5-μg injection of any of these factors was sufficient for the osteoclast recruitment in the mutant mice, although the number of osteoclasts recruited by rhVEGFs or rhPlGF-1 was 60–70% of that by rhM-CSF. The antagonistic anti–c-Fms mAb, AFS98 27
, decreased osteoclast recruitment by rhM-CSF to ~25%, but not that by rhVEGFs or rhPlGF-1, confirming that c-Fms mediates the response of osteoclast precursor cells to M-CSF, but not the response to VEGFs or PlGF-1.
Capacity of rhM-CSF, rhVEGFs, and rhPlGF-1 to Recruit Osteoclasts in op/op Mice
As shown in A, osteoclasts were strongly stained with rabbit anti–mouse VEGFR-1 polyclonal antibody, whereas endothelial cells were weakly positive for VEGFR-1. In contrast, osteoclasts were not stained with AVAS12 anti–mouse VEGFR-2 mAb 28
, while endothelial cells were positively stained for VEGFR-2 ( B). Neither normal rabbit IgG ( C) nor rat IgG2a (data not shown) stained any cell types. rhM-CSF–induced osteoclasts in op/op
mice showed the same staining pattern as described above (data not shown). These results demonstrate that osteoclasts predominantly express VEGFR-1, in a manner similar to monocyte/macrophage lineage cells 2223
. VEGF121 does not bind neuropilin-1 21
. PlGF-1 binds VEGFR-1, but not VEGFR-2 or neuropilin-1 2122232425
. The results that both rhVEGF121 and rhPlGF-1 showed activities comparable to rhVEGF165 in the support of osteoclast recruitment () confirm that the response of osteoclast precursor cells to VEGF is mediated by VEGFR-1.
Figure 1 Immunohistochemical staining of femur sections for VEGFRs. Longitudinal sections of femurs of 3-wk-old +/? mice were stained with either anti–VEGFR-1 polyclonal antibody (A), AVAS12 anti–VEGFR-2 mAb (B), or rabbit IgG (C). Arrowheads indicate (more ...)
Next, we examined the capacity of VEGF and M-CSF to support the survival of mature osteoclasts by neutralizing VEGF endogenously produced in op/op
mice with injections of VEGFR-1/Fc chimeric protein. Consistent with our previous observations 1213
, osteoclast number reached a plateau at 3 d after a single rhM-CSF injection and was maintained up to day 7 ( and ). Consecutive injections of the chimeric protein at 12-h intervals during days 4–6 decreased osteoclasts to ~25%, whereas injections of human IgG1 did not affect osteoclast number (). In contrast, when rhM-CSF was injected together with VEGFR-1/Fc, osteoclast number increased to the levels observed in mice consecutively injected with rhM-CSF alone. These results indicate that survival of osteoclasts recruited after a single rhM-CSF injection was supported by endogenously produced VEGF in op/op
mice and that M-CSF can support the survival of mature osteoclasts without the help of VEGF.
Effect of Injections of VEGFR-1/Fc Chimeric Protein on the Survival of rhM-CSF–recruited Osteoclasts in op/op Mice
We also examined the bone resorption in the femurs of op/op
mice that had received either a single rhM-CSF injection only or consecutive injections of VEGFR-1/Fc and rhM-CSF in addition to the single rhM-CSF injection. Osteoclasts in the former group of mice are thought to perform their functions with the support of endogenous VEGF, whereas those in the latter rely on exogenous rhM-CSF. As reported previously 12
, resorption of a massive amount of bone trabeculae and replacement with bone marrow in femurs were apparent by 7 d after a single rhM-CSF injection ( and ). Bone resorption was also similarly observed in the latter group of mice ( C). These observations show that both M-CSF and VEGF can support the bone-resorbing function of osteoclasts.
Figure 2 Resorption of bone trabeculae in the femurs of op/op mice by osteoclasts with the support of endogenous VEGF or exogenous rhM-CSF. The treatment was started at 12 d of age, and the mice were killed at 19 d of age. Longitudinal sections of femurs were (more ...)
The above finding that VEGF is endogenously produced at levels sufficient for the survival of mature osteoclasts and expression of their functions prompted us to confirm that rhM-CSF can induce osteoclast recruitment without the help of endogenous VEGF. As shown in , twice the number of osteoclasts were recruited by multiple injections of rhM-CSF compared with a single injection. Concomitant injections of VEGFR-1/Fc with rhM-CSF did not affect osteoclast recruitment. These results are the first unequivocal demonstration of the capacity of M-CSF to support in vivo osteoclast differentiation.
Effect of VEGFR-1/Fc Injection on Osteoclast Recruitment by rhM-CSF in op/op Mice
It became clear that M-CSF supports osteoclast differentiation in cooperation with osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/RANKL 3132
. We examined whether rhVEGF165 can replace rhM-CSF in osteoclast generation in in vitro culture of nonadherent bone marrow cells. Consistent with previous observations 3132
, no TRAP-positive cells appeared in the presence of rhM-CSF or rhRANKL alone (data not shown). rhVEGF165 alone also failed to support the osteoclast differentiation ( A). A combination of rhVEGF165 and rhRANKL supported the generation of TRAP-positive cells ( B), although the cells were significantly smaller in size than those generated in the presence of rhM-CSF and rhRANKL ( C). Consequently, the osteoclasts supported by rhVEGF165 and rhRANKL formed smaller resorption lacunae than those supported by rhM-CSF and rhRANKL ( and ). These results demonstrate that VEGF can indeed support osteoclast differentiation in cooperation with ODF/OPGL/TRANCE/RANKL.
Figure 3 Ability of VEGF to support in vitro generation of osteoclasts. Nonadherent bone marrow cells were cultured in the presence of rhVEGF165 alone (A), rhVEGF165 and rhRANKL (B and D), or rhM-CSF and rhRANKL (C and E) in the wells of 96-well plates (A–C) (more ...)
Finally, we examined whether progressive correction of osteopetrosis with age accompanied by an increase of osteoclasts in op/op
is due to endogenously produced VEGF. As shown in A, a significantly larger number of small osteoclasts with 2–3 nuclei was observed in the femurs of 2-mo-old op/op
mice (28 ± 1 osteoclasts/section) compared with those of 2-wk-old mutants ( and ), even though the size of the femur sections of the older animals was ~1.6 times larger than that of younger ones. In addition, TRAP-positive mononuclear cells were frequently observed in the marrow space. Five consecutive injections of 100 μg goat anti-VEGF polyclonal antibody at 12-h intervals significantly decreased osteoclast number (4 ± 2 osteoclasts/section; B). Injections of goat IgG did not affect osteoclast number (data not shown). VEGFR-1/Fc injections according to the protocol applied to 2-wk-old mutant mice () failed to show any noticeable effect on osteoclast number (data not shown). A single injection of 5 μg rhVEGF165 induced further recruitment of osteoclasts (64 ± 5 osteoclasts/section; C), indicating that VEGF levels in the femurs of 2-mo-old op/op
mice are still insufficient to recruit osteoclasts at maximum level. These results demonstrate that VEGF is responsible for the spontaneous osteoclast recruitment in the absence of functional M-CSF in op/op
mice. Changes in osteoclast number with the age and difference in the amount of VEGFR-1/Fc required to neutralize endogenous VEGF activity in 2-wk- and 2-mo-old animals suggest higher levels of VEGF production in older mutant mice, although the possibility that sensitivity of osteoclast precursors to VEGF changes with age cannot be ruled out.
Figure 4 Dependence of osteoclasts in the femurs of 2-mo-old op/op mice on endogenously produced VEGF. The mice were killed 3 d after the onset of the treatment. Longitudinal sections of femurs were stained for TRAP activity and counterstained with hematoxylin. (more ...)
This study demonstrates that M-CSF and VEGF can play almost entirely overlapping roles in osteoclastic bone resorption. The presence of either of the cytokines was sufficient to support all the processes of osteoclastic bone resorption, i.e., the differentiation of osteoclasts and their survival and active bone resorption, representing a unique type of redundancy of cytokine signaling. However, osteoclasts generated in vitro with the support of rhVEGF165 and rhRANKL were significantly smaller in size and formed smaller resorption lacunae compared with those supported by rhM-CSF and rhRANKL. Osteoclasts observed in 2-mo-old op/op mice had only two to three nuclei. Nevertheless, our data indicated that progressive correction of osteopetrosis in op/op mice is due to endogenously produced VEGF.
It has been well established that VEGF is a key regulator of vasculogenesis 21
. Osteoclastic bone resorption and concomitant bone marrow formation are closely associated with active neovascularization 833
, and osteoblasts have been reported to produce VEGF 34
. Our results indicate that VEGF is produced in op/op
mice at levels sufficient for the survival and functioning of mature osteoclasts, but not for their recruitment at maximal levels. The finding that mice lacking a single VEGF allele die in utero with aberrant blood vessel formation in the yolk sac and embryo indicates that VEGF is produced at threshold levels for endothelial cell proliferation 3536
. Furthermore, mice expressing the VEGFR-1 lacking the tyrosine kinase domain 26
had no appreciable abnormality in osteoclastic bone resorption (M. Shibuya, The University of Tokyo, personal communication). Therefore, M-CSF seems to play a dominant role in osteoclastic bone resorption under physiological conditions.
Macrophages from mice with kinase-deficient VEGFR-1 exhibit a defect in their migratory response to VEGF 26
. The common feature of predominant expression of VEGFR-1 in monocytes and macrophages 212223
and in osteoclasts may provide further support for the view of shared origin of these cells. We found previously that multiple injections of rhM-CSF are required for macrophage recruitment in the femurs of op/op
. In the present study, we also failed to find any sign of macrophage recruitment in the femurs after a single injection of rhVEGFs or rhPlGF-1 (data not shown). These observations may suggest that macrophage lineage cells are less sensitive to M-CSF, VEGFs, and PlGF-1 compared with osteoclast precursors or more probably that macrophage precursors are more strictly dependent on the continuous presence of M-CSF.
The function of VEGFR-1 as a mediator of mitogenic response of endothelial cells to VEGF has yet to be clearly identified, although unequivocal evidence for such a role of VEGFR-2 has accumulated 21
. The phenotypes of the mice with VEGFR-1 deficiency 37
and those expressing kinase-deficient VEGFR-1 29
strongly suggest the role of VEGFR-1 in the negative regulation of endothelial growth in embryonic angiogenesis. Therefore, it is of interest to compare the VEGFR-1 signaling in osteoclasts and their precursor cells with that in endothelial cells.