Introduction: Vascular Endothelial Growth Factor (VEGF) is a proangiogenic cytokine that is expressed highly by many solid tumours often correlating with poor prognosis. VEGF has also been shown to interact with osteoclasts and their precursors in organ cultures to increase differentiation and survival and VEGF receptors have been found on osteoclasts in vitro. In this work we aimed to investigate the expression of VEGF and its receptors in bone metastases from primary breast tumours and further characterise its effects on osteoclasts. We performed immunolocalisation of VEGF in bone metastases and using VEGF and VEGF receptor-specific ligands we assessed their role in osteoclastogenesis in vitro.

Methods: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained with antibodies to VEGF and its receptors VEGFR1 and 2, and the macrophage marker CD68.

To investigate osteoclastogenesis in vitro Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured over a two-week period under stimulation by cytokines (RANKL, M-CSF, VEGF, PlGF, a specific ligand for VEGFR 1 and VEGF-D, a specific ligand for VEGFR 2). RAW 264.7 cells (a mouse monocyte/macrophage cell line able to differentiate into osteoclast-like cells) were cultured for seven days under stimulation by cytokines (RANKL, VEGF and M-CSF). Osteoclasts were identified by staining for Tartrate Resistant Acid Phophatase (TRAP) and numbers of multinucleated cells counted per treatment. Culture on ivory slices was performed to measure resorption activity of the osteoclasts.

Results: The immunohistochemistry demonstrated that breast cancer metastases express VEGF strongly and that the osteoclasts surrounding metastases express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

The PBMCs stimulated by VEGF and RANKL together differentiated into multinucleated TRAP positive cells in similar numbers (22±4.7) per field of view to the M-CSF and RANKL (27.3±7.2). Resorption of ivory was identified in these cultures. Stimulation with PlGF and RANKL resulted in increased osteoclastogenesis but VEGF-D with RANKL had little effect. Similar results were seen in triplicate experiments RAW 264.7 cells also differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL.

Discussion and Conclusions: VEGF is able to induce the differentiation of human and mouse osteoclast-like cells from monocyte precursors in the presence of RANKL and this seems to be mediated by VEGFR1. This may lead to an increase in bone resorption in physiological and pathological situations where there is an increase in VEGF, such as in tumours, embryogenesis and fracture repair. VEGF signalling could be a therapeutic target for osteoclast inhibtion in these situations.

Introduction and Aims: To investigate the expression of Vascular Endothelial Growth Factor (VEGF) and its receptors in bone metastases from primary breast tumors and further characterise its effects on osteoclasts in vitro.

Method and Results: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2, and the macrophage marker CD68. This demonstrated that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

To investigate osteoclastogenesis in vitro, Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured under stimulation by cytokines. Tartrate Resistant Acid Phophatase (TRAP) positive multinucleated cells were counted in duplicate per treatment and experiments repeated three times. VEGF and RANKL together induced differentiation of multinucleated TRAP-positive cells in similar numbers (22±4.7[SE]) per field of view to M-CSF and RANKL (27.3±7.2[SE]). Stimulation with PlGF (a specific ligand for VEGFR1) and RANKL induced osteoclastogenesis, but VEGF-D (a specific ligand for VEGFR2) with RANKL had little effect.

RAW 264.7 cells (mouse monocyte cell line) differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL. Culture under the same conditions on ivory disks was performed and resorption of ivory by osteoclasts from both PBMC and RAW cells was identified.

Conclusion: VEGF, the angiogenic cytokine, is expressed highly by many solid tumors often correlating with poor prognosis. We have shown that VEGF induces monocytes to differentiate into osteoclast-like cells in the presence of RANKL and this seems to be mediated by VEGFR1. VEGF may therefore play a role in physiological bone resorption and in pathological situations, such as tumor osteolysis and consequently VEGF signalling may be a therapeutic target for osteoclast inhibition.