Angiogenesis and Osteoclastogenesis Are Mutually Stimulated in Myeloma: A Role for VEGF and Osteopontin.

Angiogenesis and osteoclastogenesis occur in the bone marrow as myeloma (MM) progresses. Suppression of osteoclastogenesis by bisphosphonates reduced tumor burden as well as vascular density in the BM in myeloma animal models(Croucher et al. 2003). These evidences suggest a link between angiogenesis, osteoclastogenesis and MM tumor expansion which orchestrates a network of co-operative cellular interactions. We have previously demonstrated that a cell-cell interaction between osteoclasts (OCs) and MM cells potently enhanced MM cell growth and survival as well as angiogenesis. Such angiogenic effects were almost completely abrogated by antibodies against VEGF and osteopontin (OPN) in combination, suggesting VEGF and OPN as predominant angiogenic factors produced in MM bone marrow microenvironment. Angiogenesis is a multi-step process including migration, proliferation and sprouting in response to angiogenic stimuli. The impact of these factors on these angiogenic processes as well as on osteoclastogenesis by vascular cells is largely unknown. We therefore investigated roles for OPN and VEGF in combination in different steps in angiogenesis and in induction of osteoclastogenic activity by vascular endothelial cells. Conditioned media (CM) from co-cultures of peripheral blood mononuclear cell-derived OCs and RPMI8226 cells at 10 % enhanced survival of HUVEC under factor-deprived conditions 3-fold compared to control culture media, and transmigration of HUVEC 1.5-fold. Antibodies against VEGF and OPN each alone partially and both in combination mostly abrogated these effects of the CM. Consistently, VEGF and OPN co-operatively promoted survival and transmigration of HUVEC as well as angiogenesis in vitro. In order to further clarify a role for VEGF and OPN in a link between angiogenesis and osteoclastogenesis, we next examined osteoclastogenic activity produced by HUVEC upon VEGF and OPN stimulation using rabbit bone cell cultures on dentine slices. CM were collected from HUVEC cultured on either OPN- or type 1 collagen-coated wells in the presence or absence of VEGF. CM from OPN-coated wells with VEGF enhanced OC formation most potently(2.8-fold) compared to control culture media, while CM from OPN-coated wells without VEGF, type 1 collagen-coated wells with VEGF and type 1 collagen-coated wells without VEGF enhanced 2-, 1.8- and 1.2-fold, respectively, indicating co-operative actions of VEGF and OPN. Furthermore, such VEGF and OPN treatment was found to induce osteoclastogenic IL-8 by HUVEC most apparently among 125 different cytokines determined by cytokine antibody arrays. These results suggest that VEGF and OPN derived from MM cells and OCs cause angiogenesis as well as osteoclastogenesis at least in part through the enhancement of migration and survival and the induction of osteoclastogenic activity of vascular endothelial cells, thereby mutually stimulating angiogenesis and osteoclastogenesis. Therefore, VEGF and OPN may be a therapeutic target against MM bone marrow environment to ameliorate bone destruction along with MM expansion.