Zoledronic acid inhibits the earliest stages of vasculogenesis: blockage of the transdifferentiation of monocytes into endothelial cells induced by pleiotrophin and mCSF

4651 We have discovered a novel mechanism leading to blood vessel formation that involves the transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (Chen et al, Blood 2005; Yeh et al, Br J Haematol 2006) and breast cancer (Zhang et al, JBC 1997) cells. The nitrogen-containing bisphosphonate zoledronic acid has been shown to inhibit bone loss and recent studies also demonstrate profound anti-angiogenic effects of this drug. The inhibitory effects of zoledronic acid on blood vessel formations remain unknown. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hoursand again after 5 days with or without zoledronic acid or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding zoledronic acid at either 1.5 or 0.15 uM. In order to examine whether zoledronic acid also reduces the expression of endothelial genes and proteins when monocytes are induced to transdifferentiate in the presence of these cytokines, we used RT-PCR and Western blot analysis on the endothelial cell markers Flk-1 Tie-2 and von Willebrand factor (vWF). The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at zoledronic acid concentrations of 15uM, 1.5uM and 0.15uM. We further examined the effects of zoledronic acid on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with zoledronic acid (1.5uM) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubation for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, zoledronic acid significantly reduced new macroscopic and microscopic vessel formation. These experiments define a previously unrecognized novel mechanism by which zoledronic acid may inhibit new blood vessel formation in cancer patients_preventing the transdifferentiation of monocytes into endothelial cells induced by PTN. They also suggest a specific new mechanism through which zoledronic acid has anti-angiogenic effects which provides further support for the potential anti-cancer effects of this agent.