Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. 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 hours and again after 5 days with or without ATO 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 either the IC50 (5x10⁻⁶M) dose or low (5x10⁻⁷M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10⁻¹²M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10⁻⁶M) and lower (5x10⁻⁷M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10⁻¹²M). We further examined the effects of ATO and bortezomib 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 ATO (5x10⁻⁶M) or bortezomib (3x10⁻¹¹M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator 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, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.