Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF

Abstract

Emerging evidence suggests that angiogenic signalling pathways play important role in the patho-biology of chronic lymphocytic leukemia (CLL). Our goal was to investigate: (i) the spontaneous and hypoxia-induced production of pro-angiogenic factors, VEGF and Ang2, by Real-time PCR and ELISA, (ii) the degree of vascularization in CLL-infiltrated bone marrow (BM) compartment by CD34 immunohistochemical staining of microvessels and (iii) the direct angiogenic effect of CLL-derived VEGF and Ang2 by function-blocking experiments in Matrigel assays. The results demonstrated that CLL cells spontaneously express both VEGF and Ang2 and are able to secrete these factors in surrounding microenvironment. Full-length Ang2 mRNA and truncated form Ang2(443) were detectable. Moreover, CLL cells were shown to enhance secretion of both VEGF and Ang2 proteins when subjected to hypoxic condition. Furthermore, increased in vivo and in vitro angiogenesis was induced by CLL cells. Enhanced BM vascularity correlated with Ig-unmutated CLL subset and increased expression of Ang2. Then, we demonstrated that supernatants obtained from CLL cells significantly increase the HUVEC tube formation in Matrigel assays and that this enhanced angiogenic capacity is mediated by both CLL-derived VEGF and Ang2. Taken together, these results suggest that several simultaneous mechanisms may be involved in the CLL capacity to induce the disruption of pre-existing vessel structures to give rise to tumor neoangiogenesis. The preliminary studies in solid tumors, showing that the disruption of Ang2 function can inhibit tumor vessel density and growth, are encouraging and suggest the possibility of new future therapeutic options targeting CLL microenvironment.