Abstract 1959: Proteomics Identifies Thymidine Phosphorylase as a Key Regulator of the Angiogenic Potential of Endothelial Progenitor Cells
Background: Endothelial progenitor cells (EPCs) and colony-forming units (CFUs) have been widely studied for their therapeutic potential and as markers of vascular sufferance. Their pathophysiological importance, however, is subject to ongoing controversy. Interestingly, recent studies point towards a role in the release of pro-angiogenic factors.
Methods and Results: For the identification of secreted factors, conditioned medium of EPC cultures and CFUs was analyzed by a MALDI-TOF-TOF mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. This extensive proteomic analysis enabled the identification of 543 non-redundant proteins and revealed matrix metalloproteinase-9 (MMP-9), interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF) and thymidine phosphorylase or platelet-derived endothelial cell growth factor (PD-ECGF) as major proangiogenic factors in EPCs. These results were verified by RT-PCR and multiplex cytokine assays and complemented by a comparison of the cellular proteome of cultured EPCs and CFUs using difference in-gel electrophoresis. Functional experiments revealed thymidine phosphorylase to be a key survival factor for EPCs: inhibition of thymidine phosphorylase by 5Br-6Am-U or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, while supplementation with deoxyribose phosphate, the enzymatic product of thymidine phosphorylase, abrogated this effect. Moreover, deoxyribose phosphate released by EPCs stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene silencing experiments in transmigration and wound repair assays. Finally, the effect of thymidine phosphorylase on angiogenesis was investigated by implantation of matrigel plugs in mice. In these in vivo experiments, deoxyribose phosphate strongly promoted neovascularization.
Conclusions: This study is the first to use state-of-the art proteomic techniques to comprehensively characterize CFUs and early EPCs. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role for thymidine phosphorylase activity in the survival and proangiogenic potential of EPCs.