Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.
Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.
Pula
Giordano
G
Cardiovascular Division, King's College London School of Medicine, King's College London, United Kingdom.
Mayr
Ursula
U
Evans
Colin
C
Prokopi
Marianna
M
Vara
Dina S
DS
Yin
Xiaoke
X
Astroulakis
Zoe
Z
Xiao
Qingzhong
Q
Hill
Jonathan
J
Xu
Qingbo
Q
Mayr
Manuel
M
D000328:Adult; D042501:Angiogenic Proteins / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001976:Bromouracil / Q000031:analogs & derivatives / Q000494:pharmacology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000032:analysis / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism*; D003855:Deoxyribose / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000166:cytology*; D055018:Hemangioblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3 / Q000096:biosynthesis; D008298:Maleates / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000502:physiology*; D018384:Oxidative Stress; D040901:Proteomics; D034741:RNA, Small Interfering / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014945:Wound Healing
D042501:Angiogenic Proteins; D017077:Culture Media, Conditioned; D016207:Cytokines; D039661:Integrin beta3; D008298:Maleates; D034741:RNA, Small Interfering; D001976:Bromouracil; D003855:Deoxyribose; C039893:6-amino-5-bromouracil; D013939:Thymidine Phosphorylase; C014476:diethyl maleate