Proteomics Identifies Thymidine Phosphorylase As a Key Regulator of the Angiogenic Potential of Colony-Forming Units and Endothelial Progenitor Cell Cultures
Proteomics Identifies Thymidine Phosphorylase As a Key Regulator of the Angiogenic Potential of Colony-Forming Units and Endothelial Progenitor Cell Cultures
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DOI：
10.1161/circresaha.108.182261
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发表时间：
2009-01-02
影响因子：
20.1
通讯作者：
Mayr, Manuel
中科院分区：
文献类型：
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作者：
Pula, Giordano;Mayr, Ursula;Mayr, Manuel
内皮祖细胞（EPC）培养物和集落形成单位（CFU）已被广泛研究其治疗和诊断潜力。最近的数据表明EPCs在释放促血管生成因子中的作用。为了鉴定EPC分泌的因子，使用基质辅助激光解吸/电离串联飞行时间质谱仪结合通过纳流液相色谱法的离线肽分离来分析来自EPC培养物和CFU的条件培养基。结果通过RT-PCR和多重细胞因子测定进行验证，并通过使用差异凝胶电泳对培养的EPCs和CFU进行细胞蛋白质组学分析进行补充。这种广泛的蛋白质组学分析揭示了促血管生成因子胸苷磷酸化酶（TP）的存在。功能实验表明，5-溴-6-氨基-尿嘧啶或基因沉默抑制TP导致基础和氧化应激诱导的细胞凋亡显着增加，而补充2-脱氧-D-核糖-1-磷酸（dRP），TP的酶促产物，废除了这种效果。此外，在内皮祖细胞培养物中产生的dRP以旁分泌的方式刺激内皮细胞迁移，如在迁移和伤口修复试验中的基因沉默实验所示。RGD肽和整合素α v β 3的抑制性抗体减弱了EPC培养物条件培养基对内皮细胞迁移的影响。最后，通过在小鼠中植入Matrigel塞来研究TP对血管生成的影响。在这些体内实验中，dRP强烈促进新血管形成。我们的数据支持EPCs以旁分泌方式发挥其促血管生成活性的概念，并证明TP活性在其存活和促血管生成潜力中起关键作用。(Circ Res. 2009; 104：32-40.）
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 alpha v beta 3 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. (Circ Res. 2009; 104: 32-40.)