A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library
A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library
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DOI：
10.1074/jbc.m308681200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Alakhov, V
中科院分区：
文献类型：
--
作者：
El-Mousawi, M;Tchistiakova, L;Alakhov, V
已知血管内皮生长因子（VEGF）在肿瘤血管生成和转移形成中起主要作用，其通过与两种酪氨酸激酶受体VEGFRI（Flt-1）和VEGFRII（KDR）的相互作用介导。已知抑制肿瘤组织中的VEGF依赖性事件可增强细胞凋亡并抑制肿瘤生长。一种新的肽，SP5.2，它选择性地结合Flt-1和抑制范围广泛的VEGF介导的事件，被确定使用噬菌体展示文库筛选。荧光素标记的SP5.2特异性结合VEGF刺激的原代人脑内皮细胞（HCECs），而非刺激的HCECs，以及人神经母细胞瘤细胞（ShyY）没有显示出与肽的任何相互作用。SP5.2抑制重组人VEGF（165）诱导的培养的原代人脐静脉内皮细胞增殖，IC 50为5 μ M。SP5.2还显示拮抗VEGF和PLGF诱导的HCECs增殖，但不拮抗碱性成纤维细胞生长因子诱导的HCECs增殖。与“乱序”肽相反，还发现SP5.2选择性地抑制VEGF刺激的HCEC迁移。使用毛细血管样管形成试验对SP5.2的抗血管生成活性进行的体外分析表明，在10 μ M SP5.2存在下，生长在MatrigelTM上的HCEC的VEGF诱导的血管生成被完全抑制。进一步的研究表明，SP5.2阻止VEGF诱导的HCECs单层渗透性增加。为了探索SP5.2是否可以用作靶向剂，产生了SP5.2与报告蛋白（过氧化物酶和β-半乳糖苷酶）的化学和重组缀合物。与原始合成的SP5.2相比，所得产物显示出对重组Flt-1的结合亲和力的显著增加（SP5.2-β-gal为200倍，SP5.2-过氧化物酶为400倍），这表明基于SP5.2结构可以潜在地开发具有纳摩尔范围内的治疗活性的缀合物。
Vascular endothelial growth factor ( VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) with an IC50 of 5 muM. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF- stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF- induced angiogenesis of HCECs grown on Matrigel(TM) was completely inhibited in the presence of 10 muM SP5.2. Further studies demonstrated that SP5.2 prevented VEGF- induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins ( peroxidase and beta-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-beta-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure.