Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-A_xxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-A_xxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-A_xxxa and EGFP denotes VEGF-A_xxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

血管内皮生长因子 A ( VEGF-A ) 末端外显子的可变剪接生成两个具有不同功能的蛋白质家族。促血管生成 VEGF-A _xxx a 亚型是通过选择末端外显子的近端 3' 剪接位点产生的。使用替代的远端剪接位点会产生抗血管生成的 VEGF-A _xxxb 蛋白质。双色剪接敏感报告基因旨在模拟 VEGF-A 可变剪接，并用作进一步研究该可变剪接事件的分子工具。部分 VEGF-A 的末端外显子和前面的内含子被插入到一个小基因结构中，然后是两个荧光蛋白的编码序列。根据剪接过程中使用的外显子的哪个 3' 剪接位点表达不同的荧光蛋白（dsRED 表示 VEGF-A _xxx a，EGFP 表示 VEGF-A _xxxb). 荧光输出可用于跟踪体外和体内的剪接决定。在不同细胞系中成功报告基因验证并使用已知调节剂改变剪接后，使用 LOPAC 小分子库进行了筛选。使用荧光板阅读器检测 dsRED 和 EGFP 表达来测量报告剪接的改变。使用流式细胞术进一步验证感兴趣的化合物，并评估其对内源性VEGF-A 的影响mRNA 和蛋白质水平的选择性剪接。离体和体外血管生成测定用于证明化合物的抗血管生成作用。此外，在 Matrigel 体内模型中研究了抗血管生成活性。总之，我们已经确定了一组通过调节VEGF-A末端外显子剪接而具有抗血管生成活性的化合物。