Journal
ONCOGENESIS
Volume 10, Issue 5, Pages -Publisher
SPRINGERNATURE
DOI: 10.1038/s41389-021-00323-0
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Funding
- BBSRC [BB/J007293/2]
- British Heart Foundation [PG/15/53/31371]
- Diabetes UK [17/0005668]
- BBSRC [BB/J007293/2] Funding Source: UKRI
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By using a bichromatic splicing-sensitive reporter to mimic VEGF-A alternative splicing, a set of compounds has been identified that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing. The activity was validated in different cell lines and screened using a library of small molecules.
Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-A(xxx)a 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(xxx)b 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(xxx)a and EGFP denotes VEGF-A(xxx)b). 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.
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