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Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale.

Harris, S; Craze, M; Newton, J; Fisher, M; Shima, DT; Tozer, GM; Kanthou, C; (2012) Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale. PLOS One , 7 (5) , Article e35231. 10.1371/journal.pone.0035231. Green open access

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Abstract

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer 'tails' explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 'knock-in' vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues.

Type: Article
Title: Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale.
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0035231
Publisher version: http://dx.doi.org/10.1371/journal.pone.0035231
Language: English
Additional information: © 2012 Harris et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This work was supported by Cancer Research United Kingdom (Programme Grant Tumour Microcirculation, grant number C1276/A9993. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Keywords: Animals, Cell Line, Tumor, Exons, Humans, Male, Mice, Protein Isoforms, Rats, Reverse Transcriptase Polymerase Chain Reaction, Vascular Endothelial Growth Factor A
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/1364920
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