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.
Highlights
Vascular endothelial growth factor-A is a key regulator of angiogenesis, a process fundamental to the growth and metastasis of tumours [1,2]
To investigate expression of VEGF isoforms in our mouse fibrosarcoma cell lines, their corresponding solid tumour extracts and normal mouse and human cells/ tissues, we adopted a PCR approach using primers designed to amplify VEGFxxx and VEGFxxxb families simultaneously, consistent with the primer design strategy previously reported for the identification of human VEGF165b [26]. cDNA extracts were prepared from mouse fibrosarcoma cells and tumours as well as mouse heart, lung, liver and kidney organs from two different strains of mice (CBA and severe combined immunodeficiency (SCID)), to accommodate possible strain differences in VEGF isoform expression
Products corresponding to VEGF121, VEGF165 and VEGF189 were detected in our panel of human tissue cDNAs (Fig. 4B & C), again no VEGFxxxb isoforms were detected, consistent with our previous mouse and human data (Fig. 3A–C)
Summary
Vascular endothelial growth factor-A (hereafter referred to as VEGF) is a key regulator of angiogenesis, a process fundamental to the growth and metastasis of tumours [1,2]. Many current vascular based anti-cancer therapies are targeted to inhibiting VEGF [7,8,9,10]. Both the human and murine VEGF genes are comprised of eight exons separated by seven introns [11,12]. All VEGF isoforms contain exons 1 to 5 and 8 but differ in composition of remaining exons 6 to 7 (Fig. 1) and binding to extracellular matrix and neuropilin co-receptors
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
