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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Vascular Endothelial Growth Factor-A (VEGF-A) splicing generates angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms.
• VEGFxxxb isoforms have been reported in humans, rats, and mice, suggesting a role in regulating angiogenesis.

Purpose of the Study:

• To investigate the expression of VEGFxxxb isoforms in mouse fibrosarcoma cell lines and normal tissues.
• To identify potential artefacts in the detection of VEGFxxxb isoforms using current molecular methods.

Main Methods:

• RT-PCR was used to analyze VEGF isoform expression in mouse fibrosarcoma cell lines and normal mouse and human tissues.
• Fibrosarcoma cell lines with defined VEGF isoform expression (VEGF120, 164, 188) were used as controls, including those engineered to preclude VEGFxxxb splicing.
• Primer design and PCR conditions were critically evaluated to identify potential amplification artefacts.

Main Results:

• Primers designed for simultaneous amplification of VEGFxxx and VEGFxxxb isoforms only detected VEGFxxx variants in both species.
• Artefactual amplification of VEGFxxxb-like products was observed in mouse samples and in fibrosarcoma cells/tumors where true VEGFxxxb splicing is impossible.
• These artefacts were explained by primer annealing to VEGFxxx transcripts and amplification of exon 8b primer 'tails', particularly with specific reverse primer designs.

Conclusions:

• Current methods for detecting VEGFxxxb isoforms are prone to artefacts, leading to potential misinterpretation of expression data.
• Anti-angiogenic VEGFxxxb isoforms are not commonly expressed in mouse or human tissues.
• Further refinement of detection methodologies is crucial for accurate assessment of VEGF isoform expression and its biological significance.