Abstract
Vascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA121, VEGFA165 and VEGFA189. A second set of anti-angiogenic isoforms termed VEGFAxxxb that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFAxxxb isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFAxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7–8 junction in 10 tissues did not identify any VEGFAxxxb transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFAxxxb isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA145 and VEGFA148 isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.
Highlights
The pivotal role of Vascular endothelial growth factor (VEGFA) in angiogenesis has been recognised for many years[1] and it is one of the most extensively studied growth factors
We reasoned that if the putative VEGFAxxxb transcripts exist, they would be represented within the wealth of RNA sequencing data publicly available[27]
Another putative novel upstream site is shared by the remaining 8 Expressed Sequence Tags (ESTs). These are all from the same hepatocellular carcinoma library (LIBEST_006533)[30] and may represent a tumour-related transcript not represented in the cognate library from corresponding non-cancerous liver tissue (LIBEST_005601)
Summary
The pivotal role of VEGFA in angiogenesis has been recognised for many years[1] and it is one of the most extensively studied growth factors. The VEGFA splice isoforms (Fig. 1a) are named according to the number of amino acids they contain (e.g. VEGFA121 or VEGFA165). It has been suggested that these isoforms are implicated in human diseases such as systemic sclerosis[10], peripheral artery disease[9] and maternal gestational diabetes[12]. These findings raise the alarming possibility that many studies of VEGFA need to be re-evaluated due to potential overlap between detection of VEGFAxxx or VEGFAxxxb isoforms[13], with potential implications for VEGFA-based therapies[14]. Antibodies raised against the VEGFAxxxb c-terminus have detected signals in tissues and blood[9, 10, 12, 17,18,19] but this could be due to translational read-through of exon 8a-containing transcripts[20, 21] or cross-reactivity
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