ABSTRACT
BACKGROUND: The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. METHODS: The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. RESULTS: The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. CONCLUSION: Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.
Subject(s)
Neoplasms/prevention & control , Neovascularization, Pathologic/prevention & control , Vascular Endothelial Growth Factor A/physiology , Aged , Aged, 80 and over , Alternative Splicing , Animals , Apoptosis , Cell Line, Tumor , Cell Movement , Colon/chemistry , Colonic Neoplasms/chemistry , Endothelial Cells/physiology , Female , Humans , Male , Mice , Middle Aged , Neoplasms/pathology , Protein Isoforms , Tissue Distribution , Vascular Endothelial Growth Factor A/pharmacokineticsABSTRACT
Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.
