Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and
vascular endothelial growth factor (
VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro.
VEGF(165) has been shown to be the predominant angiogenic
isoform expressed in NB cell lines and tumours. In this study, we investigated the anti-angiogenic
isoform of
VEGF-A, generated from distal splice site selection in the terminal exon of
VEGF (
VEGF(165)b) and shown to be down-regulated in epithelial
malignancies. The expression of both the pro- (
VEGF(xxx)) and the anti-angiogenic (
VEGF(xxx)b)
isoforms was compared in a range of NB and
ganglioneuroma (GN) tumours. Whereas
VEGF(xxx)b and
VEGF(xxx) were both expressed in GN, specific up-regulation of the
VEGF(xxx)
isoforms was seen in NB at
RNA and
protein levels. Highly tumourigenic NB cell lines also showed up-regulation of the angiogenic
isoforms relative to
VEGF(xxx)b compared to less tumourigenic cell lines, and the
isoforms were differentially secreted. These results indicate that
VEGF(165) is up-regulated in NB and that there is a difference in the balance of
isoform expression from anti-angiogenic
VEGF(165)b to angiogenic
VEGF(165). Treatment with recombinant human
VEGF(165)b significantly reduced the growth rate of established xenografts of SK-N-BE(2)-C cells (4.24 +/- 1.01 fold increase in volume) compared with those treated with saline (9.76 +/- 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF(165)b-treated tumours (19.4 +/- 1.9 vessels/mm(3)) in contrast to the saline-treated tumours (45.5 +/- 8.6 vessels/mm(3)).
VEGF(165)b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK-N-BE(2)-C cells after 48 h. In conclusion,
VEGF(165)b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of
VEGF(165)b in NB and other paediatric
malignancies.