title: Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.
abstract: OBJECTIVE
  Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed.
  
  
  METHODS
  VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels.
  
  
  RESULTS
  Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively.
  
  
  CONCLUSIONS
  Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.
authors.affiliation: Paediatric Oncology Division, Beatrix Children's Hospital, University of Groningen, Groningen, the Netherlands.
authors.forename: A H
authors.initials: AH
authors.lastname: Sikkema
authors.forename: E S J M
authors.initials: ES
authors.lastname: de Bont
authors.forename: G
authors.initials: G
authors.lastname: Molema
authors.forename: A
authors.initials: A
authors.lastname: Dimberg
authors.forename: P J
authors.initials: PJ
authors.lastname: Zwiers
authors.forename: S H
authors.initials: SH
authors.lastname: Diks
authors.forename: E W
authors.initials: EW
authors.lastname: Hoving
authors.forename: W A
authors.initials: WA
authors.lastname: Kamps
authors.forename: M P
authors.initials: MP
authors.lastname: Peppelenbosch
authors.forename: W F A
authors.initials: WF
authors.lastname: den Dunnen
mesh_terms: D001254:Astrocytoma / Q000378:metabolism*; D002648:Child; D042783:Endothelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007834:Lasers; D042282:Microdissection; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000032:analysis / Q000096:biosynthesis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis / Q000096:biosynthesis*; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000032:analysis / Q000096:biosynthesis
chemical_list: D012333:RNA, Messenger; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D040321:Vascular Endothelial Growth Factor Receptor-3