title: A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors.
abstract: The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.
authors.affiliation: Tumour Angiogenesis Program, Peter MacCallum Cancer Centre; Steven.Stacker@petermac.org.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Stacker
authors.affiliation: Tumour Angiogenesis Program, Peter MacCallum Cancer Centre.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Halford
authors.affiliation: Tumour Angiogenesis Program, Peter MacCallum Cancer Centre.
authors.forename: Sally
authors.initials: S
authors.lastname: Roufail
authors.affiliation: Tumour Angiogenesis Program, Peter MacCallum Cancer Centre.
authors.forename: Carol
authors.initials: C
authors.lastname: Caesar
authors.affiliation: Tumour Angiogenesis Program, Peter MacCallum Cancer Centre.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Achen
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000235:genetics; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2