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.

Salvianolic Acid B Promotes the Survival of Random-Pattern Skin Flaps in Rats by Inducing Autophagy

“…Angiogenesis involves destruction of preexisting cell connections, mitosis, sprouting of endothelial cells, and maturation of new capillaries ( Nowak-Sliwinska et al, 2018 ). MMP9 degrades proteins related to vessel wall stability ( Li et al, 2018 ), VEGF induces mitosis of endothelial cells ( Stacker et al, 2016 ), and cadherin 5 forms intercellular junctions. Western blotting and IHC indicated that Sal B increased VEGF and cadherin 5 expression in the dermis of ischemic flaps.…”

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D

“…Bioassays employed cell lines expressing chimeric receptors consisting of the entire extracellular domain of mouse VEGFR-2 or human VEGFR-3 and the trans-membrane and cytoplasmic domains of the mouse erythropoietin receptor (25, 63). Binding and cross-linking of the chimeric receptors allows these cells to survive and proliferate in the absence of IL-3.…”

Consensus guidelines for the use and interpretation of angiogenesis assays

“…Finally, test reagents may interfere with readout chemistry; thus, compounds that affect mitochondrial function are less compatible with metabolic assays. Surrogate assays such as the VEGFR-BaF3 cell lines which bind VEGF ligands and signal through chimeric receptors in reporter cell lines have been very useful (both academically and in industry) for quantifying the presence of major angiogenic factors from human, mouse, and viral sources that stimulate the VEGFR-2 and VEGFR-3 pathways [ 69 , 70 ].…”