A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Summary

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

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.

Introduction

The vascular endothelial growth factor (VEGF) family of secreted protein growth factors and their cognate cell surface receptors is an important and diverse group of soluble ligands and membrane-embedded receptors, respectively that function in transducing signals across cellular membranes. They function mainly in endothelial cells but also in cells of epithelial origin and those of the immune system ^1,2. Signaling pathways engaged by ligand-activated VEGF receptors (VEGFRs) are critical in major pathologies, such as age-related macular degeneration and cancer, and therapeutics targeting them are in frequent clinical use (e.g., the monoclonal antibody bevac....

Protocol

Source of IL-3 and Preparation of WEHI-3D-conditioned Medium

Note: The mouse granulocytic leukemia cell line WEHI-3D is cultured to generate a conditioned medium containing IL-3.

1. Culture WEHI-3D in Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), 1% long-life glutamine supplement, 50 μg/ml gentamicin. Inoculate 5 × 10⁶ cells in the log phase of growth into 50 ml of fresh culture medium in a T175 cm³ tissue culture flask and grow for about 7 days or until the cells have passed their log phase of growth by about 24 - 48 hr (avoid excessive cell death in the culture). ....

Results

In this section, we show the results of an experiment demonstrating the essential features of a VEGFR-2-EpoR-Ba/F3 bioassay (see Figure 1 for principles of the assay). Other published studies demonstrate broader applications of the assay for alternative VEGFR-2 ligands, mutant VEGF molecules and inhibitory monoclonal antibodies ^{8,10,19,24-30}.

The data presented here represent an assay in which the VEG.......

Discussion

The assay described here relies on using cells of high viability, which are dependent on growth factors. Cells therefore need to be carefully cultured to ensure they are factor-dependent, and retain expression of the chimeric receptor. Ensuring that the medium is freshly made and not stored for an excessively long period and that WEHI-3D CM is highly active is important. Cells need to be thoroughly washed from IL-3 containing medium into the assay medium to ensure that no residual IL-3 contaminates the assay when exposin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trypan Blue	Sigma-Aldrich	T8154	0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer
G418 Sulphate (Geneticin)	Invivogen	ant-gn-5	Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.
3H-Thymidine	PerkinElmer	NET-027	This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation
Vialight Plus Kit	Lonza	LT07-221	Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent
Prestoblue Cell Viability Reagent	Invitrogen	A13261	Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell
Nunc Minitray with Nunclon Delta Surface (72 well)	Thermo Scientific	136528	Small microtitre plate
96 well Tissue Culture Plate	Falcon, Corning Inc.	353072
DMEM (1X)	Gibco	11965-92
GlutaMAX (100X)	Gibco	35050-061
Fetal Bovine Serum	Gibco	10099-141
Cell Harvester	Tomtec Life Sciences		Tomtec Harvester, 96 Mach 3M Cell Harvester
Liquid Scintillation Counter	LKB Wallac	1205	LKB Wallac 1205 Betaplate Scintillation Counter
UniFilter-96 GF/B	Perkin Elmer	6005177	White 96-well Barex Microplate with GF/B filterof 1 µm poresize
Gentamicin	Gibco, Life Technologies	15750-060
Penicillin/Streptomycin	Gibco, Life Technologies	15140-122
0.22 um pore cellulose acetate centrifuge tube filter unit	Costar, Corning Inc.	8160	Centrifuge tube filters have a 0.22 µm pore CA membrane-containing filter unit within a 500 µl capacity polypropylene microcentrifuge tube.
Fluorescence Reader	BioTek		BioTek Synergy 4 Hybrid Microplate Reader