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Angiogenesis, which is the formation of new blood vessels from pre-existing vessels, is mandatory for tumor growth. Angiogenesis is also involved in other pathological disorders such as psoriasis and macular degeneration. The process can be divided into 3 stages: endothelial cell (EC) proliferation, EC migration and vessel morphogenesis. Essen's GFP-AngioKit, an in vitro co-culture assay system, recapitulates these three stages of the angiogenic cascade. The kinetic method described herein is contrasted to currently available methods in that it provides dynamic information not available from end point experiments.
MethodsGFP-AngioKits were prepared by first expressing GFP in HUVECs and then co-culturing them in a 24-well microplate (Orange Scientific) with normal human dermal fibroblasts (NHDF) as previously described by Bishop et al. (1) and in collaboration with TCS CellWorks. The 24-well GFP-AngioKit was fed every 2–3 days and tubule formation was monitored by imaging the cultures every 12 hours in IncuCyte™ FLR. Essen's Angiogenesis Analysis Module was used to quantify tube length, tube area, and branch point formation over the time course of the experiment.
ResultsIn the presence of VEGF, GFP-labeled HUVECs proliferate and migrate to form cell clusters by day 5 (Figure 1, A and B). Further treatment with VEGF and other pro-angiogenic factors, such as bFGF and EGF, promote tubule formation and lengthening from day 5 to 14 (Figure 1E). VEGF-stimulated cultures are fully mature by day 14 (Figure 1, C and D). Neutralizing antibodies to VEGF blocked the EGF-potentiation of VEGF, which establishes that EGF is acting via the VEGF pathway (Figure 1F). In the absence of VEGF, there is greatly diminished proliferation, migration, and tubule formation.
Conclusion
Combining GFP-AngioKits with IncuCyte™ FLR enables kinetic and quantitative measurements of both the stimulation and inhibition of microvascular tubule formation. The GFP-AngioKit, in conjunction with IncuCyte™ FLR, can be used to screen for drugs affecting all stages of angiogenesis and to dissect signal transduction pathways.
