Quantitation of angiogenesis in vitro induced by VEGF-A and FGF-2 in two different human endothelial cultures - an all-in-one assay.

Angiogenic therapy is considered to be a promising tool for treatment of ischemic diseases. Many in vivo and in vitro assays have been developed to identify potential proangiogenic drugs and to investigate their mode of action. However, until now no validated system exists that would allow quantitation of angiogenesis in vitro in only one assay. Here, a previously established all-in-one in vitro assay based on staging of the angiogenic cascade was validated by quantitation of the effects of the known proangiogenic factors VEGF-A and FGF-2. Both growth factors were applied separately or in combination to human endothelial cell cultures derived from the heart and the foreskin, and angiogenesis was quantitated over 30 days of culture. Additionally, gene expression of VEGFR-1, VEGFR-2 and FGFR-1 at 3, 10, 20 or 40 days of cultivation was quantitated by RT-qPCR. In both cultures, VEGF-A as well as FGF-2 induced a run through all defined stages of angiogenesis in vitro. Application of VEGF-A only led to formation of irregular globular endothelial structures, while FGF-2 resulted in development of regular capillary-like structures. Quantitation of the angiogenic effects of VEGF-A and transcripts of VEGFR-1 and VEGFR-2 showed that a high VEGFR-1/VEGFR-2 ratio evoked deceleration of angiogenesis.