alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro

Although angiogenesis is believed to require cell-extracellular matrix interactions which are mediated in part via integrins alphav beta 3 and alphav beta 5, a formal demonstration that alphav beta 3 and alphav beta 5 are involved in endothelial-cell invasion and capillary-like tube formation is still required. This has arisen from the cellular complexities which occur in vivo and the difficulty in finding appropriate in vitro model systems. Here we have used a three-dimensional assay which employs bovine aortic and microvascular endothelial cells, to show that alphav beta 3 and alphav beta 5 regulate angiogenesis in vitro. We cloned and characterized 350-450 bp regions of the bovine homologues of alphav, beta 3 and beta 5, covering much of the beta -propeller and A-domain regions, and show that they are >95% identical to their human orthologues. We used cyclic peptides EMD 121974, 85189 and 66203, which selectively inhibit alphav beta 3 and alphav beta 5, but not gpIIbIIIa or alpha5 beta 1, to probe in vitro angiogenesis induced by angiogenic cytokines in three-dimensional fibrin or collagen gels. We found that these peptides are potent inhibitors of endothelial cell invasion and differentiation induced by vascular endothelial growth factor-A or fibroblast growth factor-2 but do not affect the unstimulated cells in 3D culture. Inhibition was greatest when cells were grown on fibrin, but also occurred on collagen I which is not a recognized ligand for alphav beta 3. These findings demonstrate the requirement for endothelial cell alphav beta 3 and alphav beta 5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of alphav beta 3 and alphav beta 5 antagonists.