αvβ3 and αvβ5 integrin antagonists inhibit angiogenesis in vitro

Although angiogenesis is believed to require cell-extracellular matrix interactions which are mediated in part via integrins αvβ3 and αvβ5, a formal demonstration that αvβ3 and αvβ5 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 αvβ3 and αvβ5 regulate angiogenesis in vitro. We cloned and characterized 350-450 bp regions of the bovine homologues of αv, β3 and β5, covering much of the β-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 αvβ3 and αvβ5, but not gpIIbIIIa or α5β1, 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 αvβ3. These findings demonstrate the requirement for endothelial cell αvβ3 and αvβ5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of αvβ3 and αvβ5 antagonists.