Cleaved kininogen (HKa) inhibits tube formation by endothelial cells in a collagen‐fibrinogen, three‐dimensional gel and is dependent on Src kinase

Abstract

We have shown that HKa as well as domain (D5) inhibit migration, proliferation and induce apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, a model of angiogenesis. Collagen and fibrinogen (1:2) were mixed with endothelial cells and an angiogenic stimulator (a mixture of bFGF, VEGF and PMA) was added to the gel in order to induce the formation of capillary structures. The anti-angiogenic effect of HKa, GST-D5 and D5 as well as peptides (300 nM) on tube formation was evaluated by measuring tube length. HKa, GST-D5 and D5 inhibit tube formation by 90%, 86%, 77% respectively. Peptides G486-K502, H475-H485 and G440-H455 inhibited by 77%, 54%, 51%, respectively. In a two dimensional cell culture system, HKa can detach endothelial cells from dishes coated with fibrinogen which is a ligand of integrin αvβ3 and α5β1. HKa can block the formation of the complex of uPAR and integrin α5β1 from 1 to 26 hours as shown by immunoprecipitation studies. Using SiRNA, we down-regulated C-terminal Src kinase expression by 80% and significantly increased tube length by 27%. The addition of HKa almost completely blocked this effect. We further found that PP2, a Src inhibitor, dose-dependently blocked tube formation in 3D gel. Since we have previously shown that uPAR can form a complex with integrins, caveolin and Src kinase, it is likely that HKa as well as D5 bound to uPAR and disrupted this complex. This outcome would further downregulate the Src kinase activity which in turn could inhibit tube formation in the 3D gel.