Integrin αvβ3 is not significantly implicated in the anti-migratory effect of anti-angiogenic urokinase kringle domain

The recombinant kringle domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the kringle domain of uPA and integrin αvβ3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin αvβ3 is involved in tumor angiogenesis, we investigated whether integrin αvβ3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-αvβ3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-α2β1 nor anti-α5β1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-αvβ3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins αvβ3, α2β1 and α5β1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin αvβ3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin αvβ3.