Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus Oil the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human Umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during Cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions (c) 2009 Elsevier Inc All rights reserved.