Endostatin-induced modulation of plasminogen activation with concomitant loss of focal adhesions and actin stress fibers in cultured human endothelial cells

Endostatin, a M-r 20,000 fragment of collagen XVIII, is able to inhibit ang iogenesis and induce apoptosis in endothelial cells in vivo. We analyzed th e effects of recombinant endostatin on human microvascular endothelial cell s, focusing on pericellular plasminogen activation and its targeting by the focal adhesion-associated cytoskeletal structures. Analysis of the proteol ytic plasminogen activator system revealed that endostatin modulates the di stribution of soluble and cell surface-associated urokinase-type plasminoge n activator (uPA) and plasminogen activator inhibitor, type 1 (PAI-1). Case in zymographic and immunoprecipitation analyses indicated that endostatin e xerts its effects by decreasing the levels of soluble uPA and PAI-1 and the ir complexes in a dose-dependent manner. Immunofluorescence analysis of cel l surface-associated uPA indicated that endostatin treatment caused the red istribution of receptor-bound uPA from focal contacts, resulting in diffuse cell surface staining. In accordance with this observation, immunofluoresc ence staining of the urokinase receptor revealed that endostatin treatment removed uPAR from focal adhesions. Accordingly, endostatin caused a rapid d isassembly of focal adhesions as observed by immunofluorescence analysis of the focal adhesion proteins vinculin and paxillin. A prominent change in t he cytoskeletal architecture was observed as the actin stress fiber network was dissociated in response to endostatin treatment. The effect of focal a dhesion disassembly was reversible, persisting from 1 h up to 6 h. Our resu lts suggest that the antiangiogenic activity of endostatin involves the mod ulation of focal adhesions and actin stress fibers and the down-regulation of the urokinase plasminogen activator system.