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Endostatin-induced Modulation of Plasminogen Activation with Concomitant Loss of Focal Adhesions and Actin Stress Fibers in Cultured Human Endothelial Cells¹

Cell Biology and Molecular/Cancer Biology Laboratories, Biomedicum Helsinki, and Haartman Institute, Departments of Virology and Pathology, University of Helsinki, FIN-00014 Helsinki, Finland [S. A. W., T. V., K. A., J. K-O.], and the Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland [M. R., T. P.]

Endostatin, a M_r 20,000 fragment of collagen XVIII, is able to inhibit angiogenesisand induce apoptosis in endothelial cells in vivo. We analyzed the effectsof recombinant endostatin on human microvascular endothelial cells, focusing on pericellular plasminogen activation and its targeting by the focal adhesion-associated cytoskeletal structures. Analysis of the proteolytic plasminogen activator system revealed that endostatin modulates the distribution of soluble and cell surface-associated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor, type 1 (PAI-1). Casein zymographic and immunoprecipitation analyses indicated that endostatin exerts its effects by decreasing the levels of soluble uPA and PAI-1 and their complexes in a dose-dependent manner. Immunofluorescence analysis of cell surface-associated uPA indicated that endostatin treatment caused the redistribution of receptor-bound uPA from focal contacts, resulting in diffuse cell surface staining. In accordance with this observation, immunofluorescence staining of the urokinase receptor revealed that endostatin treatment removed uPAR from focal adhesions. Accordingly, endostatin caused a rapid disassembly of focal adhesions as observed by immunofluorescence analysis of the focal adhesion proteins vinculin and paxillin. A prominent change in the cytoskeletal architecture was observed as the actin stress fiber network was dissociated in response to endostatin treatment. The effect of focal adhesion disassembly was reversible, persisting from 1 h up to 6 h. Our results suggest that the antiangiogenic activity of endostatin involves the modulation of focal adhesions and actin stress fibers and the down-regulation of the urokinase plasminogen activator system.

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