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Endostatin is a naturally occurring anti-angiogenic polypeptide derived from the carboxyl terminus of Type XVIII collagen, and has potential clinical utility to prevent tumor growth and metastasis. Endostatin has been shown to inhibit endothelial cell (EC) migration, to induce apoptosis of EC cells, and to block neovascularization. However, the mechanisms underlying the multiple anti-angiogenic activities of endostatin are incompletely understood. In the present study we found that continuous infusion of mice with recombinant human endostatin for 3 to 4 days prior to treatment with Vascular Endothelial Growth Factor (VEGF) blocked the sharp increase in vascular permeability normally seen in response to that angiogenic factor, as judged by extravasation of dye from skin blood vessels (Miles assay). We therefore examined effects of treatment with endostatin on components of the signal transduction pathway involved in responses of EC to VEGF in vitro. Porcine aortic EC transduced with the human VEGF receptor-2 (KDR) gene were exposed to VEGF for 30 minutes. As expected, control cells grown without endostatin showed enhanced tyrosine-specific phosphorylation of the KDR protein, indicative of receptor activation. They also showed decreased phosphorylation of the c-Src protein at the tyrosyl residue Y527, consistent with activation of protein-tyrosine kinase activity, and enhanced tyrosine-specific phosphorylation of the focal adhesion kinase (FAK), a Src substrate. In addition, immunofluorescence staining revealed enhanced organization of actin stress fibers, and showed that vascular endothelial-cadherin (VE-cadherin), a key modulator of angiogenesis and vascular permeability, migrated away from the cell membrane, presumably as a consequence of tyrosine phosphorylation. By contrast, in EC maintained in the presence of endostatin for 4 days before exposure to VEGF, neither the activation of Src (as assessed by the degree of phosphorylation at Y527) nor the enhanced phosphorylation of FAK was observed. The endostatin-treated cells contained fewer stress fibers than control cells, and showed a significantly less pronounced change in cytoskeletal organization in response to VEGF. Finally, in the endostatin-treated EC VE-cadherin remained localized at the cell membrane. These findings show that chronic exposure of EC to endostatin abrogates key steps in the signal transduction cascade normally initiated by VEGF.
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J. Vasc. Biol. 42, Sup:2 (2005) p98