Release of Soluble Urokinase Receptor from Vascular Cells*

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Abstract
Urokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1β (IL-1β), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1β stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall. *Part of this work was supported by grants (Pr 327/1-4) from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Novartis-Foundation (Nürnberg, Germany). This work is part of the MD/PhD-thesis of T.C. at the Institute for Biochemistry, Department of Medicine, Justus-Liebig-Universität, Giessen, Germany. Abbreviations: bFGF: basic fibroblast growth factor, GPI: glycosyl-phosphatidylinositol, FCS: fetal calf serum, HUVEC: human umbilical vein endothelial cells, HVSMC: human vascular smooth muscle cells, IL-1β: interleukin-1β, mAb: monoclonal antibody, PBS: phosphate-buffered saline, PDGF-BB: platelet derived growth factor-BB, piPLC: phosphatidylinositol-specific phospholipase C, piPLD: phosphatidylinositol-specific phospholipase D, PMA: phorbol myristate acetate, scuPA: single chain uPA, suPAR: soluble urokinase receptor, uPA: urokinase- type plasminogen activator, uPAR: urokinase receptor, VN: vitronectin