Anti-angiogenic Activity of the Recombinant Kringle Domain of Urokinase and Its Specific Entry into Endothelial Cells

Abstract

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp 45 -Lys 135 ) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca 2؉ flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelialcell specific internalization not mediated by the uPA receptor.

Anti-angiogenic action of plasma hyaluronan binding protein in human umbilical vein endothelial cells

“…Kringles 1-4 (angiostatin) or kringle 5 of plasminogen are known for their anti-angiogenic activity by selectively inhibiting endothelial cell growth (26). In addition, kringles derived from other molecules such as kringle 2 of prothrombin and kringles of human hepatocyte growth factor were also found to be inhibitors of endothelial cell proliferation (27,28).…”

Inhibition of migration and invasion of carcinoma cells by urokinase‐derived antagonists of αvβ5 integrin activation

“…35 Several antiangiogenic factors are derived from proteolytic processing of large molecules. 36 Interestingly, the isolated kringle region of uPA also exhibits some growth-inhibitory activity; in the report by Kim et al, 37 a recombinant kringle (UK1, residues 47-135 of human uPA) was shown to inhibit endothelial cell proliferation stimulated by bFGF, VEGF or EGF in a uPAR-independent manner. A thorough understanding of the mechanism of action of potential antiinvasive agents is required to develop efficient antimetastatic therapies as well as aiding in the rational application of combination therapies.…”

Chemotherapy of Prostate Cancer by Targeted Nanoparticles Trackable by Magnetic Resonance Imaging

“…The DNA sequence encoding the first 135 amino acids (with a C-terminal His-tag) of amino terminal fragment (ATF) of the human uPA that binds to uPAR is cloned into pET-20b(+) (Novagen) between EcoRI and XhoI sites of the vector [36]. These recombinant plasmids are transformed in competent TOP10F Escherichia coli cells (Invitrogen, CA) [36,37]. Transformants are selected on a LB-Agar-Amp plate 50 μg/mL ampicillin (LB/amp agar) which is incubated at 37 • C overnight.…”