Vascular Endothelial Growth Factor (VEGF)-Induced Endothelial Cell Migration Requires Urokinase Receptor (uPAR) for Integrin Redistribution.

VEGF activates endothelial cells to migrate and invade surrounding tissues, an initial event in the angiogenic process. For invasion, the coordinated localized formation of a proteolytic repertoir is necessary. Focusing the urokinase receptor towards the leading edge of migrating cells provides such armor and inhibition of uPA binding to its receptor inhibits invasion of endothelial cells. In addition integrins continuously have to form focal contacts at the leading edge. Thus the spatial proximity between the localized proteases and the matrix seems to be essential for matrix degradation. In order to allow cell locomotion integrins have to release their ligands when they reach the trailing end and are subsequently endocytosed and redistributed to newly formed focal adhesions in a repetitive process.

We here describe a new role of uPAR in regulating integrin redistribution. We have previously reported that stimulation of human endothelial cells by VEGF (50ng/ml) via its receptor flk-1 induces pro-uPA activation, when bound to uPAR. Subsequently a uPA/PAI-1/uPAR-complex is formed, which thereafter is endocytosed via a LDL-R family member. We now show that by this process beta-1 integrins are co-internalized in clathrin coated vesicles via a uPAR dependent mechanism. Subsequently, endocytosed uPAR recycles to focal adhesions where it co-localizes with integrin alpha-v/beta-3. Disrupting this chain of events, either by (1) RAP - a specific inhibitor of the LDL-R family - or by (2) uPAR depletion (using uPAR−/− cells or cleaving the GPI-anchor of uPAR by PI-PLC), beta-1 integrins are no longer internalized after VEGF stimulation. Under the same circumstances the migratory response of endothelial cells toward VEGF is impaired in vitro as shown by video-based migration assays and in vivo as demonstrated by matrigel angiogenesis assays. Next, we generated synthetic peptides interfering with uPAR/integrin interaction, which inhibit not only VEGF-induced integrin redistribution, but also diminish VEGF-induced endothelial cell migration, significantly.

These data suggest that in VEGF-induced cell migration uPAR plays a central role not only in focusing proteolytic activity, but also in initial integrin redistribution. Interference with this process could be a therapeutic target for diseases depending on VEGF-induced angiogenesis.