Plasticity in Endothelial Cells and Endothelial Progenitor Cells: uPAR-mediated Amoeboid Angiogenesis

Background: Mesenchymal migration requires the activity of extracellular matrix (ECM) degrading proteases and depends on Rac-driven actin cytoskeleton contractility, while amoeboid motility is a Rho-ROCKdependent movement, which allows cells to glide through, rather than degrade, ECM barriers. Here we show that mature endothelial cells (ECs) and endothelial progenitor cells (EPCs) are able to migrate by mesenchymal and amoeboid style. We used a cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteineproteases, thus mimicking the physiological environment that ECs and EPCs encounter during their migration within the angiogenesis sites. Methods: To evaluate the mesenchymal-ameboid transition we performed RhoA and Rac1 activation assay, immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers. Results: RhoA and Rac1 activation assay showed, in both EPCs and HMVECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. Then we showed that under Ph-C inhibition both cell lines acquired a round morphology and showed a Matrigel invasion that was greatly enhanced with respect to that observed in the absence of protease inhibition. We didn’t see differences in tubular-like structures formation under mesenchymal or amoeboid conditions. uPAR silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECs and EPCs, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Conclusions: The receptor of the urokinase plasminogen activator receptor (uPAR) is indispensable for ECs and EPCs to perform an efficient amoeboid angiogenesis, in terms of cell migration and capilla