Abstract
BackgroundControlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells.MethodsA cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis.ResultsIn the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions.ConclusionHere we first provide a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the “amoeboid angiogenesis”. uPAR is indispensable for ECs and ECFCs to perform an efficient amoeboid angiogenesis. Therefore, uPAR silencing or the block of its integrin-interaction, together with standard treatment against VEGF, could be a possible solution for angiogenesis inhibition.
Highlights
Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis
To be sure that the invasion capacity of endothelial cells in amoeboid conditions was independent from the compactness of the Matrigel, we tested the Endothelial Colony Forming Cells (ECFCs) and Human microvascular endothelial cells (HMVEC) invasion capacity in a Matrigel layer five times more concentrated (250 μg) than the usually used (50 μg), observing that the ratio between the percentage of migrated cells in mesenchymal or amoeboid conditions was independent of the Matrigel density (Fig. 1c)
Amoeboid style of movement: role of uPAR To investigate the role of uPAR in amoeboid angiogenesis, after validating the silencing activity of pooled small interfering RNAs targeting PLAUR mRNA, that produced an evident reduction of uPAR expression in terms of mRNA and protein in both ECFCs and HMVECs (Fig. 3a), we have studied the effect of uPAR knockdown on the small Rho-GTPases activation
Summary
Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The protease-independent amoeboid migration (named after the motility of the amoeba Dictyostelium Discoideum) is characterized by fast cycles of contraction and expansion of the cell body, which is round or ellipsoid, obtained by contraction of the cortical actin and myosin filaments with the creation of cell “blebs” [7]. This type of movement, observed in hematopoietic stem cells and certain tumor cell [1, 8], consists in a sort of “crawling” through less dense compartments of the ECM, driven by short-lived weak interaction of the amoeboid cell with the substrate. The enhanced contractility that enables cells that use the amoeboid strategy to squeeze into gaps of the ECM is promoted by the Rho/ROCK signaling pathway [9]
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