Microparticelle presentanti Sonic Hedgehog inducono angiogenesi mediante una up-regolazione di proteine di adesione in cellule endotelial

Membrane microvescicles harbouring morphogen Hedgehog promote angiogenesis and up-regulate adhesion proteins in endothelial cells Microparticles (MPs) are plasma membrane vesicles released upon cell activation or during apoptosis. This study investigated the effect of T lymphocyte-derived MPs on endothelial cells with respect to angiogenesis, migration, proliferation and adhesion. Human CEM-T cell line were activated with phytohemagglutinin for 72 h and then, stimulated with phorbol-12 myristrate-13 acetate and actinomycin D for 24 h. Under these conditions, MPs carried the morphogen Sonic Hedgehog (Hh) at their surface (Blood, 2006, 108:3012-3020). EAhy 926 endothelial cells were incubated in the absence or in the presence of 10 µg/ml MPs for 24 h. Cells were then placed in a Matrigel layer for angiogenesis studies. Migration studies were conducted using the wound healing method. Cell proliferation was determined by MTT assay. In another set of experiments, endothelial cells were fixed and permeabilized for immunostaining and confocal microscopy recording. Using the Matrigel assay, we show that MPs treatment promoted the formation of capillary-like network by endothelial cells and this effect was inhibited either by the inhibitor of Hh pathway, cyclopamine, or the Rho kinase inhibitor, Y27632. Endothelial cell migration and proliferation were inhibited in presence of MPs. MPs treatment increased both adhesion of endothelial cells by a mechanism sensitive to Y27632 and Rho A staining, a member of Rho family GTPase known to play a role in cytoskeleton regulation. Moreover, MPs enhanced the phosphorylation of the focal adhesion kinase (p-FAK), which is implicated in the regulation of cell adhesion. Treatment of endothelial cells with cyclopamine prevented the overexpression of both adhesion proteins, Rho A and p-FAK. In summary, angiogenesis, adhesion and upregulation of Rho A and p-FAK induced by MPs from T lymphocytes are mediated by Hh morphogen. These results demonstrate that Hh carried by MPs may contribute to regulation of mechanisms associated with angiogenesis.