Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro.

The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cell-derived microparticles participate in the dissemination of biological activities. However, their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMPs) from tumor necrosis factor alpha (TNFalpha)-stimulated endothelial cells served as a surface for the generation of plasmin. The generation of plasmin involved expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) at the surface of EMPs and was further increased by their ability to bind exogenous uPA on uPAR. Plasminogen was activated at the surface of EMPs in a dose-dependent, saturable, and specific manner as indicated by the inhibition of plasmin formation by epsilon-amino-caproic acid (epsilon-ACA) and carboxypeptidase B. EMP-induced plasmin generation affects tube formation mediated by endothelial progenitor cells. However, low amounts of EMPs increased tube formation, whereas higher concentrations inhibited it. Prevention of these effects by inhibitors of either uPA or plasmin underscore the key role of EMP-induced plasmin generation. In conclusion, we demonstrated that EMPs act as vectors supporting efficient plasmin generation and dissemination, a new pathway in the regulation of endothelial proteolytic activities with potential involvement in inflammation, angiogenesis, and atherosclerosis.

High urokinase expression contributes to the angiogenic properties of endothelial cells derived from circulating progenitors.

Endothelial progenitor cells (EPC) display a unique ability to repair vascular injury and promote neovascularization although the underlying molecular mechanisms remain poorly understood. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a critical role in cell migration and angiogenesis by facilitating proteolysis of extracellular matrix. The aim of the present study was to characterize the uPA/uPAR-dependent proteolytic potential of EPC outgrown from human umbilical cord blood and to analyze its contribution to their angiogenic properties in vitro. Cells derived from EPC (EPDC), presenting typical features of late outgrowth endothelial cells, were compared to mature endothelial cells, represented by human umbilical vein endothelial cells (HUVEC). Using quantitative flow cytometry, enzyme-linked immunosorbent assays and zymography, we demonstrated that EPDC displayed higher levels of uPA and uPAR. In conditioned culture media, uPA-dependent proteolytic activity was also found to be significantly increased in EPDC. This activity was paralleled by a higher secretion of pro-metalloproteinase-2 (pro-MMP-2). Inhibition of EPDC-associated uPA by monoclonal antibodies that block either uPA activity or receptor binding, significantly reduced proliferation, migration and capillary like tube formation. Moreover, tumor necrosis factor-alpha and vascular endothelial growth factor, known to be locally secreted in ischemic areas, further increased the proteolytic potential of EPDC by up-regulating uPA and uPAR expression respectively. The EPDC response to these factors was found to be more pronounced than that of HUVEC. In conclusion, these findings indicated that EPDC are characterized by high intrinsic uPA/uPAR-dependent proteolytic potential that could contribute to their invasive and angiogenic behaviour.