Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro by Romaric Lacroix, Florence Sabatier, Agnès Mialhe, Agnès Basire, Ralph Pannell, Hélène Borghi, Stephane Robert, Edouard Lamy, Laurent Plawinski, Laurence Camoin-Jau, Victor Gurewich, Eduardo Angles-Cano, and Françoise Dignat-George Blood Volume 110(7):2432-2439 October 1, 2007 ©2007 by American Society of Hematology

EMPs are able to activate plasminogen into plasmin. EMPs are able to activate plasminogen into plasmin. (A) Plot of plasmin generated versus time at varying EMP amounts per 50 μL/well (○ = 106; □ = 105; ◇ = 104; x = 103; +‖= control without EMPs) and fixed final concentrations of plasminogen (1 μM) and a plasmin-selective chromogenic substrate (0.75 mM). Representative graph of 4 independent experiments. (B) Similar experiment as in panel A expressed as change in absorbance at 405 nm per minute versus EMP amount per well (S: EMP last washing supernatant used as control). (C) Plasmin generated at varying plasminogen concentrations (0-5 μM) and a fixed amount (2.105/50 uL) of EMPs was detected with a chromogenic substrate as in panel A. Raw data (○) were fitted to the Michaelis-Menten equation allowing calculation of nonspecific activity (◇) and a Km = 0.122 μM for specific plamin generation (□). (D) Effect of various inhibitors on the generation of plasmin by EMPs (2.105/50 μL) at 0.5 μM plasminogen (α2AP = α2-antiplasmin; ϵ-ACA = ϵ-amino-caproïc acid; CPB = carboxypeptidase B; antibodies to uPA and tPA αvβ3 and α-enolase as compared with an isotype control IgG1). Results are the mean ± SD of 3 independent experiments. Romaric Lacroix et al. Blood 2007;110:2432-2439 ©2007 by American Society of Hematology

Plasminogen is activated at the surface of EMPs by uPA. Plasminogen is activated at the surface of EMPs by uPA. (A,B) A variable number of EMPs immobilized on poly-L-lysine surfaces were incubated with fixed concentrations of plasminogen (0.5 μM) and the plasmin-selective chromogenic substrate (0.75 mM). The graph in panel A shows an increase in the formation of plasmin as a function of the number of immobilized microparticles. Unbound reagents were then washed off and the chromogenic substrate added to detect plasmin that remained bound to the immobilized EMPs; the graph (B) shows an increase in bound plasmin as a function of the number of immobilized EMPs. Representative graphs (mean ± SD) of 3 independent experiments. (C,D) Protein extracts from HMEC-1 and its derived EMPs were processed for zymography (panel C) to detect plasminogen activator activity (5 μg protein/lane; S: EMP last washing supernatant) and for immunoblot (panel D) using rabbit antibodies against uPAR and purified uPAR as reference (20 μg protein/lane). The ∼50 kDa and upper bands in panel C were inhibited by antibodies to urokinase (not shown). A space has been inserted to indicate where a gel lane was cut. These gels came from different experiments as indicated by the space between the gels. Romaric Lacroix et al. Blood 2007;110:2432-2439 ©2007 by American Society of Hematology

Identification of uPA, uPAR, and uPA/uPAR complex on EMP Identification of uPA, uPAR, and uPA/uPAR complex on EMP. (A,B) Measured fluorescence intensity by flow cytometry analysis of EMPs with anti-uPA (A) and anti-uPAR (B) antibodies (black) and the corresponding isotype controls (gray). Identification of uPA, uPAR, and uPA/uPAR complex on EMP. (A,B) Measured fluorescence intensity by flow cytometry analysis of EMPs with anti-uPA (A) and anti-uPAR (B) antibodies (black) and the corresponding isotype controls (gray). (C) Representative images of immunogold labeling of uPA (top) and uPAR (bottom) analysis on EMPs by transmission electron microscopy. Slides were viewed with a JEOL 1220 transmission electron microscope (JEOL, Tokyo, Japan). Images were acquired using a MegaView III camera (Soft Imaging System, Munster, Germany) and were processed with Analysis software (Soft Imaging System) and Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA). The dimension of the bars indicate the relative small size of these EMPs, and the clusters of 15-nm gold particles (black dots) indicate the presence of uPA and uPAR at the surface of EMPs. (D) Main graph: isotherm of the binding of varying amounts of scuPA incubated with EMPs immobilized on a poly-L-lysine surface. The amount of bound scuPA was detected by its ability to activate plasminogen using a chromogenic substrate selective for plasmin. Data fitted to the Langmuir equation as indicated in “Materials and methods” allowed calculation of a dissociation constant, Kd = 0.1 nM, for the interaction of scuPA with its receptor. Representative graph (mean ± SD) of 3 independent experiments. Inset: specificity of the binding of native scuPA is demonstrated by its inhibition with a modified recombinant form of scuPA (r-scuPA, ILe159→Gly) that bind to its receptor but cannot be activated. The bars represent the mean (± SD) of 3 independent experiments. Romaric Lacroix et al. Blood 2007;110:2432-2439 ©2007 by American Society of Hematology

EMPs affect the angiogenic properties of endothelial progenitors in vitro: effects on tube formation in matrigel. EMPs affect the angiogenic properties of endothelial progenitors in vitro: effects on tube formation in matrigel. EPDC in RPMI/10% FCS and varying amounts of EMPs were plated on matrigel, and tube formation was evaluated after 24 hours of incubation. (A) Biphasic effect of EMPs on tube formation: at low EMP concentrations (≤ 2.103/well), polygon number and tube formation increased by 20% (P = .012, n = 12) over control (S: EMP last washing supernatant). However, higher amounts of EMPs (≥ 2.105 EMP/well) had the reverse effect and progressively decreased the number of tubes to 50% at 8.105 EMP/well. Stars indicate significant changes (*P < .05, **P < .01, ***P < .001). Results are the mean (± SD) of 12 independent experiments. (B) Inverted light microscope representative images (400 × magnification) showing EPDC tube formation in matrigel in the absence and presence of 2.103 or 2.105 EMPs per well. Asterisks indicate retracted cell clusters. Slides were viewed with an inverted Nikon light microscope (Nikon, Melville, NY) using an Olympus lens (40×/0.60; Olympus, Tokyo, Japan). Images were acquired using a Nikon model DXM 1200F camera and were processed with Lucia G software (both from Nikon) and Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA). (C) Experiments were performed in the presence of inhibitors of either plasmin formation (anti-uPA antibody) or activity (α2-antiplasmin, α2-AP). The biphasic effect of EMPs on tube formation (left bar panel) was abolished by the anti-uPA antibody (middle) and by α2-AP (right). Asterisks indicate significant changes (P < .05). Results are the mean (± SD) of 3 independent experiments. Romaric Lacroix et al. Blood 2007;110:2432-2439 ©2007 by American Society of Hematology

EMPs are involved in plasmin generation in matrigel. EMPs are involved in plasmin generation in matrigel. (A) Plasmin generation in matrigel is dependent on the amount of EMPs. The chromogenic substrate CBS0065 was added to the matrigel with the EMPs, and under these conditions an increase in plasmin formation was detected from 105 EMPs (P < .001) after 2 hours of incubation. Asterisks indicate significant changes (P < .05). Results are the mean (± SD) of 3 independent experiments. (B) Plasmin generation in matrigel at different EMP amounts is dependent on the supply of plasminogen by FCS. The graph shows plasmin activity detected after 3 hours of incubation ▒, medium with 10% FCS; ▓, medium with plasminogen-free FCS; ▤, medium with plasminogen-free FCS supplemented with 1 μM plasminogen). Activity in the absence of EMPs may represent low levels of plasmin and/or limited amounts of activators in matrigel. Asterisks indicate significant changes (P < .05). Results are the mean (± SD) of 3 independent experiments. Romaric Lacroix et al. Blood 2007;110:2432-2439 ©2007 by American Society of Hematology