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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02587

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TGF-ß-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen

Vladimír Leksa^1,2,*,, Samuel Godar^3,*, Herbert B. Schiller¹, Elke Fuertbauer³, Arshad Muhammad³, Katarina Slezakova², Vaclav Horejsi⁴, Peter Steinlein⁵, Ulrich H. Weidle⁶, Bernd R. Binder⁷ and Hannes Stockinger^1,3

¹ BMT, BioMolecular Therapeutics, Brunner Strasse 59, 1235 Vienna, Austria
² Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Vlarska 5 83334, Slovak Republic
³ Department of Molecular Immunology, Centre of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Lazarettgasse 19, A1090 Vienna, Austria
⁴ Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, Prague 6, CZ-16637, Czech Republic
⁵ Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria
⁶ Roche Applied Science, Nonnenwald 2, 82377 Penzberg, Germany
⁷ Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Lazarettgasse 19, A1090 Vienna, Austria

View larger version (16K): [in a new window]   Fig. 1. TGF-ß-induced apoptosis in ECs. HUVECs were cultivated in 96-well plates (5000 cells per well) in the presence of purified TGF-ß (50 ng ml-1, 10 ng ml-1 and 2 ng ml-1) in assay medium. After incubation for 24 hours, the cells were washed with cultivation medium and lysed. Intact DNA was removed by centrifugation and the presence of soluble nucleosomes in the supernatant was detected by the Death ELISA kit (Roche). The relative apoptotic response was determined using a standard included in the kit.

View larger version (29K): [in a new window]   Fig. 2. Effect of CD87 and CD222 co-expression on TGF-ß activation. Mouse CD222-/- fibroblasts (A) stably transduced with CD222 (B), CD87 (C) or both CD222 and CD87 (D) were cultivated in 96-well plates (5000 cells per well) and treated with 20 µg ml-1 Plg or 1 µg ml-1 suPA for 12 hours. The interfering agents were used in following concentrations: aprotinin (apro), 2 µg ml-1; pepB or pepC, 20 µg ml-1. After incubation, the culture supernatant was analysed for the presence of active TGF-ß by the MLEC assay. Results are expressed as concentration of active TGF-ß in ng ml-1.

View larger version (39K): [in a new window]   Fig. 3. Analysis of TGF-ß activation in culture supernatants of ECs and SMCs by Plg fragments. (A) K1-3 and mini-Plg purity was analysed by Coomassie stain (CB) and by western blotting using the K1-3-specific mAb 2PG or the mini-Plg-specific mAb 4PG (top). The plasmin activity in the preparations was analysed using the plasmin-specific colorimetric substrate S-2251 before and after activation with active uPA (bottom). HUVECs (B) or HUVSMCs (C) were cultivated in 96-well plates (5000 cells per well) and treated with 240 nM Plg and 60 nM, 120 nM or 240 nM mini-Plg (B), or with 240 nM mini-Plg (C) in assay medium for 12 hours. For analysis of total TGF-ß (LTGF-ß plus active TGF-ß), culture supernatants were heated for 10 minutes at 80°C. For analysis of active TGF-ß alone, untreated supernatants were used. Active or total TGF-ß was then analysed by the MLEC assay.

View larger version (34K): [in a new window]   Fig. 4. Analysis of the mini-Plg/CD222 interaction. (A) Plg fragments were coated (10 µg ml-1) on 96-well immunosorbent plates, which were blocked with 1% BSA. Then, the wells were incubated with purified CD222 for 4 hours at 4°C in BB. After washing twice with BB, bound CD222 was eluted using SDS-PAGE sample buffer and visualized by western blotting using the anti-CD222 mAb MEM-238. (B) Effect of CD222 expression on cell binding to mini-Plg. CD222-/- mouse fibroblasts expressing human CD222 or human CD87 were allowed to attach to 96-well plates coated with Plg, mini-Plg or K1-3. The number of cells displayed is the number of cells attached to wells coated with the indicated proteins after subtraction of the cell binding to BSA. (C) Determination of the binding affinity of Plg fragments to CD222 by surface plasmon resonance. Purified CD222 was immobilized on a Biacore chip. The equilibrium dissociation constant Kd was calculated from the ratio of dissociation rate constant kd and association rate constant ka. Standard deviation was obtained from three measurements.

View larger version (30K): [in a new window]   Fig. 5. Analysis of mini-Plg-induced apoptosis in ECs. (A) HUVECs cultivated in 20% serum medium in 96-well plates (5000 cells per well) were treated with 240 nM Plg or Plg fragments for 24 hours. After incubation for 24 hours, the cells were washed with cultivation medium and lysed. Intact DNA was removed by centrifugation and the presence of soluble nucleosomes in the supernatant was detected by the Death ELISA kit (Roche). The relative apoptotic response was determined using a standard included in the kit. HUVECs were treated with mini-Plg with or without the serine-protease inhibitor aprotinin (Apro, 10 µg ml-1), the caspase-3 inhibitor DEVD-FMK (1 µM) or a neutralizing anti-TGF-ß antibody (10 µg ml-1) for 24 hours. (B) Cells were treated for 24 hours with 240 nM Plg or mini-Plg in medium containing 1% serum. After incubation, the detached cells were pooled with the trypsinized adherent cells and assayed for the occurrence of apoptosis by FACS analysis using an apoptosis detection kit (Calbiochem, La Jolla, CA, USA). Viable cells were annexin-V negative and PI negative; nonviable necrotic cells or late apoptotic were annexin-V positive and PI positive; early apoptotic cells were annexin-V positive and PI negative. Similar results were obtained in three different experiments.

View larger version (72K): [in a new window]   Fig. 6. Effects of CD222-derived peptides on mini-Plg-induced apoptosis in ECs. (A) HUVECs were incubated in medium containing 1% serum for 24 hours with mini-Plg (240 nM) in the presence of the CD222-derived peptides and control peptides (20 µg ml-1), then stained with annexin-V/FITC and analysed under a fluorescence microscope. Apoptotic cells are indicated by arrows. Scale bar, 100 µm. (B) Cells were treated as in A. After incubation, the detached cells were pooled with the trypsinized adherent cells and assayed for the occurrence of apoptosis by FACS analysis using an apoptosis detection kit (Calbiochem, La Jolla, CA, USA). Viable cells were annexin-V negative and PI negative; nonviable necrotic cells or late apoptotic were annexin-V positive and PI positive; early apoptotic cells were annexin-V positive and PI negative. Similar results were obtained in three different experiments.

View larger version (83K): [in a new window]   Fig. 7. Analysis of CD222 and CD87 expression and complex formation in ECs versus SMCs. (A) FACS analysis using specific mAbs. Cell-surface expression of CD222, CD87 and CD147 on HUVECs and HUVSMCs was analysed by immunofluorescence analysis and flow cytometry using the anti-CD222 mAb MEM-238, the anti-CD87 mAb C8 and the anti-CD147 mAb MEM-M6/1. (B) Surface-biotinylated HUVECs and HUVSMCs were subjected to immunoprecipitation using MEM-238. The precipitate was immunoblotted with MEM-238, the anti-CD87mAb H2 or the control mAb AFP-01. (C) HUVECs and SMCs were grown in gelatin-coated coverslips, washed, fixed, blocked and stained with the specific fluorescently labeled antibodies MEM-238/AF555 (CD222, red) and E4-AF488 (CD87, green). The slides were analysed under a confocal laser-scanning microscope. Colocalization structures (yellow) are indicated by arrows. All images show a region of 110x110 µm.

View larger version (20K): [in a new window]   Fig. 8. Model of min-Plg-mediated apoptosis in ECs. Our findings indicate that the complex between CD87 and CD222 is a platform for the activation of LTGF-ß. We have shown that mini-Plg, a proteolytic fragment of Plg, can induce TGF-ß activation followed by apoptosis in ECs. Based on our data and the studies of other laboratories (Cornelius et al., 1998; Godar et al., 1999; Morikawa et al., 2000; Scapini et al., 2002), we propose that, under certain pathological conditions, such as inflammation or cancer, the inactive proenzyme Plg is converted to mini-Plg upon binding to CD222 (e.g. by elastase or cathepsin D). Because CD222 associates with CD87 in ECs, the CD87-bound uPA can convert the CD222-bound mini-Plg to the active protease mini-Plm. This leads to partial digestion of LTGF-ß bound to CD222, followed by the release of the active TGF-ß, inducing apoptosis in ECs.

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