Microparticles of Human Atherosclerotic Plaques Enhance the Shedding of the Tumor Necrosis Factor-α Converting Enzyme/ADAM17 Substrates, Tumor Necrosis Factor and Tumor Necrosis Factor Receptor-1
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070021
ISSN1525-2191
AutoresMatthias Canault, Aurélie S. Leroyer, Franck Peiretti, Guy Lesèche, Alain Tedgui, Bernadette Bonardo, Marie‐Christine Alessi, Chantal M. Boulanger, Gilles Nalbone,
Tópico(s)Platelet Disorders and Treatments
ResumoHuman atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-α converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 ± 2007 TACE/ADAM17+ MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304TNF), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304TNF cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17+ MPs could regulate the inflammatory balance in the culprit lesion. Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-α converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 ± 2007 TACE/ADAM17+ MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304TNF), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304TNF cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17+ MPs could regulate the inflammatory balance in the culprit lesion. Atherosclerosis is a chronic inflammatory disease of the vessel wall resulting from the interactions between modified lipoproteins, monocytes/macrophages, lymphocytes, and vascular cells.1Hansson GK Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (7239) Google Scholar The development and the progression of atherosclerotic plaques are associated with apoptotic cell death and accumulation of microparticles (MPs) within the lesion.1Hansson GK Inflammation, atherosclerosis, and coronary artery disease.N Engl J Med. 2005; 352: 1685-1695Crossref PubMed Scopus (7239) Google Scholar, 2Kockx MM De Meyer GRY Muhring J Jacob W Bult H Herman AG Apoptosis and related proteins in different stages of human atherosclerotic plaques.Circulation. 1998; 97: 2307-2315Crossref PubMed Scopus (369) Google Scholar, 3Mallat Z Tedgui A Current perspective on the role of apoptosis in atherothrombotic disease.Circ Res. 2001; 88: 998-1003Crossref PubMed Scopus (177) Google Scholar MPs are submicron plasma membrane vesicles released during cell activation or apoptosis and harbor at their surface transmembrane proteins initially present at the parent cell surface, conferring to MPs a dynamic storage pool of bioactive molecules.4Morel O Toti F Hugel B Freyssinet JM Cellular microparticles: a disseminated storage pool of bioactive vascular effectors.Curr Opin Hematol. 2004; 11: 156-164Crossref PubMed Scopus (286) Google Scholar, 5Distler JH Pisetsky DS Huber LC Kalden JR Gay S Distler O Microparticles as regulators of inflammation: novel players of cellular crosstalk in the rheumatic diseases.Arthritis Rheum. 2005; 52: 3337-3348Crossref PubMed Scopus (207) Google Scholar, 6Boulanger CM Amabile N Tedgui A Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease.Hypertension. 2006; 48: 180-186Crossref PubMed Scopus (341) Google Scholar MPs have been isolated from human atherosclerotic plaque but are absent in healthy blood vessels.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar Human plaque MPs originate mainly from leukocytes, red blood cells, endothelial cells, and smooth muscle cells.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar They also express a procoagulant activity associated with the presence of phosphatidylserine and tissue factor at their surface, which could lead to thrombus formation at the time of plaque rupture.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar, 8Mallat Z Hugel B Ohan J Leseche G Freyssinet J-M Tedgui A Shed membrane microparticles with procoagulant potential in human atherosclerotic plaques: a role for apoptosis in plaque thrombogenicity.Circulation. 1999; 99: 348-353Crossref PubMed Scopus (605) Google Scholar, 9Bonderman D Teml A Jakowitsch J Adlbrecht C Gyongyosi M Sperker W Lass H Mosgoeller W Glogar DH Probst P Maurer G Nemerson Y Lang IM Coronary no-reflow is caused by shedding of active tissue factor from dissected atherosclerotic plaque.Blood. 2002; 99: 2794-2800Crossref PubMed Scopus (132) Google Scholar Inflammatory processes are regulated by the balance between pro- and anti-inflammatory mediators or cytokines. Sheddases also modulate this equilibrium by cleaving transmembrane proteins (cytokines, receptors, adhesion molecules, and so forth) at the cell surface, releasing soluble ectodomains with altered function.10Garton KJ Gough PJ Raines EW Emerging roles for ectodomain shedding in the regulation of inflammatory responses.J Leukoc Biol. 2006; 79: 1105-1116Crossref PubMed Scopus (196) Google Scholar The typical example is the tumor necrosis factor (TNF)-α converting enzyme (TACE). Initially discovered as the protease that cleaves the 26-kDa proform of TNF (pro-TNF) to yield the TNF soluble form (sTNF),11Black RA Rauch CT Kozlosky CJ Peschon JJ Slack JL Wolfson MF Castner BJ Stocking KL Reddy P Srinivasan S Nelson N Boiani N Schooley KA Gerhart M Davis R Fitzner JN Johnson RS Paxton RJ March CJ Cerretti DP A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature. 1997; 385: 729-733Crossref PubMed Scopus (2767) Google Scholar, 12Moss ML Jin SL Milla ME Bickett DM Burkhart W Carter HL Chen WJ Clay WC Didsbury JR Hassler D Hoffman CR Kost TA Lambert MH Leesnitzer MA McCauley P McGeehan G Mitchell J Moyer M Pahel G Rocque W Overton LK Schoenen F Seaton T Su JL Warner J Willard D Becherer JD Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.Nature. 1997; 385: 733-736Crossref PubMed Scopus (1504) Google Scholar TACE also cleaves ectodomains of several other transmembrane proteins13Smalley DM Ley K L-selectin: mechanisms and physiological significance of ectodomain cleavage.J Cell Mol Med. 2005; 9: 255-266Crossref PubMed Scopus (190) Google Scholar such as TNFR-1 and TNFR-2.14Reddy P Slack JL Davis R Cerretti DP Kozlosky CJ Blanton RA Shows D Peschon JJ Black RA Functional analysis of the domain structure of tumor necrosis factor-α converting enzyme.J Biol Chem. 2000; 275: 14608-14614Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 15Solomon KA Pesti N Wu G Newton RC Cutting edge: a dominant negative form of TNF-α converting enzyme inhibits ProTNF and TNFRII secretion.J Immunol. 1999; 163: 4105-4108Crossref PubMed Google Scholar TACE belongs to the ADAM family (ADAM17) and is synthesized as an inactive proform that is further cleaved into an active form by proprotein convertases, such as furin.16Peiretti F Canault M Deprez-Beauclair P Berthet V Bonardo B Juhan-Vague I Nalbone G Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme.Exp Cell Res. 2003; 285: 278-285Crossref PubMed Scopus (69) Google Scholar, 17Srour N Lebel A McMahon S Fournier I Fugere M Day R Dubois CM TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity.FEBS Lett. 2003; 554: 275-283Crossref PubMed Scopus (89) Google Scholar We recently reported that TACE/ADAM17 is expressed in both cellular and acellular areas of lesions from apoE−/− mice and in human atherosclerotic plaques.18Canault M Peiretti F Kopp F Bonardo B Bonzi M-F Coudeyre J-C Alessi M-C Juhan-Vague I Nalbone G The TNF alpha converting enzyme (TACE/ADAM17) is expressed in the atherosclerotic lesions of apolipoprotein E-deficient mice: possible contribution to elevated plasma levels of soluble TNF alpha receptors.Atherosclerosis. 2006; 187: 82-91Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar We therefore hypothesized that MPs present in the plaque are potential carriers of TACE/ADAM17. MPs were isolated from human atherosclerotic plaques and analyzed for their content in TACE/ADAM17 protein and activity. Results showed that MPs carry TACE/ADAM17, mainly in its mature active form, catalyze in vitro hydrolysis of a mimetic peptide containing the cleavage site of pro-TNF, and activate the shedding of TACE/ADAM17 substrates such as TNF, TNFR-1, and endothelial protein C receptor (EPCR). MPs were isolated from human atherosclerotic plaques removed from 25 patients undergoing carotid endarterectomy (73 ± 2 years of age; 79% male), as recently reported.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar Plaques were obtained either from symptomatic patients (70% with ischemic attacks and 30% with stroke, n = 10) or from asymptomatic patients (n = 15) with critical asymptomatic stenosis of the carotid artery (>75% narrowing). As control experiments, healthy human internal mammary arteries (n = 3, obtained as surgical waste) were submitted to the same isolation protocol. All patients gave their informed consent to the study, which was approved by our local ethical committee. Surgical samples obtained within 90 minutes after excision were rapidly rinsed in ice-cold sterile phosphate-buffered saline (PBS) solution supplemented with streptomycin and penicillin (100 U/ml each). Atherosclerotic lesions were then mechanically separated from the apparently healthy vessel wall. Plaques were thoroughly minced for 15 minutes into 1-mm3 tissue fragments using fine scissors in a volume of Dulbecco's modified Eagle's medium (supplemented with 10 μg/ml polymyxin B, streptomycin, and penicillin, and filtered through a 0.22-μm membrane) corresponding to the respective weight of each lesion. The resulting preparations were centrifuged first at 400 × g (15 minutes) and then at 12,500 × g (5 minutes) to remove cells and cell debris. The resulting supernatants referred to as plaque homogenates were subsequently used for flow cytometry analysis of plaque microparticle cellular origins.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar The remaining plaque homogenate was further centrifuged at 20,500 × g for 150 minutes at 4°C to pellet MPs. The supernatant was discarded, and MP pellets were gently suspended in fresh Dulbecco's modified Eagle's medium (1/10 of volume corresponding to the respective weight of each lesion) and were used for in vitro purposes (activity and cell stimulation). Endothelial MPs were obtained from human umbilical vein endothelial cells (HUVECs) (fourth passage) maintained in serum-deprived medium for 72 hours. The medium was first centrifuged at 300 × g for 10 minutes to eliminate cell debris and then at 20,500 × g for 150 minutes at 4°C to pellet MPs that were subsequently resuspended in fresh Dulbecco's modified Eagle's medium. All analyses were performed on homogenates prepared from atherosclerotic plaques or normal arteries and were performed on a Coulter EPICS XL flow cytometer (Beckman Coulter, Villepinte, France) as recently reported.7Leroyer AS Isobe H Leseche G Castier Y Wassef M Mallat Z Binder BR Tedgui A Boulanger CM Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques.J Am Coll Cardiol. 2007; 49: 772-777Crossref PubMed Scopus (332) Google Scholar MP gate was defined as events with a 0.1- to 1-μm diameter and then plotted on a FL/FSC fluorescence dot plot to determinate positively labeled MPs by specific antibodies. MP concentration was assessed by comparison to Flowcount calibrator beads. MPs bearing phosphatidylserine were labeled using fluorescein isothiocyanate (FITC)-conjugated Annexin V (Roche Diagnostics, Meylan, France) in the presence or in the absence of CaCl2 (5 mmol/L). The cellular origin of human plaque MPs was determined as follows. We incubated 10 μl of plaque homogenates with different fluorochrome-labeled antibodies or their corresponding isotype-matched IgG controls at room temperature for 30 minutes. Anti-CD4-phycoerythrin (PE) was provided by BD Biosciences Pharmingen (Le Pont-de-Claise, France); anti-CD41-PE-cyanin5 (PC5), anti-CD66b-FITC, anti-CD144-PE, and anti-CD235a-FITC were obtained from Beckman Coulter; anti-CD14-PE was from Caltag Laboratories (Burlingame, CA). MPs derived from endothelial cells, monocytes/macrophages, lymphocytes, erythrocytes, and granulocytes were identified as CD144+, CD14+, CD4+, CD235a+, and CD66b+, respectively. The presence of intracellular smooth muscle cell actin was assayed after MP fixation in paraformaldehyde (2%) and permeabilization by saponin (0.1%). Anti-smooth muscle cell actin antibodies (rabbit IgG, dilution 1:2; LabVision, Runcorn, UK), or rabbit IgG (as a negative control), were incubated for 1 hour at room temperature. MP samples were washed once in PBS, and Alexa Fluor 555 donkey anti-rabbit IgGs (Invitrogen, Cergy-Pontaise, France) were then added for 30 minutes at room temperature. To investigate the presence of TACE/ADAM17 at the surface of plaque MPs, we incubated 10 μl of homogenate with 5 μl of monoclonal anti-human TACE/ADAM17-PE (clone no. 111633) or its corresponding isotype-matched IgG control (R&D Systems, Lille, France) at room temperature for 20 minutes in the dark. The same anti-TACE-PE antibody (5 μl) was used in co-labeling with the anti-CD66b-FITC (20 μl) and the anti-235a-FITC (20 μl). For the co-labeling experiments with PE-conjugated antibodies (20 μl each of anti-CD4, -CD14, and -CD144), we used the monoclonal antibody anti-human TACE/ADAM17 conjugated to fluorescein (clone no. 111633) (5 μl). All subsequent in vitro experiments were performed using the 20,500 × g-pelleted plaque MPs. In these assays, plaque MPs reached a final concentration of 14,250 ± 4990 Annexin V+ MPs/μl, which corresponded to 11% of the average MP concentration in the plaque. Plaque MPs (n = 4) were incubated with the classical pro-TNF mimetic fluorogenic peptide (peptide III; R&D Systems) harboring the consensus sequence A-V (Mca-PLAQA↓V-Dpa-RSSSR-NH2) cleaved by TACE/ADAM17.19Black RA Doedens JR Mahimkar R Johnson R Guo L Wallace A Virca D Eisenman J Slack J Castner B Sunnarborg SW Lee DC Cowling R Jin G Charrier K Peschon JJ Paxton R Substrate specificity and inducibility of TACE (tumour necrosis factor α-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity.Biochem Soc Symp. 2003; 70: 39-52Crossref PubMed Scopus (68) Google Scholar MPs (10 μl) were suspended in 100 μl of the final activity buffer (25 mmol/L Tris/HCl, pH 8.0, containing 2.5 μmol/L ZnCl2). Fluorogenic peptide I (substrate of MMP-1, -2, -7, -8, -9, -12, -13, -14, -15, and -16), peptide II (substrate of MMP-3 and -10) (R&D Systems), and peptide III were diluted in the activity buffer at the final concentration of 10 μmol/L. Recombinant ectodomain of human TACE/ADAM17 (R&D Systems), used as the positive control, or MPs were extemporaneously mixed with the substrate in a final volume of 100 μl at room temperature to initiate the reaction. TACE/ADAM17 inhibitor was premixed with MPs or recombinant TACE/ADAM17 at 4°C for 15 minutes. Mixtures were immediately delivered in a 96-well black plate and read in a microplate fluorescence reader (Chameleon; Hidex, Turku, Finland). For all substrates, fluorescence-related enzymatic cleavage was monitored at 320-nm excitation and 405-nm emission wavelength for 2 to 3 hours. Blank (buffer and MPs and substrate, separately) was subtracted from sample measurements for calculations. HUVECs were isolated and cultured as previously described.20Peiretti F Alessi MC Henry M Anfosso F Juhan-Vague I Nalbone G Intracellular calcium mobilization suppresses the TNF-α-stimulated synthesis of PAI-1 in human endothelial cells. Indications that calcium acts at a translational level.Arterioscler Thromb Vasc Biol. 1997; 17: 1550-1560Crossref PubMed Scopus (22) Google Scholar They were used at the third passage for MP stimulation experiments. The human endothelial cell line ECV-304 and ECV-304 cells stably overexpressing TNF (ECV-304TNF) were cultured as described.21Peiretti F Canault M Bernot D Bonardo B Deprez-Beauclair P Juhan-Vague I Nalbone G Proteasome inhibition activates the transport and the ectodomain shedding of TNF-α receptors in human endothelial cells.J Cell Sci. 2005; 118: 1061-1070Crossref PubMed Scopus (17) Google Scholar Monocytic mouse cell line homozygous for TACE/ADAM17 mutation, which deletes the Zn2+ binding domain (TACE/ADAM17ΔZn/ΔZn cells), and monocytic mouse TACE/ADAM17 cells expressing active TACE/ADAM17 were kindly provided by Dr. J. Peschon (Amgen Inc., Thousand Oaks, CA).11Black RA Rauch CT Kozlosky CJ Peschon JJ Slack JL Wolfson MF Castner BJ Stocking KL Reddy P Srinivasan S Nelson N Boiani N Schooley KA Gerhart M Davis R Fitzner JN Johnson RS Paxton RJ March CJ Cerretti DP A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature. 1997; 385: 729-733Crossref PubMed Scopus (2767) Google Scholar In HUVECs, the constitutive release of TNF or TNFR-1 was less than the detection limit in the present experimental conditions. Thus, HUVECs were first exposed to phorbol ester (PMA; 20 nmol/L, 4 hours), washed with serum-free medium (Dulbecco's modified Eagle's medium, 0.2% bovine serum albumin, 0.1 μmol ZnCl2, 1% penicillin/streptomycin, and l-Glu) and incubated for 2 hours in this medium with plaque MPs with or without TMI-2 (1.0 μmol/L) (VF 0.4 ml). ECV-304TNF cells were exposed for 2 hours to plaque-pelleted MPs in the presence or the absence of TMI-2 (1.0 μmol/L) in the above serum-free medium (VF 0.4 ml). Conditioned medium was collected and centrifuged at 20,500 × g to remove the MPs. Cells were lysed with a lysis buffer (PBS, 0.2% Triton X-100, and 1 μg/ml Pefabloc) for subsequent cellular protein assay (enzyme-linked immunosorbent assay and total proteins). In some cases, the 20,500 × g supernatant cleared of the MPs was divided in two parts: one was stored at 4°C, and the other one was centrifuged at 170,000 × g for 16 hours at 4°C (Beckman Optima TLX ultracentrifuge, TLA-100.2 rotor) to sediment exosome-like particles that could be potentially released by cells.22Hawari FI Rouhani FN Cui X Yu Z-X Buckley C Kaler M Levine SJ Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors.Proc Natl Acad Sci USA. 2004; 101: 1297-1302Crossref PubMed Scopus (183) Google Scholar TNF or TNFR-1 shedding was expressed as the ratio of TNF or TNFR-1 in the culture medium over cellular TNF or TNFR-1, respectively, to take into account variations in cellular protein level. MP pellets were lysed with the above lysis buffer containing TMI-2 to prevent the autocleavage of TACE/ADAM17. MP proteins were first submitted to concanavalin A column separation and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% acrylamide or NuPAGE 10% acrylamide; Invitrogen) followed by immunoblotting as previously described.21Peiretti F Canault M Bernot D Bonardo B Deprez-Beauclair P Juhan-Vague I Nalbone G Proteasome inhibition activates the transport and the ectodomain shedding of TNF-α receptors in human endothelial cells.J Cell Sci. 2005; 118: 1061-1070Crossref PubMed Scopus (17) Google Scholar TACE/ADAM17 and ADAM10 were revealed with the rabbit polyclonal anti-human TACE/ADAM17 (R&D Systems) and the rabbit polyclonal anti-human ADAM10 (eBioscience, Montrouge, France), respectively. The potential presence of exosomes in MP preparations was analyzed using the anti-TSG-101 antibody (Sigma, L'Isles d'Abeau Chesnes, France) and the anti-lactadherin antibody.23Silvestre J-S Thery C Hamard G Boddaert J Aguilar B Delcayre A Houbron C Tamarat R Blanc-Brude O Heeneman S Clergue M Duriez M Merval R Levy B Tedgui A Amigorena S Mallat Z Lactadherin promotes VEGF-dependent neovascularization.Nat Med. 2005; 11: 499-506Crossref PubMed Scopus (252) Google Scholar The MW was estimated with the BenchMark ladder (Invitrogen). Total RNA from HUVECs was extracted with the RNeasy mini kit from Qiagen (Courtaboeuf, France). Primer pairs for human TNFR-1 and eEF1α and RT-PCR conditions were described previously.24Lopez S Peiretti F Bonardo B Juhan-Vague I Nalbone G Tumor necrosis factor alpha up-regulates in an autocrine manner the synthesis of plasminogen activator inhibitor type-1 during induction of monocytic differentiation of human HL-60 leukemia cells.J Biol Chem. 2000; 275: 3081-3087Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Total protein content of cell lysates was assayed using the bicinchoninic acid protein assay kit from Sigma-Aldrich. Levels of human sTNF, sTNFR-1, sICAM-1, and MCP-1, and murine sTNFR-1 were determined by enzyme-linked immunosorbent assay according to the specifications of the supplier (R&D Systems). Soluble endothelial protein C receptor (sEPCR) was assayed by enzyme-linked immunosorbent assay using the specification of the Asserachrom sEPCR kit (Diagnostica Stago, Asnières, France). Culture media and reagents were from Gibco BRL (Invitrogen). Bovine serum albumin and Pefabloc were from Sigma. TACE/ADAM17 inhibitor TMI-2 was kindly donated by Dr. J. Levin (Wyeth Research, Cambridge, MA). Recombinant active human TACE/ADAM17 was from R&D Systems. Results were expressed as mean ± SD or SEM where indicated. Differences of the means between two groups were evaluated by the Mann-Whitney U-test, with P < 0.05 considered as significant. Flow cytometry analysis of plaque homogenates (Figure 1A) detected the presence of 119,639 ± 26,325 Annexin V+ MPs, and 12,867 ± 2007 TACE/ADAM17+ MPs per mg of plaque (Figure 1B) (mean ± SEM, n = 25). In contrast, isolation of MPs from healthy human internal mammary arteries did not yield detectable levels of TACE/ADAM17+ MPs (n = 3) (Figure 1B). TACE+ MP abundance was not different between asymptomatic and symptomatic plaques (13,487 ± 2529 versus 11,975 ± 3397 TACE+ MPs/mg plaque, respectively; P = 0.48). The cellular origin of TACE/ADAM17+ MPs was determined by positive co-labeling of TACE/ADAM17 and cellular markers (n = 12, Figure 1C). TACE/ADAM17+ MPs mostly originated from leukocytes (lymphocytes, granulocytes, and monocytes/macrophages) and also from erythrocytes and endothelial cells. None seems to be of smooth muscle cell origin because TACE/ADAM17+ MPs did not co-label with the smooth muscle cell actin antibody. There was no difference in the cellular origin of TACE/ADAM17+ MPs between symptomatic and asymptomatic plaques (data not shown). Co-pelleting of plaque MPs with exosomes that sediment at much higher centrifugation speeds than MPs was excluded in the 20,500 × g pellet because there was no significant labeling for the exosomal markers TSG-10125Stoorvogel W Kleijmeer MJ Geuze HJ Raposo G The biogenesis and functions of exosomes.Traffic. 2002; 3: 321-330Crossref PubMed Scopus (679) Google Scholar, 26Théry C Zitvogel L Amigorena S Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (4027) Google Scholar (Figure 1D) or lactadherin (data not shown).22Hawari FI Rouhani FN Cui X Yu Z-X Buckley C Kaler M Levine SJ Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors.Proc Natl Acad Sci USA. 2004; 101: 1297-1302Crossref PubMed Scopus (183) Google Scholar, 27Théry C Regnault A Garin J Wolfers J Zitvogel L Ricciardi-Castagnoli P Raposo G Amigorena S Molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73.J Cell Biol. 1999; 147: 599-610Crossref PubMed Scopus (889) Google Scholar, 28Stoeck A Keller S Riedle S Sanderson MP Runz S Le Naour F Gutwein P Ludwig A Rubinstein E Altevogt P A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44.Biochem J. 2006; 393: 609-618Crossref PubMed Scopus (208) Google Scholar In plaque MP pellets, the mature form of TACE/ADAM17 was primarily predominant over the proform. Only a minor band of the proform was detected when a preparation exceptionally rich in MPs was examined (Figure 1E). The presence of the mature form of TACE/ADAM17 on MPs prompted us to investigate whether MPs could be catalytically active in vitro. The human recombinant TACE/ADAM17 ectodomain (10 ng/assay), which contains the active catalytic site, time dependently cleaved the pro-TNF mimetic peptide (peptide III). TMI-2, a preferential inhibitor of TACE/ADAM17,29Zhang Y Hegen M Xu J Keith JC Jin G Du X Cummons T Sheppard BJ Sun L Zhu Y Rao VR Wang Q Xu W Cowling R Nickerson-Nutter C Gibbons J Skotnicki J Lin L-L Levin J Characterization of (2R,3S)-2-([4-(2-butynyloxy)phenyl]sulfonyl amino)-N,3-dihydroxybutanamide, a potent and selective inhibitor of TNF-α converting enzyme.Int Immunopharmacol. 2004; 4: 1845-1857Crossref PubMed Scopus (58) Google Scholar inhibited this cleavage by 87 and 100% at 5 and 50 nmol/L, respectively (Figure 2A). The natural endogenous TACE/ADAM17 inhibitor TIMP-330Amour A Slocombe PM Webster A Butler M Knight CG Smith BJ Stephens PE Shelley C Hutton M Knauper V Docherty AJ Murphy G TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.FEBS Lett. 1998; 435: 39-44Crossref PubMed Scopus (551) Google Scholar (100 nmol/L) inhibited by 85% the TACE/ADAM17-dependent cleavage of the peptide. Plaque MPs pelleted from homogenates cleaved the fluorogenic peptide in a time-dependent manner (Figure 2B). The MP-induced increase in fluorescence was strongly reduced by TMI-2, in a concentration-dependent manner. Inhibition by TMI-2 averaged 63% at 5 nmol/L and was optimal (87%) at 500 nmol/L, after 120 minutes of incubation (Figure 2B). TIMP-3 (100 nmol/L) inhibited the MP-induced increase in fluorescence to the same extent as TMI-2 (5 nmol/L) did. The TACE/ADAM17-dependent hydrolysis of the peptide augmented with the increasing concentrations of MPs (Figure 2C). No TACE/ADAM17 activity could be detected in the supernatant resulting from MP pelleting (45 minutes; 20,500 × g), indicating that the activity is carried by MPs and not by smaller vesicular structures. We also investigated whether plaque MPs hydrolyze two other fluorogenic peptides that are substrates of several MMPs. Peptide I is cleaved by a large panel of MMPs (MMP-1, -2, -7, -8, -9, -12, -13, -14, -15, and -16), whereas peptide II is preferentially cleaved by MMP-3 and MMP-10. Peptides I and II were not hydrolyzed by recombinant TACE/ADAM17 because the increase in fluorescence after 120 minutes resulting from their cleavage was less than 10% of the initial fluorescence value (data not shown). However, peptides I and II were cleaved by plaque MPs (Figure 2D), but TMI-2 (1 μmol/L) inhibited their cleavage by only 28 and 20%, respectively, indicating that plaque MPs carry other active protease(s)31Taraboletti G D'Ascenzo S Borsotti P Giavazzi R Pavan A Dolo V Shedding of the matrix metalloproteinases MMP-2, MMP-9, an
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