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Comparative Proteomics Profiling Reveals Role of Smooth Muscle Progenitors in Extracellular Matrix Production

Originally publishedhttps://doi.org/10.1161/ATVBAHA.110.204651Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:1325–1332

Abstract

Objective— Recent studies on cardiovascular progenitors have led to a new appreciation that paracrine factors may support the regeneration of damaged tissues.

Methods and Results— We used a shotgun proteomics strategy to compare the secretome of peripheral blood–derived smooth muscle progenitors (SPCs) with human aortic smooth muscle cells. The late-outgrowth SPCs produced fewer proteolytic enzymes and inflammatory cytokines and showed reduced invasive capacity. Similar to smooth muscle cells, SPCs secreted extracellular matrix. However, SPCs produced different matrix proteins, as evidenced by the truncation of proangiogenic domains in collagen α-1 (I) and increased production of periostin. Moreover, SPCs retained serum proteins, including proteoglycans, regulating collagen assembly; and pigment epithelium–derived factor, a potent inhibitor of angiogenesis. As a functional consequence, their conditioned medium was less angiogenic, as demonstrated by endothelial tube formation assays in vitro and implantation of Matrigel plugs into nude, severe combined immunodeficient mice (NOD/SCID).

Conclusion— The present study represents an important conceptual development, suggesting that SPCs may contribute to extracellular matrix production.

Several studies1,2 have shown that vascular progenitor cells are present in circulating blood. This new concept in vascular biology resulted in a rapid translation into a clinical context. Trials have already been conducted to evaluate the therapeutic potential of bone marrow–derived and circulating progenitor cells in patients.3–5 Yet, the contribution of cell therapy to cardiovascular repair is still debated. Much of the controversy arises from the fact that there is little evidence to suggest that these cells are present in large numbers and permanently incorporate into the vessel wall.6–8 Most cells are immediately lost after injection, with additional loss occurring in the months that follow. Therefore, cell therapy might stimulate vessel formation and functional improvement in a paracrine manner.9

Our proteomic study of the secretome of colony-forming units and endothelial progenitor cell cultures (EPCs)10 confirmed that their conditioned medium is proangiogenic but revealed that the markers used to define their endothelial potential may arise from an uptake of platelet microparticles by adherent mononuclear cells and that platelet microparticles contribute to the angiogenic activity of the conditioned medium.11 In addition, platelet factors may induce an angiogenic monocyte/macrophage phenotype.10 These studies demonstrated how the use of proteomics can provide new insights, which were not obtained by conventional techniques. Little is known about secreted factors produced by other putative progenitor cell populations. The aim of the present study is to compare the secretome of late-outgrowth smooth muscle progenitors (SPCs) with early angiogenic cells (EACs), previously referred to as EPCs, and human aortic smooth muscle cells (SMCs).

Methods

An expanded supplemental Methods section is available online (http://atvb.ahajournals.org).

Cell Culture

The study was approved by the institutional review board of the Phoenix VA Health Care System, Phoenix, Ariz, and the ethics review board of J. W. Goethe University, University of Frankfurt, Frankfurt, Germany and King’s College London, London, England. Cell culture of peripheral blood mononuclear cells was performed as previously described.2,9 Before sampling, cells were washed carefully 3 times and placed in serum-free medium. After 24 hours, the conditioned media were harvested, centrifuged for 2 hours at 20 000g to remove particulates, and frozen at −80°C. Four replicates were obtained for late-outgrowth SPCs and EACs; 3 replicates were analyzed for human aortic SMCs.

Liquid Chromatography-Tandem Mass Spectrometry Analysis

Proteins in the conditioned media were subject to an in-solution digest with trypsin. Tryptic peptides were separated by reverse-phase nanoflow liquid chromatography (Easy-nanoLC) and analyzed online by tandem mass spectrometry using a linear ion trap with high mass accuracy (LTQ-Orbitrap). The resultant mass spectra were matched to database entries (UniProtKB/Swiss-Prot, release version 10.5) using the version of the SEQUEST algorithm contained in Bioworks 3.3 and imported into Scaffold, version 1.7. Assignments were accepted when the Xcorr score was greater than 1.9 for singly charged ions, greater than 2.5 for doubly charged ions, and greater than 3.0 for triply charged ions, along with a peptide probability of less than 1e−3. Results were further filtered for 2 or more independent peptides per protein identification. Protocols are available at http://www.vascular-proteomics.com.

Methods for immunoblotting, RT-PCR, immunofluorescence staining, matrix metalloproteinase (MMP) 1 ELISA, 27-plex cytokine measurements, cell invasion, tube formation, and Matrigel plug assays are available online at http://atvb.ahajournals.org.

Results

Comparison of SPCs With EACs

Late-outgrowth SPCs were generated by removing vascular endothelial growth factor and adding platelet-derived growth factor when first primordial outgrowth colonies formed in cultured cells. Differences in secreted proteins were investigated using liquid chromatography-tandem mass spectrometry. Label-free differential expression analysis returned protein features distinguishing EACs from SPCs (supplemental Table I and supplemental II; available online at http://atvb.ahajournals.org). Reconstructed ion chromatograms for selected proteins are shown in supplemental Figure I (available online at http://atvb.ahajournals.org). The proteomic data confirmed that EACs are of monocytic origin (CD14) and that their conditioned medium is rich in platelet proteins (CXCL7).11 Consistent with previous microarray analysis,9 EACs secreted high levels of cathepsins, including cathepsin L. The latter has been implicated for neovascularization.12 In contrast, CD14, platelet proteins, and cathepsins were not detected in SPCs staining positive for smooth muscle α-actin (Figure 1A). Their conditioned medium contained a variety of collagen chains (supplemental Figure II). Thus, the 2 cell populations showed a distinct protein profile in their conditioned medium. To validate the proteomic findings, the differential expression of CD14 and selected extracellular matrix proteins was verified at the mRNA level (Figure 1B).

Figure 1. Late-outgrowth SPCs vs EACs. A, Morphological features of SPCs and immunofluorescent staining for smooth muscle α-actin (red). Nuclei were counterstained with Hoechst 33258 (blue). B, RT-PCR for mRNA expression of CD14 and extracellular matrix proteins.

Comparison of SPCs With Aortic SMCs

To further characterize SPCs, we compared their secretome with human aortic SMCs. The classification of all identified proteins according to the Gene Ontology Annotation returned “extracellular matrix” and “proteinaceous extracellular matrix” as major categories in SMCs and SPCs, but not in EACs (supplemental Figure III). The consensus report for proteins present in both SMCs and SPCs contained 100 proteins (supplemental Table III). Proteins predominantly found in either SMCs or SPCs are listed in supplemental Table IV and supplemental Table V. Proteolytic enzymes and extracellular matrix proteins are highlighted in the Table.

Table. Extracellular Components in the Conditioned Medium of SPC and SMC Cultures

Protein Name*Entry NameMolecular Weight, kDaSMC (n=3)SPC (n=4)P Value
Proteases and protease inhibitors
    Matrix metalloproteinase 1MMP1_HUMAN54158±2812±80.03§
    72-kDa type IV collagenaseMMP2_HUMAN7410±28±20.60
    Metalloproteinase inhibitor 1TIMP1_HUMAN2322±21±10.004§
    Metalloproteinase inhibitor 2TIMP2_HUMAN245±16±10.90
    Procollagen C–endopeptidase enhancer 1PCOC1_HUMAN487±33±00.30
    Pappalysin-1 (IGF-dependent IGFBP-4 protease)PAPP1_HUMAN1812±1ND0.20
Collagen chains
    Collagen α-1 (I)CO1A1_HUMAN13932±9114±240.04§
    Collagen α-1 (III)CO3A1_HUMAN13920±616±20.61
    Collagen α-1 (V)CO5A1_HUMAN1842±011±20.03§
    Collagen α-1 (VI)CO6A1_HUMAN10923±315±40.16
    Collagen α-1 (XIV)COEA1_HUMAN194ND4±10.04§
    Collagen α-2 (I)CO1A2_HUMAN12959±1767±90.70
    Collagen α-2 (IV)CO4A2_HUMAN1682±11±10.13
    Collagen α-2 (V)CO5A2_HUMAN1456±213±30.17
    Collagen α-2 (VI)CO6A2_HUMAN1096±28±20.52
    Collagen α-3 (VI)CO6A3_HUMAN34413±3ND0.06
Laminin subunits
    Laminin α-4LAMA4_HUMAN20320±3ND0.02§
    Laminin α-5LAMA5_HUMAN4001±02±00.09
    Laminin β-1LAMB1_HUMAN19817±53±00.11
    Laminin γ-1LAMC1_HUMAN17822±88±20.20
Proteoglycans and glycoproteins
    FibronectinFINC_HUMAN26327±868±120.04§
    VersicanCSPG2_HUMAN37320±3ND0.03§
    PeriostinPOSTN_HUMAN93ND8±40.18
    PerlecanPGBM_HUMAN46977±1517±30.06
    DecorinPGS2_HUMAN409±22±00.09
    LumicanLUM_HUMAN383±2ND0.19
    BiglycanPGS1_HUMAN426±1ND0.03§
    Fibrillin-1FBN1_HUMAN31223±137±140.18
    EGF-containing fibulinlike ECM protein 1 (fibulin 3)FBLN3_HUMAN553±1ND0.15
    Follistatin-related protein 1FSTL1_HUMAN3514±58±20.33
    SPARC (osteonectin)SPRC_HUMAN3514±411±20.52
    Nidogen-1NID1_HUMAN1362±11±00.30
    Nidogen-2NID2_HUMAN1511±01±10.52
    EMILIN-1EMIL1_HUMAN1075±31±00.23
    Thrombospondin 1TSP1_HUMAN1301±11±10.95
    Thrombospondin 2TSP2_HUMAN13016±9ND0.20
    Galectin 1LEG1_HUMAN157±24±10.22
IGF-binding proteins
    4IBP4_HUMAN284±1ND0.06
    6IBP6_HUMAN254±11±00.08
    7 (PGI2-stimulating factor)IBP7_HUMAN2917±24±10.009§
(Continued)

Table. Continued

Protein Name*Entry NameMolecular Weight, kDaSMC (n=3)SPC (n=4)P Value
CXCL indicates chemokine (C-X-C motif) ligand; ECM, extracellular matrix; EGF, epidermal growth factor; IGFBP, insulin growth factor–binding protein; MMP, matrix metalloproteinase; ND, not detected; PGI, prostacyclin; PTX, pentraxin; SMC, smooth muscle cell; SPARC, secreted protein acidic and rich in cysteine; SPC, smooth muscle progenitor; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases.
*The table highlights proteins that were chosen for their potential importance in the ECM. The complete list of identified proteins and their normalized spectral counts (percentage of total spectra) are available in supplemental Tables III through V (available online at http://atvb.ahajournals.org).
†Data are given as mean±SE number of assigned tryptic peptides, determined by liquid chromatography–tandem mass spectrometry.
‡Derived from t tests.
§P<0.05.
Other ECM-associated proteins
    Growth-regulated protein α (CXCL1)GROA_HUMAN1136±84±30.04§
    Pentraxin-related protein PTX3PTX3_HUMAN424±2ND0.15
    Extracellular matrix protein 1ECM1_HUMAN614±15±10.36
    EGF-like repeat and discoidin I-like domain-containing protein 3EDIL3_HUMAN54ND3±10.02§
    Galectin-3-binding proteinLG3BP_HUMAN652±2ND0.42
    TGF-β–induced protein ig-h3BGH3_HUMAN7512±4ND0.12
    Latent TGF β–binding protein 2LTBP2_HUMAN1953±1ND0.03§
    Latent TGF-binding protein, isoform 1SLTB1S_HUMAN1535±51±10.48
    Pregnancy zone proteinPZP_HUMAN1642±34±40.53
    Sulfhydryl oxidase 1QSCN6_HUMAN834±2ND0.22
    Lysyl oxidase homolog 2LOXL2_HUMAN872±14±10.39

SPCs Produce Extracellular Matrix

A significant degree of overlap was observed among the matrix proteins identified in the conditioned medium of SPCs and SMCs. Similar to SMCs, SPCs express fibronectin, collagen α-1 (I), and collagen α-1 (V) (Figure 2A). However, unlike SMCs, there was no spectral evidence for the N-terminal domains of collagen α-1 (I) in SPCs that contain a von Willebrand factor type C module and a heparin-binding domain implicated in endothelial tube formation13 (supplemental Figure IV). Matrix proteins predominantly expressed in SPCs included collagen α-1 (XIV), which plays an adhesive role by integrating collagen bundles; and periostin, which is required for maturation and extracellular matrix stabilization.14

Figure 2. Expression of extracellular matrix proteins. A, RT-PCR for mRNA levels of selected extracellular matrix proteins and pigment epithelium–derived factor (PEDF) in SMCs and SPCs. B, Serum-free culture media conditioned by SMCs and SPCs were probed with antibodies to periostin, thrombospondin 2, biglycan, PEDF, and insulin-like growth factor–binding protein (IGFBP-2). The antibody against biglycan is specific for the human protein, as confirmed by the absence of staining with purified bovine biglycan (data not shown); the antibodies against PEDF and IGFBP-2 recognize both the human and the bovine protein. The additional PEDF band at greater than 200 kDa is likely the result of binding to other proteins. Irreversible reduction and alkylation with dithiothreitol and iodoacetamide abolished the high-molecular-weight staining and increased the intensity of the 50-kDa band. The presence of PEDF in both bands was confirmed by liquid chromatography-tandem mass spectrometry (data not shown).

SPCs Retain Serum Proteins

Although all cell lines were cultured in serum-free media before sampling and the carryover for high abundant serum proteins was identical in both cell types (bovine albumin and fetuin A represented on average 5.0% and 0.85% of the total spectra in SPCs, respectively), and 5.3% and 0.86% of the total spectra in SPCs, respectively; the conditioned medium of SPCs contained additional matrix proteins, which were not of human, but of bovine, origin. For 3 proteoglycans (biglycan, decorin, and lumican), there was clear spectral evidence for the bovine protein in SPCs and the human homologue in SMCs (supplemental Table VI). Consistent with the observed reduction of human-specific peptides, mRNA levels of biglycan, decorin, and lumican were downregulated in SPCs (Figure 2A). Similarly, other matrix-binding proteins (ie, pigment epithelium–derived factor [PEDF], a potent inhibitor of angiogenesis; and insulin-like growth factor–binding protein 2) were detected as bovine proteins in the conditioned medium of SPCs in the absence of corresponding mRNA expression (Figure 2A [PEDF] and data not shown [insulin-like growth factor–binding protein 2]), suggesting cell type–specific retention from bovine serum. Differences for periostin, thrombospondin 2, biglycan, PEDF, and insulin-like growth factor–binding protein 2 were verified by immunoblotting (Figure 2B).

Low Proteolytic and Inflammatory Activity in SPCs

In agreement with the spectral counts in the proteomic data set (Table), ELISA measurements established that the concentrations of MMP-1 in the conditioned medium of serum-starved SMCs exceeded 100 ng/mL, whereas MMP-1 levels in the supernatant of SPCs were less than 1 ng/mL (n=3, data not shown). Comparable results were obtained by immunoblotting (Figure 3A). Differences became less pronounced if antibodies for active MMP-1 were used (top panel) because tissue inhibitor of metalloproteinases-1 (TIMP-1), a potent MMP inhibitor, was enriched in the conditioned medium of SMCs. Yet, N-cadherin, an established MMP target on the SMC surface,15 was shedded in their conditioned medium (supplemental Table IV). Similarly, the cytokine secretion of SPCs was remarkably low given their origin from the myeloid lineage. This was confirmed at the mRNA (Figure 3B) and protein levels (supplemental Table VII). More important, SPCs produced less vascular endothelial growth factor and showed reduced invasive capacity (Figure 3C), suggesting that SPCs are unlikely to promote angiogenesis.

Figure 3. Proteolytic and inflammatory activity. A, Cell culture supernatants of SMCs and SPCs were probed with antibodies to active MMP-1, total MMP-1, and TIMP-1. Differences for total MMP-1 are more pronounced than for active MMP-1. Because there are no “housekeeping proteins” in conditioned medium, secreted protein acidic and rich in cysteine (SPARC) was chosen as the loading control based on the proteomic data presented in supplemental Table III (available online at http://atvb.ahajournals.org; protein No. 20, 0.20% vs 0.14% of total spectra in SMCs and SPCs, respectively). B, Cytokine expression evaluated by RT-PCR. Corresponding protein levels are listed in supplemental Table VII. SPCs express platelet-derived growth factor receptor beta (PDGFR-B). C, Invasion assay comparing SMCs with SPCs. Results were derived from 3 independent experiments, each performed in triplicate. *Significant difference by paired t test, P<0.05.

Functional Validation

To explore whether the observed changes in SPCs were functionally important, we assessed endothelial tube formation in vitro. As shown in Figure 4A and B, the formation of new tubelike structures in Matrigel was supported by conditioned medium from SMCs, but not from SPCs. Supplementing cultures of human umbilical vein endothelial cells with conditioned medium of SMCs or SPCs did not alter endothelial proliferation and survival (Figure 4C). However, endothelial cell numbers were higher if dishes were precoated with conditioned medium of SPCs than SMCs (Figure 4D), suggesting that the matrix of SPCs has distinct functional properties. Thus, it was interesting to observe that endothelial growth factor–like repeat and discoidin I–like domain-containing protein 3, which promotes adhesion of endothelial cells through interaction with the αvβ3integrin receptor and inhibits formation of vascular-like structures,16 was only identified in the conditioned medium of SPCs (Table). Finally, the angiogenic effect was investigated by implanting Matrigel plugs injected into nude, severe immunodeficient mice (NOD.CB17-Prkdcscid). Compared with the conditioned medium of SMCs, plugs treated with the conditioned medium of SPCs showed reduced vascularization over the implantation period (Figure 5).

Figure 4. Angiogenic activity in vitro. A, Endothelial tube formation in Matrigel was quantified in the presence of conditioned medium of SMCs and SPCs. B, Reduced tube surface in the presence of conditioned medium of SPCs. Data are given as mean±SE of 3 independent experiments, each performed in duplicate. C, Conditioned medium of SMCs or SPCs was added to human umbilical vein endothelial cells (HUVECs) at a dilution of 1:1. The supplementation had no effect on cell numbers (n=3). D, In contrast, coating dishes with conditioned medium of SPCs before seeding of HUVECs increased endothelial cell numbers by 20% vs conditioned medium of SMCs (n=4). Experiments were performed in triplicate. *Significant difference by a t test, P<0.05.

Figure 5. Angiogenesis in vivo. A, DMEM or conditioned medium from SMCs or SPCs was mixed with Matrigel and injected into mice with severe combined immunodeficiency. Sections of the plugs were stained with hematoxylin-eosin. B and C, Matrigel plugs with conditioned medium from SMCs, but not SPCs, showed a significant increase in the number of neovessels. Data are representative of at least 3 independent experiments. *Significant difference by a t test, P<0.05.

Discussion

Although vascular progenitor cells may contribute to tissue repair, the mechanisms by which they act remain unsettled. Herein, we demonstrated that late-outgrowth SPCs show a distinct secretion profile compared with EACs. Similar to their SMC counterparts, SPCs express a range of extracellular matrix and matricellular proteins, some unique to SPCs only, but released less proteases and inflammatory cytokines. In this respect, SPCs displayed properties that were also distinct from aortic SMCs.

SPCs Secrete Fewer Proteolytic Enzymes

One of the most prominent differences between SMCs and SPCs was observed for the interstitial collagenase MMP-1. Several lines of evidence suggest that SMCs constitutively express MMPs; however, when stimulated by cytokines, SMCs also produce activated forms of MMPs, which is essential for their invasive capacity.17,18 Because cytokines augment the production of MMPs without appreciably affecting the synthesis of TIMPs, they can tip the regional balance of MMP activity in favor of vascular matrix degradation, making atherosclerotic plaques vulnerable to rupture.19 Notably, baseline secretion of MMP-1 in SMCs appears to be influenced by age: human newborn but not adult SMCs produced high amounts of MMP-1 in vitro, which correlated with the presence of avb3 integrin on their surface.20 In contrast, SPCs produced less MMP-1, a finding consistent with a previous report21 that SPCs express high levels of β1, but less of αvβ3 integrin. The β3 integrin receptor mRNA has been shown to be upregulated early after injury of the rat carotid artery, with a time course correlating to SMC proliferation and migration to the intima, whereas a β3 integrin–blocking antibody almost completely blocked SMC migration from the media to the intima.22 Thus, the lack of β3 integrin on SPCs provides a likely explanation for their reduced MMP secretion and impaired invasive capacity, as observed in the present study.

SPCs Produce Extracellular Matrix

Consistent with their integrin profile, SPCs show increased adherence to collagen type I21; this matrix protein was also found in abundance in their conditioned medium. EACs produced predominantly proteases and inflammatory molecules, which would facilitate the breakdown of existing matrix and the recruitment of mononuclear cells to the site of injury. Late-outgrowth SPCs secreted barely any inflammatory cytokines or matrix-degrading enzymes; however, they expressed extracellular matrix components. For example, levels of periostin, one of the most abundant mRNAs associated with vascular injury23 and essential for cardiac repair,24,25 were higher in SPCs than in SMCs. These findings provide a mechanistic underpinning for recent observations that injections of SPCs have beneficial effects on atherosclerosis by promoting changes in plaque composition toward a stable phenotype26: when human EPCs or SPCs were injected every other week in apolipoprotein E−/− recombination activating gene-2 (RAG2)−/− mice, injection of SPCs, but not EPCs, increased collagen and SMC content and reduced the number of macrophages in atherosclerotic plaques. Furthermore, an interaction between EPCs and SPCs was recently shown to enhance the formation of a mature and functional vascular network after cell-based therapy.27 Consistent with these in vivo data, our proteomic findings provided direct evidence that late-outgrowth SPCs express extracellular matrix and are likely to play a distinct role from EACs, referred to as EPCs in the publications previously mentioned.

SPCs Retain Serum Proteins

Although all cells were cultured in serum-free media before sampling to ensure that the collected conditioned media contain no other extraneous proteins, except for the secreted or shed proteins, SPCs selectively retained specific bovine proteins from the serum supplement. This conclusion was supported by 3 independent lines of evidence: (1) The proteins were identified as bovine rather than human by tandem mass spectrometry. (2) Gene expression was absent or markedly downregulated at the transcriptional level. (3) There was positive or negative immunostaining, depending on whether the antibody recognizes both the human and the bovine protein or the human protein only. Examples included PEDF, a potent inhibitor of angiogenesis28 that binds to newly formed collagen and counters the effects of vascular endothelial growth factor; and biglycan, which is involved in collagen assembly and is essential for the functional integrity of the vascular wall.29 The fact that proteins from the bovine serum supplement can bind to extracellular matrix clearly illustrates the advantage of a proteomics approach, which can distinguish between proteins from different species.

Limitations of the Study

Although mass spectrometry is a valuable tool to array secreted proteins, minor components can remain undetected. Moreover, it is possible that SPCs are specialist myeloid cells,30 bone marrow mesenchymal stem cells, or pericyte-like cells (CD14, SM α-actin+, and platelet-derived growth factor receptor B+). Similarly, SMCs do not undergo terminal differentiation, and heterogeneity is observed among different SMC isolates from human aortas, in particular for cytokine secretion (data not shown); their differentiation state is plastic and rapidly influenced by external stimuli. As with any in vitro study, findings in cultured cells may not allow a straightforward translation onto their phenotype in vivo. However, without expansion in culture, it is impossible to sample their secretome and obtain sufficient material for proteomic analysis.

Conclusion

In conclusion, proteomics is an evolving field in cardiovascular research and proteomic techniques offer an unbiased approach to phenotype putative progenitor cell populations.31,32 As demonstrated in this study, a comprehensive description of their secretome can advance our understanding of their biological potential.

Received on: January 2, 2009; final version accepted on: April 15, 2010.

We thank Xiaoke Yin, PhD; Qiuru Xing, MSc; Anthony Sullivan, PhD; and Gary Woffendin, PhD for their technical assistance.

Sources of Funding

This study was supported by the European Vascular Genomics Network (LSHM-CT-2003-503254) as part of the 6th European Framework Programme; a grant from the British Heart Foundation; a grant from the Oak Foundation; a grant from the Juvenile Diabetes Research Foundation (Dr Simper); and resources and facilities at the Phoenix VA Health Care System (Dr Simper). Dr Mayr is a Senior Research Fellow from the British Heart Foundation.

Disclosures

None.

Footnotes

Correspondence to Manuel Mayr, MD, PhD, King’s British Heart Foundation Centre, King’s College London, 125 Coldharbour Ln, London SE5 9NU, England. E-mail

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