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Bone Marrow Mesenchymal Stem Cells Stabilize Already-formed Aortic Aneurysms More Efficiently than Vascular Smooth Muscle Cells in a Rat Model

  • F. Schneider
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France

    Department of Vascular Surgery, University Hospital, Poitiers, France
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  • F. Saucy
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France

    Department of Vascular and Thoracic Surgery, University Hospital Lausanne, Switzerland
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  • R. de Blic
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France
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  • J. Dai
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France
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  • F. Mohand
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France
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  • H. Rouard
    Affiliations
    Etablissement Français du Sang, Université Paris-Est Créteil, France
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  • J.-B. Ricco
    Affiliations
    Department of Vascular Surgery, University Hospital, Poitiers, France
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  • J.-P. Becquemin
    Affiliations
    Department of Vascular Surgery, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris, Créteil, France
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  • M. Gervais
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France
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  • E. Allaire
    Correspondence
    Corresponding author. E. Allaire, Department of Vascular Surgery, Henri Mondor Hospital, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil Cedex, France. Tel.: +33 01 45 84 15 34.
    Affiliations
    CNRS EAC 7054, Centre de Recherches Chirurgicales Dominique Chopin, Faculty of Medicine, Université Paris-Est Créteil, France

    Department of Vascular Surgery, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris, Créteil, France
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Open ArchivePublished:April 17, 2013DOI:https://doi.org/10.1016/j.ejvs.2013.03.007

      Purpose

      Abdominal aortic aneurysms (AAAs) expand because of aortic wall destruction. Enrichment in Vascular Smooth Muscle Cells (VSMCs) stabilizes expanding AAAs in rats. Mesenchymal Stem Cells (MSCs) can differentiate into VSMCs. We have tested the hypothesis that bone marrow-derived MSCs (BM-MSCs) stabilizes AAAs in a rat model.

      Material and methods

      Rat Fischer 344 BM-MSCs were isolated by plastic adhesion and seeded endovascularly in experimental AAAs using xenograft obtained from guinea pig. Culture medium without cells was used as control group. The main criteria was the variation of the aortic diameter at one week and four weeks. We evaluated the impact of cells seeding on inflammatory response by immunohistochemistry combined with RT-PCR on MMP9 and TIMP1 at one week. We evaluated the healing process by immunohistochemistry at 4 weeks.

      Results

      The endovascular seeding of BM-MSCs decreased AAA diameter expansion more powerfully than VSMCs or culture medium infusion (6.5% ± 9.7, 25.5% ± 17.2 and 53.4% ± 14.4; p = .007, respectively). This result was sustained at 4 weeks. BM-MSCs decreased expression of MMP-9 and infiltration by macrophages (4.7 ± 2.3 vs. 14.6 ± 6.4 mm2 respectively; p = .015), increased Tissue Inhibitor Metallo Proteinase-1 (TIMP-1), compared to culture medium infusion. BM-MSCs induced formation of a neo-aortic tissue rich in SM-alpha active positive cells (22.2 ± 2.7 vs. 115.6 ± 30.4 cells/surface units, p = .007) surrounded by a dense collagen and elastin network covered by luminal endothelial cells.

      Conclusions

      We have shown in this rat model of AAA that BM-MSCs exert a specialized function in arterial regeneration that transcends that of mature mesenchymal cells. Our observation identifies a population of cells easy to isolate and to expand for therapeutic interventions based on catheter-driven cell therapy.

      Keywords

      Stabilization of aneurysmal growth using mesenchymal stem cells in an already-formed abdominal aortic aneurysm model has not been published. Direct cellular seeding using catheter-based procedures could be an alternative to treat aortic expansion associated with type II endoleak after endovascular aortic aneurysm repair.

      Introduction

      Abdominal aortic aneurysms (AAAs) expand because of wall atrophy, a consequence of proteolytic injury of extracellular matrix (ECM), disappearance of vascular smooth muscle cells (VSMCs), and absence of compensatory aortic reconstruction.
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      Abdominal aortic aneurysm.
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      New insight in aetiopathogenesis of aortic diseases.
      In expanding experimental AAAs, correction of depletion in VSMCs triggers wall reconstruction and stabilizes the diameter of the diseased aorta.
      • Allaire E.
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      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      • Dai J.
      • Losy F.
      • Guinault A.M.
      • Pages C.
      • Anegon I.
      • Desgranges P.
      • et al.
      Overexpression of transforming growth factor-beta 1 stabilizes already-formed aortic aneurysms. A first approach to induction of unctional healing by endovascular gene therapy.
      • Dai J.
      • Michineau S.
      • Franck G.
      • Desgranges P.
      • Becquemin J.P.
      • Gervais M.
      • et al.
      Long term stabilization of expanding aortic aneurysms by a short course of cyclosporine A through transforming growth factor-beta induction.
      Accordingly, increasing VSMC content by endovascular cell seeding could be a therapeutic strategy. It requires the identification of cells easy to isolate and to expand.
      A subset of bone marrow (BM) cells, referred to as Mesenchymal Stem Cells (MSCs) can be isolated by adherence to plastic wells and can easily be expanded.
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      • Deriglasova U.F.
      • Kulagina N.N.
      Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.
      As MSCs are multipotent and can home at sites of injury, it has been proposed that BM-MSCs contribute to post-natal tissue repair.
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      Marrow stromal cells as stem cells for nonhematopoietic tissues.
      In vitro, MSCs have been shown to differentiate into VSMC-like cells upon PDGF-BB stimulation.
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      • San H.
      • Gordon D.
      • Haudenschild C.C.
      • et al.
      Recombinant platelet-derived growth factor B gene expression in porcine arteries induce intimal hyperplasia in vivo.
      In vivo, MSCs have been shown to contribute to healing of mechanically injured arteries.
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      • Prockop D.J.
      Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair-current views.
      This repair process results in intimal hyperplasia, a VSMC-rich tissue hypertrophy that contrasts with the VSMC-deprived atrophy of the aneurysmal wall. Previous reports have suggested that MSCs may be of interest in AAAs.
      • Hashizume R.
      • Yamawaki-Ogata A.
      • Ueda Y.
      • Wagner W.
      • Narita Y.
      Mesenchymal stem cells attenuate angiotensin II-induced aortic aneurysm growth in apolipoprotein E-deficient mice.
      However, there is no demonstration that MSCs stabilize expanding AAAs. This demonstration requires a model of AAA with statistically significant diameter expansion after cell seeding, with thrombus at wall/blood interface.
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      • Dai J.
      • Losy F.
      • Guinault A.M.
      • Pages C.
      • Anegon I.
      • Desgranges P.
      • et al.
      Overexpression of transforming growth factor-beta 1 stabilizes already-formed aortic aneurysms. A first approach to induction of unctional healing by endovascular gene therapy.
      The xenograft model, in which aneurysmal degeneration is driven by inflammation, matrix metalloprotease activity regulated by the plasmin pathway,
      • Allaire E.
      • Hasenstab D.
      • Kenagy R.D.
      • Starcher B.
      • Clowes M.M.
      • Clowes A.W.
      Prevention of aneurysm development and rupture by local overexpression of plasminogen activator inhibitor-1.
      • Allaire E.
      • Forough R.
      • Clowes M.
      • Starcher B.
      • Clowes A.W.
      Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.
      fulfills these requirements.
      In this study, we have tested the hypothesis that local endovascular seeding of BM-MSCs induces aortic tissue growth and stabilizes the diameter of expanding experimental AAAs, a strategy compatible with future catheter-based cell therapy approaches in aortic aneurysm disease.

      Material and Methods

      BM-MSC isolation

      Cells were isolated from 12 week old male Fischer 344 rats (Charles River, St Quentin Fallavier, France). Aortic VSMCs were isolated
      • Dai J.
      • Michineau S.
      • Franck G.
      • Desgranges P.
      • Becquemin J.P.
      • Gervais M.
      • et al.
      Long term stabilization of expanding aortic aneurysms by a short course of cyclosporine A through transforming growth factor-beta induction.
      and grown in RPMI 1640 and Medium 199 (1:1), with l-glutamine and 10% fetal calf serum (FCS) (Invitrogen, Corporation, Paisley, UK). BM-MSCs were isolated from femurs and tibias by centrifugation and selected by plastic adhesion after overnight incubation in α-MEM with 20% FCS. After this step, medium was removed after 72 h. Antibiotics and anti-fungic were added in each culture medium. Adherent cells were passaged when reaching 80% confluence. BM-MSCs phenotypes were shown to express MSC markers (CD44, CD73, CD90, CD105), but not leukocyte markers (CD45, CD11b).

      Surgery

      Animals were housed and taken care of according to the European Union Standards. They received analgesia and were anesthetized with .1 ml/100 g body weight pentobarbital i.p. AAAs were generated in 250 g-12 week old, male Fischer 344 rats by implanting a segment of Hartley guinea pig aorta (xenograft) as previously described.
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      Guinea pig (Charles River) infrarenal aortas were decellularized using .1% sodium dodecyl sulfate (Sigma, St-Louis, USA) to obtain intact tubes of aortic ECM which were orthotopically implanted into rats with 10-0 sutures. Fourteen days after xenograft implantation, a chimeric AAA (>50% diameter increase) had developed from the degraded guinea pig ECM infiltrated by cells from rat, with a luminal thrombus,
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      as previously described. AAAs were isolated from blood flow by clamps. The lumen was rinsed with culture medium through a PE10 catheter introduced by an aortotomy performed downstream in the native aorta. Passage 5 to 6 BM-MSCs (n = 6 for the time point at one week and n = 5 for the time point at one month) or VSMCs (n = 5 for a unique time point at one week) suspended in culture medium with 5% FCS were injected into the AAA as previously described.
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      To summarize, animals were perfused 2 min on each side, so perfusion took 8 min. In another set of experiments BM-MSCs were stained before seeding with the fluorescent dye PKH26 (Sigma). These xenograft were harvested at 48 h and one week to get sufficient fluorescent signal. As controls, AAAs were infused with culture medium with 5% FCS with no cell (n = 5 at one week and n = 5 at one month).

      AAA assessment

      AAA maximum transverse diameter was measured at endovascular infusion and before euthanasia 1 or 4 weeks later using a graduated scale under dissecting microscopy. Percentage of diameter increase was calculated as follows: (diameter at evaluation time − diameter at seeding) × 100/diameter at seeding. After inclusion into paraffin, 5 μm cross sections were generated from the center of AAAs. Elastin and collagen were stained by orcein and sirius red, respectively. For immunohistochemistry, primary antibodies were mouse anti rat monoclonals: ED1 clone for monocytes/macrophages, RECA for endothelial cells (Serotec, Oxford, England) and smooth muscle alpha-actin (clone 1A4, Sigma). An alkaline phosphatase–antialkaline phosphatase technique was used (Dakopatts). Control sections were generated by omission of the primary antibody and with a nonrelevant primary antibody. Quantitative analyses used Image-Pro Plus Software (Media, Cybernetics, Bethesda, Md). Two blinded observers recorded the percentage of the total area for each section.

      mRNA semi-quantification

      MMP-9 and TIMP-1 mRNA contents were analyzed using reverse transcription polymerase chain reaction (RT-PCR), comparative to the domestic gene 18S (QuantumRNA™18s Internal Standards kit, Ambion, Montrouge, France) (primers: MMP-9: forward: 5′-CTGCGTATTTCCATTCATCTT-3′; reverse: 5′-ATGCCTTTTATGTCGTCTTCA-3′; TIMP-1: forward: 5′-CCCCAGAAATCAACGAGAGACCA-3′; reverse: 5′-ACACCCCACAGCCAGCACTAT-3′). Intima, and media plus adventitia were separated by micro dissection and pooled by layers and groups. Total RNA was extracted with TRIzol™(Life Technologies, Paisley, UK) and treated with grade I DNase (Roche Molecular Biomedicals, Rosny, France). Reverse transcription was done with random primers, and Reverse transcriptase M-MLV, dNTP, dithiothreitol, and ribonuclease inhibitor (Eurobio, Les Ulis, France). PCR was performed in a PCR Express thermo cycler (Hybaid, UK) with DNA Taq polymerase (Eurobio, Les Ulis, France). Bands of amplified sequences corresponding to the gene of interest and to 18s were quantified with Gel Analysis (Iconix, Marnes La Coquette, France). Results were expressed as ratios between signals corresponding to the gene of interest and 18s.

      Statistical analysis

      Results were expressed as mean ± SD. For independent qualitative parameters a Chi-square test was used (biostat TGV). The nonparametric Mann–Whitney U and Kruskal Wallis tests (Statview, version 4.5) were used for statistical comparisons between two and three groups, respectively. P < .05 was considered significant.

      Results

      Endovascular seeding of BM-MSCs stabilizes the diameter of expanding AAAs

      BM-MSCs isolated by plastic adhesion were seeded onto the lumen of formed and expanding AAAs with luminal thrombus. On cross sections made from AAAs harvested 48 h (n = 2) and one week after seeding (n = 2), fusion images showed co-localization of nuclei stained with DAPI and red fluorescence in the intermediate area of the intima, indicating presence of seeded MSCs or daughter cells (Fig. 1). No PKH26 labeling could be seen four weeks after BM-MSC seeding.
      Figure thumbnail gr1
      Figure 1Cross-sectional views of rat AAAs one week after endovascular seeding of PKH26 labeled MSCs. Luminal side is indicated by (*). a: nuclear staining with DAPI; c: PKH26-labeling; b: fusion of a and c; d: high power field view of b. Nuclear staining (→) is co-localized with PKH26 staining (red). e: Hematoxylin-Eosin staining of section cut. Original magnification: a, b, c, e: ×20; d: ×100. Thr: thrombus. Med: media.
      One million BM-MSCs were seeded onto the lumen of already formed AAAs. In the control group infused with culture medium with no cell, the diameter of AAAs continued to increase at one week (n = 5, 48.2 ± 17.0%) and at four weeks (n = 5, 88.3 ± 61.6%) after cell infusion. In contrast, endovascular injection of one million BM-MSCs stopped AAA diameter expansion at one (n = 6, 6.6 ± 9.7%) and at four weeks (n = 5, 9.2 ± 30.1%). Statistical analysis shown that one and four week growth rate differences between control and BM-MSC groups were significant (p = .006 and p = .028 respectively) (Fig. 2).
      Figure thumbnail gr2
      Figure 2A. Diameter expansion of formed AAAs. Y axis is diameter increase (%). B: Macroscopic view of xenograft 28 days after culture medium perfusion as controls (upper panel) and MSCs perfusion (lower panel). The dotted green line indicate the exact site of outer to outer wall diameter measurement. Standard deviation is indicated by bar errors.

      BM-MSCs decrease inflammation and further MMP-driven wall destruction, and trigger tissue regeneration in formed AAAs

      Addition of MSCs decreased infiltration of AAAs by monocyte/macrophages at one week (ED1 positive cells: 14.6 ± 6.4 vs. 4.7 ± 2.3/mm2 in control and BM-MSC groups, respectively, p = .015) (Fig. 3A). Semi-quantitative analysis by RT-PCR expression in AAAs over the domestic gene 18s showed a decrease in MMP-9, and an increase in TIMP-1, mRNAs, one week after endovascular injection of BM-MSCs, with different expression patterns according to localization in thrombus-intima or media-adventitia (Fig. 3B) (n = 3). Immunostaining with an anti-MMP-9 antibody confirmed a sharp decrease in MMP-9 protein accumulation upon MSC endovascular seeding.
      Figure thumbnail gr3
      Figure 3A: Representative view of immunohistochemistry against ED-1 (original magnification: ×4). B: Semi-quantitative RT-PCR of mRNAs encoding for MMP-9 and TIMP-1 in AAA extracts one week after endovascular infusion, over the domestic gene 18s. Standard deviation is indicated by bar errors.
      Four weeks after BM-MSC seeding, collagen content, expressed as the percentage of total vessel wall area, and elastin content, expressed as the percentage of medial area, were significantly higher than in controls (collagen: 7.35 ± 1.9 vs. 22.5 ± 9.1 Surface-%, p = .007; elastin: 4.25 ± 4.30 vs. 28.52 ± 16.5 Surface-%, p = .008) (Fig. 4a, b, d, e).
      Figure thumbnail gr4
      Figure 4Representative cross-sectional views of AAAs four weeks after endovascular treatment. Upper panel: control group (a, b, c, g); lower panel: MSC treated AAA (d, e, f, h). a, d: Sirius red; b, e: orcein; c, f: anti-smooth muscle alpha-actin immunostatining. Orginal magnification: ×4. Thr: luminal thrombus; Ih: intimal hyperplasia. g, h: immunostaining of rat endothelial cells (arrow: endothelium lining on the luminal aspect; Lu: lumen), original magnification ×40.
      After BM-MSC seeding, a tissue developed on the luminal aspect of AAAs and replaced the thrombus which persisted in the control group (Fig. 4a–f). Cell and extracellular matrix accumulating on the luminal surface of the aneurismal wall recapitulated the organization of an aortic wall. The neo aortic wall contained a dense extracellular network with collagen (Fig. 4a, d) and elastin (1.42 ± .9 vs. 6.6 ± 1.9 Surface-%, p = .007, in control and BM-MSC groups, respectively, p = .007) (Fig. 4b, e). The neo vessel wall contained abundant SM-alpha-active positive cells (22.2 ± 2.7 vs. 115.6 ± 30.4 cells/surface units, in control and MSC groups, respectively, p = .007) (Fig. 4c, f). Surface units were calculated by measurement of media's surface. Rat Endothelial Cell Antigen (RECA) immunostaining showed endothelial cells covering VSMC-like cells at the interface with circulating blood. In contrast the luminal surface of the AAAs infused with culture medium was covered with thrombus devoid of endothelium (Fig. 4g, h).

      Rat BM-MSCs stabilize expanding AAAs more efficiently than VSMCs

      After having shown previously that aortic VSMCs stabilize expanding rat AAAs,
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      we designed a dose–response experiment to compare the impact of BM-MSC and VSMC seeding on this clinically relevant parameter at one week. Whereas five million VSMCs were needed to curb diameter expansion, only one million BM-MSCs were sufficient to stabilize AAA diameter (Fig. 5). The mean diameter expansion rate was 48.2 ± 17.0% after culture medium infusion (controls), 25.5 ± 17.2% after VSMC seeding (p = .11 in comparison with controls) and 6.6 ± 9.7% after MSC infusion (p = .006 in comparison with controls) (Kruskall–Wallis test between the three groups: p = .007).
      Figure thumbnail gr5
      Figure 5Dose–response experiment comparing a single quantity of seeded MSCs with increasing quantities of VSMCs one week after perfusion. Standard deviation is indicated by bar errors.

      Discussion

      Using a rat model of formed, expanding, AAAs with a luminal thrombus, we demonstrate that the endovascular seeding of BM-MSCs down-regulates inflammation and proteolysis, triggers aortic regeneration and stops diameter expansion. In this setting, BM-MSCs appear to be more efficient than aortic VSMCs in stabilizing formed AAAs. Our data add credit to the hypothesis that BM-MSCs have unique tissue healing properties that could be utilized to gain stability of VSMC-deprived AAAs.
      AAAs expand inexorably once they have reached 40 mm in diameter,
      • The UK Small Trial Participants
      Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms.
      a suggestion that no efficient repair occurs in these aortic lesions. Healing of injured arteries usually occurs on their luminal aspect, resulting in cell and ECM accumulation resulting with an increase of the artery wall mass. Cells contributing to this repair process may be VSMCs, blood-derived MSCs or medial and adventitial
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      Mesenchymal stem cells attenuate angiotensin II-induced aortic aneurysm growth in apolipoprotein E-deficient mice.
      In human AAAs and in the xenograft model, this luminal repair does not occur. In particular, significant intimal hyperplasia does not develop. As an explanation for this absence of endoluminal healing, it can be proposed that the migration of repair cells through the aneurysmal wall toward the intima faces limiting factors, such as inflammation-driven apoptosis
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      We have observed elsewhere that the luminal surface of the thrombus in human AAAs encloses factors instrumental for MSC recruitment, platelet-derived growth factors, and MSC-like cells, a suggestion that healing aborts on the luminal aspect of AAAs. To support this hypothesis, other have shown that MSCs do not expand in contact with human AAA thrombus, due to polymorphonuclear cells-derived proteases.
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      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
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      • Losy F.
      • Guinault A.M.
      • Pages C.
      • Anegon I.
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      • et al.
      Overexpression of transforming growth factor-beta 1 stabilizes already-formed aortic aneurysms. A first approach to induction of unctional healing by endovascular gene therapy.
      • Dai J.
      • Michineau S.
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      • Desgranges P.
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      • et al.
      Long term stabilization of expanding aortic aneurysms by a short course of cyclosporine A through transforming growth factor-beta induction.
      with polymorphonuclears, a recapitulation of a structural feature important for the evolution of human AAAs.
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      We have documented in this study, using PKH26 labeling, that the addition of an important quantity of BM-MSCs resulted in cell attachment and survival up to one week after endovascular seeding.
      The expansion and rupture of AAAs in the xenograft model occur after aortic ECM injury by inflammation and MMP-driven proteolysis.
      • Allaire E.
      • Hasenstab D.
      • Kenagy R.D.
      • Starcher B.
      • Clowes M.M.
      • Clowes A.W.
      Prevention of aneurysm development and rupture by local overexpression of plasminogen activator inhibitor-1.
      • Allaire E.
      • Forough R.
      • Clowes M.
      • Starcher B.
      • Clowes A.W.
      Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.
      We have shown that endovascular addition of BM-MSCs down-regulates monocyte/macrophage accumulation, and decreases the MMP proteolytic burden in the wall of formed AAAs. These results are in line with other studies showing that MSCs control inflammation in host tissues.
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      BM-MSCs addition stopped aneurysmal wall injury presumably through paracrine secretion of protease inhibitors as suggested by TIMP-1 mRNA accumulation at sites of BM-MSC seeding in our in vivo experiments.
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      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      In a report on MSC peri-adventitial seeding in an ApoE-Angiotensin II model, Hashizume et al. have observed that prevention of AAA degeneration and preservation of medial elastin was associated with an increased expression of IGF-1 and TIMP-1.
      • Hashizume R.
      • Yamawaki-Ogata A.
      • Ueda Y.
      • Wagner W.
      • Narita Y.
      Mesenchymal stem cells attenuate angiotensin II-induced aortic aneurysm growth in apolipoprotein E-deficient mice.
      Another important finding of this study was that endovascular seeding of BM-MSCs resulted in the formation of a structured arterial tissue in the intima in replacement of the luminal thrombus, a validation of our hypothesis that BM-MSCs induce aortic healing in the context of AAAs and increase wall mass. An endothelium separated VSMC-like cells from blood, contrasting with the surface of the luminal thrombus in control AAAs as well as in human atherosclerotic AAAs, devoid of endothelium. BM-MSC seeding was followed by the accumulation of SM-alpha actin-positive cells surrounded by ECM with collagen and elastin, a recapitulation of artery wall structure. These data are concordant with other data demonstrating production of ECM by MSCs.
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      • Mayr U.
      • Urbich C.
      • Zampetaki A.
      • Prokopi M.
      • Didangelos A.
      • et al.
      Comparative proteomics profiling reveals role of smooth muscle progenitors in extracellular matrix production.
      BM-MSC seeding was followed by generation of elastin in the intimal outgrowth, which has not been observed after VSMC seeding.
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      Elastin is a highly specialized molecule that determines important mechanical properties in arteries.
      • Robins S.
      • Farquharson C.
      Connective tissue components of the blood vessel wall in health and disease.
      It is generally accepted that provisional elastin is produced by VSMCs in the artery wall during ontogeny. Our present observations suggest that in arterial repair, progenitor cells induce the generation of a more specialized tissue than differentiated VSMCs. In order to compare BM-MSCs and VSMCs in their ability to stabilize expanding AAAs, we have designed a dose–response experiment. Five fold less BM-MSCs than VSMCs were needed to gain diameter stabilization. Further investigations are needed to unravel mechanisms by which undifferentiated BM-MSCs perform better than VSMCs in stabilizing AAAs. Importantly, neither BM-MSC nor VSMC
      • Allaire E.
      • Muscatelli-Groux B.
      • Guinault A.M.
      • Pagès C.
      • Goussard A.
      • Mancet C.
      • et al.
      Vascular smooth muscle cell endovascular therapy stabilizes already developed aneurysms in a model of aortic injury elicited by inflammation and proteolysis.
      addition triggered repair of ECM in the media layer of AAAs. The absence of PKH-labeled cells in the AAA wall itself demonstrates that BM-MSCs did not penetrate into the injured vessel wall. Direct differentiation of MSCs into SM-like cells has been suggested in previous experiments in which MSCs were seeded on vascular prostheses.
      • Mirza A.
      • Hyvelin J.M.
      • Rochefort G.Y.
      • Lermusiaux P.
      • Antier D.
      • Awede B.
      • et al.
      Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiological vascular wall.
      However, accumulating data suggest that healing elicited by MSCs in injured tissues involves paracrine mechanisms rather than direct differentiation and incorporation of progenitors in scar tissues.
      • Ortiz L.A.
      • Dutreil M.
      • Fattman C.
      • Pandey A.C.
      • Torres G.
      • Go K.
      • et al.
      Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury.
      Previous attemps to regenerate tissues in the cardiovascular system have been limited by the poor survival of MSCs after seeding.
      • Strauer B.E.
      • Brehm M.
      • Zeus T.
      • Köstering M.
      • Hernandez A.
      • Sorg R.V.
      • et al.
      Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans.
      • Assmus B.
      • Schachinger V.
      • Teupe C.
      • Britten M.
      • Lehmann R.
      • Döbert N.
      • et al.
      Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI).
      • Bel A.
      • Messas E.
      • Agbulut O.
      • Richard P.
      • Samuel J.L.
      • Bruneval P.
      • et al.
      Transplantation of autologous fresh bone marrow into infarcted myocardium: a word of caution.
      In our experiments, survival of BM-MSCs documented by PKH labeling might have been facilitated by direct contact of seeded cells with circulating blood. A previous work has already shown promising results to stabilize the growth of an AAA model in pig by peri adventitial MSCs application.
      • Turnbull I.C.
      • Hadri L.
      • Rapti K.
      • Sadek M.
      • Liang L.
      • Shin H.J.
      • et al.
      Aortic implantation of mesenchymal stem cells after aneurysm injury in a porcine model.
      This finding, together with the development of catheter-based technologies in current vascular interventions, suggests that endovascular, rather than peri-aortic delivery, offers perspectives for an appealing clinical transfer of technology. The most valuable clinical application could be BM MSCs seeding to treat aortic expansion secondary to type II endoleak. BM-MSCs could be easily isolated and expanded in vitro from a patient after bone marrow harvesting. These cells could be injected as an autograft process in the space between the native aortic wall and the stent graft through feeding inferior mesenteric artery catheterism, or direct sac puncture guided by computerized tomography.

      Conclusions

      We have demonstrated in this study that BM-MSCs exert a specialized function in arterial regeneration and functional recovery that transcends mature mesenchymal cells. Endovascular seeding of BM-MSCs induces accumulation of aortic tissue and stabilizes the diameter of expanding AAAs. This finding opens a new field for therapeutic intervention based on catheter-driven cell therapy.

      Funding

      This work was funded by la Fondation de France (grant 2005 005341), la Fondation de l'Avenir pour la Recherche Médicale, and “Fighting aneurysmal Disease”, a grant from the European Union.

      Conflict of Interest

      None.

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