Therapeutic benefits of young, but not old, adipose-derived mesenchymal stem cells in a chronic mouse model of bleomycin-induced pulmonary fibrosis

Published:September 17, 2015DOI:
      The observation that pulmonary inflammatory lesions and bleomycin (BLM)-induced pulmonary fibrosis spontaneously resolve in young mice, whereas remaining irreversible in aged mice suggests that impairment of pulmonary regeneration and repair is associated with aging. Because mesenchymal stem cells (MSCs) may promote repair after injury, we postulated that differences in MSCs from aged mice may underlie postinjury fibrosis in aging. The potential for young-donor MSCs to inhibit BLM-induced pulmonary fibrosis in aged male mice (>22 months) has not been studied. Adipose-derived MSCs (ASCs) from young (4 months) and old (22 months) male mice were infused 1 day after intratracheal BLM administration. At 21-day sacrifice, aged BLM mice demonstrated lung fibrosis by Ashcroft score, collagen content, and αv-integrin messenger RNA (mRNA) expression. Lung tissue from aged BLM mice receiving young ASCs exhibited decreased fibrosis, matrix metalloproteinase (MMP)-2 activity, oxidative stress, and markers of apoptosis vs BLM controls. Lung mRNA expression of tumor necrosis factor-alpha was also decreased in aged BLM mice receiving young-donor ASCs vs BLM controls. In contrast, old-donor ASC treatment in aged BLM mice did not reduce fibrosis and related markers. On examination of the cells, young-donor ASCs had decreased mRNA expression of MMP-2, insulin-like growth factor (IGF) receptor, and protein kinase B (AKT) activation compared with old-donor ASCs. These results show that the BLM-induced pulmonary fibrosis in aged mice could be blocked by young-donor ASCs and that the mechanisms involve changes in collagen turnover and markers of inflammation.


      IPF (idiopathic pulmonary fibrosis), BLM (bleomycin), MMP (matrix metalloproteinase), MSC (mesenchymal stem cell), ASC (adipose-derived mesenchymal stem cell), BM-MSC (bone marrow-derived mesenchymal stem cell), oASC (old-donor ASC), yASC (young-donor ASC), AKT (protein kinase B), TGF-β (transforming growth factor-β), TNF-α (tumor necrosis factor-alpha), LPS (lipopolysaccharide), PE (phycoerythrin), FITC (fluorescein isothiocyanate), VEGF (vascular endothelial growth factor), IGF (insulin-like growth factor), PCR (polymerase chain reaction), pAKT (phosphorylated AKT), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling), ROS (reactive oxygen species), IgG (immunoglobulin G), ANOVA (analysis of variance), FACS (fluorescence activated cell sorting), ARDS (acute respiratory distress syndrome)
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        • Raghu G.
        • Collard H.R.
        • Egan J.J.
        • et al.
        An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.
        Am J Respir Crit Care Med. 2011; 183: 788-824
        • Fernandez I.E.
        • Eickelberg O.
        New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis.
        Lancet. 2012; 380: 680-688
        • Raghu G.
        • Weycker D.
        • Edelsberg J.
        • Bradford W.Z.
        • Oster G.
        Incidence and prevalence of idiopathic pulmonary fibrosis.
        Am J Respir Crit Care Med. 2006; 174: 810-816
        • Hunninghake G.M.
        A new hope for idiopathic pulmonary fibrosis.
        N Engl J Med. 2014; 370: 2142-2143
        • Izbicki G.
        • Segel M.J.
        • Christensen T.G.
        • Conner M.W.
        • Breuer R.
        Time course of bleomycin-induced lung fibrosis.
        Int J Exp Pathol. 2002; 83: 111-119
        • Ortiz L.A.
        • Gambelli F.
        • McBride C.
        • et al.
        Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.
        Proc Natl Acad Sci U S A. 2003; 100: 8407-8411
        • Rojas M.
        • Xu J.
        • Woods C.R.
        • et al.
        Bone marrow-derived mesenchymal stem cells in repair of the injured lung.
        Am J Respir Cell Mol Biol. 2005; 33: 145-152
        • Chaudhary N.I.
        • Schnapp A.
        • Park J.E.
        Pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model.
        Am J Respir Crit Care Med. 2006; 173: 769-776
        • Ortiz L.A.
        • Dutreil M.
        • Fattman C.
        • et al.
        Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury.
        Proc Natl Acad Sci U S A. 2007; 104: 11002-11007
        • Aguilar S.
        • Scotton C.J.
        • McNulty K.
        • et al.
        Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis.
        PLoS One. 2009; 4: e8013
        • Degryse A.L.
        • Tanjore H.
        • Xu X.C.
        • et al.
        Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis.
        Am J Physiol Lung Cell Mol Physiol. 2010; 299: L442-L452
        • Redente E.F.
        • Jacobsen K.M.
        • Solomon J.J.
        • et al.
        Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis.
        Am J Physiol Lung Cell Mol Physiol. 2011; 301: L510-L518
        • Sueblinvong V.
        • Neujahr D.C.
        • Mills S.T.
        • et al.
        Predisposition for disrepair in the aged lung.
        Am J Med Sci. 2012; 344: 41-51
        • Foskett A.M.
        • Bazhanov N.
        • Ti X.
        • et al.
        Phase-directed therapy: TSG-6 targeted to early inflammation improves bleomycin-injured lungs.
        Am J Physiol Lung Cell Mol Physiol. 2014; 306: L120-L131
        • Leach H.G.
        • Chrobak I.
        • Han R.
        • Trojanowska M.
        Endothelial cells recruit macrophages and contribute to a fibrotic milieu in bleomycin lung injury.
        Am J Respir Cell Mol Biol. 2013; 49: 1093-1101
        • Hecker L.
        • Logsdon N.J.
        • Kurundkar D.
        • et al.
        Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.
        Sci Transl Med. 2014; 6: 231ra47
        • Peng R.
        • Sridhar S.
        • Tyagi G.
        • et al.
        Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for “active” disease.
        PLoS One. 2013; 8: e59348
        • Redente E.F.
        • Keith R.C.
        • Janssen W.
        • et al.
        Tumor necrosis factor-α accelerates the resolution of established pulmonary fibrosis in mice by targeting profibrotic lung macrophages.
        Am J Respir Cell Mol Biol. 2014; 50: 825-837
        • Bustos M.L.
        • Huleihel L.
        • Kapetanaki M.G.
        • et al.
        Aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response.
        Am J Respir Crit Care Med. 2014; 189: 787-798
        • Barkauskas C.E.
        • Cronce M.J.
        • Rackley C.R.
        • et al.
        Type 2 alveolar cells are stem cells in adult lung.
        J Clin Invest. 2013; 123: 3025-3036
        • Selman M.
        • Pardo A.
        Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers.
        Proc Am Thorac Soc. 2006; 3: 364-372
        • Richeldi L.
        • du Bois R.M.
        • Raghu G.
        • et al.
        Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis.
        N Engl J Med. 2014; 370: 2071-2082
        • Kotton D.N.
        • Morrisey E.E.
        Lung regeneration: mechanisms, applications and emerging stem cell populations.
        Nat Med. 2014; 20: 822-832
        • Huang K.
        • Kang X.
        • Wang X.
        • et al.
        Conversion of bone marrow mesenchymal stem cells into type II alveolar epithelial cells reduces pulmonary fibrosis by decreasing oxidative stress in rats.
        Mol Med Rep. 2015; 11: 1685-1692
        • Garcia O.
        • Carraro G.
        • Turcatel G.
        • et al.
        Amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via CCL2 modulation in bronchoalveolar lavage.
        PLoS One. 2013; 8: e71679
        • Villeda S.A.
        • Plambeck K.E.
        • Middeldorp J.
        • et al.
        Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.
        Nat Med. 2014; 20: 659-663
        • Paul S.M.
        • Reddy K.
        Young blood rejuvenates old brains.
        Nat Med. 2014; 20: 582-583
        • Feng Z.
        • Plati A.R.
        • Cheng Q.L.
        • et al.
        Glomerular aging in females is a multi-stage reversible process mediated by phenotypic changes in progenitors.
        Am J Pathol. 2005; 167: 355-363
        • Furuichi K.
        • Shintani H.
        • Sakai Y.
        • et al.
        Effects of adipose-derived mesenchymal cells on ischemia-reperfusion injury in kidney.
        Clin Exp Nephrol. 2012; 16: 679-689
        • Saito S.
        • Nakayama T.
        • Hashimoto N.
        • et al.
        Mesenchymal stem cells stably transduced with a dominant-negative inhibitor of CCL2 greatly attenuate bleomycin-induced lung damage.
        Am J Pathol. 2011; 179: 1088-1094
        • Wang Q.
        • Zhu H.
        • Zhou W.G.
        • et al.
        N-acetylcysteine-pretreated human embryonic mesenchymal stem cell administration protects against bleomycin-induced lung injury.
        Am J Med Sci. 2013; 346: 113-122
        • Ashcroft T.
        • Simpson J.M.
        • Timbrell V.
        Simple method of estimating severity of pulmonary fibrosis on a numerical scale.
        J Clin Pathol. 1988; 41: 467-470
        • Karl M.
        • Berho M.
        • Pignac-Kobinger J.
        • Striker G.E.
        • Elliot S.J.
        Differential effects of continuous and intermittent 17beta-estradiol replacement and tamoxifen therapy on the prevention of glomerulosclerosis: modulation of the mesangial cell phenotype in vivo.
        Am J Pathol. 2006; 169: 351-361
        • Potier M.
        • Karl M.
        • Zheng F.
        • et al.
        Estrogen-related abnormalities in glomerulosclerosis-prone mice: reduced mesangial cell estrogen receptor expression and prosclerotic response to estrogens.
        Am J Pathol. 2002; 160: 1877-1885
        • Glassberg M.K.
        • Choi R.
        • Manzoli V.
        • et al.
        17beta-estradiol replacement reverses age-related lung disease in estrogen-deficient C57BL/6J mice.
        Endocrinology. 2014; 155: 441-448
        • Henderson N.C.
        • Arnold T.D.
        • Katamura Y.
        • et al.
        Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.
        Nat Med. 2013; 19: 1617-1624
        • Sueblinvong V.
        • Neveu W.A.
        • Neujahr D.C.
        • et al.
        Aging promotes pro-fibrotic matrix production and increases fibrocyte recruitment during acute lung injury.
        Adv Biosci Biotechnol. 2014; 5: 19-30
        • Parker M.W.
        • Rossi D.
        • Peterson M.
        • et al.
        Fibrotic extracellular matrix activates a profibrotic positive feedback loop.
        J Clin Invest. 2014; 124: 1622-1635
        • Chung M.P.
        • Monick M.M.
        • Hamzeh N.Y.
        • et al.
        Role of repeated lung injury and genetic background in bleomycin-induced fibrosis.
        Am J Respir Cell Mol Biol. 2003; 29: 375-380
        • Lee S.H.
        • Lee E.J.
        • Lee S.Y.
        • et al.
        The effect of adipose stem cell therapy on pulmonary fibrosis induced by repetitive intratracheal bleomycin in mice.
        Exp Lung Res. 2014; 40: 117-125
        • Cargnoni A.
        • Gibelli L.
        • Tosini A.
        • et al.
        Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis.
        Cell Transplant. 2009; 18: 405-422
        • Moodley Y.
        • Atienza D.
        • Manuelpillai U.
        • et al.
        Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury.
        Am J Pathol. 2009; 175: 303-313
        • Park S.A.
        • Kim M.J.
        • Park S.Y.
        • et al.
        EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-β/Smad and ROS signaling.
        Cell Mol Life Sci. 2015; 72: 2023-2039
        • Nkyimbeng T.
        • Ruppert C.
        • Shiomi T.
        • et al.
        Pivotal role of matrix metalloproteinase 13 in extracellular matrix turnover in idiopathic pulmonary fibrosis.
        PLoS One. 2013; 8: e73279
        • Kapetanaki M.G.
        • Mora A.L.
        • Rojas M.
        Influence of age on wound healing and fibrosis.
        J Pathol. 2013; 229: 310-322
        • Elliot S.J.
        • Catanuto P.
        • Espinosa-Heidmann D.G.
        • et al.
        Estrogen receptor beta protects against in vivo injury in RPE cells.
        Exp Eye Res. 2010; 90: 10-16
        • Glassberg M.K.
        • Elliot S.J.
        • Fritz J.
        • et al.
        Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness.
        J Clin Endocrinol Metab. 2008; 93: 1625-1633
        • Kassira N.
        • Glassberg M.K.
        • Jones C.
        • et al.
        Estrogen deficiency and tobacco smoke exposure promote matrix metalloproteinase-13 activation in skin of aging B6 mice.
        Ann Plast Surg. 2009; 63: 318-322
        • Cai Y.
        • Zhu L.
        • Zhang F.
        • et al.
        Noninvasive monitoring of pulmonary fibrosis by targeting matrix metalloproteinases (MMPs).
        Mol Pharm. 2013; 10: 2237-2247
        • Knoblauch A.
        • Will C.
        • Goncharenko G.
        • Ludwig S.
        • Wixler V.
        The binding of Mss4 to alpha-integrin subunits regulates matrix metalloproteinase activation and fibronectin remodeling.
        FASEB J. 2007; 21: 497-510
        • Rajashekhar G.
        • Shivanna M.
        • Kompella U.B.
        • Wang Y.
        • Srinivas S.P.
        Role of MMP-9 in the breakdown of barrier integrity of the corneal endothelium in response to TNF-α.
        Exp Eye Res. 2014; 122: 77-85
        • Choo S.
        • Papandria D.
        • Zhang Y.
        • et al.
        Outcomes analysis after percutaneous abdominal drainage and exploratory laparotomy for necrotizing enterocolitis in 4,657 infants.
        Pediatr Surg Int. 2011; 27: 747-753
        • Russo R.C.
        • Garcia C.C.
        • Barcelos L.S.
        • et al.
        Phosphoinositide 3-kinase gamma plays a critical role in bleomycin-induced pulmonary inflammation and fibrosis in mice.
        J Leukoc Biol. 2011; 89: 269-282
        • Park S.
        • Ahn J.Y.
        • Lim M.J.
        • et al.
        Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts.
        J Mol Med (Berl). 2010; 88: 807-816
        • Cheresh P.
        • Kim S.J.
        • Tulasiram S.
        • Kamp D.W.
        Oxidative stress and pulmonary fibrosis.
        Biochim Biophys Acta. 2013; 1832: 1028-1040
        • Nelson K.K.
        • Melendez J.A.
        Mitochondrial redox control of matrix metalloproteinases.
        Free Radic Biol Med. 2004; 37: 768-784
        • Smith N.J.
        • Chan H.W.
        • Osborne J.E.
        • Thomas W.G.
        • Hannan R.D.
        Hijacking epidermal growth factor receptors by angiotensin II: new possibilities for understanding and treating cardiac hypertrophy.
        Cell Mol Life Sci. 2004; 61: 2695-2703
        • Ray P.D.
        • Huang B.W.
        • Tsuji Y.
        Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.
        Cell Signal. 2012; 24: 981-990
        • Stadtman E.R.
        Role of oxidant species in aging.
        Curr Med Chem. 2004; 11: 1105-1112
        • Schiller H.B.
        • Fernandez I.E.
        • Burgstaller G.
        • et al.
        Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair.
        Mol Syst Biol. 2015; 11: 819
        • Tatsumi K.
        • Ohashi K.
        • Matsubara Y.
        • et al.
        Tissue factor triggers procoagulation in transplanted mesenchymal stem cells leading to thromboembolism.
        Biochem Biophys Res Commun. 2013; 431: 203-209


      Jun Tashiro, MD, MPH, is a research fellow in the Department of Surgery at the University of Miami Miller School of Medicine. His article is based on a presentation given at the 2015 Combined Annual Meeting of the Central Society for Clinical and Translational Research and the Midwestern Section American Federation for Medical Research, held in Chicago, Ill. Dr. Tashiro is interested in optimizing mesenchymal stem cell-based therapy and identifying its most active components in the setting of pulmonary fibrosis and other processes mediated by inflammation.