Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis

S. Zanotti, S. Gibertini, F. Blasevich, C. Bragato, A. Ruggieri, S. Saredi, M. Fabbri, P. Bernasconi, L. Maggi, R. Mantegazza, M. Mora

Research output: Contribution to journalArticle

Abstract

Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis. © 2018 International Society of Matrix Biology
Original languageEnglish
Pages (from-to)77-100
Number of pages24
JournalMatrix Biology
Volume74
DOIs
Publication statusPublished - 2018

Fingerprint

Exosomes
MicroRNAs
Skeletal Muscle
Fibrosis
Fibroblasts
Muscles
Duchenne Muscular Dystrophy
Collagen
Cardiotoxins
Caveolin 1
Untranslated RNA
Messenger RNA
Myofibroblasts
MAP Kinase Signaling System
Myoblasts
Fibronectins
Cell Communication
Actins

Keywords

  • Duchenne muscle dystrophy
  • Exosomes
  • Fibroblasts
  • Fibrosis
  • miRNAs
  • Myofibroblasts
  • alpha smooth muscle actin
  • cardiotoxin
  • caveolin 1
  • ceramide
  • collagen
  • fibronectin
  • microRNA
  • microRNA 199a 5p
  • mitogen activated protein kinase
  • protein kinase B
  • unclassified drug
  • adult
  • Akt signaling
  • animal experiment
  • animal model
  • animal tissue
  • Article
  • cell proliferation
  • cell transdifferentiation
  • cell transfer
  • cellular distribution
  • controlled study
  • Duchenne muscular dystrophy
  • electric potential
  • enzyme activation
  • exosome
  • fibroblast
  • genetic transfection
  • human
  • human cell
  • MAPK signaling
  • microarray analysis
  • mouse
  • mRNA expression level
  • muscle biopsy
  • myoblast
  • myofibroblast
  • nonhuman
  • organelle biogenesis
  • pathogenesis
  • phenotype
  • priority journal
  • protein expression
  • skeletal muscle
  • tibialis anterior muscle
  • transcription regulation
  • upregulation

Cite this

Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis. / Zanotti, S.; Gibertini, S.; Blasevich, F.; Bragato, C.; Ruggieri, A.; Saredi, S.; Fabbri, M.; Bernasconi, P.; Maggi, L.; Mantegazza, R.; Mora, M.

In: Matrix Biology, Vol. 74, 2018, p. 77-100.

Research output: Contribution to journalArticle

@article{d8ff5c7590144649b0eed2f7772cdb67,
title = "Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis",
abstract = "Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis. {\circledC} 2018 International Society of Matrix Biology",
keywords = "Duchenne muscle dystrophy, Exosomes, Fibroblasts, Fibrosis, miRNAs, Myofibroblasts, alpha smooth muscle actin, cardiotoxin, caveolin 1, ceramide, collagen, fibronectin, microRNA, microRNA 199a 5p, mitogen activated protein kinase, protein kinase B, unclassified drug, adult, Akt signaling, animal experiment, animal model, animal tissue, Article, cell proliferation, cell transdifferentiation, cell transfer, cellular distribution, controlled study, Duchenne muscular dystrophy, electric potential, enzyme activation, exosome, fibroblast, genetic transfection, human, human cell, MAPK signaling, microarray analysis, mouse, mRNA expression level, muscle biopsy, myoblast, myofibroblast, nonhuman, organelle biogenesis, pathogenesis, phenotype, priority journal, protein expression, skeletal muscle, tibialis anterior muscle, transcription regulation, upregulation",
author = "S. Zanotti and S. Gibertini and F. Blasevich and C. Bragato and A. Ruggieri and S. Saredi and M. Fabbri and P. Bernasconi and L. Maggi and R. Mantegazza and M. Mora",
note = "Cited By :1 Export Date: 5 February 2019 CODEN: MTBOE Correspondence Address: Mora, M.; Muscle Cell Biology Lab, Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Via Temolo 4, Italy; email: marina.mora@istituto-besta.it Chemicals/CAS: collagen, 9007-34-5; fibronectin, 86088-83-7; mitogen activated protein kinase, 142243-02-5; protein kinase B, 148640-14-6 Funding details: University of Southampton Funding details: GTB12001F Funding details: Associazione Italiana per la Ricerca sul Cancro, AIRC, 10007 Funding text 1: We thank Dr. Antonello Maruotti of the Dipartimento di Giurisprudenza, Economia, Politica e Lingue Moderne, LUMSA Universit{\`a}, Roma and Centre for Innovation and Leadership in Health Sciences, University of Southampton, Southampton, UK, for help with statistics. We thank the EuroBioBank and Telethon Network of Genetic Biobanks (GTB12001F to MM) for providing biological samples. This work was supported by grants for current research from the Italian Ministry of Health , years 2015–2017. M.F. is supported by funds from AIRC 5 × 1000 n. 10007 . References: Smith, L.R., Barton, E.R., Regulation of fibrosis in muscular dystrophy (2018) Matrix Biol., 68-69, pp. 602-615. , (Review); Tidball, J.G., Mechanisms of muscle injury, repair, and regeneration (2011) Compr. Physiol., 1, pp. 2029-2062; Schaefer, L., Decoding fibrosis: mechanisms and translational aspects (2018) Matrix Biol., 68-69, pp. 1-7; Martinez, F.J., Collard, H.R., Pardo, A., Raghu, G., Richeldi, L., Selman, M., Swigris, J.J., Wells, A.U., Idiopathic pulmonary fibrosis (2017) Nat. Rev. Dis. Prim, 3; Schuppan, D., Ashfaq-Khan, M., Yang, A.T., Kim, Y.O., Liver fibrosis: direct antifibrotic agents and targeted therapies (2018) Matrix Biol., 68-69, pp. 435-451; Gewin, L.S., Renal fibrosis: primacy of the proximal tubule (2018) Matrix Biol., 68-69, pp. 248-262; Li, L., Zhao, Q., Kong, W., Extracellular matrix remodeling and cardiac fibrosis (2018) Matrix Biol., 68-69, pp. 490-506; Schulz, J.-N., Plomann, M., Sengle, G., Gullberg, D., Krieg, T., Eckes, B., New developments on skin fibrosis - essential signals emanating from the extracellular matrix for the control of myofibroblasts (2018) Matrix Biol., 68-69, pp. 522-532; Oliveira Dias, D., G{\"o}ritz, C., Fibrotic scarring following lesions to the central nervous system (2018) Matrix Biol., 68-69, pp. 561-570; Wynn, T.A., Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases (2007) J. Clin. Invest., 117, pp. 524-529; Kramann, R., Di Rocco, D.P., Humphreys, B.D., Understanding the origin, activation and regulation of matrix-producing myofibroblasts for treatment of fibrotic disease (2013) J. Pathol., 231, pp. 273-289; Piersma, B., R.A. Bank, Boersema, M., Signaling in fibrosis: TGF-β, WNT, and YAP/TAZ converge (2015) Front. Med. (Lausanne), 2, p. 59; Thannickal, V.J., Lee, D.Y., White, E.S., Cui, Z., Larios, J.M., Chacon, R., Horowitz, J.C., Thomas, P.E., Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase (2003) J. Biol. Chem., 278, pp. 12384-12389; Pakshir, P., Hinz, B., The big five in fibrosis: macrophages, myofibroblasts, matrix, mechanics, and miscommunication (2018) Matrix Biol., 68-69, pp. 81-93; Kalluri, R., Weinberg, R.A., The basics of epithelial–mesenchymal transition (2009) J. Clin. 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year = "2018",
doi = "10.1016/j.matbio.2018.07.003",
language = "English",
volume = "74",
pages = "77--100",
journal = "Matrix Biology",
issn = "0945-053X",
publisher = "Elsevier B.V.",

}

TY - JOUR

T1 - Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis

AU - Zanotti, S.

AU - Gibertini, S.

AU - Blasevich, F.

AU - Bragato, C.

AU - Ruggieri, A.

AU - Saredi, S.

AU - Fabbri, M.

AU - Bernasconi, P.

AU - Maggi, L.

AU - Mantegazza, R.

AU - Mora, M.

N1 - Cited By :1 Export Date: 5 February 2019 CODEN: MTBOE Correspondence Address: Mora, M.; Muscle Cell Biology Lab, Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Via Temolo 4, Italy; email: marina.mora@istituto-besta.it Chemicals/CAS: collagen, 9007-34-5; fibronectin, 86088-83-7; mitogen activated protein kinase, 142243-02-5; protein kinase B, 148640-14-6 Funding details: University of Southampton Funding details: GTB12001F Funding details: Associazione Italiana per la Ricerca sul Cancro, AIRC, 10007 Funding text 1: We thank Dr. Antonello Maruotti of the Dipartimento di Giurisprudenza, Economia, Politica e Lingue Moderne, LUMSA Università, Roma and Centre for Innovation and Leadership in Health Sciences, University of Southampton, Southampton, UK, for help with statistics. We thank the EuroBioBank and Telethon Network of Genetic Biobanks (GTB12001F to MM) for providing biological samples. This work was supported by grants for current research from the Italian Ministry of Health , years 2015–2017. M.F. is supported by funds from AIRC 5 × 1000 n. 10007 . References: Smith, L.R., Barton, E.R., Regulation of fibrosis in muscular dystrophy (2018) Matrix Biol., 68-69, pp. 602-615. , (Review); Tidball, J.G., Mechanisms of muscle injury, repair, and regeneration (2011) Compr. Physiol., 1, pp. 2029-2062; Schaefer, L., Decoding fibrosis: mechanisms and translational aspects (2018) Matrix Biol., 68-69, pp. 1-7; Martinez, F.J., Collard, H.R., Pardo, A., Raghu, G., Richeldi, L., Selman, M., Swigris, J.J., Wells, A.U., Idiopathic pulmonary fibrosis (2017) Nat. Rev. Dis. Prim, 3; Schuppan, D., Ashfaq-Khan, M., Yang, A.T., Kim, Y.O., Liver fibrosis: direct antifibrotic agents and targeted therapies (2018) Matrix Biol., 68-69, pp. 435-451; Gewin, L.S., Renal fibrosis: primacy of the proximal tubule (2018) Matrix Biol., 68-69, pp. 248-262; Li, L., Zhao, Q., Kong, W., Extracellular matrix remodeling and cardiac fibrosis (2018) Matrix Biol., 68-69, pp. 490-506; Schulz, J.-N., Plomann, M., Sengle, G., Gullberg, D., Krieg, T., Eckes, B., New developments on skin fibrosis - essential signals emanating from the extracellular matrix for the control of myofibroblasts (2018) Matrix Biol., 68-69, pp. 522-532; Oliveira Dias, D., Göritz, C., Fibrotic scarring following lesions to the central nervous system (2018) Matrix Biol., 68-69, pp. 561-570; Wynn, T.A., Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases (2007) J. Clin. Invest., 117, pp. 524-529; Kramann, R., Di Rocco, D.P., Humphreys, B.D., Understanding the origin, activation and regulation of matrix-producing myofibroblasts for treatment of fibrotic disease (2013) J. Pathol., 231, pp. 273-289; Piersma, B., R.A. Bank, Boersema, M., Signaling in fibrosis: TGF-β, WNT, and YAP/TAZ converge (2015) Front. Med. (Lausanne), 2, p. 59; Thannickal, V.J., Lee, D.Y., White, E.S., Cui, Z., Larios, J.M., Chacon, R., Horowitz, J.C., Thomas, P.E., Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase (2003) J. Biol. Chem., 278, pp. 12384-12389; Pakshir, P., Hinz, B., The big five in fibrosis: macrophages, myofibroblasts, matrix, mechanics, and miscommunication (2018) Matrix Biol., 68-69, pp. 81-93; Kalluri, R., Weinberg, R.A., The basics of epithelial–mesenchymal transition (2009) J. Clin. 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PY - 2018

Y1 - 2018

N2 - Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis. © 2018 International Society of Matrix Biology

AB - Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis. © 2018 International Society of Matrix Biology

KW - Duchenne muscle dystrophy

KW - Exosomes

KW - Fibroblasts

KW - Fibrosis

KW - miRNAs

KW - Myofibroblasts

KW - alpha smooth muscle actin

KW - cardiotoxin

KW - caveolin 1

KW - ceramide

KW - collagen

KW - fibronectin

KW - microRNA

KW - microRNA 199a 5p

KW - mitogen activated protein kinase

KW - protein kinase B

KW - unclassified drug

KW - adult

KW - Akt signaling

KW - animal experiment

KW - animal model

KW - animal tissue

KW - Article

KW - cell proliferation

KW - cell transdifferentiation

KW - cell transfer

KW - cellular distribution

KW - controlled study

KW - Duchenne muscular dystrophy

KW - electric potential

KW - enzyme activation

KW - exosome

KW - fibroblast

KW - genetic transfection

KW - human

KW - human cell

KW - MAPK signaling

KW - microarray analysis

KW - mouse

KW - mRNA expression level

KW - muscle biopsy

KW - myoblast

KW - myofibroblast

KW - nonhuman

KW - organelle biogenesis

KW - pathogenesis

KW - phenotype

KW - priority journal

KW - protein expression

KW - skeletal muscle

KW - tibialis anterior muscle

KW - transcription regulation

KW - upregulation

U2 - 10.1016/j.matbio.2018.07.003

DO - 10.1016/j.matbio.2018.07.003

M3 - Article

VL - 74

SP - 77

EP - 100

JO - Matrix Biology

JF - Matrix Biology

SN - 0945-053X

ER -