title: Unacylated ghrelin promotes skeletal muscle regeneration following hindlimb ischemia via SOD-2-mediated miR-221/222 expression.
abstract: BACKGROUND
  Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated-ghrelin (UnAG) to reduce ischemia-induced tissue damage in a mouse model of peripheral artery disease.
  
  
  RESULTS
  We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen-actived protein kinase signaling UnAG protected against reactive oxygen species-induced cell injuries by inducing the expression of superoxide dismutase-2 (SOD-2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR-221/222, previously linked to muscle regeneration processes, was up-regulated and negatively correlated with p57(Kip2) expression in UnAG-treated mice. UnAG, unlike AG, promoted cell-cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG-induced p38/mitogen-actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD-2-depleted SCs. By siRNA technology, we also demonstrated that SOD-2 is the antioxidant enzyme involved in the control of miR-221/222-driven posttranscriptional p57(Kip2) regulation. Loss-of-function experiments targeting miR-221/222 and local pre-miR-221/222 injection in vivo confirmed a role for miR-221/222 in driving skeletal muscle regeneration after ischemia.
  
  
  CONCLUSIONS
  These results indicate that UnAG-induced skeletal muscle regeneration after ischemia depends on SOD-2-induced miR-221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species-mediated skeletal muscle damage.
authors.affiliation: Department of Medical Sciences, University of Turin, Turin, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Togliatto
authors.forename: Antonella
authors.initials: A
authors.lastname: Trombetta
authors.forename: Patrizia
authors.initials: P
authors.lastname: Dentelli
authors.forename: Paolo
authors.initials: P
authors.lastname: Cotogni
authors.forename: Arturo
authors.initials: A
authors.lastname: Rosso
authors.forename: Matthias H
authors.initials: MH
authors.lastname: Tschöp
authors.forename: Riccarda
authors.initials: R
authors.lastname: Granata
authors.forename: Ezio
authors.initials: E
authors.lastname: Ghigo
authors.forename: Maria F
authors.initials: MF
authors.lastname: Brizzi
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000378:metabolism; D004195:Disease Models, Animal; D054439:Ghrelin / Q000031:analogs & derivatives / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology*; D006614:Hindlimb; D007511:Ischemia / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000098:blood supply / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D017570:MyoD Protein / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D051763:PAX7 Transcription Factor / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000172:deficiency / Q000235:genetics; D012038:Regeneration / Q000187:drug effects*; D032496:Satellite Cells, Skeletal Muscle / Q000187:drug effects / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099156:Superoxide Dismutase 2; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000975:Antioxidants; D050761:Cyclin-Dependent Kinase Inhibitor p57; D054439:Ghrelin; D035683:MicroRNAs; D017570:MyoD Protein; D051763:PAX7 Transcription Factor; D017382:Reactive Oxygen Species; D054440:Receptors, Ghrelin; D013482:Superoxide Dismutase; D048051:p38 Mitogen-Activated Protein Kinases; D000099156:Superoxide Dismutase 2; C081026:CD68 protein, mouse; D000097382:CD68 Molecule; C493965:Cdkn1c protein, mouse; C546122:MIRN221 microRNA, mouse; C546123:MIRN222 microRNA, mouse; C057363:MyoD1 myogenic differentiation protein; C497322:Pax7 protein, mouse
keywords: ROS; UnAG; miRNAs; satellite cells; superoxide dismutase‐2