title: Myoblast-conditioned media improve regeneration and revascularization of ischemic muscles in diabetic mice.
abstract: BACKGROUND
  Diabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties. In myoblasts and muscle satellite cells HO-1 improves survival, proliferation and production of proangiogenic growth factors. Induction of HO-1 in injured tissues facilitates neovascularization, the process impaired in diabetes. We aimed to examine whether conditioned media from the HO-1 overexpressing myoblast cell line can improve a blood-flow recovery in ischemic muscles of diabetic mice.
  
  
  METHODS
  Analysis of myogenic markers was performed at the mRNA level in primary muscle satellite cells, isolated by a pre-plate technique from diabetic db/db and normoglycemic wild-type mice, and then cultured under growth or differentiation conditions. Hind limb ischemia was performed by femoral artery ligation in db/db mice and blood recovery was monitored by laser Doppler measurements. Mice were treated with a single intramuscular injection of conditioned media harvested from wild-type C2C12 myoblast cell line, C2C12 cells stably transduced with HO-1 cDNA, or with unconditioned media.
  
  
  RESULTS
  Expression of HO-1 was lower in muscle satellite cells isolated from muscles of diabetic db/db mice when compared to their wild-type counterparts, what was accompanied by increased levels of Myf5 or CXCR4, and decreased Mef2 or Pax7. Such cells also displayed diminished differentiation potential when cultured in vitro, as shown by less effective formation of myotubes and reduced expression of myogenic markers (myogenic differentiation antigen - myoD, myogenin and myosin). Blood flow recovery after induction of severe hind limb ischemia was delayed in db/db mice compared to that in normoglycemic individuals. To improve muscle regeneration after ischemia, conditioned media collected from differentiating C2C12 cells (control and HO-1 overexpressing) were injected into hind limbs of diabetic mice. Analysis of blood flow revealed that media from HO-1 overexpressing cells accelerated blood-flow recovery, while immunohistochemical staining assessment of vessel density in injected muscle confirmed increased angiogenesis. The effect might be mediated by stromal-cell derived factor-1α proangiogenic factor, as its secretion is elevated in HO-1 overexpressing cells.
  
  
  CONCLUSIONS
  In conclusion, paracrine stimulation of angiogenesis in ischemic skeletal muscle using conditioned media may be a safe approach exploiting protective and proangiogenic properties of HO-1 in diabetes.
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. m.kozakowska@uj.edu.pl.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kozakowska
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. j.kotlinowski@uj.edu.pl.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Kotlinowski
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. anna.grochot-przeczek@uj.edu.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Grochot-Przeczek
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. maciek.ciesla@uj.edu.pl.
authors.forename: Maciej
authors.initials: M
authors.lastname: Ciesla
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. bartosz.pilecki@uj.edu.pl.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Pilecki
authors.affiliation: R&D Department, Adamed Ltd, Pienkow 149, Czosnow, 05-152, Poland. rderlacz@biol.uw.edu.pl.|Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, University of Warsaw, Miecznikowa 1, Warsaw, 02-096, Poland. rderlacz@biol.uw.edu.pl.
authors.forename: Rafal
authors.initials: R
authors.lastname: Derlacz
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. jozef.dulak@uj.edu.pl.|Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland. jozef.dulak@uj.edu.pl.
authors.forename: Jozef
authors.initials: J
authors.lastname: Dulak
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Krakow, 30-387, Poland. alicja.jozkowicz@uj.edu.pl.
authors.forename: Alicja
authors.initials: A
authors.lastname: Jozkowicz
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000032:analysis; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000378:metabolism / Q000473:pathology*; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D007511:Ischemia / Q000150:complications / Q000378:metabolism / Q000473:pathology; D064326:MEF2 Transcription Factors / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000502:physiology*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5 / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D051763:PAX7 Transcription Factor / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000378:metabolism; D012038:Regeneration / Q000187:drug effects*; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
chemical_list: C514936:CXCR4 protein, mouse; D054377:Chemokine CXCL12; D017077:Culture Media, Conditioned; D064326:MEF2 Transcription Factors; C497418:Myf5 protein, mouse; D051787:Myogenic Regulatory Factor 5; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; D019718:Receptors, CXCR4; D042461:Vascular Endothelial Growth Factor A; D051547:Heme Oxygenase-1