title: Mechanisms of paracrine cardioprotection by cord blood mesenchymal stromal cells.
abstract: OBJECTIVE
  Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways.
  
  
  METHODS
  Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex(®) immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells (poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic).
  
  
  RESULTS
  CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs.
  
  
  CONCLUSIONS
  The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors.forename: Andreas Matthaeus
authors.initials: AM
authors.lastname: Bader
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors.forename: Andreja
authors.initials: A
authors.lastname: Brodarac
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Klose
authors.affiliation: Institute of Transfusion Medicine and Immunology, Mannheim, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Bieback
authors.affiliation: Heart Center, University of Cologne, Cologne, Germany.
authors.forename: Yeong-Hoon
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kurtz
authors.affiliation: Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany Deutsches Herzzentrum Berlin, Berlin, Germany stamm@dhzb.de.
authors.forename: Christof
authors.initials: C
authors.lastname: Stamm
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology; D020935:MAP Kinase Signaling System; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008955:Models, Cardiovascular; D017202:Myocardial Ischemia; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D017077:Culture Media, Conditioned; D050796:STAT3 Transcription Factor; D051057:Proto-Oncogene Proteins c-akt
keywords: Cardiomyocyte; Cell therapy; Cord blood; Ischaemia; Stem cell