title: Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart.
abstract: BACKGROUND
  Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium.
  
  
  OBJECTIVE
  To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene.
  
  
  METHODS
  Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants.
  
  
  RESULTS
  Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05).
  
  
  CONCLUSIONS
  The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.
authors.affiliation: Institute of Human Genetics, Polish Academy of Sciences. kurpimac@man.poznan.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Janeczek
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zimna
authors.forename: Natalia
authors.initials: N
authors.lastname: Rozwadowska
authors.forename: Monika
authors.initials: M
authors.lastname: Fraczek
authors.forename: Paulina
authors.initials: P
authors.lastname: Kucharzewska
authors.forename: Marek
authors.initials: M
authors.lastname: Ruciński
authors.forename: Tomasz
authors.initials: T
authors.lastname: Mietkiewski
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kolanowski
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Malcher
authors.forename: Maciej
authors.initials: M
authors.lastname: Kurpisz
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D018274:Electroporation; D042783:Endothelial Cells / Q000166:cytology; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D032390:Myoblasts, Smooth Muscle / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D009203:Myocardial Infarction / Q000628:therapy*; D018919:Neovascularization, Physiologic / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D018384:Oxidative Stress / Q000235:genetics; D012038:Regeneration / Q000235:genetics; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A
chemical_list: D042461:Vascular Endothelial Growth Factor A; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III