title: Activation of diverse signaling pathways by ex-vivo delivery of multiple cytokines for myocardial repair.
abstract: We tested the hypothesis that simultaneous transgenic overexpression of a select quartet of growth factors activates diverse signaling pathways for mobilization and participation of various stem/progenitor cells for cardiogenesis in the infarcted heart. Human insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1a), and hepatocyte growth factor (HGF) plasmids were synthesized and transfected into skeletal myoblasts (SM) from young male wild-type or transgenic rats expressing green fluorescent protein (GFP). Overexpression of growth factors in transfected SM ((Trans)SM) was confirmed by reverse transcription polymerase chain reaction, western blotting, and fluorescence immunostaining. Using our custom-made growth factor array and western blotting, multiple angiogenic and prosurvival factors were detected in (Trans)SM, including secreted frizzled related protein-1,2,4,5, matrix metalloproteinases-3 and 9, connexin-43, netrin-1, Nos-2, Wnt-3, Akt, MAPK42/44, Stat3, nuclear factor kappa B (NFκB), hypoxia-inducible factor 1 (HIF-1α), and protein kinase C (PKC).   The conditioned medium (CM) from (Trans)SM was cytoprotective for cardiomyocytes following H(2)O(2) treatment [P<0.01 vs. CM from native SM ((Nat)SM)], promoted a higher transwell migration of human umbilical cord vein endothelial cells (223.3±1.8, P<0.01) and in vitro tube formation (47.8±1.9, P<0.01). Intramyocardial transplantation of 1.5×10(6) (Trans)SM (group-3) in a rat model of acute myocardial infarction induced extensive mobilization of cMet(+), ckit(+), ckit(+)/GATA(4+), CXCR4(+), CD44(+), CD31(+), and CD59(+) cells into the infarcted heart on day 7 and improved integration of (Trans)SM in the heart compared to (Nat)SM (group 2) (P<0.05). Extensive neomyogenesis and angiogenesis in group-3 (P<0.01 vs. group-2), with resultant attenuation of infarct size (P<0.01 vs. group-2) and improvement in global heart function (P<0.01 vs. group-2) was observed at 8 weeks. In conclusion, simultaneous activation of diverse signaling pathways by overexpression of multiple growth factors caused massive mobilization and homing of stem/progenitor cells from peripheral circulation, the bone marrow, and the heart for accelerated repair of the infarcted myocardium.
authors.affiliation: Department of Pathology, University of Cincinnati, 231Albert Sabin Way, Cincinnati, OH 45267, USA.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Konoplyannikov
authors.forename: Khawaja Husnain
authors.initials: KH
authors.lastname: Haider
authors.forename: Vien Khach
authors.initials: VK
authors.lastname: Lai
authors.forename: Rafeeq P H
authors.initials: RP
authors.lastname: Ahmed
authors.forename: Shujia
authors.initials: S
authors.lastname: Jiang
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ashraf
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D019610:Cytoprotection; D005260:Female; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006334:Heart Function Tests / Q000379:methods; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D008297:Male; D024510:Muscle Development; D032448:Myoblasts, Skeletal / Q000378:metabolism / Q000637:transplantation; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D017077:Culture Media, Conditioned; C066855:HGF protein, human; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D017228:Hepatocyte Growth Factor; D007334:Insulin-Like Growth Factor I; D006861:Hydrogen Peroxide