title: TGF-β2 treatment enhances cytoprotective factors released from embryonic stem cells and inhibits apoptosis in infarcted myocardium.
abstract: We investigated whether factors released from mouse embryonic stem (ES) cells primed with and without transforming growth factor (TGF)-β2 inhibit iodoacetic acid (IAA)- and H(2)O(2)-induced apoptosis in the cell culture system as well as after transplantation in the infarcted heart. We generated conditioned media (CMs) from ES cells primed with and without TGF-β2 and determined their effects on IAA- and H(2)O(2)-induced apoptosis in H9c2 cells. We also transplanted both ES-CMs in the infarcted heart to determine the effects on apoptosis and cardiac function after myocardial infarction (MI) at day (D)1 and D14. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, apoptotic ELISA, and cell viability data demonstrated significantly (P < 0.05) reduced apoptosis with ES-CM compared with controls in both cell culture models. Moreover, TGF-β2-primed ES-CM (T-ES-CM) demonstrated enhanced beneficial effects, with further reduced (P < 0.05) apoptosis compared with ES-CM, suggesting the a presence of additional cytoprotective released factors after TGF-β2 treatment. Next, our in vivo apoptosis data suggested significant decrease in apoptosis with both ES-CMs compared with MI alone at D1 and D14. Notably, T-ES-CM demonstrated significant (P < 0.05) inhibition of apoptosis and fibrosis with improved cardiac function compared with ES-CM at D14, whereas no such effects were observed at D1. Next, we confirmed that apoptosis is mediated through a prosurvival Akt pathway. Moreover, we determined that after TGF-β2 treatment there was a two- to fivefold increase in cytoprotective released factors (interleukin-10, stem cell factor, tissue inhibitor of matrix metalloproteinase-1, and VEGF) with T-ES-CM compared with ES-CM. In conclusion, we suggest that factors released from ES cells with and without TGF-β2 treatment contain antiapoptotic factors that inhibit apoptosis in vitro and in vivo. We also suggest that T-ES-CM demonstrates additional beneficial effects that provide useful information for future therapeutic applications in regenerative medicine.
authors.affiliation: Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, 32816, USA. dsingla@mail.ucf.edu
authors.forename: Dinender K
authors.initials: DK
authors.lastname: Singla
authors.forename: Reetu D
authors.initials: RD
authors.lastname: Singla
authors.forename: Stephanie
authors.initials: S
authors.lastname: Lamm
authors.forename: Carley
authors.initials: C
authors.lastname: Glass
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005260:Female; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000494:pharmacology; D016753:Interleukin-10 / Q000378:metabolism; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000188:drug therapy* / Q000628:therapy; D019089:Stem Cell Factor / Q000378:metabolism; D033581:Stem Cell Transplantation; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D007210:Indoleacetic Acids; D019089:Stem Cell Factor; D019715:Tissue Inhibitor of Metalloproteinase-1; D053781:Transforming Growth Factor beta2; D042442:Vascular Endothelial Growth Factors; D016753:Interleukin-10; C030737:indoleacetic acid; D006861:Hydrogen Peroxide; D020781:Matrix Metalloproteinase 1