title: Hypoxic preconditioning enhances survival of human adipose-derived stem cells and conditions endothelial cells in vitro.
abstract: To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.
authors.affiliation: O'Brien Institute, Melbourne, Australia.
authors.forename: Samantha Licy
authors.initials: SL
authors.lastname: Stubbs
authors.forename: Sarah Tzu-Feng
authors.initials: ST
authors.lastname: Hsiao
authors.forename: Hitesh Mahendrabhai
authors.initials: HM
authors.lastname: Peshavariya
authors.forename: Shiang Yong
authors.initials: SY
authors.lastname: Lim
authors.forename: Gregory James
authors.initials: GJ
authors.lastname: Dusting
authors.forename: Rodney James
authors.initials: RJ
authors.lastname: Dilley
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D015687:Cell Hypoxia; D048430:Cell Shape; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000378:metabolism; D019610:Cytoprotection; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism; D008875:Middle Aged; D009025:Morpholines / Q000494:pharmacology; D018919:Neovascularization, Physiologic; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D061251:Primary Cell Culture; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D002867:Chromones; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D013778:Tetrazolium Salts; D013844:Thiazoles; D042461:Vascular Endothelial Growth Factor A; D048051:p38 Mitogen-Activated Protein Kinases; C497442:HIF1A protein, human; C467484:VEGFA protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C022616:thiazolyl blue