title: Hypoxia Enhances Proliferation of Human Adipose-Derived Stem Cells via HIF-1ɑ Activation.
abstract: BACKGROUND
  Adipose tissue-derived stem cells (ASCs) have been recently isolated from human subcutaneous adipose tissue. ASCs may be useful in regenerative medicine as an alternative to bone marrow-derived stem cells. Changes in the oxygen concentration influence physiological activities, such as stem cell proliferation. However, the effects of the oxygen concentration on ASCs remain unclear. In the present study, the effects of hypoxia on ASC proliferation were examined.
  
  
  METHODS
  Normal human adipose tissue was collected from the lower abdomen, and ASCs were prepared with collagenase treatment. The ASCs were cultured in hypoxic (1%) or normoxic (20%) conditions. Cell proliferation was investigated in the presence or absence of inhibitors of various potentially important kinases. Hypoxia inducible factor (HIF)-1α expression and MAP kinase phosphorylation in the hypoxic culture were determined with western blotting. In addition, the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in hypoxic or normoxic conditions were determined with real-time RT-PCR. The effects of these growth factors on ASC proliferation were investigated. Chromatin immunoprecipitation (ChIP) of the HIF-1α-binding hypoxia responsive element in FGF-2 was performed. HIF-1α was knocked down by siRNA, and FGF-2 expression was investigated.
  
  
  RESULTS
  ASC proliferation was significantly enhanced in the hypoxic culture and was inhibited by ERK and Akt inhibitors. Hypoxia for 5-15 minutes stimulated the phosphorylation of ERK1/2 among MAP kinases and induced HIF-1α expression. The levels of VEGF and FGF-2 mRNA and protein in the ASCs were significantly enhanced in hypoxia, and FGF-2 increased ASC proliferation. The ChIP assay revealed an 8-fold increase in the binding of HIF-1α to FGF-2 in hypoxia. HIF-1α knockdown by siRNA partially inhibited the FGF-2 expression of ASCs induced by hypoxia.
  
  
  CONCLUSIONS
  ASC proliferation was enhanced by hypoxia. HIF-1α activation, FGF-2 production, and the ERK1/2 and Akt pathway were involved in this regulatory mechanism.
authors.affiliation: Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan.
authors.forename: Natsuko
authors.initials: N
authors.lastname: Kakudo
authors.affiliation: Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Morimoto
authors.affiliation: Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ogawa
authors.affiliation: Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Taketani
authors.affiliation: Department of Plastic and Reconstructive Surgery, Kansai Medical University, Osaka, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kusumoto
mesh_terms: D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2