title: Impact of low oxygen on the secretome of human adipose-derived stromal/stem cell primary cultures.
abstract: Tissue fibrosis can lead to organ dysfunction, patient morbidity, and mortality. Adipose-derived Stromal/stem Cells (ASCs) represent a potential therapeutic. Immediately following grafting, ASCs would reside in a lower O2 environment. ASC secretome was examined under 5% O2 ("low O2") and 21% O2 ("ambient O2") culture conditions. ASCs from five female donors were cultured in low or ambient O2 conditions for 3 days and pooled conditioned medium was compared by two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MS/MS). Of 71 proteins identified, five proteins involved in extracellular matrix (ECM) remodeling exhibited ≥2-fold decrease under low O2 culture and were confirmed by Western immunoblot and qRT-PCR: fibronectin 1, TGF-β1-induced protein (βig-h3), osteonectin, and collagens type 1α1 and α2. ELISAs performed using 10 donors also confirmed significant decreases during low O2 culture in 4-6 ASC donors. For low abundant proteins, a 36 cytokine/chemokine array was performed. Fifteen cytokines/chemokines including Type 2 cytokines IL-13, MCP-1, and CD40 ligand were detected in ambient O2 ASC medium. IL-6 was detected in low O2 but not ambient O2 ASC medium. These findings demonstrate that low O2 ASC exposure resulted in reduced ECM protein and Type 2 cytokine secretions that are significant with regard to inflammation in fibrosis.
authors.affiliation: Department of Structural and Cellular Biology, Tulane University, New Orleans, LA, USA.
authors.forename: Trivia P
authors.initials: TP
authors.lastname: Frazier
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Gimble
authors.forename: Indu
authors.initials: I
authors.lastname: Kheterpal
authors.forename: Brian G
authors.initials: BG
authors.lastname: Rowan
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology; D000328:Adult; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D005355:Fibrosis / Q000209:etiology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D008875:Middle Aged; D010100:Oxygen / Q000008:administration & dosage*; D020543:Proteome / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D016326:Extracellular Matrix Proteins; D015850:Interleukin-6; D020543:Proteome; D010100:Oxygen
keywords: 2-D; ASCs; CD40 L; CD40 ligand; COL1α1; COL1α2; DMEM; Dulbecco's modified Eagle's medium; ECM; Extracellular matrix (ECM) remodeling protein secretions and ASCs; FBS; FDR; HIF-1α; IFN-γ; IL-1β; IL-4,6,10,13; LC–MS/MS; Low oxygen culture and adipose-derived stromal/stem cell (ASC) secretome; MCP-1; PAI-1; ROC curve; SPARC/osteonectin; SVF; TGF-β1-induced protein; TNF-α; Tissue fibrosis and inflammation; WAT; adipose tissue-derived stromal/stem cells; collagen type 1α1; collagen type α2; extracellular matrix; false discovery rate; fetal bovine serum; hypoxia-inducible factor-1α; interferon-gamma; interleukin-1β; interleukins-4, -6, -10, and -13; liquid chromatography and tandem mass spectrometry; monocyte chemotactic protein-1; plasminogen activator inhibitor-type 1; qRT-PCR; quantitative reverse-transcriptase polymerase chain reaction; receiver operating characteristic curve; secreted protein acidic, cysteine-rich; serine protease inhibitors; serpins; stromal vascular fraction; tumor necrosis factor-α; two-dimensional; white adipose tissue; βig-h3