Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells.
OBJECTIVE
Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media.


METHODS
The effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined.


RESULTS
Trypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media.


CONCLUSIONS
Optimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.
Rasmussen
Jeppe Grøndahl
JG
Department of Pharmacology, Aarhus University, Aarhus, Denmark. jgr1@dadlnet.dk
Frøbert
Ole
O
Pilgaard
Linda
L
Kastrup
Jens
J
Simonsen
Ulf
U
Zachar
Vladimir
V
Fink
Trine
T
D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D051381:Rats; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D043925:Angiogenesis Inducing Agents; D017077:Culture Media, Conditioned; D014357:Trypsin