Toll-like receptor 4 ablation improves stem cell survival after hypoxic injury.
BACKGROUND
Mesenchymal stem cell (MSC) therapy improves cardiac function after ischemia/reperfusion injury, but its effectiveness is limited by MSC survival in hypoxic environments. Toll-like receptor 4 (TLR4) contributes to pro-apoptotic signaling under hypoxic conditions. Activation of intracellular AKT and ERK pathways opposes this signal and improves cell survival. It is unknown whether ablation of TLR4 affects these pathways after hypoxic injury in MSCs. We hypothesized that: 1) TLR4 knockout (TLR4KO) in MSCs improves survival after hypoxic injury; and 2) this survival difference is due to improved signaling in the AKT and ERK pathways.


METHODS
Murine wild-type (WT) and TLR4KO MSCs were harvested from bone marrow and grown in vitro. A total of 0.1 × 10(6) cells/well were incubated in hypoxic conditions versus normoxic controls. After 24 h, these groups were examined for cell survival via counting and compared using a t-test with P < 0.05 = statistical significance. AKT and ERK concentrations were measured in lysate using Western blot analysis.


RESULTS
The morphology of WT and TLR4KO MSCs was similar. In line with our previous findings, hypoxia did significantly increase cell death in WT cells (1.79 × 10(5) living cells/mL control versus 0.88 × 10(5) hypoxia, P < 0.05). Hypoxic injury did not increase cell death in the TLR4KO group (1.68 × 10(5) control versus 1.82 × 10(5) hypoxia, P < 0.05). Increased AKT activation was observed in all TLR4KO groups. TLR4 did not affect phosphorylated ERK levels.


CONCLUSIONS
TLR4-knockout MSCs show improved survival after hypoxic injury because of increased AKT pathway signal. Use of TLR4-knockout MSCs in ischemia/reperfusion studies results in enhanced cardioprotection; improved stem cell survival was likely a contributing factor.
Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Benjamin D
BD
Brewster
Joshua D
JD
Rouch
Meijing
M
Wang
Daniel R
DR
Meldrum
D000818:Animals; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D000860:Hypoxia / Q000503:physiopathology*; D020935:MAP Kinase Signaling System; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000502:physiology*
C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D051057:Proto-Oncogene Proteins c-akt