Toll-like receptor 4 ablation improves stem cell survival after hypoxic injury

Abstract

IntroductionMesenchymal 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. Materials and methodsMurine wild-type (WT) and TLR4KO MSCs were harvested from bone marrow and grown in vitro. A total of 0.1 × 106 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. ResultsThe 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 × 105 living cells/mL control versus 0.88 × 105 hypoxia, P < 0.05). Hypoxic injury did not increase cell death in the TLR4KO group (1.68 × 105 control versus 1.82 × 105 hypoxia, P < 0.05). Increased AKT activation was observed in all TLR4KO groups. TLR4 did not affect phosphorylated ERK levels. ConclusionTLR4-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.