The low survival rate of endothelial progenitor cells (EPCs) in vivo which are susceptible to adverse microenvironments including inflammation and oxidative stress has become one primary challenge of EPCs transplantation for regenerative therapy. Recent studies reported functional expression of toll-like receptor (TLR) 4 on EPCs and dose-dependent effects of lipopolysaccharide (LPS) on cellular oxidative stress and angiogenic properties. However, the involved mechanism has not yet been elucidated well, and the influence of TLR4 signaling on EPCs survival and function in vivo is unknown. In the present study, we observed the effects of LPS and TLR4/SIRT3 on EPCs mitochondrial permeability and intracellular mitochondrial superoxide. We employed the monocrotaline-induced pulmonary arteriolar injury model to observe the effects of TLR4/SIRT3 on the recruitment and survival of transplanted EPCs. We found the destructive effects of 10 μg/mL LPS on mitochondrial homeostasis, and cellular viability was mediated by TLR4/SIRT3 signals at least partially, and the TLR4 mediates the early-stage recruitment of transplanted EPCs in pulmonary arteriolar inflammation injury; however, SIRT3 has more contribution to the survival of incorporated EPCs and ameliorated arteriolar remodeling in lung vascular tissue. The study provides insights for the critical role of TLR4/SIRT3 in LPS-induced oxidative stress and mitochondrial disorder in EPCs in vitro and in vivo. The TLR4/SIRT3 signaling is important for EPCs resistance against inflammation and oxidative stress and may represent a new manipulating target for developing efficient cell therapy strategy.

Background Information Endothelial progenitor cells (EPCs) can exert angiogenic effects by a paracrine mechanism, where exosomes work as an important mediator. Recent studies reported functional expression of toll‐like receptor (TLR) 4 on human EPCs and dose‐dependent effects of lipopolysaccharide (LPS) on EPC angiogenic properties. To study the effects of TLR4/LPS signaling on EPC‐derived exosomes (Exo) and clarify the mechanism, we investigated the role of LPS on exosomes secretion from human EPCs and tested their anti‐oxidation/senescence functions. We employed the inhibitors of the plasma membrane Ca2+‐ATPase (PMCA), endoplasmic reticulum Ca2+‐ATPase (ERCA), PLC‐IP3 pathway and store‐operated calcium entry to assess the effects of LPS on EPC intracellular calcium signalings which critical for exosome secretion. Results LPS induced the release of Exo in a TLR4‐dependent manner in vitro, which effect can be partly abrogated by an membrane‐permeable IP 3 R antagonist, 2‐aminoethyl diphenylborinate (2‐APB), but not PLC inhibitor, U‐73122. The LPS can significantly delay the fallback of [Ca2+]i after isolating the cellular PMCA activity, and disturb PMCA 1/4 expression. The distribution of elevated intracellular calcium seemed coincident with the development of the multivesicular bodies (MVBs). furthermore, the anti‐oxidation/senescence properties of LPS‐induced Exo were validated by the senescence‐associated β‐galactosidase activity assay and reactive oxygen species (ROS) related H2DCF‐DA assay. Conclusions The mechanism of PMCA downregulation and IP3R‐dependent ER Ca2+ release may contribute to the pro‐exosomal effects of LPS on EPCs. Significance This study provides new insights into the potential role of LPS/TLR4 pathway in regulating EPC‐derived exosomes, which may help to develop some feasible approach to manipulate the Exo secretion and promote the clinical application of EPCs therapy in future.