Therapeutic Potential And Mechanisms Of Action Of Implanted Adipose-Derived Stem Cells In Rat Small-for-Size Liver Graft Injury

Aim:Graft injury after small-for-size liver transplantation impairs graft function and threatens the survival of the recipients. The use of adipose-derived stem cell (ADSC) for liver injury protection and repair is promising. It was showed that ADSC secreted a physiologically relevant level of vascular endothelial growth factor (VEGF), which is an important growth factor for vascular repair and regeneration, and is showed to promote liver regeneration. Our aim in this study is to investigate the therapeutic effect of ADSC implantation and potential role of ADSC-derived VEGF in the setting of small-for-size liver graft injury after transplantation.Methods and method:ADSCs were harvested from rat inguinal fat pad and were cultured in vitro for expansion. The contribution of ADSCs secreted VEGF on liver injury protection was determined by specific silencing of VEGF gene expression using RNA interference technology. Two types of lentivirus either containing green fluorescent protein (GFP) and small hairpin RNA specifically targeting VEGF gene expression (VEGFi-shRNA) or containing GFP and a negative control sequence (NC-shRNA) were used in this study. The liver sinusoidal endothelial cells (LSECs) were isolated and co-cultured with either VEGFi-shRNA ADSCs or NC-shRNA ADSCs. Apoptosis assay by flow cytometry was performed after24hours of co-culture.35%partial liver transplantation was performed and2×106either VEGFi-shRNA ADSCs or NC-shRNA ADSCs per rat were injected through portal vein right after the transplantation procedure. Blood and liver tissue samples on post-operative day1,3,7were collected for further analysis. Liver frozen sections on post-operative day1,7,14and30were examined for GFP fluorescence.Results:In vitro apoptosis assay showed apoptosis rate of cultured LSECs was much lower in LSECs co-cultured with3×105NC-shRNA ADSCs. In Vivo, we found VEGF secreted by implanted ADSCs improved graft microcirculatory disturbances, serum liver function parameters and survival. The improved microcirculatory status was also reflected by reduced hepatocellular damage, especially LSEC apoptosis, and improved liver regeneration. These effects were accompanied by decreased expression of endothelin receptor type A, increased Bcl-2/Bax ratio, decreased expression of Bad and elevated proportion of phosphorylated Bad. In vivo tracking of implanted ADSCs showed GFP-positive ADSCs were visible on early post-operative days and vanished two weeks later.Conclusions:Implanted syngeneic ADSCs attenuated small-for-size liver graft injuries and subsequently enhanced liver regeneration in a rat35%liver transplantation model. The VEGF secreted by implanted ADSCs played a crucial role in this process.