The Mechanism Of SIRT1 Mediated Endothelial Injury In The Pathogenesis Of Transplant Associated Thrombotic Microangiopathy And Retrospective Clinical Study In TA-TMA

Objective: Transplant-associated thrombotic microangiopathy(TA-TMA)is a serious complication of hematopoietic stem cell transplantation(HSCT)with a high mortality rate.Previous studies have suggested that complement activation and vascular endothelial injury play essential roles in TA-TMA.SIRT1(Silent Information Regulator 1)is thought to be a nicotinamide adenine dinucleotide-dependent(NAD+)deacetylase that contributes to oxidative stress,complement,and platelet function.As a result,the mechanism of SIRT1 action in TA-TMA will be researched in this study.Additionally,the clinical data of TATMA patients and non-TA-TMA patients were retrospectively analyzed to explore the risk factors of TA-TMA.Methods:(1)Our study obtained peripheral blood samples from 10 consecutive patients at TA-TMA clinical diagnosis and without any treatment from January 2018 to January 2022 at the First Affiliated Hospital of Soochow University.20 patients with a GVHD and non-complications patients after HSCT were also collected.(2)RNA-seq was performed to analysis the expression of SIRT1 in TA-TMA and CTR.SIRT1 protein levels were quantified in human samples by the ELISA Kit.(3)HUVECs were provided with TATMA patients plasma to construct an in vitro model of TA-TMA.Flow cytometry and Immunofluorescence were used to observe the expression of incubated C3 and C5b-9 in HUVECs.Also,the level of SIRT1 in TA-TMA model in vitro was detected by RT-PCR and Western-blot.Then,RES and SRT1720 were added and their effects on complement and reactive oxygen species(ROS)were investigated by the above methods.To isolate platelets from healthy donors and further explore the effect of TA-TMA on plateletHUVECs adhesion and platelet aggregation.Western-blot was used to explore the downstream mechanism of SIRT1.(4)TA-TMA and non-TA-TMA(control group)patients were matched 1:1 by PSM(Propensity Score Matching).Clinical data of patients were retrospectively analyzed to explore the risk factors and median survival time of TA-TMA patients.Results:(1)SIRT1 levels were decreased in TA-TMA patients compared to those after HSCT without complications and acute graft-versus-host disease(a GVHD).(2)HUVECs were then incubated overnight in vitro with TA-TMA plasma and CTR.In the HUVECs of the TA-TMA group,C3 and C5b-9 were activated,as were ROS production,plateletHUVECs adhesion and aggregation.SIRT1 stimulators could restore the above processes,including inhibition of C3 and C5b-9 deposition,reduction of ROS levels,and attenuation of platelet-HUVECs adhesion and aggregation.In addition,TA-TMA plasma treatment decreased the protein expression of SIRT1,P-AKT/AKT,and total Nrf-2 in HUVECs.In contrast,upregulated SIRT1 enhanced P-AKT /AKT and Nrf-2.(3)Clinical statistics showed that early EBV infection after allo-HSCT could lead to the development of TATMA.The median time(months)of EBV infection in TA-TMA and non-TA-TMA patients were 5(1-43)and 39(1-46),respectively.Also,the median survival time(days)of TA-TMA patients was 180(125,235),and the mean survival time(days)of non-TA-TMA patients was1148(950,1346).Conclusion:(1)Overexpression of SIRT1 reduces endothelial dysfunction by inhibiting complement activation and ROS while inhibiting platelet adhesion.(2)Meanwhile,SIRT1 attenuated endothelial injury by regulating the SIRT1/Nrf-2 and AKT axes,suggesting that SIRT1 is a promising protective therapeutic target for TA-TMA.(3)The early onset of EBV after HSCT leads to TA-TMA and the median survival time of TA-TMA is significantly reduced compared with non-TA-TMA patients.