The Effects Of Endothelial Calpain On Rejection Response During Heart Xenotransplantation

Heart transplantation is currently the only hope for patients with heart failure.However,the shortage of donors has become a bottleneck in the development of allogeneic transplantation.Although xenotransplantation has theoretically broad application prospects due to its biogenicity and modifiability,the complex immune rejection is the main obstacle for the clinical application.The rejection of xenotransplantation mainly includes hyper acute rejection(HAR),delayed xenograft rejection(DXR)or acute vascular rejection(AVR)and chronic cell and humoral rejection.Gene editing technology combined with immunotherapy can effectively control the occurrence of HAR.However,the following DXR period has become the main obstacle due to its unclear mechanism.The mechanisms of DXR are generally recognized as follows:activated inflammatory cells infiltrate donor organs continuously,extensive platelet aggregation and fibrin deposition in microvessels and activation of donor vascular endothelial cells.Due to the poor therapeutic effect on platelet aggregation,the occurrence of DXR is closely related to the activation of inflammatory cells and donor endothelial cells.Calpains are a group of intracellular calcium-dependent hydrolytic enzymes,which shows the activity of proteolytic enzymes through Ca2+ nactivation and autolysis.The Calpain system currently consists of 15 isozymes.Calpain 1 and Calpain 2 are widely distributed in tissues.Of note,they are uniquely expressed in endothelial cells.Calpastatin(CAST)is an endogenous specific inhibitor protein of Calpain.However,the effect of Calpain activity on the activation of endothelial cells and inflammatory cells during xenotransplantation have not been reported.Objective:To explore the role of endothelial Calpain in xenotransplantation,xenotransplantation models in vivo and in vitro were built.Methods:In vivo experiments,endothelial Canps1-knockout(Canpsl-KO)mice and wild-type mice were used to establish a mouse-rat heart xenotransplantation models,and the survival time of xenograft hearts was observed.HE and immunohistochemical staining were used to detect the morphological changes and the infiltration of inflammatory cells.In vitro experiments,Calpain activity in mouse microvascular endothelial cells(MCECs)was inhibited by transfecting with adenovirus packed with CAST.After rat serum or lymphocytes stimulated MCECs,to mimic DXR,the changes of endothelial cells were observed and detected.In further mechanism study,TNF-?,one of the most common inflammatory factors that plays an important role in xenotransplantation,was used to stimulate endothelial cells.Endothelial cell activation,proliferation,migration and tube formation ability,as well as intracellular I-?B and ?-catenin expression were detected after stimulation of TNF-?.Results:In the mouse-rat heart xenotransplantation model,we found that there was no significant difference in the survival time of transplanted heart between the two groups without immunosuppressant intervention.After immunosuppressant intervention,the mean survival time of donor hearts from Canpsl-KO mice was significantly longer than that of wild-type mic.Pathological examination displayed that the infiltration of inflammatory cells in the transplanted hearts from Canps1-KO mice was significantly less than that of wild-type mice.In vitro experiments,we found calpain activity was effectively suppressed by Ad-CAST transfection in MCECs,which reduced the expression levels of adhesion molecules(ICAM-1 and VCAM-1,endothelial cell activation marker protein)and protected the morphological integrity in endothelial cells.Moreover,TNF-? administration induced endothelial activation and promoted the proliferation,migration and tube formation in endothelial cells.The effects of TNF-? on endothelial activation depended on Calpain activity,which is related to the regulation of intracellular expression of I-?B and ?-catenin.Conclusion:During the DXR xenotransplantation period,Calpain activity was increased in the endothelial cells.Inhibition of Calpain reduced the expression of adhesion molecules and the infiltration of inflammatory cells in xenograft hearts,thus significantly prolonging the survival time of the grafts.Furthermore,inhibition of calpain activity reduced the proliferation,migration and tube formation in endothelial cells under inflammatory stress,which mechanically is related to Calpain hydrolyzing I-?B and promoting the expression of?-catenin.