| Background:Cryopreserved allogenic valves have been clinically used for years,revealing good hemodynamics and a high resistance to endocarditis.However,a proportion of these valves suffer from tissue degeneration after transplantation,which is now thought to be closely related to the immunogenicity of the remaining tissue and extracellular matrix damage caused by the cryopreservation procedure.Recently,vitrification protocols were reported to preserve the matrix integrity of allograft valves,and concomitantly result in a low tissue immunogenicity.Nevertheless,the precise mechanism of presenting immunodulatory effect on vitrified tissues remains unclear,and the relation between allogeneic cell survival and tissue immunogenicity is controversial.Besides,the VS83 vitrification protocol has been shown to result in excellent preservation of heart valve allograft matrices,but poor retention of tissue viability.Further improvements should be explored.Objective:(1)to determine whether vitrification with VS55 formuation leads to a reduction in tissue immunogenicity and the association between the vitrification method and the activation of the NLRP3 inflammasome.Besides,to study the relation between allogeneic cell survival and tissue immunogenicity.(2)to investigated the effect of adding MitoQ to the VS83 formulation in terms of the post-warming viability of aortic valve tissues,as well as mitochondrial morphology and the expression of proteins associated with mitochondrial function.Methods and results:In the first part of this study,endothelial cells were removed from New Zealand rabbit aortic root tissue to diminish the influence of endothelium residua.Similar levels of tissue viability and ATP content were observed after conventional freezing cryopreservation(CFC)or ice-free cryopreservation(IFC).After implanting subcutaneously into Chinese rabbits,a significant reduction in TNF? and IL-6 was detected in the IFC group.We also studied the activation of the NLRP3 inflammasome 6 hours after tissue culture.It revealed a high level of NLRP3,Interleukin-1? and caspase-1 expression in the CFC tissues after thawing,and a low level in vitrified tissues treated with VS55.In the second arm,aortic heart valve tissues(leaflets and associated aortic root)were obtained from New Zealand White rabbits and randomly divided to four groups: Group A,control;Group B,conventional freezing cryopreservation;Group C,ice-free cryopreservation using VS83 formulation;or Group D,ice-free cryopreservation using VS83 with the addition of MitoQ(20 nM).Samples were then cryopreserved for 2 months.A reduction in the production of ROS was observed in MitoQ-treated samples after thawing.Furthermore,the addition of MitoQ to the formulation lead to a limited but significant improvement in cell viability,ATP content and mitochondrial morphology,which were seriously impaired during cryopreservation with VS83 alone.The MitoQ group also demonstrated a marked increase in the expression of mitofusin 2(Mfn2)after 3 h of incubation,whereas dynamin-related protein 1(Drp1)was suppressed.Low cryosurvival and severe mitochondrial damage were associated with the VS83 vitrification method.The addition of MitoQ lead to an improvement in tissue viability post-cryopreservation,which might due to enhanced mitochondrial fusion in early post-thaw stages.Conclusion:Based on these result we conclude that:(1)vitrification with VS55 formulation suppresses NLRP3 inflammasome activation in post-thaw aortic valve homografts,and alleviates the inflammatory response after transplantation.Additionally,the survival of allogeneic cells may not be the main factor affecting immunogenicity of vitrified allografts.(2)Both cell viability and mitochondrial morphology were seriously impaired during cryopreservation with VS83 formulation.Some improvement was observed after supplementing with MitoQ,which might be related to an enhancement of mitochondrial fusion in early post-warming stages. |
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