Introduction
Solid organ transplantation (SOT) is the most effective therapy for end-stage organ disease. Crucial advances in organ preservation, immunosuppression therapies, anesthesiology management, and surgical techniques have contributed to the success of its implementation. Dynamic cumulative injury of the graft is primarily associated with donor type, age, comorbidities, and maintenance, but ischemia-reperfusion injury (IRI) could worsen its development. Sustained injuries, surgical complications, recipient comorbidities, and host vs. graft immune response could collectively affect the SOT outcome. Around 10% of early organ failures are attributed to IRI during liver transplantation (LT), leading to primary graft dysfunction and a higher incidence of acute and chronic rejection.
Epigenetic Changes in Liver Transplantation
Epigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. This study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors.
Methods
Twelve liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq.
Results
Significant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-a, NFKB, and interleukin signaling. Cell enrichment analysis identified macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response.
Discussion
DNA methylation is a common epigenetic mechanism cells use to «switch off» genes. CpG islands (the sequences where methylation occurs) are usually near upstream transcription start sites, with increased methylation correlating with low to no transcription. This study reveals that even relatively short periods of stress can alter the DNA methylation patterns, which subsequently was reflected in the transcriptome. The DNA methylation data at the promoter level during cold ischemia indicates shifts in biological and metabolic processes, activating genes involved in coagulation, clot formation, and energy production.
Cold Ischemia and Reperfusion
Interestingly, we detected SERPINE1 (PAI-1) expression upregulation, the main inhibitor of tissue-type plasminogen activator (tPA) and urokinase (uPA), reducing and preventing fibrinolysis. SERPINE1 modulation in IRI, as we observed, has been described earlier in the literature. LT procedures involve vascular manipulation in a complex scenario where coagulopathy may occur due to several factors (temperature changes, hemodilution, calcium and acid-base imbalance, etc.). This is particularly evident during the anhepatic phase (the time from the physical removal of the liver from the recipient to recirculation of the graft), where fibrin formation can initiate from the absence of coagulation factor synthesis and clearance of activated fibrinolytic factors.
Transcriptome Analysis
In contrast, the DNA methylation signature obtained during reperfusion, and after cold ischemia demonstrated shifts from hypomethylation to hypermethylation at the promoter level. Interestingly, gene clusters related to coagulation cascades, blood microparticles formation, and platelet degranulation have been deactivated. This finding might help understand over-coagulation processes seen in many LT cases, which are responsible for non-technical factors related to early arterial thrombosis or changes in the intrahepatic vascular pattern observed when early CT scans are performed for other clinical reasons.
Conclusions
In conclusion, despite these limitations, our study highlights the relevance of methylation in the IRI during LT, even seeming complementary to transcriptomics analysis and providing a complete picture of the ongoing biomolecular processes. Moreover, the DNA methylation datasets generated in our study are the early literature of IRI and will be essential for other researchers’ future integration of omics information. Further methylome studies in the LT outcome are expected soon to understand better how IRI or the donor-recipient environment affects the modulation of whole gene expression comparing early and late post-transplant periods. It remains to be seen if the putative epigenetic changes in the hepatocytes have a long-term effect on the phenotype of the transplanted organ. Cumulative injury dealt with by epigenetic modifications might have a lasting impact on the overall LT outcome.
References
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