Primary cilia as a targetable node between biliary injury, senescence and regeneration in liver transplantation.

BACKGROUND & AIMS: Biliary complications are a major cause of morbidity and mortality in liver transplantation. Up to 25% of patients that develop biliary complications require additional surgical procedures, re-transplantation or die in the absence of a suitable regraft. Here, we investigate the role of the primary cilium, a highly-specialised sensory organelle, in biliary injury leading to post-transplant biliary complications. METHODS: Human biopsies were used to study the structure and function of primary cilia in liver transplant recipients that develop biliary complications (N=7) in comparison with recipients without biliary complications (N=12). To study the biological effects of the primary cilia during transplantation, we generated murine models that recapitulate liver procurement and cold storage, and assessed the elimination of the primary cilia in biliary epithelial cells in the K19CreERTKif3aflox/flox mouse model. To explore the molecular mechanisms responsible for the observed phenotypes we used in vitro models of ischemia, cellular senescence and primary cilia ablation. Finally, we used pharmacological and genetic approaches to target cellular senescence and the primary cilia, both in mouse models and discarded human donor livers. RESULTS: Prolonged ischemic periods before transplantation result in ciliary shortening and cellular senescence, an irreversible cell cycle arrest that blocks regeneration. Our results indicate that primary cilia damage results in biliary injury and a loss of regenerative potential. Senescence negatively impacts primary cilia structure and triggers a negative feedback loop that further impairs regeneration. Finally, we explore how targeted interventions for cellular senescence and/or the stabilisation of the primary cilia improve biliary regeneration following ischemic injury. CONCLUSIONS: Primary cilia play an essential role in biliary regeneration and we demonstrate that senolytics and cilia-stabilising treatments provide a potential therapeutic opportunity to reduce the rate of biliary complications and improve clinical outcomes in liver transplantation. IMPACT AND IMPLICATIONS: Up to 25% of liver transplants result in biliary complications, leading to additional surgery, retransplants, or death. We found that the incidence of biliary complications is increased by damage to the primary cilium, an antenna that protrudes from the cell and is key to regeneration. Here, we show that treatments that preserve the primary cilia during the transplant process provide a potential solution to reduce the rates of biliary complications.

The BAR Score Predicts and Stratifies Outcomes Following Liver Retransplantation: Insights From a Retrospective Cohort Study.

Liver retransplantation (reLT) yields poorer outcomes than primary liver transplantation, necessitating careful patient selection to avoid futile reLT. We conducted a retrospective analysis to assess reLT outcomes and identify associated risk factors. All adult patients who underwent a first reLT at the Medical University of Innsbruck from 2000 to 2021 (N = 111) were included. Graft- and patient survival were assessed via Kaplan-Meier plots and log-rank tests. Uni- and multivariate analyses were performed to identify independent predictors of graft loss. Five-year graft- and patient survival rates were 64.9% and 67.6%, respectively. The balance of risk (BAR) score was found to correlate with and be predictive of graft loss and patient death. The BAR score also predicted sepsis (AUC 0.676) and major complications (AUC 0.720). Multivariate Cox regression analysis identified sepsis [HR 5.179 (95% CI 2.575-10.417), p < 0.001] as the most significant independent risk factor for graft loss. At a cutoff of 18 points, the 5 year graft survival rate fell below 50%. The BAR score, a simple and easy to use score available at the time of organ acceptance, predicts and stratifies clinically relevant outcomes following reLT and may aid in clinical decision-making.

Small bowel neuroendocrine tumors: An analysis of clinical presentation, diagnostic workup and surgical approach-A single center retrospective study.

Background: Neurocrine neoplasms (NEN) of the small bowel (SBNEN) are a rare entity and mostly asymptomatic. The aim of this study was to explore trends in the clinical presentation, diagnostic workup, surgical approach and oncological outcome in patients with SBNEN at our surgical department. Materials and methods: All patients who underwent surgical resection for SBNEN from 2004 to 2020 at our department were enrolled in this single center retrospective study. Results: A total of 32 patients were included in this study. In most cases, the diagnosis was based on incidental findings during endoscopy or radiographic imaging (n = 23; 72%). Twenty cases had a G1 tumor and 12 cases a G2 tumor. The 1-, 3- and 5-year overall survival (OS) were 96%, 86% and 81%, respectively. Patients with a tumor more than 30 mm had a significantly lower OS (p = 0.01). For G1 tumors, the estimated disease-free survival (DFS) was 109 months. Again, the DFS was significantly lower when the tumor had more than 30 mm in diameter (p = 0.013). Conclusion: Due to the mostly asymptomatic presentation, the diagnostic workup can be difficult. An aggressive approach and a strict follow-up seem to be important for the oncological outcome.

Senolytic treatment preserves biliary regenerative capacity lost through cellular senescence during cold storage.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)-dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

Reassessment of Relevance and Predictive Value of Parameters Indicating Early Graft Dysfunction in Liver Transplantation: AST Is a Weak, but Bilirubin and INR Strong Predictors of Mortality.

Introduction: Early graft dysfunction (EAD) complicates liver transplantation (LT). The aim of this analysis was to discriminate between the weight of each variable as for its predictive value toward patient and graft survival. Methods: We reviewed all LT performed at the Medical University of Innsbruck between 2007 and 2018. EAD was recorded when one of the following criteria was present: (i) aspartate aminotransferase (AST) levels >2,000 IU/L within the first 7 days, (ii) bilirubin levels ≥10mg/dL or (iii) international normalized ratio (INR) ≥1.6 on postoperative day 7. Results: Of 616 LT, 30.7% developed EAD. Patient survival did not differ significantly (P = 0.092; log rank-test = 2.87), graft survival was significantly higher in non-EAD patients (P = 0.008; log rank-test = 7.13). Bilirubin and INR on postoperative day 7 were identified as strong mortality predictors (Bilirubin HR = 1.71 [1.34, 2.16]; INR HR = 2.69 [0.51, 14.31]), in contrast to AST (HR = 0.91 [0.75, 1.10]). Similar results were achieved for graft loss estimation. A comparison with the Model for Early Allograft Function (MEAF) and the Liver Graft Assessment Following Transplantation (L-GrAFT) score identified a superior discrimination potential but lower specificity. Conclusion: Contrarily to AST, bilirubin and INR have strong predictive capacity for patient and graft survival. This fits well with the understanding, that bile duct injury and deprivation of synthetic function rather than hepatocyte injury are key factors in LT.

Cell-Based Regeneration and Treatment of Liver Diseases.

The liver, in combination with a functional biliary system, is responsible for maintaining a great number of vital body functions. However, acute and chronic liver diseases may lead to irreversible liver damage and, ultimately, liver failure. At the moment, the best curative option for patients suffering from end-stage liver disease is liver transplantation. However, the number of donor livers required by far surpasses the supply, leading to a significant organ shortage. Cellular therapies play an increasing role in the restoration of organ function and can be integrated into organ transplantation protocols. Different types and sources of stem cells are considered for this purpose, but highly specific immune cells are also the focus of attention when developing individualized therapies. In-depth knowledge of the underlying mechanisms governing cell differentiation and engraftment is crucial for clinical implementation. Additionally, novel technologies such as ex vivo machine perfusion and recent developments in tissue engineering may hold promising potential for the implementation of cell-based therapies to restore proper organ function.

Expression of MICA in Zero Hour Biopsies Predicts Graft Survival After Liver Transplantation.

In search for novel biomarkers to assess graft quality, we investigated whether defined candidate genes are predictive for outcome after liver transplantation (LT). Zero-hour liver biopsies were obtained from 88 livers. Gene expression of selected candidate markers was analyzed and correlated with clinical parameters as well as short and long-term outcomes post LT. Whereas both, the calculated Eurotransplant Donor-Risk-Index and the donor body mass index, had either a poor or no predictive value concerning serum levels indicative for liver function (ALT, AST, GGT, bilirubin) after 6 months, chronological donor age was weakly predictive for serum bilirubin (AUC=0.67). In contrast, the major histcompatibility complex class I related chain A (MICA) mRNA expression demonstrated a high predictive value for serum liver function parameters revealing an inverse correlation (e.g. for ALT: 3 months p=0.0332; 6 months p=0.007, 12 months 0.0256, 24 months p=0.0098, 36 months, p=0.0153) and proved significant also in a multivariate regression model. Importantly, high expression of MICA mRNA revealed to be associated with prolonged graft survival (p=0.024; log rank test) after 10 years of observation, whereas low expression was associated with the occurrence of death in patients with transplant related mortality (p=0.031). Given the observed correlation with short and long-term graft function, we suggest MICA as a biomarker for pre-transplant graft evaluation.

Liver regeneration and inflammation: from fundamental science to clinical applications.

Liver regeneration is a complex process involving the crosstalk of multiple cell types, including hepatocytes, hepatic stellate cells, endothelial cells and inflammatory cells. The healthy liver is mitotically quiescent, but following toxic damage or resection the cells can rapidly enter the cell cycle to restore liver mass and function. During this process of regeneration, epithelial and non-parenchymal cells respond in a tightly coordinated fashion. Recent studies have described the interaction between inflammatory cells and a number of other cell types in the liver. In particular, macrophages can support biliary regeneration, contribute to fibrosis remodelling by repressing hepatic stellate cell activation and improve liver regeneration by scavenging dead or dying cells in situ. In this Review, we describe the mechanisms of tissue repair following damage, highlighting the close relationship between inflammation and liver regeneration, and discuss how recent findings can help design novel therapeutic approaches.

Noninvasive Detection of Ischemic Vascular Damage in a Pig Model of Liver Donation After Circulatory Death.

BACKGROUND AND AIMS: Liver graft quality is evaluated by visual inspection prior to transplantation, a process highly dependent on the surgeon's experience. We present an objective, noninvasive, quantitative way of assessing liver quality in real time using Raman spectroscopy, a laser-based tool for analyzing biomolecular composition. APPROACH AND RESULTS: A porcine model of donation after circulatory death (DCD) with normothermic regional perfusion (NRP) allowed assessment of liver quality premortem, during warm ischemia (WI) and post-NRP. Ten percent of circulating blood volume was removed in half of experiments to simulate blood recovery for DCD heart removal. Left median lobe biopsies were obtained before circulatory arrest, after 45 minutes of WI, and after 2 hours of NRP and analyzed using spontaneous Raman spectroscopy, stimulated Raman spectroscopy (SRS), and staining. Measurements were also taken in situ from the porcine liver using a handheld Raman spectrometer at these time points from left median and right lateral lobes. Raman microspectroscopy detected congestion during WI by measurement of the intrinsic Raman signal of hemoglobin in red blood cells (RBCs), eliminating the need for exogenous labels. Critically, this microvascular damage was not observed during WI when 10% of circulating blood was removed before cardiac arrest. Two hours of NRP effectively cleared RBCs from congested livers. Intact RBCs were visualized rapidly at high resolution using SRS. Optical properties of ischemic livers were significantly different from preischemic and post-NRP livers as measured using a handheld Raman spectrometer. CONCLUSIONS: Raman spectroscopy is an effective tool for detecting microvascular damage which could assist the decision to use marginal livers for transplantation. Reducing the volume of circulating blood before circulatory arrest in DCD may help reduce microvascular damage.

How to Handle Arterial Conduits in Liver Transplantation? Evidence From the First Multicenter Risk Analysis.

OBJECTIVE: The aims of the present study were to identify independent risk factors for conduit occlusion, compare outcomes of different AC placement sites, and investigate whether postoperative platelet antiaggregation is protective. BACKGROUND: Arterial conduits (AC) in liver transplantation (LT) offer an effective rescue option when regular arterial graft revascularization is not feasible. However, the role of the conduit placement site and postoperative antiaggregation is insufficiently answered in the literature. STUDY DESIGN: This is an international, multicenter cohort study of adult deceased donor LT requiring AC. The study included 14 LT centers and covered the period from January 2007 to December 2016. Primary endpoint was arterial occlusion/patency. Secondary endpoints included intra- and perioperative outcomes and graft and patient survival. RESULTS: The cohort was composed of 565 LT. Infrarenal aortic placement was performed in 77% of ACs whereas supraceliac placement in 20%. Early occlusion (≤30 days) occurred in 8% of cases. Primary patency was equivalent for supraceliac, infrarenal, and iliac conduits. Multivariate analysis identified donor age >40 years, coronary artery bypass, and no aspirin after LT as independent risk factors for early occlusion. Postoperative antiaggregation regimen differed among centers and was given in 49% of cases. Graft survival was significantly superior for patients receiving aggregation inhibitors after LT. CONCLUSION: When AC is required for rescue graft revascularization, the conduit placement site seems to be negligible and should follow the surgeon's preference. In this high-risk group, the study supports the concept of postoperative antiaggregation in LT requiring AC.

Toll-like receptor 3 mediates ischaemia/reperfusion injury after cardiac transplantation.

OBJECTIVES: Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS: Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic-polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3-/- mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS: IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3-/- animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS: We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation.

Preoperative Assessment of Muscle Mass Using Computerized Tomography Scans to Predict Outcomes Following Orthotopic Liver Transplantation.

BACKGROUND: Sarcopenia is an established risk factor predicting survival in chronically ill and trauma patients. We herein examine the assessment and clinical implication of sarcopenia in liver transplantation (LT). METHODS: Computerized tomography scans from 172 patients waitlisted for LT were analyzed by applying 6 morphometric muscle scores, including 2 density indices (psoas density [PD] and skeletal muscle density [SMD]) and 4 scores based on muscle area (total psoas area, psoas muscle index, skeletal muscle area, and skeletal muscle index). RESULTS: The prevalence of sarcopenia in our cohort ranged from 7.0% to 37.8%, depending on the score applied. Only sarcopenia as defined by the density indices PD and SMD (but not total psoas area, psoas muscle index, skeletal muscle area, or skeletal muscle index) revealed clinical relevance since it correlates significantly with postoperative complications (≥Grade III, Clavien-Dindo classification) and sepsis. Furthermore, sarcopenia predicted inferior patient and graft survival, with low muscle density (PD: <38.5 HU or SMD: <30 HU) representing an independent risk factor in a multivariate regression model (P < 0.05). Importantly, the widely used Eurotransplant donor risk index had a predictive value in nonsarcopenic patients but failed to predict graft survival in patients with sarcopenia. CONCLUSIONS: Sarcopenia revealed by low muscle density correlates with major complications following LT and acts as an independent predictor for patient and graft survival. Therefore, the application of a simple computerized tomography-morphologic index can refine an individual recipient's risk estimate in a personalized approach to transplantation.