Extracellular Vesicles in Liver Transplantation: Current Evidence and Future Challenges.

Extracellular vesicles (EVs) are emerging as a promising field of research in liver disease. EVs are small, membrane-bound vesicles that contain various bioactive molecules, such as proteins, lipids, and nucleic acids and are involved in intercellular communication. They have been implicated in numerous physiological and pathological processes, including immune modulation and tissue repair, which make their use appealing in liver transplantation (LT). This review summarizes the current state of knowledge regarding the role of EVs in LT, including their potential use as biomarkers and therapeutic agents and their role in graft rejection. By providing a comprehensive insight into this emerging topic, this research lays the groundwork for the potential application of EVs in LT.

Seroconversion After Coronavirus Disease 2019 Vaccination in Patients Awaiting Liver Transplantation: Fact or Fancy?

Chronic liver disease increased the risk of severe coronavirus disease 2019 (COVID-19). Trials to assess efficacy/safety of COVID-19 vaccines in liver disease are underway. We evaluated the humoral immune response and safety of anti-COVID-19 vaccination among patients waiting liver transplantation (LT). We enrolled all pre-LT adults who completed anti-COVID-19 vaccination between January 2021-August 2021 as study group. Patients with histories of COVID-19 received 1 vaccine dose, and all others received 2 doses. Patients were tested for COVID-19 immunoglobulin G (IgG) within 1 and 2 months after vaccination. Safety was evaluated with telephone interviews/outpatient visits. A control group of 30 healthcare workers who underwent vaccination in January 2021 and tested for IgG after 4 months was included. In the 89 pre-LT patients, at T1 (23 days after vaccination), seroconversion rate was 94.4%, and median IgG value was 1980 binding antibody units/mL (interquartile range 646-2080), and at T2 (68 days after vaccination) was 92.0%, with IgG value of 1450 (577-2080); (T1 versus T2, P = 0.38). In the 10/89 patients who received 1 vaccine dose, the median IgG value was 274 (68-548) before vaccine (T0), 2080 (1165-2080) at T1, and 2030 (964-2080) at T2 (T0 versus T1, P = 0.03; T1 versus T2, P = 0.99). All controls tested positive at 4 months after vaccination, with a median value of 847 (509-1165; P < 0.001 versus T1 and P = 0.04 versus T2 in the study group). No serious adverse event was reported in both groups. Our data from 89 pre-LT patients suggest a high rate of immunization (94.4%) after a median time of 23 days from safe COVID-19 vaccine. None of the patients developed COVID-19.

State-of-the-Art and Future Directions in Organ Regeneration with Mesenchymal Stem Cells and Derived Products during Dynamic Liver Preservation.

Transplantation is currently the treatment of choice for end-stage liver diseases but is burdened by the shortage of donor organs. Livers from so-called extended-criteria donors represent a valid option to overcome organ shortage, but they are at risk for severe post-operative complications, especially when preserved with conventional static cold storage. Machine perfusion technology reduces ischemia-reperfusion injury and allows viability assessment of these organs, limiting their discard rate and improving short- and long-term outcomes after transplantation. Moreover, by keeping the graft metabolically active, the normothermic preservation technique guarantees a unique platform to administer regenerative therapies ex vivo. With their anti-inflammatory and immunomodulatory properties, mesenchymal stem cells are among the most promising sources of therapies for acute and chronic liver failure, but their routine clinical application is limited by several biosafety concerns. It is emerging that dynamic preservation and stem cell therapy may supplement each other if combined, as machine perfusion can be used to deliver stem cells to highly injured grafts, avoiding potential systemic side effects. The aim of this narrative review is to provide a comprehensive overview on liver preservation techniques and mesenchymal stem cell-based therapies, focusing on their application in liver graft reconditioning.

Human liver stem cell-derived extracellular vesicles reduce injury in a model of normothermic machine perfusion of rat livers previously exposed to a prolonged warm ischemia.

Livers from donors after circulatory death (DCD) are a promising option to increase the donor pool, but their use is associated with higher complication rate and inferior graft survival. Normothermic machine perfusion (NMP) keeps the graft at 37°C, providing nutrients and oxygen supply. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) are able to reduce liver injury and promote regeneration. We investigated the efficacy of a reconditioning strategy with HLSC-EVs in an experimental model of NMP. Following total hepatectomy, rat livers were divided into 4 groups: (i) healthy livers, (ii) warm ischemic livers (60 min of warm ischemia), (iii) warm ischemic livers treated with 5 × 108 HLSC-EVs/g-liver, and (iv) warm ischemic livers treated with a 25 × 108 HLSC-EVs/g-liver. NMP lasted 6 h and HLSC-EVs (Unicyte AG, Germany) were administered within the first 15 min. Compared to controls, HLSC-EV treatment significantly reduced transaminases release. Moreover, HLSC-EVs enhanced liver metabolism by promoting phosphate utilization and pH self-regulation. As compared to controls, the higher dose of HLSC-EV was associated with significantly higher bile production and lower intrahepatic resistance. Histologically, this group showed reduced necrosis and enhanced proliferation. In conclusion, HLSC-EV treatment during NMP was feasible and effective in reducing injury in a DCD model with prolonged warm ischemia.

Renal damage in Hepatorenal Syndrome: A still unsolved issue.

Acute kidney injury (AKI) is a common complication of cirrhosis, burdened by high morbidity and mortality rates and progression to chronic kidney disease. Hepatorenal syndrome (HRS) is a peculiar type of functional AKI observed in cirrhotic patients with ascites. HRS diagnosis is still clinical, once pre-renal azotemia and intrinsic kidney damage have been excluded by applying well-established and internationally adopted criteria. HRS is considered reversible because of the absence of intrinsic renal damage. However, HRS reversibility has been questioned, due to the lack of response to treatment with vasoconstrictors plus albumin in a relevant percentage of patients and to the persistence of renal dysfunction in HRS patients who underwent liver transplantation (LT). Indeed, LT is the only ultimate treatment, as it solves both liver failure and portal hypertension. Thus, the presence of renal damage in HRS can be hypothesized. In this scenario, neutrophil gelatinase-associated lipocalin (NGAL), one of the most promising biomarkers, may help in characterizing the type of renal injury, distinguishing between HRS and acute tubular necrosis. This review gathers the available evidence in favor and against the presence of structural lesions in HRS in terms of either renal histology and urinary biomarkers with a particular focus on NGAL. The ability to properly characterize which component of renal dysfunction prevails - functional rather than structural - entails a relevant clinical impact for the treatment of these patients, both in terms of medical therapy and liver vs. combined liver-kidney transplantation.

Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model: An Experimental Setting for Organ Recondition and Pharmacological Intervention.

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. The ex vivo NMP hypoxic Rat Liver Perfusion Model represents a feasible approach that allow pharmacological intervention on isolated rat livers by using a combination of NMP and infusion of a number of drugs and/or biological material (cells, microvesicles, etc.). The combination of these two techniques may not only be applied for tissue preservation purposes, but also to investigate the biological effects of molecules and treatment useful in tissue protection. The protocol describes an ex vivo murine model of NMP capable of maintaining liver function despite an ongoing hypoxic injury induced by hemodilution. Furthermore, with this NMP system it is possible to deliver cells treatment or pharmacological intervention to an ex vivo perfused liver and suggests that could represent an innovative approach to recondition organs.

Protective Effects of Human Liver Stem Cell-Derived Extracellular Vesicles in a Mouse Model of Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (IRI) is observed in liver transplantation and hepato-biliary surgery and is associated with an inflammatory response. Human liver stem cell-derived extracellular vesicles (HLSC-EV) have been demonstrated to reduce liver damage in different experimental settings by accelerating regeneration and by modulating inflammation. The aim of the present study was to investigate whether HLSC-EV may protect liver from IRI in a mouse experimental model. Segmental IRI was obtained by selective clamping of intrahepatic pedicles for 90 min followed by 6 h of reperfusion. HLSC-EV were administered intravenously at the end of the ischemic period and histopathological and biochemical alterations were evaluated in comparison with controls injected with vehicle alone. Intra liver localization of labeled HLSC-EV was assessed by in in vivo Imaging System (IVIS) and the internalization into hepatocytes was confirmed by fluorescence analyses. As compared to the control group, administration of 3 × 109 particles (EV1 group) significantly reduced alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, necrosis extension and cytokines expression (TNF-α, CCL-2 and CXCL-10). However, the administration of an increased dose of HLSC-EV (7.5 × 109 particles, EV2 group) showed no significant improvement in respect to controls at enzyme and histology levels, despite a significantly lower cytokine expression. In conclusion, this study demonstrated that 3 × 109 HLSC-EV were able to modulate hepatic IRI by preserving tissue integrity and by reducing transaminases release and inflammatory cytokines expression. By contrast, a higher dose was ineffective suggesting a restricted window of biological activity. Graphical abstract.