The ubiquitin ligase Uhrf2 is a master regulator of cholesterol biosynthesis and is essential for liver regeneration.

Fibroblast growth factors (FGFs) are key regulators of the remarkable regenerative capacity of the liver. Mice lacking FGF receptors 1 and 2 (Fgfr1 and Fgfr2) in hepatocytes are hypersensitive to cytotoxic injury during liver regeneration. Using these mice as a model for impaired liver regeneration, we identified a critical role for the ubiquitin ligase Uhrf2 in protecting hepatocytes from bile acid accumulation during liver regeneration. During regeneration after partial hepatectomy, Uhrf2 expression increased in an FGFR-dependent manner, and Uhrf2 was more abundant in the nuclei of liver cells in control mice compared with FGFR-deficient mice. Hepatocyte-specific Uhrf2 knockout or nanoparticle-mediated Uhrf2 knockdown caused extensive liver necrosis and impaired hepatocyte proliferation after partial hepatectomy, resulting in liver failure. In cultured hepatocytes, Uhrf2 interacted with several chromatin remodeling proteins and suppressed the expression of cholesterol biosynthesis genes. In vivo, the loss of Uhrf2 resulted in cholesterol and bile acid accumulation in the liver during regeneration. Treatment with a bile acid scavenger rescued the necrotic phenotype, hepatocyte proliferation, and the regenerative capacity of the liver in Uhrf2-deficient mice subjected to partial hepatectomy. Our results identify Uhrf2 as a key target of FGF signaling in hepatocytes and its essential function in liver regeneration and highlight the importance of epigenetic metabolic regulation in this process.

Large-Scale Quantitative Proteomics Identifies the Ubiquitin Ligase Nedd4-1 as an Essential Regulator of Liver Regeneration.

The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice.

OBJECTIVE: Fibroblast growth factors (Fgfs) are key orchestrators of development, and a role of Fgfs in tissue repair is emerging. Here we studied the consequences of inducible loss of Fgf receptor (Fgfr) 4, the major Fgf receptor (Fgfr) on hepatocytes, alone or in combination with Fgfr1 and Fgfr2, for liver regeneration after PH. DESIGN: We used siRNA delivered via nanoparticles combined with liver-specific gene knockout to study Fgfr function in liver regeneration. Liver or blood samples were analysed using histology, immunohistochemistry,real-time RT-PCR, western blotting and ELISA. RESULTS: siRNA-mediated knockdown of Fgfr4 severely affected liver regeneration due to impairment of hepatocyte proliferation combined with liver necrosis.Mechanistically, the proliferation defect resulted from inhibition of an Fgf15-Fgfr4-Stat3 signalling pathway,which is required for injury-induced expression of the Foxm1 transcription factor and subsequent cell cycle progression, while elevated levels of intrahepatic toxicbile acids were identified as the likely cause of the necrotic damage. Failure of liver mass restoration in Fgfr4 knockdown mice was prevented at least in part by compensatory hypertrophy of hepatocytes. Most importantly, our data revealed partially redundant functions of Fgf receptors in the liver, since knock down of Fgfr4 in mice lacking Fgfr1 and Fgfr2 in hepatocytes caused liver failure after PH due to severe liver necrosis and a defect in regeneration. CONCLUSIONS: These results demonstrate that Fgfr signalling in hepatocytes is essential for liver regeneration and suggest activation of Fgfr signalling asa promising approach for the improvement of the liver's regenerative capacity.

Knockdown and knockout of ß1-integrin in hepatocytes impairs liver regeneration through inhibition of growth factor signalling.

The liver has a unique regenerative capability, which involves extensive remodelling of cell-cell and cell-matrix contacts. Here we study the role of integrins in mouse liver regeneration using Cre/loxP-mediated gene deletion or intravenous delivery of ß1-integrin siRNA formulated into nanoparticles that predominantly target hepatocytes. We show that although short-term loss of ß1-integrin has no obvious consequences for normal livers, partial hepatectomy leads to severe liver necrosis and reduced hepatocyte proliferation. Mechanistically, loss of ß1-integrin in hepatocytes impairs ligand-induced phosphorylation of the epidermal growth factor and hepatocyte growth factor receptors, thereby attenuating downstream receptor signalling in vitro and in vivo. These results identify a crucial role and novel mechanism of action of ß1-integrins in liver regeneration and demonstrate that protein depletion by nanoparticle-based delivery of specific siRNA is a powerful strategy to study gene function in the regenerating liver.

The use of a reversible proteasome inhibitor in a model of Reduced-Size Orthotopic Liver transplantation in rats.

Ischemia/reperfusion injury (IRI), inherent in liver transplantation (LT), is the main cause of initial deficiencies and primary non-function of liver allografts. Living-related LT was developed to alleviate the mortality resulting from the scarcity of suitable deceased grafts. The main problem in using living-related LT for adults is graft size disparity. In this study we propose for the first time that the use of a proteasome inhibitor (Bortezomib) treatment could improve liver regeneration and reduce IRI after Reduced-Size Orthotopic Liver transplantation (ROLT). Rat liver grafts were reduced by removing the left lateral lobe and the two caudate lobes and preserved in UW or IGL-1 preservation solution for 1h liver and then subjected to ROLT with or without Bortezomib treatment. Our results show that Bortezomib reduces IRI after LT and is correlated with a reduction in mitochondrial damage, oxidative stress and endoplasmic reticulum stress. Furthermore, Bortezomib also increased liver regeneration after reduced-size LT and increased the expression of well-known ischemia/reperfusion protective proteins such as nitric oxide synthase, heme oxigenase 1 (HO-1) and Heat Shock Protein 70. Our results open new possibilities for the study of alternative therapeutic strategies aimed at reducing IRI and increasing liver regeneration after LT. It is hoped that the results of our study will contribute towards improving the understanding of the molecular processes involved in IRI and liver regeneration, and therefore help to improve the outcome of this type of LT in the future.

Ubiquitin-proteasome system inhibitors and AMPK regulation in hepatic cold ischaemia and reperfusion injury: possible mechanisms.

In the present Hypothesis article, we summarize and present data from the literature that support our hypothesis on the potential mechanisms by which UPS (ubiquitin-proteasome system) inhibitors reduce I/R (ischaemia/reperfusion) injury in the liver. I/R is the main cause of primary liver failure and, consequently, minimizing the detrimental effects of this process could increase the number of suitable transplantation grafts and also enhance the survival rate of patients after liver transplantation. A potential strategy to reduce I/R injury is the use of UPS inhibitors either as additives to preservation solutions or as drugs administered to patients. However, there is still controversy over whether the use of UPS inhibitors is beneficial or deleterious with regard to liver injury. From our experience and the few studies that have investigated the role of UPS in hepatic I/R, we believe that the use of UPS inhibitors is a potential strategy to reduce I/R injury in liver transplantation and graft preservation. We hypothesize that one of the main mechanisms of action of UPS inhibitors may be the up-regulation of AMPK (AMP-activated protein kinase) activity and the consequent down-regulation of mTOR (mammalian target of rapamycin), which may finally influence autophagy and preserve the energy state of the cell.

Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury.

Chronic organ-donor shortage has required the acceptance of steatotic livers for transplantation purposes despite the higher risk of graft dysfunction or nonfunction associated with the cold ischemia-reperfusion injury. This study evaluated the use of melatonin as an additive to Institute Georges Lopez (IGL-1) solution for protecting nonsteatotic and steatotic liver grafts against cold ischemia-reperfusion injury. In the current investigation, we used an ex vivo isolated perfused rat liver model. Steatotic and nonsteatotic livers were preserved for 24 hr (4°C) in University of Wisconsin or IGL-1 solutions with or without melatonin, as well as in University of Wisconsin solution alone. Thereafter, livers were subjected to 2-hr reperfusion (37°C). We assessed hepatic injury (transaminases) and function [bile production and sulfobromophthalein (BSP) clearance, vascular resistance], as well as other factors potentially implicated in the high vulnerability of steatotic livers against ischemia-reperfusion injury (oxidative stress and related inflammatory mediators including nitric oxide and cytokines). We also evaluated well-known cytoprotective factors as hemeoxygenase 1 (HO-1). Fatty livers preserved in IGL-1 solution enriched with melatonin showed lower transaminase levels and higher bile production and BSP clearance when compared to those obtained for livers maintained in IGL-1 solution alone. A significant diminution of vascular resistance was also observed when melatonin was added to the IGL-1 solution. The melatonin benefits correlated with the generation of nitric oxide (through constitutive e-NOS activation) and the prevention of oxidative stress and inflammatory cytokine release including tumor necrosis factor and adiponectin, respectively. The addition of melatonin to IGL-1 solution improved nonsteatotic and steatotic liver graft preservation, limiting their risk against cold ischemia-reperfusion injury.

Insulin like growth factor-1 increases fatty liver preservation in IGL-1 solution.

AIM: To investigate the benefits of insulin like growth factor-1 (IGF-1) supplementation to serum-free institut georges lopez-1 (IGL-1) solution to protect fatty liver against cold ischemia reperfusion injury. METHODS: Steatotic livers were preserved for 24 h in IGL-1 solution supplemented with or without IGF-1 and then perfused "ex vivo" for 2 h at 37degrees C. We examined the effects of IGF-1 on hepatic damage and function (transaminases, percentage of sulfobromophthalein clearance in bile and vascular resistance). We also studied other factors associated with the poor tolerance of fatty livers to cold ischemia reperfusion injury such as mitochondrial damage, oxidative stress, nitric oxide, tumor necrosis factor-α (TNF-α) and mitogen-activated protein kinases. RESULTS: Steatotic livers preserved in IGL-1 solution supplemented with IGF-1 showed lower transaminase levels, increased bile clearance and a reduction in vascular resistance when compared to those preserved in IGL-1 solution alone. These benefits are mediated by activation of AKT and constitutive endothelial nitric oxide synthase (eNOS), as well as the inhibition of inflammatory cytokines such as TNF-α. Mitochondrial damage and oxidative stress were also prevented. CONCLUSION: IGL-1 enrichment with IGF-1 increased fatty liver graft preservation through AKT and eNOS activation, and prevented TNF-α release during normothermic reperfusion.

Improved rat steatotic and nonsteatotic liver preservation by the addition of epidermal growth factor and insulin-like growth factor-I to University of Wisconsin solution.

This study examined the effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) supplementation to University of Wisconsin solution (UW) in steatotic and nonsteatotic livers during cold storage. Hepatic injury and function were evaluated in livers preserved for 24 hours at 4 degrees C in UW and in UW with EGF and IGF-I (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. AKT was inhibited pharmacologically. In addition, hepatic injury and survival were evaluated in recipients who underwent transplantation with steatotic and nonsteatotic livers preserved for 6 hours in UW and UW with EGF and IGF-I (separately or in combination). The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3beta (GSK3(beta)) inhibition in nonsteatotic livers and PPAR gamma overexpression in steatotic livers. When AKT was inhibited, the effects of EGF and IGF-I on GSK3(beta), PPAR gamma, hepatic injury and function disappeared. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the liver transplantation model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. In conclusion, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation, whereas a combination of both seems unnecessary.

Pharmacological strategies against cold ischemia reperfusion injury.

IMPORTANCE OF THE FIELD: Good organ preservation is a determinant of graft outcome after revascularization. The necessity of increasing the quality of organ preservation, as well as of extending cold storage time, has made it necessary to consider the use of pharmacological additives. AREAS COVERED IN THIS REVIEW: The complex physiopathology of cold-ischemia-reperfusion (I/R) injury--and in particular cell death, mitochondrial injury and endoplasmic reticulum stress--are reviewed. Basic principles of the formulation of the different preservation solutions are discussed. WHAT THE READER WILL GAIN: Current strategies and new trends in static organ preservation using additives such as trimetazidine, polyethylene glycols, melatonin, trophic factors and endothelin antagonists in solution are presented and discussed. The benefits and mechanisms responsible for enhancing organ protection against I/R injury are also discussed. Graft preservation was substantially improved when additives were added to the preservation solutions. TAKE HOME MESSAGE: Enrichment of preservation solutions by additives is clinically useful only for short periods. For longer periods of cold ischemia, the use of such additives becomes insufficient because graft function deteriorates as a result of ischemia. In such conditions, the preservation strategy should be changed by the use of machine perfusion in normothermic conditions.

Insulin-like growth factor and epidermal growth factor treatment: new approaches to protecting steatotic livers against ischemia-reperfusion injury.

Hepatic steatosis is a major risk factor in ischemia-reperfusion (I/R). IGF-binding proteins (IGFBPs) modulate IGF-I action by transporting circulating IGF-I to its sites of action. Epidermal growth factor (EGF) stimulates IGF-I synthesis in vitro. We examined the effect of IGF-I and EGF treatment, separately or in combination, on the vulnerability of steatotic livers to I/R. Our results indicated that I/R impaired IGF-I synthesis only in steatotic livers. Only when a high dose of IGF-I (400 microg/kg) was given to obese animals did they show high circulating IGF-I:IGFBP levels, increased hepatic IGF-I levels, and protection against damage. In lean animals, a dose of 100 microg/kg IGF-I protected nonsteatotic livers. Our results indicated that the combined administration of IGF-I and EGF resulted in hepatic injury parameters in both liver types similar to that obtained by IGF-I and EGF separately. IGF-I increased egf expression in both liver types. The beneficial role of EGF on hepatic I/R injury may be attributable to p38 inhibition in nonsteatotic livers and to PPAR gamma overexpression in steatotic livers. In conclusion, IGF-I and EGF may constitute new pharmacological strategies to reduce the inherent susceptibility of steatotic livers to I/R injury.

Effect of angiotensin II and bradykinin inhibition in rat reduced-size liver transplantation.

This study examined whether angiotensin II (Ang II) blockers [Ang II type I receptor antagonist, Ang II type II receptor antagonist, and angiotensin converting enzyme (ACE) inhibitor] could reduce hepatic injury and improve regeneration in reduced-size orthotopic liver transplantation (ROLT) and whether the beneficial effects of ischemic preconditioning (PC) in ROLT could be explained by changes in Ang II. We show that small liver grafts generated Ang II after ROLT and that this was associated with increased angiotensinogen and ACE messenger RNA expression. Furthermore, inhibition of Ang II did not contribute to PC-induced protection in ROLT. All Ang II blockers reduced hepatic injury, but none of them promoted liver regeneration. Bradykinin (BK) receptor antagonist improved liver regeneration but did not reduce hepatic injury in ROLT. Finally, the combination of Ang II blockers and BK receptor antagonists in ROLT reduced hepatic injury and improved liver regeneration. In conclusion, treatments with either Ang II blockers or BK receptor antagonists cannot, on their own, improve the outcome of ROLT. Although Ang II blockers can reduce hepatic ischemia-reperfusion injury and BK receptor antagonists can promote liver regeneration, neither confers both benefits at the same time. Consequently, it may be of clinical interest to apply both treatments simultaneously.

Inhibition of angiotensin II action protects rat steatotic livers against ischemia-reperfusion injury.

OBJECTIVE: We examined whether pharmacologic strategies blocking angiotensin II actions protect steatotic livers against ischemia-reperfusion (I/R) injury. The effects of ischemic preconditioning (PC) on angiotensin II were also evaluated. DESIGN: Randomized and controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Zucker rats. INTERVENTIONS: The following experimental groups were studied: I/R, ischemia-reperfusion + angiotensin-converting enzyme inhibitor (I/R+ACE inhibitor), ischemia-reperfusion + angiotensin II type I receptor antagonist (I/R+AT1R antagonist), ischemia-reperfusion + angiotensin II type II receptor antagonist (I/R+AT2R antagonist), and PC (5 mins of ischemia + 10 mins of reperfusion before I/R). In some of these groups, the action of bradykinin (BK) and/or peroxisome-proliferator-activated receptor-gamma (PPARgamma) was altered pharmacologically. MEASUREMENTS AND MAIN RESULTS: I/R+ACE inhibitor, I/R+AT1R antagonist, and I/R+AT2R antagonist reduced hepatic injury in steatotic livers compared with the I/R group. PC reduced angiotensin II generation and hepatic injury in steatotic livers in comparison to I/R group. Our results revealed that I/R+ACE inhibitor, I/R+AT1R antagonist, I/R+AT2R antagonist, and PC increased BK compared with the I/R group. In addition, the effects of PC on BK and hepatic injury were abolished when angiotensin II was administered. Furthermore, administration of BK receptor antagonists to the I/R+ACE inhibitor, I/R+AT1R antagonist, I/R+AT2R antagonist, and PC groups resulted in hepatic injury similar to the I/R group, indicating that the benefits of ACE inhibitor, AT1R antagonist, AT2R antagonist, and PC were abolished when the action of BK was inhibited. Experiments aimed at investigating why BK was protective in steatotic livers indicated that BK acts as a positive regulator of PPARgamma. If PPARgamma action was inhibited, BK did not protect steatotic livers against hepatic injury. CONCLUSIONS: Pharmacologic blockers of angiotensin II action (ACE inhibitors, AT1R antagonists, and AT2R antagonists) and PC, which reduced angiotensin II generation, increased BK generation in steatotic livers after I/R. This in turn increased PPARgamma and protected this type of liver against I/R injury.

Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.

UNLABELLED: Hepatic steatosis is a major risk factor in ischemia-reperfusion (I/R). Adiponectin acts as an antiobesity and anti-inflammatory hormone. Adiponectin activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a transcription factor that regulates inflammation in liver disease. Ischemic preconditioning (PC) based on brief periods of I/R protects steatotic livers against subsequent sustained I/R injury, but just how this is achieved is poorly understood. This study explains the role of PPAR-alpha and adiponectin in the vulnerability shown by steatotic livers to I/R and the benefits of PC in this situation. PPAR-alpha and adiponectin levels in nonsteatotic livers undergoing I/R were similar to those found in the sham group. However, reduced PPAR-alpha and increased adiponectin levels, particularly the high molecular weight isoform, were observed in steatotic livers as a consequence of I/R. Our results suggest that mitogen-activated protein kinases (MAPKs) may be positive regulators of adiponectin accumulation in steatotic livers. The addition of adiponectin small interfering RNA (siRNA) before I/R protected steatotic livers against oxidative stress and hepatic injury. The induction of PC before I/R increased PPAR-alpha and reduced adiponectin levels in steatotic livers. PC, which increased PPAR-alpha, as well as PPAR-alpha agonist pretreatment reduced MAPK expression, adiponectin, oxidative stress, and hepatic injury that follows I/R. In addition, the administration of a PPAR-alpha antagonist in preconditioned steatotic livers eliminated the beneficial effects of PC on MAPKs, adiponectin, oxidative stress, and hepatic injury. CONCLUSION: Steatotic livers are more predisposed to down-regulate PPAR-alpha and overexpress adiponectin when subjected to I/R. PPAR-alpha agonists and adiponectin siRNA are promising candidates to protect steatotic livers. PPAR-alpha agonists as well as PC, through PPAR-alpha, inhibited MAPK expression following I/R. This in turn inhibited adiponectin accumulation in steatotic livers and adiponectin-worsening effects on oxidative stress and hepatic injury.