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Unfortunately, there is a gap of understanding in the pathophysiology of chronic liver disease due to the lack of experimental models that exactly mimic the human disease. Additionally, the diagnosis of patients is very poor due to the lack of biomarkers than can detect the disease in early stages. Thus, it is of utmost interest the generation of a multidisciplinary consortium from different countries with a direct translation. The present reports the meeting of the 2021 Iberoamerican Consortium for the study of liver Cirrhosis, held online, in October 2021. The meeting, was focused on the recent advancements in the field of chronic liver disease and cirrhosis with a specific focus on cell pathobiology and liver regeneration, molecular and cellular targets involved in non-alcoholic hepatic steatohepatitis, alcoholic liver disease (ALD), both ALD and western diet, and end-stage liver cirrhosis and hepatocellular carcinoma. In addition, the meeting highlighted recent advances in targeted novel technology (-omics) and opening therapeutic avenues in this field of research.

Animal models are crucial for improving our understanding of human pathogenesis, enabling researchers to identify therapeutic targets and test novel drugs. In the current review, we provide a comprehensive summary of the most widely used experimental models of chronic liver disease, starting from early stages of fatty liver disease (non-alcoholic and alcoholic) to steatohepatitis, advanced cirrhosis and end-stage primary liver cancer. We focus on aspects such as reproducibility and practicality, discussing the advantages and weaknesses of available models for researchers who are planning to perform animal studies in the near future. Additionally, we summarise current and prospective models based on human tissue bioengineering.

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol‐associated and metabolic‐associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol‐associated liver disease plus metabolic‐associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA‐sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets., DUAL model perfectly mimics all histological, metabolic and transcriptomic gene signatures of human advanced steatohepatitis, and thus serve as a preclinical tool for the development of therapeutic targets.

Background Metabolic diseases are risk factors for severe Coronavirus disease (COVID-19), which have a close relationship with metabolic dysfunction-associated fatty liver disease (MAFLD). Aims To evaluate the presence of MAFLD and fibrosis in patients with COVID-19 and its association with prognosis. Methods Retrospective cohort study. In hospitalized patients with COVID-19, the presence of liver steatosis was determined by computed tomography scan (CT). Liver fibrosis was assessed using the NAFLD fibrosis score (NFS score), and when altered, the AST to platelet ratio index (APRI) score. Mann-Whitney U, Student´s t-test, logistic regression analysis, Kaplan-Meier curves and Cox regression analysis were used. Results 432 patients were analyzed, finding steatosis in 40.6%. No differences in pulmonary involvement on CT scan, treatment, or number of days between the onset of symptoms and hospital admission were found between patients with and without MAFLD. The presence of liver fibrosis was associated with higher severity scores, higher levels of inflammatory markers, requirement of mechanical ventilation, incidence of acute kidney injury (AKI), and higher mortality than patients without fibrosis. Conclusion The presence of fibrosis rather than the presence of MAFLD is associated with increased risk for mechanical ventilation, development of AKI, and higher mortality in COVID-19 patients.

The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1-S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. Conclusion: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo. (Hepatology 2014;59:651–660)

The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8Δhepa mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Background & Aims c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl 4 )-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. Methods We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 ( Jnk1 Δhepa ) or combination of Jnk1 and Jnk2 ( Jnk Δhepa ), as well as Jnk1- floxed C57BL/6 (control) mice, were given injections of CCl 4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. Results Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk Δhepa mice produced a greater level of liver injury than that observed in Jnk1 Δhepa or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl 4 also induced stronger hepatic injury in Jnk Δhepa mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1 Δhepa or control mice. Hepatocytes from Jnk Δhepa mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate−activated protein kinase, total protein adenosine monophosphate−activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk Δhepa and control mice from liver injury. Conclusions In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl 4 - and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.

Liver fibrosis is a chronic wound healing process leading to liver scarring by excessive accumulation of extracellular matrix proteins including collagen. The main producers of liver collagen are myofibroblasts derived from activated hepatic stellate cells (HSC). Additionally, other cell types such as portal fibroblasts and bone marrow derived cells may contribute to extracellular matrix (ECM) production. Liver fibrosis develops on the basis of chronic liver injury induced e.g. by chronic viral hepatitis B or C infection, excessive alcohol abuse or fatty liver disease frequently associated with obesity (1). Although immune cells play an essential role in the modulation of liver fibrosis, its pathogenesis implicitly involves injury and proliferation of hepatic stellate cells, hepatocytes and potentially other cell species. Upon liver damage, dying hepatocytes stimulate remnant hepatocytes to re-enter the cell cycle in order to restore the original liver mass and function (2). Liver injury also stimulates HSC activation via complex mechanisms. This involves the conversion of a resting, vitamin A storing cell into a proliferating HSC without vitamin A droplets but capable of producing pro-inflammatory cytokines and ECM components such as collagen (3). The transition from quiescent (G0) cells into the active phase of the cell cycle is predominantly controlled by E-type cyclins and their associated kinase, Cdk2 (4). In mammals, two E-cyclins are known termed Cyclin E1 (CcnE1) and Cyclin E2 (CcnE2), respectively (5, 6). Despite their anticipated essential function for developmental and regenerative processes, single genetic inactivation of CcnE1, CcnE2 or Cdk2 does not affect viability or development in mice (7–10). However, fibroblasts deficient for both E-cyclins are unable to re-enter the cell cycle from G0 (9). We recently demonstrated that CcnE1 and CcnE2 play antagonistic roles in the regenerating liver following partial hepatectomy (11). Accordingly, CcnE2−/− livers show increased and prolonged CcnE1/Cdk2 activity, resulting in earlier and sustained DNA synthesis, hepatomegaly and excessive endoreplication of hepatocytes, whereas ablation of CcnE1 provoked only a moderate delay of hepatocyte proliferation. Earlier work using rat HSC indicated that HSC activation is associated with increased gene expression of CcnE, Cyclin D and induction of polyploidy (12). However, the precise role of E-type cyclins for activation and proliferation of HSC and subsequent liver fibrogenesis has remained elusive. In the present study, we aimed to investigate the contribution of E-type cyclins for liver fibrosis in vivo using constitutive CcnE1−/− and CcnE2−/− knockout mice and derived primary HSC. Our current work demonstrates that CcnE1, but not CcnE2 is essential for HSC survival, proliferation and liver fibrogenesis.

Background & Aims Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. Methods Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc tg ) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. Results Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc tg mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc tg mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. Conclusions Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.

The human gene PTPN11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene, as dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylenenitrite (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a sub-fraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant active mutants, PTPN11/Shp2 has a tumor suppressor function in liver.

Background & Aims E-cyclins control the transition of quiescent cells into the cell cycle. Two E-cyclins, CcnE1 and CcnE2, have been described, but their specific contributions to cell cycle reentry in vivo are poorly understood. Liver regeneration following partial hepatectomy is an excellent in vivo model for the study of cell cycle reentry of quiescent cells. We investigated the relevance of E-cyclins in directing resting hepatocytes into the cell cycle after partial hepatectomy using CcnE1 and CcnE2 knockout mice. Methods Partial hepatectomy (70%) was performed in CcnE1 (E1 −/− ) and CcnE2 (E2 −/− ) knockout and wild-type mice. Liver regeneration was monitored by cell cycle markers for G 1 /S phase, S phase, and M phase as well as by determining the liver/body weight ratio after partial hepatectomy. Ploidy of hepatocytes was determined by fluorescence-activated cell sorting and fluorescent in situ hybridization. Results CcnE1 deletion resulted in normal liver regeneration with a slight delay of the G 1 /S-phase transition and a defect in endoreplication of otherwise polyploid hepatocytes. Surprisingly, E2 −/− mice displayed accelerated and sustained DNA synthesis after partial hepatectomy, excessive endoreplication in hepatocytes, and a liver mass that was 45% greater than that of wild-type mice after termination of the regeneration process. CcnE2 depletion induced overexpression of CcnE1 and prolonged cdk2 kinase activity after partial hepatectomy. Conclusions CcnE2 has an unexpected role in repressing CcnE1; the phenotype of E2 −/− mice appears to result from CcnE1 overexpression and cdk2 hyperactivation. CcnE1 and CcnE2 therefore have nonredundant functions for S-phase entry and endoreplication during liver regeneration.

Cyclin E1 controls G1/S phase transition of the eukaryotic cell cycle. We report the impact of alternative spliced cyclin E1 isoforms on cell cycle regulation in hepatocytes. We show that expression of new cyclin E1 mRNA variants IN3, Delta4, and Delta5 is associated with retarded proliferation in murine hepatocellular carcinoma. Additionally, we demonstrate that a new cyclin E1 isoform Delta3/8 lacking the central part of wild-type mRNA is expressed predominantly in nonproliferating murine hepatocytes. Following partial hepatectomy, Delta3/8 is downregulated when hepatocytes enter the cell cycle from quiescence. The Delta3/8 protein does not exhibit any cyclin box motif but binds cyclin-dependent kinase 2 without stimulating kinase activity. We demonstrate that Delta3/8 lacks any nuclear localization signal and is exclusively located in the cytoplasm. Overexpression of Delta3/8 in cultured cells leads to a delayed G0-G1 transition, indicating that this splice variant helps to maintain a quiescent state of hepatocytes. In conclusion, we identified an isoform of cyclin E1 involved in G0 maintenance and suggest an additional mechanism for cell cycle control.

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.

Background and Aims: Liver regeneration is impaired in obese, diabetic rodents developing spontaneous steatohepatitis such as ob/ob and KK-A mice, in which hepatic NKT cells are depleted. In this study, we investigated the differences in hepatic regeneration after partial hepatectomy (PH) in mice selectively depleted NK and/or NKT cells. Methods: Male, 12 week-old CD1d-knockout (KO) mice, which lack NKT cells systemically, and wild type (WT; C57Bl/6) mice were used. Some mice were pretreated with NK1.1 or asialo-GM1 antibody 24hr prior to experiments to deplete NK/NKT cells or NK cells alone, respectively. Mice underwent the 2/3 PH, and the uptake of BrdU and the expression of PCNA in hepatocyte nuclei were detected by immunohistochemistry. Hepatic expression levels of cyclin D1 and IFNg mRNA were analyzed quantitatively by Western blotting and real time RT-PCR, respectively. Results: In CD1d-KO mice, BrdU uptake and PCNA expression were almost similar to those in WT mice 48hr after PH, the labeling indices being nearly 20% and 30%, respectively. In sharp contrast, both parameters after PH was decreased remarkably in WT mice pretreated with an NK1.1 antibody, values reaching only 1.7% and 11.9%, respectively (P < 0.01 vs. WT after PH). Further, expression of cylin D1 in the liver 48hr after PH was nearly normal in CD1d-KO mice, but the levels were almost completely blunted in mice given an NK1.1 antibody. Interestingly, CD1d-KO mice given an asialoGM1 antibody showed impaired regenerative responses as similar to WT mice given an NK1.1 antibody. Hepatic IFNg mRNA was elevated nearly 5-fold in WT mice 6hr after PH, which was not affected by pretreatment with an NK1.1 antibody. Conclusions: These findings clearly indicated that liver regeneration is markedly impaired when both NKT and NK cells are depleted in two different preparations. NK cell-function under regeneration appears to be paradoxical in the presence or absence of NKT cells, and it is unlikely that IFNg plays a regulatory role in regeneration failure caused by simultaneous depletion of NK and NKT cells. It is therefore concluded that NKT and NK cells are coordinately involved in normal regenerative responses in the liver. 1084 CYCLIN E1 AND D1 CAN DRIVE LIVER REGENERATION IN MICE WITHOUT CDK2 W. Hu, Y.A. Nevzorova, J.R. Nowak, U. Haas, N. Moro, Y. Geng, P. Sicinski, M. Barbacid, C. Trautwein, C. Liedtke. Department of Medicine III, University Hospital Aachen, RWTH Aachen University, Aachen, Germany; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid, Spain E-mail: whu@ukaachen.de

Background & Aims Chronic liver injury triggers complications such as liver fibrosis and hepatocellular carcinoma (HCC), which are associated with alterations in distinct signalling pathways. Of particular interest is the interaction between mechanisms controlled by IKKγ/NEMO, the regulatory IKK subunit, and Jnk activation for directing cell death and survival. In the present study, we aimed to define the relevance of Jnk in hepatocyte-specific NEMO knockout mice ( NEMO Δhepa ), a genetic model of chronic inflammatory liver injury. Methods We generated Jnk1 −/− /NEMO Δhepa and Jnk2 −/− /NEMO Δhepa mice by crossing NEMO Δhepa mice with Jnk1 and Jnk2 global deficient animals, respectively, and examined the progression of chronic liver disease. Moreover, we investigated the expression of Jnk during acute liver injury, evaluated the role of Jnk1 in bone marrow-derived cells, and analysed the expression of NEMO and p-JNK in human diseased-livers. Results Deletion of Jnk1 significantly aggravated the progression of liver disease, exacerbating apoptosis, compensatory proliferation and carcinogenesis in NEMO Δhepa mice. Conversely, Jnk2 −/− /NEMO Δhepa displayed hepatic inflammation. By using bone marrow transfer, we observed that Jnk1 in haematopoietic cells had an impact on the progression of chronic liver disease in NEMO Δhepa livers. These findings are of clinical relevance since NEMO expression was downregulated in hepatocytes of patients with HCC whereas NEMO and p-JNK were expressed in a large amount of infiltrating cells. Conclusions A synergistic function of Jnk1 in haematopoietic cells and hepatocytes might be relevant for the development of chronic liver injury. These results elucidate the complex function of Jnk in chronic inflammatory liver disease.