Serum ferritin levels can predict long-term outcomes in patients with metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: Hyperferritinaemia is associated with liver fibrosis severity in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), but the longitudinal implications have not been thoroughly investigated. We assessed the role of serum ferritin in predicting long-term outcomes or death. DESIGN: We evaluated the relationship between baseline serum ferritin and longitudinal events in a multicentre cohort of 1342 patients. Four survival models considering ferritin with confounders or non-invasive scoring systems were applied with repeated five-fold cross-validation schema. Prediction performance was evaluated in terms of Harrell's C-index and its improvement by including ferritin as a covariate. RESULTS: Median follow-up time was 96 months. Liver-related events occurred in 7.7%, hepatocellular carcinoma in 1.9%, cardiovascular events in 10.9%, extrahepatic cancers in 8.3% and all-cause mortality in 5.8%. Hyperferritinaemia was associated with a 50% increased risk of liver-related events and 27% of all-cause mortality. A stepwise increase in baseline ferritin thresholds was associated with a statistical increase in C-index, ranging between 0.02 (lasso-penalised Cox regression) and 0.03 (ridge-penalised Cox regression); the risk of developing liver-related events mainly increased from threshold 215.5 µg/L (median HR=1.71 and C-index=0.71) and the risk of overall mortality from threshold 272 µg/L (median HR=1.49 and C-index=0.70). The inclusion of serum ferritin thresholds (215.5 µg/L and 272 µg/L) in predictive models increased the performance of Fibrosis-4 and Non-Alcoholic Fatty Liver Disease Fibrosis Score in the longitudinal risk assessment of liver-related events (C-indices>0.71) and overall mortality (C-indices>0.65). CONCLUSIONS: This study supports the potential use of serum ferritin values for predicting the long-term prognosis of patients with MASLD.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration.

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

c-Rel-dependent Chk2 signaling regulates the DNA damage response limiting hepatocarcinogenesis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. APPROACH AND RESULTS: Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. CONCLUSION: Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.

Innate and Adaptive Immunopathogeneses in Viral Hepatitis; Crucial Determinants of Hepatocellular Carcinoma.

Viral hepatitis B (HBV) and hepatitis C (HCV) infections remain the most common risk factors for the development of hepatocellular carcinoma (HCC), and their heterogeneous distribution influences the global prevalence of this common type of liver cancer. Typical hepatitis infection elicits various immune responses within the liver microenvironment, and viral persistence induces chronic liver inflammation and carcinogenesis. HBV is directly mutagenic but can also cause low-grade liver inflammation characterized by episodes of intermittent high-grade liver inflammation, liver fibrosis, and cirrhosis, which can progress to decompensated liver disease and HCC. Equally, the absence of key innate and adaptive immune responses in chronic HCV infection dampens viral eradication and induces an exhausted and immunosuppressive liver niche that favors HCC development and progression. The objectives of this review are to (i) discuss the epidemiological pattern of HBV and HCV infections, (ii) understand the host immune response to acute and chronic viral hepatitis, and (iii) explore the link between this diseased immune environment and the development and progression of HCC in preclinical models and HCC patients.

Sulfatase-2 from Cancer Associated Fibroblasts: An Environmental Target for Hepatocellular Carcinoma?

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, ß-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/ß-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFß1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFß1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRß/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKß/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/ß-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKß/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

Caucasian lean subjects with non-alcoholic fatty liver disease share long-term prognosis of non-lean: time for reappraisal of BMI-driven approach?

OBJECTIVE: The full phenotypic expression of non-alcoholic fatty liver disease (NAFLD) in lean subjects is incompletely characterised. We aimed to investigate prevalence, characteristics and long-term prognosis of Caucasian lean subjects with NAFLD. DESIGN: The study cohort comprises 1339 biopsy-proven NAFLD subjects from four countries (Italy, UK, Spain and Australia), stratified into lean and non-lean (body mass index (BMI) 10 483 person-years), 4.7% of lean vs 7.7% of non-lean patients reported liver-related events (p=0.37). No difference in survival was observed compared with non-lean NAFLD (p=0.069). CONCLUSIONS: Caucasian lean subjects with NAFLD may progress to advanced liver disease, develop metabolic comorbidities and experience cardiovascular disease (CVD) as well as liver-related mortality, independent of longitudinal progression to obesity and PNPLA3 genotype. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD where the disease manifests at lower overall BMI thresholds. LAY SUMMARY: NAFLD may affect and progress in both obese and lean individuals. Lean subjects are predominantly males, have a younger age at diagnosis and are more prevalent in some geographic areas. During the follow-up, lean subjects can develop hepatic and extrahepatic disease, including metabolic comorbidities, in the absence of weight gain. These patients represent one end of a wide spectrum of phenotypic expression of NAFLD.

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 µL media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Long-term outcomes and predictive ability of non-invasive scoring systems in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-invasive scoring systems (NSS) are used to identify patients with non-alcoholic fatty liver disease (NAFLD) who are at risk of advanced fibrosis, but their reliability in predicting long-term outcomes for hepatic/extrahepatic complications or death and their concordance in cross-sectional and longitudinal risk stratification remain uncertain. METHODS: The most common NSS (NFS, FIB-4, BARD, APRI) and the Hepamet fibrosis score (HFS) were assessed in 1,173 European patients with NAFLD from tertiary centres. Performance for fibrosis risk stratification and for the prediction of long-term hepatic/extrahepatic events, hepatocarcinoma (HCC) and overall mortality were evaluated in terms of AUC and Harrell's c-index. For longitudinal data, NSS-based Cox proportional hazard models were trained on the whole cohort with repeated 5-fold cross-validation, sampling for testing from the 607 patients with all NSS available. RESULTS: Cross-sectional analysis revealed HFS as the best performer for the identification of significant (F0-1 vs. F2-4, AUC = 0.758) and advanced (F0-2 vs. F3-4, AUC = 0.805) fibrosis, while NFS and FIB-4 showed the best performance for detecting histological cirrhosis (range AUCs 0.85-0.88). Considering longitudinal data (follow-up between 62 and 110 months), NFS and FIB-4 were the best at predicting liver-related events (c-indices>0.7), NFS for HCC (c-index = 0.9 on average), and FIB-4 and HFS for overall mortality (c-indices >0.8). All NSS showed limited performance (c-indices <0.7) for extrahepatic events. CONCLUSIONS: Overall, NFS, HFS and FIB-4 outperformed APRI and BARD for both cross-sectional identification of fibrosis and prediction of long-term outcomes, confirming that they are useful tools for the clinical management of patients with NAFLD at increased risk of fibrosis and liver-related complications or death. LAY SUMMARY: Non-invasive scoring systems are increasingly being used in patients with non-alcoholic fatty liver disease to identify those at risk of advanced fibrosis and hence clinical complications. Herein, we compared various non-invasive scoring systems and identified those that were best at identifying risk, as well as those that were best for the prediction of long-term outcomes, such as liver-related events, liver cancer and death.

A PDCD1 Role in the Genetic Predisposition to NAFLD-HCC?

Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver (PNPLA3 rs738409; TM6SF2 rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes (MICA rs2596542; CD44 rs187115; PDCD1 rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. PNPLA3 and TM6SF2 SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while PDCD1 SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. PDCD1 rs7421861 was independently associated with NAFLD-HCC development, while PDCD1 rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis.

TREM-2 defends the liver against hepatocellular carcinoma through multifactorial protective mechanisms.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer usually arising on a background of chronic liver injury involving inflammatory and hepatic regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM-2) is predominantly expressed in hepatic non-parenchymal cells and inhibits Toll-like receptor signalling, protecting the liver from various hepatotoxic injuries, yet its role in liver cancer is poorly defined. Here, we investigated the impact of TREM-2 on liver regeneration and hepatocarcinogenesis. DESIGN: TREM-2 expression was analysed in liver tissues of two independent cohorts of patients with HCC and compared with control liver samples. Experimental HCC and liver regeneration models in wild type and Trem-2-/- mice, and in vitro studies with hepatic stellate cells (HSCs) and HCC spheroids were conducted. RESULTS: TREM-2 expression was upregulated in human HCC tissue, in mouse models of liver regeneration and HCC. Trem-2-/- mice developed more liver tumours irrespective of size after diethylnitrosamine (DEN) administration, displayed exacerbated liver damage, inflammation, oxidative stress and hepatocyte proliferation. Administering an antioxidant diet blocked DEN-induced hepatocarcinogenesis in both genotypes. Similarly, Trem-2-/- animals developed more and larger tumours in fibrosis-associated HCC models. Trem-2-/- livers showed increased hepatocyte proliferation and inflammation after partial hepatectomy. Conditioned media from human HSCs overexpressing TREM-2 inhibited human HCC spheroid growth in vitro through attenuated Wnt ligand secretion. CONCLUSION: TREM-2 plays a protective role in hepatocarcinogenesis via different pleiotropic effects, suggesting that TREM-2 agonism should be investigated as it might beneficially impact HCC pathogenesis in a multifactorial manner.

c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

cRel expression regulates distinct transcriptional and functional profiles driving fibroblast matrix production in systemic sclerosis.

OBJECTIVES: NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. METHODS: Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. RESULTS: cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. CONCLUSION: cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.

Presence of Serum Antinuclear Antibodies Does Not Impact Long-Term Outcomes in Nonalcoholic Fatty Liver Disease.

INTRODUCTION: We investigated the longitudinal impact of antinuclear antibody (ANA) on clinical outcomes and survival in nonalcoholic fatty liver disease (NAFLD). METHODS: ANA were found in 16.9% of 923 biopsy-proven NAFLD patients, but none of them had histologic autoimmune hepatitis (AIH) or developed AIH after a mean follow-up of 106±50 months. RESULTS: Although ANA-positive cases had a higher prevalence of nonalcoholic steatohepatitis at baseline, the occurrence of liver-related events, hepatocellula carcinoma, cardiovascular events, extrahepatic malignancy, and overall survival were similar to ANA-negative. DISCUSSION: Once AIH has been ruled out, the long-term outcomes and survival are unaffected by the presence of ANA in patients with NAFLD.

Fibrogenic Activity of MECP2 Is Regulated by Phosphorylation in Hepatic Stellate Cells.

BACKGROUND & AIMS: Methyl-CpG binding protein 2, MECP2, which binds to methylated regions of DNA to regulate transcription, is expressed by hepatic stellate cells (HSCs) and is required for development of liver fibrosis in mice. We investigated the effects of MECP2 deletion from HSCs on their transcriptome and of phosphorylation of MECP2 on HSC phenotype and liver fibrosis. METHODS: We isolated HSCs from Mecp2-/y mice and wild-type (control) mice. HSCs were activated in culture and used in array analyses of messenger RNAs and long noncoding RNAs. Kyoto Encyclopedia of Genes and Genomes pathway analyses identified pathways regulated by MECP2. We studied mice that expressed a mutated form of Mecp2 that encodes the S80A substitution, MECP2S80, causing loss of MECP2 phosphorylation at serine 80. Liver fibrosis was induced in these mice by administration of carbon tetrachloride, and liver tissues and HSCs were collected and analyzed. RESULTS: MECP2 deletion altered expression of 284 messenger RNAs and 244 long noncoding RNAs, including those that regulate DNA replication; are members of the minichromosome maintenance protein complex family; or encode CDC7, HAS2, DNA2 (a DNA helicase), or RPA2 (a protein that binds single-stranded DNA). We found that MECP2 regulates the DNA repair Fanconi anemia pathway in HSCs. Phosphorylation of MECP2S80 and its putative kinase, HAS2, were induced during transdifferentiation of HSCs. HSCs from MECP2S80 mice had reduced proliferation, and livers from these mice had reduced fibrosis after carbon tetrachloride administration. CONCLUSIONS: In studies of mice with disruption of Mecp2 or that expressed a form of MECP2 that is not phosphorylated at S80, we found phosphorylation of MECP2 to be required for HSC proliferation and induction of fibrosis. In HSCs, MECP2 regulates expression of genes required for DNA replication and repair. Strategies to inhibit MECP2 phosphorylation at S80 might be developed for treatment of liver fibrosis.

A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices.

Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono- or co-cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme-linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFß1) and platelet-derived growth factor (PDGFßß) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α-smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor-cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell-cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFß1/PDGFßß stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor-like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFß1/PDGFßß-stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor-cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies.

Hepatocellular Carcinoma in Obesity, Type 2 Diabetes, and NAFLD.

Hepatocellular carcinoma (HCC) is the second commonest cause of cancer death worldwide. Rather than falling as a result of prevention and treatments for viral hepatitis, an increase is evident in developed nations consequent to the rising prevalence of obesity and type 2 diabetes mellitus (T2DM)-the two major risk factors for nonalcoholic fatty liver disease (NAFLD). The majority of patients with HCC complicating these conditions present with advanced disease as the tools for surveillance are inadequate, and the "at-risk" population is not well characterized. This review will summarize the epidemiological evidence linking obesity, T2DM, and NAFLD with HCC, what is known about the pathogenic mechanisms involved, as well as their relevance for clinicians managing patients at risk. There will also be an overview of the "unmet needs" surrounding this topic, with suggestions for the direction translational research should take in order to prevent progression of NAFLD to HCC, to improve early detection of HCC in those with NAFLD, as well as to improve outcomes for those affected.