Unresolved basic issues in hepatologyBasic research and comparative human medical advances during the last decades and centuries have significantly improved diagnostic and treatment modalities in Hepatology.However, the evolution of this area has sparked not only scientific and clinical benefits but also controversies.The focused series "Unresolved Basic Issues in Hepatology" contains 13 reviews from leading experts from Australia, Austria, Canada, China, France, Germany, India, UK and USA highlighting current perspectives on hot and often controversial discussed topics in the field of Hepatology (Figure 1).In their article "Revisiting hemochromatosis: genetic vs. phenotypic manifestations", Anderson and Bardou-Jacquet report about the high phenotypic variability in hereditary hemochromatosis that can be largely explained by a range of environmental, genetic and physiological factors.They strengthen the point that variations in penetrance between individuals might be due to the fact that some genes involved in the pathogenesis of iron homeostasis disorders are still not know yet and that both co-morbidities and genetic polymorphisms that do not affect iron levels per se could influence or determine the propensity of the disease.The review "Autoimmune hepatitis: is histology conclusive?" by Beer and Dienes critically discuss the diagnostic value of liver biopsy in the diagnosis and management of autoimmune hepatitis.The authors provide some clues on the histological difference between clinical subtypes of autoimmune hepatitis showing that a definitive diagnosis of autoimmune hepatitis is not possible by histology alone and should be better based on combined criteria including the presence of autoantibodies, immunoglobulin G elevations, and graded histopathology.Cai and Boyer discuss "The role of bile acids in cholestatic liver injury" focusing on their direct effects on hepatocytes, cholangiocytes, stellate cells and immune cells.From their contribution it became clear that despite the fundamental insights in the pathogenesis of cholestatic liver injury, important mechanistic details on the effects of bile acids on specific liver cells are still not understood.Ding and colleagues highlight the current knowledge on "Exosomes as therapeutic vehicles in liver diseases".They describe the role of exosomes and their cargo as potential therapeutic tools to treat or to alleviate liver injury caused by various factors.As the authors discuss, there are still many problems to be solved before the wealth of encouraging findings obtained with these membrane-bound extracellular vesicles in preclinical studies can be applied clinically.In the review "It's all about the spaces between cells: Role of extracellular matrix in liver fibrosis" Khurana and colleagues critically discuss the advances in the understanding of the physiological role of factors contributing to extracellular matrix homeostasis, turnover and impairment.Particularly, it is discussed how the knowledge of individual mediators can be harnessed for the therapy of hepatic fibrosis.In their article "Liver failure-future challenges and remaining questions" Lemmer and colleagues describe controversial aspects in the classification of liver failure.They propose that it is necessary to differentiate three types of liver failure, namely acute liver failure, acute-on-chronic liver failure, and acute-on-liver-cirrhosis that mainly can be discriminated by changes in coagulation factors and inhibitors.Melnik reviewed molecular mechanisms that may explain dairy-induced hepatocancerogenesis.In his article entitled "Dairy consumption and hepatocellular carcinoma risk" he highlights how abundant constitutes of milk proteins and milk exosomes and their cargo might contribute to the pathogenesis of hepatocellular carcinoma.Based on accumulating evidence from a large number of epidemiological and translational studies, he concluded that several milk products need to be listed as dairyrelated risk factors for hepatocellular carcinoma.The article "Non-alcoholic fatty liver diseases: current challenges and future directions" written by Roeb provides information about critical issues in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).It summarizes comprehensively the actual knowledge about relevant risk factors and management strategies.In her article she nicely illustrates that key questions related to disease definition, diagnosis, therapy, and long-term management are still only partially answered.In line with this assumption, Rosso and colleagues argued that there is an urgent need to identify better biomarkers for the diagnosis of NAFLD.In their contribution "Diagnostic management of nonalcoholic fatty liver disease: a

In vertebrates, members of the cysteine-rich protein (CRP) family are characterized by the presence of two LIM domains linked to short glycine-rich repeats. These proteins mediate protein-protein interactions and are of fundamental importance for cell differentiation, cytoskeletal remodeling, and transcriptional regulation. To date, a vast amount of information about vertebrate CRPs has become available, including their biological functions, interacting partners, and three-dimensional structures. Compatible with a molecular adapter role, structural data reveal that the LIM domains within these proteins represent completely independent folded units bridged by flexible linker regions. The physiological roles for individual CRPs was determined by targeted gene disruption analysis and by identification of common and specific binding partners by means of yeast and mammalian two-hybrid screens. Several CRP-like LIM domain proteins with close structural and sequence similarity were identified in arthropods, protozoas and plants, supporting the notion that this subset of LIM domain proteins has been highly conserved over the span of evolution thereby emphasizing the importance of their function.

This review focus on sex-related differences in Metabolic dysfunction-associated steatotic liver disease (MASLD), the most recent term used to describe the range of “nonalcoholic” fatty liver disorders with metabolic causes. The review discusses data on epidemiology, mechanisms, and treatment outcomes. Men are more susceptible to MASLD than women, although premenopausal women have a lower risk that increases with age and is influenced by reproductive status. Additionally, women have higher rates of MASLD-related cardiovascular disease and extrahepatic cancers compared to men. Both hormones and chromosomes play a role in MASLD susceptibility. Hormone imbalances, such as hypogonadism, polycystic ovary syndrome, and chromosomal abnormalities like Turner and Klinefelter syndromes, contribute to hepatic steatosis through metabolic and lipid dysregulation. Sex hormones also modulate key molecular and cellular pathways in MASLD, impacting liver-resident cells, gut microbiota, and innate immunity. Reproductive factors, including age at menarche, menopause, hormone therapy, and lactation, further influence disease risk. Breastfeeding not only lowers maternal metabolic risk but also protects infants from MASLD-related complications. Sex also influences how individuals respond to lifestyle changes and pharmacotherapy for obesity, diabetes, and dyslipidemia. While testosterone replacement therapy may benefit liver health in hypogonadal men, evidence on other hormone therapies is limited. More research is needed to better understand the roles of sex hormones and chromosomes in MASLD development and progression at different life stages. Clinical trials should routinely include sex-based analyses to enhance generalizability and ensure equitable care.

Bis-choline-tetrathiomolybdate, introduced as WTX101 (now known as ALXN1840), is a first-in-class copper-protein-binding agent for oral therapy of Wilson’s disease. In contrast to other decoppering agents such as trientine or D-penicillamine it acts by forming a tripartite complex with copper and albumin, thereby detoxifying excess liver and blood copper through biliary excretion. Preclinical animal experimentation with this drug was typically done with the alternative ammonium salt of tetrathiomolybdate, which is expected to have identical properties in terms of copper binding. Here, we comparatively analyzed the therapeutic efficacy of ALXN1840, D-penicillamine and trientine in lowering hepatic copper content in Atp7b−/− mouse. Liver specimens were subjected to laser ablation inductively conductively plasma mass spectrometry and electron microscopic analysis. We found that ALXN1840 caused a massive increase of hepatic copper and molybdenum during early stages of therapy. Prolonged treatment with ALXN1840 reduced hepatic copper to an extent that was similar to that observed after administration of D-penicillamine and trientine. Electron microscopic analysis showed a significant increase of lysosomal electron-dense particles in the liver confirming the proposed excretory pathway of ALXN1840. Ultrastructural analysis of mice treated with dosages comparable to the bis-choline-tetrathiomolybdate dosage used in an ongoing phase III trial in Wilson’s disease patients, as well as D-penicillamine and trientine, did not show relevant mitochondrial damage. In contrast, a high dose of ALXN1840 applied for four weeks triggered dramatic structural changes in mitochondria, which were notably characterized by the formation of holes with variable sizes. Although these experimental results may not be applicable to patients with Wilson’s disease, the data suggests that ALXN1840 should be administered at low concentrations to prevent mitochondrial dysfunction and overload of hepatic excretory pathways.

Liver fibrosis is an excessive scarring process resulting in progressive disruption of the normal tissue architecture and impaired organ function. It comprises many different etiologies and its consequences present a substantial medical and economic burden worldwide Mechanistically, fibrosis is initiated by parenchymal cell destruction resulting in tissue damage that is associated with an inflammatory response. This in turn provokes the local activation of mesenchymal cells which have the capacity to produce extracellular matrix compounds such as collagens. Most important in this process are hepatic stellate cells (HSCs) that are pericytes in the perisinusoidal space of the liver. Upon activation, these cells lose their quiescent phenotype and transit in a process called transdifferentiation into proliferative fibrogenic -smooth muscle positive (-SMA) myofibroblasts. These are the central cellular drivers of hepatic fibrogenesis in experimental and human liver injury. Although the cellular and molecular mechanisms of hepatic fibrosis including important pro-inflammatory and pro-fibrogenic soluble mediators (chemokines, cytokines) and signaling pathways are identified, there exist no specific and effective antifibrotic pharmacological therapies.

Activated hepatic stellate cells (aHSCs) are key players in the fibrotic cascade in inflamed livers, with transforming growth factor-beta 1 (TGF-β1) being the most potent pro-fibrotic cytokine. Therefore, TGF-β1 downregulation in aHSCs may serve as an interesting therapeutic approach for the treatment of liver fibrosis. However, excessive collagen deposition in the extracellular matrix (ECM) hinders drug delivery to aHSCs. Chitosan nanoparticles (CS-NPs) show intrinsic affinity for collagen, holding potential for drug delivery to fibrotic livers. In this study, CS-NPs were used for the in vivo delivery of anti-TGF-β1 siRNA. To promote delivery into aHSCs, CS-NPs were modified with different densities of platelet-derived growth factor receptor-β (PDGF-β) binding peptides as the targeting moiety. The CS-NPs showed an average hydrodynamic diameter of 103 ± 7 nm, a zeta potential of 24 ± 1 mV, and an siRNA encapsulation efficiency of 92.39 ± 6.4%. In healthy mice, biweekly treatment of siRNA-free unmodified CS-NPs up to a concentration of 120 mg/kg for 4 weeks was well tolerated and caused no organ-specific toxicity. The biodistribution of the unmodified and peptide-modified CS-NPs was carried out to identify potential sites of accumulation in healthy and fibrotic mice, and histopathological studies confirmed the safety of the formulations in nontarget sites in the case of fibrotic animals. Cell culture experiments confirmed the affinity of unmodified CS-NPs to the collagenated ECM, whereas collagen density reduction with collagenase-loaded CS-NPs (Coll-NPs) was necessary to promote cellular uptake of the peptide-modified CS-NPs. When loaded with anti-TGF-β1 siRNA and used for the treatment of the CCl4 liver fibrosis model in mice, CS-NPs decorated with a high density of PDGF-β binding peptide could significantly reduce the hepatic TGF-β1 (by approximately 65%) and fibronectin (by approximately 63%) levels. Pretreatment with Coll-NPs could contribute to a further reduction of both markers by around 10%. Histopathological evaluations with Masson Trichrome staining revealed a reduction in the aggregation of the portal inflammatory cells, an absence in the proliferation of the fibroblastic cells in between hepatocytes, and a decrease in collagen deposition in the liver following treatment with anti-TGF-β1 siRNA-loaded peptide-modified CS-NPs, both with and without Coll-NP pretreatment. Accordingly, the results demonstrate the plausibility of anti-TGF-β1-siRNA delivery to aHSCs using PDGF-β binding peptide-modified CS-NPs for the treatment of liver fibrosis.

Due to its profibrogenic actions, TGF-beta1 and the subsequent steps of the TGF-beta1 induced signaling cascade are prefered targets for antifibrogenic therapies. The fact that Smad-dependent TGF-beta signaling is modulated by reactive oxygen species (ROS) prompted us to investigate the influence of L-cysteine and its derivative N-acetylcysteine (NAC), a scavenger of ROS and precursor of glutathion, on TGF-beta signaling in culture-activated hepatic stellate cells (HSC). In a previous study, we have shown that NAC is able to block TGF-beta? signaling in HSC, but is ineffective when the signal is transduced through a constitutive active TGF-beta? receptor (TbetaR) type I. Analysis of the three known TbetaRs (type I, II and III) in HSC by means of ligand crosslinking and western blot experiments revealed that only receptor type III is affected by NAC treatment. We now show, that in contrast to NAC, L-cysteine exhibits no influence on TGF-beta signaling or TbetaRIII protein (betaglycan) integrity, when applied in concentrations, which do not impair cell viability. In vitro data imply that the sensitivity of TbetaRIII for reducing agents is due to a premodification of this receptor in HSC by limited proteolysis. This proteolytic modification seems to be specific for HSC, since the reductant sensitivity is not observed in cultured cells of various species expressing TbetaRIII heterologously. By further dissecting the NAC sensitivity in the initial step of TGF-beta signaling, we were able to identify a so far unknown member of the TbetaR-family in HSC, i.e. endoglin, an endothelial cell homologue of betaglycan. We currently investigate if this disulfide-linked homodimer is involved in the NAC dependent blockade of TGF-beta signaling in HSC.

% of body weight changes, clinical chemistry parameters and White blood cell count were anaylsed

Liver fibrosis is a significant challenge in hepatology, as it represents the common pathway of chronic liver injury due to various causes such as viral hepatitis, metabolic dysfunction-associated steatotic liver disease (MASLD), intoxication, alcohol-related liver disease, autoimmune conditions, and genetic disorders [...]