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Summary Cellular senescence is a state of irreversible cell cycle arrest that has important physiological functions. However, cellular senescence is also a hallmark of ageing and has been associated with several pathological conditions. A wide range of factors including genotoxic stress, mitogens and inflammatory cytokines can induce senescence. Phenotypically, senescent cells are characterised by short telomeres, an enlarged nuclear area and damaged genomic and mitochondrial DNA. Secretion of proinflammatory proteins, also known as the senescence-associated secretory phenotype, is a characteristic of senescent cells that is thought to be the main contributor to their disease-inducing properties. In the past decade, the role of cellular senescence in the development of non-alcoholic fatty liver disease (NAFLD) and its progression towards non-alcoholic steatohepatitis (NASH) has garnered significant interest. Until recently, it was suggested that hepatocyte cellular senescence is a mere consequence of the metabolic dysregulation and inflammatory phenomena in fatty liver disease. However, recent work in rodents has suggested that senescence may be a causal factor in NAFLD development. Although causality is yet to be established in humans, current evidence suggests that targeting senescent cells has therapeutic potential for NAFLD. We aim to provide insights into the quality of the evidence supporting a causal role of cellular senescence in the development of NAFLD in rodents and humans. We will elaborate on key cellular and molecular features of senescence and discuss the efficacy and safety of novel senolytic drugs for the treatment or prevention of NAFLD.

Background and Aims As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease. Methods To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo. Results Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries. Conclusions We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease., Graphical abstract

Background & Aims Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types. Methods Sox9EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence. Results BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFPhigh BECs. Single Sox9EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation. Conclusions Our data demonstrate that Sox9EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver., Graphical abstract

Background & Aims Liver stiffness is increased in advanced chronic liver disease (ACLD) and accurately predicts prognosis in this population. Recent data suggest that extracellular matrix stiffness per se may modulate the phenotype of liver cells. We aimed at investigating the effect of matrix stiffness on the phenotype of liver cells of rats with cirrhosis, assessing its influence on their response to antifibrotic strategies and evaluating associated molecular mechanisms. Methods Hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells were isolated from healthy rats or rats with cirrhosis (carbon tetrachloride or thioacetamide), and cultured on polyacrylamide gels with different physiologically relevant stiffness for 72 h. Results All cell types of rats with cirrhosis cultured at low stiffness showed a significant phenotype amelioration vs. rigid matrix (assessed by quantitative morphology, mRNA expression, protein synthesis, and electron microscopy imaging). Additionally, stiffness modified the antifibrotic effects of liraglutide in stellate cells of rats with cirrhosis. Finally, evaluation of nuclear morphology revealed that high stiffness induced nuclei deformation in all cell types, an observation confirmed in cells from human livers. Disconnecting the nucleus from the cytoskeleton by cytoskeleton disruption or a defective form of nesprin 1 significantly recovered spherical nuclear shape and quiescent phenotype of cells. Conclusions The environment's stiffness per se modulates the phenotype of healthy rats and liver cells of rats with cirrhosis by altering the nuclear morphology through cytoskeleton-derived mechanical forces. The reversibility of this mechanism suggests that targeting the stiffness-mediated intracellular mechanical tensions may represent a novel therapeutic strategy for ACLD. Lay summary During cirrhosis, the liver becomes scarred, stiff, and unable to perform its normal functions efficiently. In this study, we demonstrated that cells from diseased (stiff) livers recovered their functionality when placed in a soft environment (as that of a healthy liver). Furthermore, treatments aimed at tricking liver cells into believing they are in a healthy, soft liver improved their function and could potentially contribute to treat cirrhosis., Graphical abstract, Highlights • Low stiffness directly improves the phenotype of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells. • Stiffness modulates sinusoidal crosstalk in advanced chronic liver disease (ACLD). • Stiffness may determine the efficacy of antifibrotic strategies. • Mechanical forces applied to the nucleus through the LINC complex mediate the effects of stiffness on liver cell phenotype.

While the liver demonstrates remarkable resilience during aging, there is growing evidence that it undergoes all the cellular hallmarks of aging, which increases the risk of liver and systemic disease. The aging process in the liver is driven by alterations of the genome and epigenome that contribute to dysregulation of mitochondrial function and nutrient sensing pathways, leading to cellular senescence and low-grade inflammation. These changes promote multiple phenotypic changes in all liver cells (hepatocytes, liver sinusoidal endothelial, hepatic stellate and Küpffer cells) and impairment of hepatic function. In particular, age-related changes in the liver sinusoidal endothelial cells are a significant but under-recognized risk factor for the development of age-related cardiometabolic disease., Graphical Abstract Unlabelled Image, Highlights • Liver aging is driven by transcription and metabolic epigenome alterations. • This leads to cellular senescence and low-grade inflammation. • Hepatocyte, sinusoidal endothelial, stellate and Küpffer cells undergoes the hallmarks of aging. • Each cell type demonstrates phenotypical cellular changes with age.

The incidence of hepatocellular carcinoma (HCC) and the mortality resulting from HCC are both increasing. Most patients with HCC are diagnosed at advanced stages when curative treatments are impossible. Current drug therapy extends mean overall survival by only a short period of time. Genetic mutations associated with HCC vary widely. Therefore, transgenic and mutant animal models are needed to investigate the molecular effects of specific mutations, classify them as drivers or passengers, and develop targeted treatments. Cirrhosis, however, is the premalignant state common to 90% of HCC patients. Currently, no specific therapies are available to halt or reverse the progression of cirrhosis to HCC. Understanding the genetic drivers of HCC as well as the biochemical, mechanical, hormonal, and metabolic changes associated with cirrhosis could lead to novel treatments and cancer prevention strategies. Although additional therapies recently received Food and Drug Administration approval, significant clinical breakthroughs have not emerged since the introduction of the multikinase inhibitor sorafenib, necessitating alternate research strategies. Zebrafish (Danio rerio) are effective for disease modeling because of their high degree of gene and organ architecture conservation with human beings, ease of transgenesis and mutagenesis, high fecundity, and low housing cost. Here, we review zebrafish models of HCC and identify areas on which to focus future research efforts to maximize the advantages of the zebrafish model system. Keywords: Cirrhosis, Tumor Microenvironment, Model Organisms, Inflammation, Drug-Induced Liver Injury, Autophagy

Background Dysfunction of liver sinusoidal endothelial cells (LSECs) is permissive for the progression of liver fibrosis and cirrhosis and responsible for its clinical complications. Here, we have mapped the spatial distribution of heterogeneous liver ECs in normal vs cirrhotic mouse livers and identified zone-specific transcriptomic changes of LSECs associated with liver cirrhosis using scRNA-seq technology. Approach & Results Cirrhosis was generated in endothelial specific green fluorescent protein (GFP) reporter mice through carbon tetrachloride inhalation for 12 weeks. GFP-positive liver EC populations were isolated from control and cirrhotic mice by FACS. We identified 6 clusters of liver EC populations including 3 clusters of LSECs, 2 clusters of vascular ECs and 1 cluster of lymphatic ECs. Based on previously reported LSEC-landmarks, we mapped the 3 clusters of LSECs in zones 1, 2, and 3, and determined phenotypic changes in each zone between control and cirrhotic mice. We found genes representing capillarization of LSECs (eg, CD34) as well as extracellular matrix genes were most upregulated in LSECs of zone 3 in cirrhotic mice, which may contribute to the development of basement membranes. LSECs in cirrhotic mice also demonstrated decreased expression of endocytic receptors, most remarkably in zone 3. Transcription factors (Klf2 [Kruppel-like factor-2], Klf4 [Kruppel-like factor-4], and AP-1) that induce nitric oxide production in response to shear stress were downregulated in LSECs of all zones in cirrhotic mice, implying increased intrahepatic vascular resistance. Conclusion This study deepens our knowledge of the pathogenesis of liver cirrhosis at a spatial, cell-specific level, which is indispensable for the development of novel therapeutic strategies to target the most dysfunctional liver ECs., Graphical abstract

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

In vitro models of the human liver are important for the following: (1) mitigating the risk of drug-induced liver injury to human beings, (2) modeling human liver diseases, (3) elucidating the role of single and combinatorial microenvironmental cues on liver cell function, and (4) enabling cell-based therapies in the clinic. Methods to isolate and culture primary human hepatocytes (PHHs), the gold standard for building human liver models, were developed several decades ago; however, PHHs show a precipitous decline in phenotypic functions in 2-dimensional extracellular matrix–coated conventional culture formats, which does not allow chronic treatment with drugs and other stimuli. The development of several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment for enhancing the function of both PHHs and induced pluripotent stem cell–derived human hepatocyte-like cells; long-term (4+ weeks) stabilization of hepatocellular function typically requires co-cultivation with liver-derived or non–liver-derived nonparenchymal cell types. In addition, the recent development of liver organoid culture systems can provide a strategy for the enhanced expansion of therapeutically relevant cell types. Here, we discuss advances in engineering approaches for constructing in vitro human liver models that have utility in drug screening and for determining microenvironmental determinants of liver cell differentiation/function. Design features and validation data of representative models are presented to highlight major trends followed by the discussion of pending issues that need to be addressed. Overall, bioengineered liver models have significantly advanced our understanding of liver function and injury, which will prove useful for drug development and ultimately cell-based therapies.

Aldosterone, with pro-oxidation and pro-autophagy capabilities, plays a key role in liver fibrosis. However, the mechanisms underlying aldosterone-promoted liver sinusoidal endothelial cells (LSECs) defenestration remain unknown. Caveolin 1 (Cav1) displays close links with autophagy and fenestration. Hence, we aim to investigate the role of Cav1-related autophagy in LSECs defenestration. We found the increase of aldosterone/MR (mineralocorticoid receptor) level, oxidation, autophagy, and defenestration in LSECs in the human fibrotic liver, BDL or hyperaldosteronism models; while antagonizing aldosterone or inhibiting autophagy relieved LSECs defenestration in BDL-induced fibrosis or hyperaldosteronism models. In vitro, fenestrae of primary LSECs gradually shrank, along with the down-regulation of the NO-dependent pathway and the augment of the AMPK-dependent autophagy; these effects were aggravated by rapamycin (an autophagy activator) or aldosterone treatment. Additionally, aldosterone increased oxidation mediated by Cav1, reduced ATP generation, and subsequently induced the AMPK-dependent autophagy, leading to the down-regulation of the NO-dependent pathway and LSECs defenestration. These effects were reversed by MR antagonist spironolactone, antioxidants or autophagy inhibitors. Besides, aldosterone enhanced the co-immunoprecipitation of Cav1 with p62 and ubiquitin, and induced Cav1 co-immunofluorescence staining with LC3, ubiquitin, and F-actin in the perinuclear area of LSECs. Furthermore, aldosterone treatment increased the membrane protein level of Cav1, whereas decrease the cytoplasmic protein level of Cav1, indicating that aldosterone induced Cav1-related selective autophagy and F-actin remodeling to promote defenestration. Consequently, Cav1-related selective autophagy initiated by aldosterone-induced oxidation promotes LSECs defenestration via activating the AMPK-ULK1 pathway and inhibiting the NO-dependent pathway.

The lymphatic vascular system has been minimally explored in the liver despite its essential functions including maintenance of tissue fluid homeostasis. The discovery of specific markers for lymphatic endothelial cells has advanced the study of lymphatics by methods including imaging, cell isolation, and transgenic animal models and has resulted in rapid progress in lymphatic vascular research during the last decade. These studies have yielded concrete evidence that lymphatic vessel dysfunction plays an important role in the pathogenesis of many diseases. This article reviews the current knowledge of the structure, function, and markers of the hepatic lymphatic vascular system as well as factors associated with hepatic lymphangiogenesis and compares liver lymphatics with those in other tissues. Keywords: VEGF, Inflammation, Cirrhosis, Portal Hypertension

Background & Aims Liver inflammation is a common extraintestinal manifestation of inflammatory bowel disease (IBD), but whether liver involvement is a consequence of a primary intestinal defect or results from alternative pathogenic processes remains unclear. Therefore, we sought to determine the potential pathogenic mechanism(s) of concomitant liver inflammation in an established murine model of IBD. Methods Liver inflammation and immune cell subsets were characterized in ileitis-prone SAMP1/YitFc (SAMP) and AKR/J (AKR) control mice, lymphocyte-depleted SAMP (SAMPxRag-1−/−), and immunodeficient SCID recipient mice receiving SAMP or AKR donor CD4+ T cells. Proliferation and suppressive capacity of CD4+ T-effector (Teff) and T-regulatory (Treg) cells from gut-associated lymphoid tissue (GALT) and livers of SAMP and AKR mice were measured. Results Surprisingly, prominent inflammation was detected in 4-week-old SAMP livers before histologic evidence of ileitis, whereas both disease phenotypes were absent in age-matched AKR mice. SAMP liver disease was characterized by abundant infiltration of lymphocytes, required for hepatic inflammation to occur, a TH1-skewed environment, and phenotypically activated CD4+ T cells. SAMP intrahepatic CD4+ T cells also had the ability to induce liver and ileal inflammation when adoptively transferred into SCID recipients, whereas GALT-derived CD4+ T cells produced milder ileitis but not liver inflammation. Interestingly, SAMP intrahepatic CD4+ Teff cells showed increased proliferation compared with both SAMP GALT- and AKR liver-derived CD4+ Teff cells, and SAMP intrahepatic Tregs were decreased among CD4+ T cells and impaired in in vitro suppressive function compared with AKR. Conclusions Activated intrahepatic CD4+ T cells induce liver inflammation and contribute to experimental ileitis via locally impaired hepatic immunosuppressive function.

Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors., Hepcidin, the central regulator of iron homoeostasis, is itself regulated by many factors including iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation.

Pathologic angiogenesis appears to be intrinsically associated with the fibrogenic progression of chronic liver diseases, which eventually leads to the development of cirrhosis and related complications, including hepatocellular carcinoma. Several laboratories have suggested that this association is relevant for chronic liver disease progression, with angiogenesis proposed to sustain fibrogenesis. This minireview offers a synthesis of relevant findings and opinions that have emerged in the last few years relating liver angiogenesis to fibrogenesis. We discuss liver angiogenesis in normal and pathophysiologic conditions with a focus on the role of hypoxia and hypoxia-inducible factors and assess the evidence supporting a clear relationship between angiogenesis and fibrogenesis. A section is dedicated to the critical interactions between liver sinusoidal endothelial cells and either quiescent hepatic stellate cells or myofibroblast-like stellate cells. Finally, we introduce the unusual, dual (profibrogenic and proangiogenic) role of hepatic myofibroblasts and emerging evidence supporting a role for specific mediators like vasohibin and microparticles and microvesicles.

Background & Aims The liver is an organ with paradoxic immunologic properties and is known for its tolerant microenvironment, which holds important implications for hepatic diseases. The molecular basis for this local immune suppression, however, is poorly understood. In this study, we aimed to determine the role of liver sinusoidal endothelial cell lectin (LSECtin), a recently identified member of the dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) family, in the regulation of hepatic T-cell immune response. Methods The regulation of T-cell effector function by LSECtin was determined by co-stimulated T cells with anti-CD3/CD28 monoclonal antibody and LSECtin protein, or co-culture of T-cell receptor transgenic T cells with mouse LSECs in vitro. We generated LSECtin knockout mice and prepared recombinant LSECtin protein and complementary DNA plasmids to analyze the role of LSECtin in hepatic T-cell immune regulation in vivo. Results We showed that LSECtin specifically recognized activated T cells and negatively regulated their immune responses. In mice with T-cell–mediated acute liver injury, the lack of LSECtin accelerated the disease owing to an increased T-cell immune response, whereas the exogenous administration of recombinant LSECtin protein or plasmid ameliorated the disease via down-regulation of T-cell immunity. Conclusions Our results reveal that LSECtin is a novel regulator of T cells and expose a crucial mechanism for hepatic T-cell immune suppression, perhaps opening up a new approach for treatment of inflammatory diseases in the liver.