Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

The Origin and Fate of Liver Myofibroblasts.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.

SREBP Regulation of Lipid Metabolism in Liver Disease, and Therapeutic Strategies.

Sterol regulatory element-binding proteins (SREBPs) are master transcription factors that play a crucial role in regulating genes involved in the biogenesis of cholesterol, fatty acids, and triglycerides. As such, they are implicated in several serious liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). SREBPs are subject to regulation by multiple cofactors and critical signaling pathways, making them an important target for therapeutic interventions. In this review, we first introduce the structure and activation of SREBPs, before focusing on their function in liver disease. We examine the mechanisms by which SREBPs regulate lipogenesis, explore how alterations in these processes are associated with liver disease, and evaluate potential therapeutic strategies using small molecules, natural products, or herb extracts that target these pathways. Through this analysis, we provide new insights into the versatility and multitargets of SREBPs as factors in the modulation of different physiological stages of liver disease, highlighting their potential targets for therapeutic treatment.

Alternatives to animal testing to assess MASH drugs and hepatotoxicity.

The Food and Drug Administration (FDA) Modernization Act 2.0 "allows for alternatives to animal testing for purposes of drug and biological product applications." This provides an opportunity to develop and improve alternatives to animal studies to assess drugs in the liver. Two-dimensional cultures of liver cells fail to maintain their differentiated state and fail to reproduce liver disease phenotypes. Therefore, several platforms using human liver cells are being developed either to (1) assess hepatotoxicity of drugs or (2) create "diseases in a dish" to assess the effectiveness of drugs in treating liver diseases, primarily focused on treating MASH. The technological approaches include precision cut liver slices, human liver spheroids, human liver organoids, bioprinted human liver tissues, and microphysiological systems. This review evaluates each of these technologies and their role in providing alternatives to testing in animals.

Isolation of primary human liver cells from normal and nonalcoholic steatohepatitis livers.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Developing a highly-reliable learning health system.

Multiple independent frameworks to support continuous improvement have been proposed to guide healthcare organizations. Two of the most visible are High-reliability Health care, (Chassin et al., 2013) which is emphasized by The Joint Commission, and Learning Health Systems, (Institute of Medicine, 2011) highlighted by the National Academy of Medicine. We propose that organizations consider tightly linking these two models, creating a "Highly-reliable Learning Health System." We describe several efforts at our organization that has resulted from this combined model and have helped our organization weather the COVID-19 pandemic. The organizational changes created using this framework will enable our health system to support a culture of quality across our teams and better fulfill our tripartite mission of high-quality care, effective education of trainees, and dissemination of important innovations.

Human Hepatic Stellate Cells: Isolation and Characterization.

Liver fibrosis of different etiologies is characterized by activation of hepatic stellate cells (aHSCs) into collagen type I secreting myofibroblasts, which produce fibrous scar and make the liver fibrotic. aHSCs are the major source of myofibroblasts and, therefore, the primary targets of anti-fibrotic therapy. Despite extensive studies, targeting of aHSCs in patients provides challenges. The progress in anti-fibrotic drug development relies on translational studies but is limited by the availability of primary human HSCs. Here we describe a perfusion/gradient centrifugation-based method of the large-scale isolation of highly purified and viable human HSCs (hHSCs) from normal and diseased human livers and the strategies of hHSC cryopreservation.

Candida albicans-specific Th17 cell-mediated response contributes to alcohol-associated liver disease.

Alcohol-associated liver disease is accompanied by intestinal mycobiome dysbiosis, yet the impacts on liver disease are unclear. We demonstrate that Candida albicans-specific T helper 17 (Th17) cells are increased in circulation and present in the liver of patients with alcohol-associated liver disease. Chronic ethanol administration in mice causes migration of Candida albicans (C. albicans)-reactive Th17 cells from the intestine to the liver. The antifungal agent nystatin decreased C. albicans-specific Th17 cells in the liver and reduced ethanol-induced liver disease in mice. Transgenic mice expressing T cell receptors (TCRs) reactive to Candida antigens developed more severe ethanol-induced liver disease than transgene-negative littermates. Adoptively transferring Candida-specific TCR transgenic T cells or polyclonal C. albicans-primed T cells exacerbated ethanol-induced liver disease in wild-type mice. Interleukin-17 (IL-17) receptor A signaling in Kupffer cells was required for the effects of polyclonal C. albicans-primed T cells. Our findings indicate that ethanol increases C. albicans-specific Th17 cells, which contribute to alcohol-associated liver disease.

The Role of Mesothelin in Activation of Portal Fibroblasts in Cholestatic Liver Injury.

Fibrosis is a common consequence of abnormal wound healing, which is characterized by infiltration of myofibroblasts and formation of fibrous scar. In liver fibrosis, activated Hepatic Stellate Cells (aHSCs) and activated Portal Fibroblasts (aPFs) are the major contributors to the origin of hepatic myofibroblasts. aPFs are significantly involved in the pathogenesis of cholestatic fibrosis, suggesting that aPFs may be a primary target for anti-fibrotic therapy in cholestatic injury. aPFs are distinguishable from aHSCs by specific markers including mesothelin (Msln), Mucin 16 (Muc16), and Thymus cell antigen 1 (Thy1, CD90) as well as fibulin 2, elastin, Gremlin 1, ecto-ATPase nucleoside triphosphate diphosphohydrolase 2. Msln plays a critical role in activation of PFs, via formation of Msln-Muc16-Thy1 complex that regulates TGFß1/TGFßRI-mediated fibrogenic signaling. The opposing pro- and anti-fibrogenic effects of Msln and Thy1 are key components of the TGFß1-induced activation pathway in aPFs. In addition, aPFs and activated lung and kidney fibroblasts share similarities across different organs with expression of common markers and activation cascade including Msln-Thy1 interaction. Here, we summarize the potential function of Msln in activation of PFs and development of cholestatic fibrosis, offering a novel perspective for anti-fibrotic therapy targeting Msln.

Multiplex short tandem repeat profiling of immortalized hepatic stellate cell line Col-GFP HSC.

Misidentification, cross-contamination and genetic drift of continuous animal cell lines are persistent problems in biomedical research, leading to erroneous results and inconsistent or invalidated studies. The establishment of immortalized hepatic stellate cell line Col-GFP HSC was reported in PLoS One in the year 2013. In the present study a multi loci short tandem repeat signature for this cell line was established that allows for unique cell line authentication.

Metabolic Injury of Hepatocytes Promotes Progression of NAFLD and AALD.

Nonalcoholic liver disease is a component of metabolic syndrome associated with obesity, insulin resistance, and hyperlipidemia. Excessive alcohol consumption may accelerate the progression of steatosis, steatohepatitis, and fibrosis. While simple steatosis is considered a benign condition, nonalcoholic steatohepatitis with inflammation and fibrosis may progress to cirrhosis, liver failure, and hepatocellular cancer. Studies in rodent experimental models and primary cell cultures have demonstrated several common cellular and molecular mechanisms in the pathogenesis and regression of liver fibrosis. Chronic injury and death of hepatocytes cause the recruitment of myeloid cells, secretion of inflammatory and fibrogenic cytokines, and activation of myofibroblasts, resulting in liver fibrosis. In this review, we discuss the role of metabolically injured hepatocytes in the pathogenesis of nonalcoholic steatohepatitis and alcohol-associated liver disease. Specifically, the role of chemokine production and de novo lipogenesis in the development of steatotic hepatocytes and the pathways of steatosis regulation are discussed.

Aberrant iron distribution via hepatocyte-stellate cell axis drives liver lipogenesis and fibrosis.

Hepatocytes have important roles in liver iron homeostasis, abnormalities in which are tightly associated with liver steatosis and fibrosis. Here, we show that non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are characterized by iron-deficient hepatocytes and iron overload in hepatic stellate cells (HSCs). Iron deficiency enhances hepatocyte lipogenesis and insulin resistance through HIF2α-ATF4 signaling. Elevated secretion of iron-containing hepatocyte extracellular vesicles (EVs), which are normally cleared by Kupffer cells, accounts for hepatocyte iron deficiency and HSC iron overload in NAFLD/NASH livers. Iron accumulation results in overproduction of reactive oxygen species that promote HSC fibrogenic activation. Conversely, blocking hepatocyte EV secretion or depleting EV iron cargo restores liver iron homeostasis, concomitant with mitigation of NAFLD/NASH-associated liver steatosis and fibrosis. Taken together, these studies show that iron distribution disorders contribute to the development of liver metabolic diseases.

MiR-690 treatment causes decreased fibrosis and steatosis and restores specific Kupffer cell functions in NASH.

Nonalcoholic steatohepatitis (NASH) is a liver disease associated with significant morbidity. Kupffer cells (KCs) produce endogenous miR-690 and, via exosome secretion, shuttle this miRNA to other liver cells, such as hepatocytes, recruited hepatic macrophages (RHMs), and hepatic stellate cells (HSCs). miR-690 directly inhibits fibrogenesis in HSCs, inflammation in RHMs, and de novo lipogenesis in hepatocytes. When an miR-690 mimic is administered to NASH mice in vivo, all the features of the NASH phenotype are robustly inhibited. During the development of NASH, KCs become miR-690 deficient, and miR-690 levels are markedly lower in mouse and human NASH livers than in controls. KC-specific KO of miR-690 promotes NASH pathogenesis. A primary target of miR-690 is NADK mRNA, and NADK levels are inversely proportional to the cellular miR-690 content. These studies show that KCs play a central role in the etiology of NASH and raise the possibility that miR-690 could emerge as a therapeutic for this condition.

CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease.

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-ß-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-ß increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-ß. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-ß induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-ß treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

Immunotherapy-based targeting of MSLN+ activated portal fibroblasts is a strategy for treatment of cholestatic liver fibrosis.

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln-/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1-/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN+ aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis.

Mutation of the 5'-untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice.

Arterial stiffening, a characteristic feature of obesity and type 2 diabetes, contributes to the development and progression of cardiovascular diseases (CVD). Currently, no effective prophylaxis or therapeutics is available to prevent or treat arterial stiffening. A better understanding of the molecular mechanisms underlying arterial stiffening is vital to identify newer targets and strategies to reduce CVD burden. A major contributor to arterial stiffening is increased collagen deposition. In the 5'-untranslated regions of mRNAs encoding for type I collagen, an evolutionally conserved stem-loop (SL) structure plays an essential role in its stability and post-transcriptional regulation. Here, we show that feeding a high-fat/high-sucrose (HFHS) diet for 28 wk increases adiposity, insulin resistance, and blood pressure in male wild-type littermates. Moreover, arterial stiffness, assessed in vivo via aortic pulse wave velocity, and ex vivo using atomic force microscopy in aortic explants or pressure myography in isolated femoral and mesenteric arteries, was also increased in those mice. Notably, all these indices of arterial stiffness, along with collagen type I levels in the vasculature, were reduced in HFHS-fed mice harboring a mutation in the 5'SL structure, relative to wild-type littermates. This protective vascular phenotype in 5'SL-mutant mice did not associate with a reduction in insulin resistance or blood pressure. These findings implicate the 5'SL structure as a putative therapeutic target to prevent or reverse arterial stiffening and CVD associated with obesity and type 2 diabetes.NEW & NOTEWORTHY In the 5'-untranslated (UTR) regions of mRNAs encoding for type I collagen, an evolutionally conserved SL structure plays an essential role in its stability and posttranscriptional regulation. We demonstrate that a mutation of the SL mRNA structure in the 5'-UTR decreases collagen type I deposition and arterial stiffness in obese mice. Targeting this evolutionarily conserved SL structure may hold promise in the management of arterial stiffening and CVD associated with obesity and type 2 diabetes.

Nondegradable Collagen Increases Liver Fibrosis but Not Hepatocellular Carcinoma in Mice.

Although hepatocellular cancer (HCC) usually occurs in the setting of liver fibrosis, the causal relationship between liver fibrosis and HCC is unclear. in vivo and in vitro models of HCC involving Colr/r mice (that produce a collagenase-resistant type I collagen) or wild-type (WT) mice were used to assess the relationship between type I collagen, liver fibrosis, and experimental HCC. HCC was either chemically induced in WT and Colr/r mice or Hepa 1-6 cells were engrafted into WT and Colr/r livers. The effect of hepatic stellate cells (HSCs) from WT and Colr/r mice on the growth of Hepa 1-6 cells was studied by using multicellular tumor spheroids and xenografts. Collagen type I deposition and fibrosis were increased in Colr/r mice, but they developed fewer and smaller tumors. Hepa 1-6 cells had reduced tumor growth in the livers of Colr/r mice. Although Colr/r HSCs exhibited a more activated phenotype, Hepa 1-6 growth and malignancy were suppressed in multicellular tumor spheroids and in xenografts containing Colr/r HSCs. Treatment with vitronectin, which mimics the presence of degraded collagen fragments, converted the Colr/r phenotype into a WT phenotype. Although Colr/r mice have increased liver fibrosis, they exhibited decreased HCC in several models. Thus, increased liver type I collagen does not produce increased experimental HCC.

Nonalcoholic Steatohepatitis and HCC in a Hyperphagic Mouse Accelerated by Western Diet.

BACKGROUND & AIMS: How benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH-HCC progression, as well as regression. METHODS: Hyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development. RESULTS: Foz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment. CONCLUSIONS: The Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure.