PNPLA3 rs738409, age, diabetes, sex, and advanced fibrosis jointly contribute to the risk of major adverse liver outcomes in metabolic-associated steatotic liver disease.

BACKGROUND AND AIMS: The patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 variant is associated with Steatotic Liver Disease (SLD) and its progression. We examined the association between PNPLA3 and the development of major adverse liver outcomes (MALO) and how non-modifiable and modifiable conditions modify this relationship. APPROACH AND RESULTS: 2,075 adults with biopsy-confirmed Metabolic-Associated SLD (MASLD) were enrolled in the MASLD CRN studies and followed prospectively until death, transplant, or withdrawal of consent. 104 MALOs were recorded during an average of 4.3 years. PNPLA3 G-allele (Adj. SubHazard ratio (sHR): 1.4, 95% confidence interval (CI): 1.07-1.8), advanced fibrosis (AF) (Adj. sHR: 7.8, 95% CI: 4.4-13.8), age >60 years (Adj. sHR: 2.9, 95% CI: 1.3-6.8), and type 2 diabetes mellitus (T2DM) (Adj. sHR: 2.8, 95% CI: 1.8-4.2) were associated with MALO. Among participants with AF, those carrying the G-allele displayed the highest cumulative incidence of MALO (85%) vs. noncarriers (53%), p=0.03, and p value for interaction<0.01. The strength of the association between PNPLA3 and MALO was statistically significantly greater among older than 60 years (sHR: 2.1, 95% CI: 1.5-2.8), women (sHR: 1.4, 95% CI: 1.1-1.9), and those with AF (sHR: 1.9, 95% CI: 1.5-2.4) or T2DM (sHR: 2.1, 95% CI: 1.5-2.8) as compared with their counterparts, p value for interaction between PNPLA3 and each factor<0.01. CONCLUSIONS: The deleterious effects of PNPLA3 rs738409 on the risk of MALO are significantly worsened by advanced fibrosis, age, T2DM, and sex.

Plasticity, heterogeneity, and multifunctionality of hepatic stellate cells in liver pathophysiology.

HSCs, the resident pericytes of the liver, have consistently been at the forefront of liver research due to their crucial roles in various hepatic pathological processes. Prior literature often depicted HSCs in a binary framework, categorizing them as either quiescent or activated. However, recent advances in HSC research, particularly the advent of single-cell RNA-sequencing, have revolutionized our understanding of these cells. This sophisticated technique offers an unparalleled, high-resolution insight into HSC populations, uncovering a spectrum of diversity and functional heterogeneity across various physiological states of the liver, ranging from liver development to the liver aging process. The single-cell RNA-sequencing revelations have also highlighted the intrinsic plasticity of HSCs and underscored their complex roles in a myriad of pathophysiological processes, including liver injury, repair, and carcinogenesis. This review aims to integrate and clarify these recent discoveries, focusing on how the inherent plasticity of HSCs is central to their dynamic roles both in maintaining liver homeostasis and orchestrating responses to liver injury. Future research will clarify whether findings from rodent models can be translated to human livers and guide how these insights are harnessed to develop targeted therapeutic interventions.

A humanized mouse model for adeno-associated viral gene therapy.

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg-/-/Rag2-/-/Fah-/-/Aavr-/- (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Alterations in zonal distribution and plasma membrane localization of hepatocyte bile acid transporters in patients with NAFLD.

BACKGROUND: NAFLD is highly prevalent with limited treatment options. Bile acids (BAs) increase in the systemic circulation and liver during NAFLD progression. Changes in plasma membrane localization and zonal distribution of BA transporters can influence transport function and BA homeostasis. However, a thorough characterization of how NAFLD influences these factors is currently lacking. This study aimed to evaluate the impact of NAFLD and the accompanying histologic features on the functional capacity of key hepatocyte BA transporters across zonal regions in human liver biopsies. METHODS: A novel machine learning image classification approach was used to quantify relative zonal abundance and plasma membrane localization of BA transporters (bile salt export pump [BSEP], sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [OATP] 1B1 and OATP1B3) in non-diseased (n = 10), NAFL (n = 9), and NASH (n = 11) liver biopsies. Based on these data, membrane-localized zonal abundance (MZA) measures were developed to estimate transporter functional capacity. RESULTS: NAFLD diagnosis and histologic scoring were associated with changes in transporter membrane localization and zonation. Increased periportal BSEPMZA (mean proportional difference compared to non-diseased liver of 0.090) and decreased pericentral BSEPMZA (-0.065) were observed with NASH and also in biopsies with higher histologic scores. Compared to Non-diseased Liver, periportal OATP1B3MZA was increased in NAFL (0.041) and NASH (0.047). Grade 2 steatosis (mean proportional difference of 0.043 when compared to grade 0) and grade 1 lobular inflammation (0.043) were associated with increased periportal OATP1B3MZA. CONCLUSIONS: These findings provide novel mechanistic insight into specific transporter alterations that impact BA homeostasis in NAFLD. Changes in BSEPMZA likely contribute to altered BA disposition and pericentral microcholestasis previously reported in some patients with NAFLD. BSEPMZA assessment could inform future development and optimization of NASH-related pharmacotherapies.

Hepatic NLRP3-Derived Hsp70 Binding to TLR4 Mediates MASLD to MASH Progression upon Inhibition of PP2A by Harmful Algal Bloom Toxin Microcystin, a Second Hit.

Harmful algal bloom toxin microcystin has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) progression and hepatocellular carcinoma, though the mechanisms remain unclear. Using an established mouse model of MASLD, we show that the NLRP3-Hsp70-TLR4 axis drives in part the inflammation of the liver lobule that results in the progression of MASLD to metabolic dysfunction-associated steatohepatitis (MASH). Results showed that mice deficient in NLRP3 exhibited decreased MASH pathology, blocked Hsp70 expression, and co-binding with NLRP3, a crucial protein component of the liver inflammasome. Hsp70, both in the liver lobule and extracellularly released in the liver vasculature, acted as a ligand to TLR4 in the liver, primarily in hepatocytes to activate the NF-κB pathway, ultimately leading to hepatic cell death and necroptosis, a crucial pathology of MASH progression. The above studies show a novel insight into an inflammasome-triggered Hsp70-mediated inflammation that may have broader implications in MASLD pathology. MASLD to MASH progression often requires multiple hits. One of the mediators of progressive MASLD is environmental toxins. In this research report, we show for the first time a novel mechanism where microcystin-LR, an environmental toxin, advances MASLD to MASH by triggering the release of Hsp70 as a DAMP to activate TLR4-induced inflammation in the liver.

Hedgehog Signaling: Implications in Liver Pathophysiology.

The purpose of this review is to summarize current knowledge about the role of the Hedgehog signaling pathway in liver homeostasis and disease. Hedgehog is a morphogenic signaling pathway that is active in development. In most healthy tissues, pathway activity is restricted to stem and/or stromal cell compartments, where it enables stem cell self-renewal and tissue homeostasis. Aberrant over-activation of Hedgehog signaling occurs in many cancers, including hepatocellular and cholangio-carcinoma. The pathway is also activated transiently in stromal cells of injured tissues and orchestrates normal wound healing responses, including inflammation, vascular remodeling, and fibrogenesis. In liver, sustained Hedgehog signaling in stromal cells plays a major role in the pathogenesis of cirrhosis. Hedgehog signaling was thought to be silenced in healthy hepatocytes. However, recent studies show that targeted disruption of the pathway in hepatocytes dysregulates lipid, cholesterol, and bile acid metabolism, and promotes hepatic lipotoxicity, insulin resistance, and senescence. Hepatocytes that lack Hedgehog activity also produce a secretome that activates Hedgehog signaling in cholangiocytes and neighboring stromal cells to induce inflammatory and fibrogenic wound healing responses that drive progressive fibrosis. In conclusion, Hedgehog signaling must be precisely controlled in adult liver cells to maintain liver health.

Serum Metabolites Are Associated With HFpEF in Biopsy-Proven Nonalcoholic Fatty Liver Disease.

Background Nonalcoholic fatty liver disease (NAFLD) and heart failure with preserved ejection fraction (HFpEF) share common risk factors, including obesity and diabetes. They are also thought to be mechanistically linked. The aim of this study was to define serum metabolites associated with HFpEF in a cohort of patients with biopsy-proven NAFLD to identify common mechanisms. Methods and Results We performed a retrospective, single-center study of 89 adult patients with biopsy-proven NAFLD who had transthoracic echocardiography performed for any indication. Metabolomic analysis was performed on serum using ultrahigh performance liquid and gas chromatography/tandem mass spectrometry. HFpEF was defined as ejection fraction >50% plus at least 1 echocardiographic feature of HFpEF (diastolic dysfunction, abnormal left atrial size) and at least 1 heart failure sign or symptom. We performed generalized linear models to evaluate associations between individual metabolites, NAFLD, and HFpEF. Thirty-seven out of 89 (41.6%) patients met criteria for HFpEF. A total of 1151 metabolites were detected; 656 were analyzed after exclusion of unnamed metabolites and those with >30% missing values. Fifty-three metabolites were associated with the presence of HFpEF with unadjusted P value <0.05; none met statistical significance after adjustment for multiple comparisons. The majority (39/53, 73.6%) were lipid metabolites, and levels were generally increased. Two cysteine metabolites (cysteine s-sulfate and s-methylcysteine) were present at significantly lower levels in patients with HFpEF. Conclusions We identified serum metabolites associated with HFpEF in patients with biopsy-proven NAFLD, with increased levels of multiple lipid metabolites. Lipid metabolism could be an important pathway linking HFpEF to NAFLD.

Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Fibrosis Progression Rate in Biopsy-Proven Nonalcoholic Fatty Liver Disease Among People With Diabetes Versus People Without Diabetes: A Multicenter Study.

BACKGROUND & AIMS: There are limited data regarding fibrosis progression in biopsy-proven nonalcoholic fatty liver disease (NAFLD) in people with type 2 diabetes mellitus (T2DM) compared with people without T2DM. We assessed the time to fibrosis progression in people with T2DM compared with people without T2DM in a large, multicenter, study of people with NAFLD who had paired liver biopsies. METHODS: This study included 447 adult participants (64% were female) with NAFLD who had paired liver biopsies more than 1 year apart. Liver histology was systematically assessed by a central pathology committee blinded to clinical data. The primary outcome was the cumulative incidence of a ≥1-stage increase in fibrosis in participants with T2DM compared with participants without T2DM. RESULTS: The mean (SD) age and body mass index (calculated as weight in kilograms divided by the square of the height in meters) were 50.9 (11.5) years and 34.7 (6.3), respectively. The median time between biopsies was 3.3 years (interquartile range, 1.8-6.1 years). Participants with T2DM had a significantly higher cumulative incidence of fibrosis progression at 4 years (24% vs 20%), 8 years (60% vs 50%), and 12 years (93% vs 76%) (P = .005). Using a multivariable Cox proportional hazards model adjusted for multiple confounders, T2DM remained an independent predictor of fibrosis progression (adjusted hazard ratio, 1.69; 95% CI, 1.17-2.43; P = .005). The cumulative incidence of fibrosis regression by ≥1 stage was similar in participants with T2DM compared with participants without T2DM (P = .24). CONCLUSIONS: In this large, multicenter cohort study of well-characterized participants with NAFLD and paired liver biopsies, we found that fibrosis progressed faster in participants with T2DM compared with participants without T2DM. These data have important implications for clinical practice and trial design.

Targeting senescent hepatocytes using the thrombomodulin-PAR1 inhibitor vorapaxar ameliorates NAFLD progression.

BACKGROUND AND AIMS: Senescent hepatocytes accumulate in parallel with fibrosis progression during NASH. The mechanisms that enable progressive expansion of nonreplicating cell populations and the significance of that process in determining NASH outcomes are unclear. Senescing cells upregulate thrombomodulin-protease-activated receptor-1 (THBD-PAR1) signaling to remain viable. Vorapaxar blocks the activity of that pathway. We used vorapaxar to determine if and how THBD-PAR1 signaling promotes fibrosis progression in NASH. APPROACH AND RESULTS: We evaluated the THBD-PAR1 pathway in liver biopsies from patients with NAFLD. Chow-fed mice were treated with viral vectors to overexpress p16 in hepatocytes and induce replicative senescence. Effects on the THBD-PAR1 axis and regenerative capacity were assessed; the transcriptome of p16-overexpressing hepatocytes was characterized, and we examined how conditioned medium from senescent but viable (dubbed "undead") hepatocytes reprograms HSCs. Mouse models of NASH caused by genetic obesity or Western diet/CCl 4 were treated with vorapaxar to determine effects on hepatocyte senescence and liver damage. Inducing senescence upregulates the THBD-PAR1 signaling axis in hepatocytes and induces their expression of fibrogenic factors, including hedgehog ligands. Hepatocyte THBD-PAR1 signaling increases in NAFLD and supports sustained hepatocyte senescence that limits effective liver regeneration and promotes maladaptive repair. Inhibiting PAR1 signaling with vorapaxar interrupts this process, reduces the burden of 'undead' senescent cells, and safely improves NASH and fibrosis despite ongoing lipotoxic stress. CONCLUSION: The THBD-PAR1 signaling axis is a novel therapeutic target for NASH because blocking this pathway prevents accumulation of senescing but viable hepatocytes that generate factors that promote maladaptive liver repair.

Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Targeting YAP-mediated HSC death susceptibility and senescence for treatment of liver fibrosis.

BACKGROUND AND AIMS: Liver fibrosis results from the accumulation of myofibroblasts (MFs) derived from quiescent HSCs, and yes-associated protein (YAP) controls this state transition. Although fibrosis is also influenced by HSC death and senescence, whether YAP regulates these processes and whether this could be leveraged to treat liver fibrosis are unknown. APPROACH AND RESULTS: YAP activity was manipulated in MF-HSCs to determine how YAP impacts susceptibility to pro-apoptotic senolytic agents or ferroptosis. Effects of senescence on YAP activity and susceptibility to apoptosis versus ferroptosis were also examined. CCl 4 -treated mice were treated with a ferroptosis inducer or pro-apoptotic senolytic to determine the effects on liver fibrosis. YAP was conditionally disrupted in MFs to determine how YAP activity in MF-HSC affects liver fibrosis in mouse models. Silencing YAP in cultured MF-HSCs induced HSC senescence and vulnerability to senolytics, and promoted ferroptosis resistance. Conversely, inducing HSC senescence suppressed YAP activity, increased sensitivity to senolytics, and decreased sensitivity to ferroptosis. Single-cell analysis of HSCs from fibrotic livers revealed heterogeneous sensitivity to ferroptosis, apoptosis, and senescence. In mice with chronic liver injury, neither the ferroptosis inducer nor senolytic improved fibrosis. However, selectively depleting YAP in MF-HSCs induced senescence and decreased liver injury and fibrosis. CONCLUSION: YAP determines whether MF-HSCs remain activated or become senescent. By regulating this state transition, Yap controls both HSC fibrogenic activity and susceptibility to distinct mechanisms for cell death. MF-HSC-specific YAP depletion induces senescence and protects injured livers from fibrosis. Clarifying determinants of HSC YAP activity may facilitate the development of novel anti-fibrotic therapies.

Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH), a leading cause of cirrhosis, strongly associates with the metabolic syndrome, an insulin-resistant proinflammatory state that disrupts energy balance and promotes progressive liver degeneration. We aimed to define the role of Smoothened (Smo), an obligatory component of the Hedgehog signaling pathway, in controlling hepatocyte metabolic homeostasis and, thereby, susceptibility to NASH. METHODS: We conditionally deleted Smo in hepatocytes of healthy chow-fed mice and performed metabolic phenotyping, coupled with single-cell RNA sequencing (RNA-seq), to characterize the role of hepatocyte Smo in regulating basal hepatic and systemic metabolic homeostasis. Liver RNA-seq datasets from 2 large human cohorts were also analyzed to define the relationship between Smo and NASH susceptibility in people. RESULTS: Hepatocyte Smo deletion inhibited the Hedgehog pathway and promoted fatty liver, hyperinsulinemia, and insulin resistance. We identified a plausible mechanism whereby inactivation of Smo stimulated the mTORC1-SREBP1c signaling axis, which promoted lipogenesis while inhibiting the hepatic insulin cascade. Transcriptomics of bulk and single Smo-deficient hepatocytes supported suppression of insulin signaling and also revealed molecular abnormalities associated with oxidative stress and mitochondrial dysfunction. Analysis of human bulk RNA-seq data revealed that Smo expression was (1) highest in healthy livers, (2) lower in livers with NASH than in those with simple steatosis, (3) negatively correlated with markers of insulin resistance and liver injury, and (4) declined progressively as fibrosis severity worsened. CONCLUSIONS: The Hedgehog pathway controls insulin sensitivity and energy homeostasis in adult livers. Loss of hepatocyte Hedgehog activity induces hepatic and systemic metabolic stress and enhances susceptibility to NASH by promoting hepatic lipoxicity and insulin resistance.

Defining the serum proteomic signature of hepatic steatosis, inflammation, ballooning and fibrosis in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Despite recent progress, non-invasive tests for the diagnostic assessment and monitoring of non-alcoholic fatty liver disease (NAFLD) remain an unmet need. Herein, we aimed to identify diagnostic signatures of the key histological features of NAFLD. METHODS: Using modified-aptamer proteomics, we assayed 5,220 proteins in each of 2,852 single serum samples from 636 individuals with histologically confirmed NAFLD. We developed and validated dichotomized protein-phenotype models to identify clinically relevant severities of steatosis (grade 0 vs. 1-3), hepatocellular ballooning (0 vs. 1 or 2), lobular inflammation (0-1 vs. 2-3) and fibrosis (stages 0-1 vs. 2-4). RESULTS: The AUCs of the four protein models, based on 37 analytes (18 not previously linked to NAFLD), for the diagnosis of their respective components (at a clinically relevant severity) in training/paired validation sets were: fibrosis (AUC 0.92/0.85); steatosis (AUC 0.95/0.79), inflammation (AUC 0.83/0.72), and ballooning (AUC 0.87/0.83). An additional outcome, at-risk NASH, defined as steatohepatitis with NAFLD activity score ≥4 (with a score of at least 1 for each of its components) and fibrosis stage ≥2, was predicted by multiplying the outputs of each individual component model (AUC 0.93/0.85). We further evaluated their ability to detect change in histology following treatment with placebo, pioglitazone, vitamin E or obeticholic acid. Component model scores significantly improved in the active therapies vs. placebo, and differential effects of vitamin E, pioglitazone, and obeticholic acid were identified. CONCLUSIONS: Serum protein scanning identified signatures corresponding to the key components of liver biopsy in NAFLD. The models developed were sufficiently sensitive to characterize the longitudinal change for three different drug interventions. These data support continued validation of these proteomic models to enable a "liquid biopsy"-based assessment of NAFLD. CLINICAL TRIAL NUMBER: Not applicable. IMPACT AND IMPLICATIONS: An aptamer-based protein scan of serum proteins was performed to identify diagnostic signatures of the key histological features of non-alcoholic fatty liver disease (NAFLD), for which no approved non-invasive diagnostic tools are currently available. We also identified specific protein signatures related to the presence and severity of NAFLD and its histological components that were also sensitive to change over time. These are fundamental initial steps in establishing a serum proteome-based diagnostic signature of NASH and provide the rationale for using these signatures to test treatment response and to identify several novel targets for evaluation in the pathogenesis of NAFLD.

The imprinted gene Zac1 regulates steatosis in developmental cadmium-induced nonalcoholic fatty liver disease.

Cadmium (Cd) exposure in adulthood is associated with nonalcoholic fatty liver disease (NAFLD), characterized by steatosis, inflammation, and fibrosis. The prevalence of NAFLD in children is increasing, suggesting a role for the developmental environment in programming susceptibility. However, the role of developmental Cd exposure in programming NAFLD and the underlying mechanisms remain unclear. We have proposed that imprinted genes are strong candidates for connecting the early life environment and later life disease. In support of this, we previously identified roles for the Imprinted Gene Network (IGN) and its regulator Zac1 in programming NAFLD in response to maternal metabolic dysfunction. Here, we test the hypothesis that developmental Cd exposure is sufficient to program NAFLD, and further, that this process is mediated by Zac1 and the IGN. Using mice, we show that developmental cadmium chloride (CdCl2) exposure leads to histological, biochemical, and molecular signatures of steatosis and fibrosis in juveniles. Transcriptomic analyses comparing livers of CdCl2-exposed and control mice show upregulation of Zac1 and the IGN coincident with disease presentation. Increased hepatic Zac1 expression is independent of promoter methylation and imprinting statuses. Finally, we show that over-expression of Zac1 in cultured hepatocytes is sufficient to induce lipid accumulation in a Pparγ-dependent manner and demonstrate direct binding of Zac1 to the Pparγ promoter. Our findings demonstrate that developmental Cd exposure is sufficient to program NAFLD in later life, and with our previous work, establish Zac1 and the IGN as key regulators of prosteatotic and profibrotic pathways, two of the major pathological hallmarks of NAFLD.

Deep learning-based quantification of NAFLD/NASH progression in human liver biopsies.

Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).

Dynamics of hepatocyte-cholangiocyte cell-fate decisions during liver development and regeneration.

The immense regenerative potential of the liver is attributed to the ability of its two key cell types - hepatocytes and cholangiocytes - to trans-differentiate to one another either directly or through intermediate progenitor states. However, the dynamic features of decision-making between these cell-fates during liver development and regeneration remains elusive. Here, we identify a core gene regulatory network comprising c/EBPα, TGFBR2, and SOX9 which is multistable in nature, enabling three distinct cell states - hepatocytes, cholangiocytes, and liver progenitor cells (hepatoblasts/oval cells) - and stochastic switching among them. Predicted expression signature for these three states are validated through multiple bulk and single-cell transcriptomic datasets collected across developmental stages and injury-induced liver repair. This network can also explain the experimentally observed spatial organization of phenotypes in liver parenchyma and predict strategies for efficient cellular reprogramming. Our analysis elucidates how the emergent dynamics of underlying regulatory networks drive diverse cell-fate decisions in liver development and regeneration.

Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents non-alcoholic steatohepatitis progression.

Spermidine is a natural polyamine that has health benefits and extends life span in several species. Deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are key enzymes that utilize spermidine to catalyze the post-translational hypusination of the translation factor EIF5A (EIF5AH). Here, we have found that hepatic DOHH mRNA expression is decreased in patients and mice with non-alcoholic steatohepatitis (NASH), and hepatic cells treated with fatty acids. The mouse and cell culture models of NASH have concomitant decreases in Eif5aH and mitochondrial protein synthesis which leads to lower mitochondrial activity and fatty acid ß-oxidation. Spermidine treatment restores EIF5AH, partially restores protein synthesis and mitochondrial function in NASH, and prevents NASH progression in vivo. Thus, the disrupted DHPS-DOHH-EIF5AH pathway during NASH represents a therapeutic target to increase hepatic protein synthesis and mitochondrial fatty acid oxidation (FAO) and prevent NASH progression.