Liver stiffness as a cornerstone in heart disease risk assessment.

Metabolic dysfunction-associated steatotic liver disease (MASLD) typically presents with hepatic fibrosis in advanced disease, resulting in increased liver stiffness. A subset of patients further develops liver cirrhosis and hepatocellular carcinoma. Cardiovascular disease is a common comorbidity in patients with MASLD and its prevalence is increasing in parallel. Recent evidence suggests that especially liver stiffness, whether or not existing against a background of MASLD, is associated with heart diseases. We conducted a narrative review on the role of liver stiffness in the prediction of highly prevalent heart diseases including heart failure, cardiac arrhythmias (in particular atrial fibrillation), coronary heart disease, and aortic valve sclerosis. Research papers were retrieved from major scientific databases (PubMed, Web of Science) until September 2023 using 'liver stiffness' and 'liver fibrosis' as keywords along with the latter cardiac conditions. Increased liver stiffness, determined by vibration-controlled transient elastography or hepatic fibrosis as predicted by biomarker panels, are associated with a variety of cardiovascular diseases, including heart failure, atrial fibrillation, and coronary heart disease. Elevated liver stiffness in patients with metabolic liver disease should lead to considerations of cardiac workup including N-terminal pro-B-type natriuretic peptide/B-type natriuretic peptide determination, electrocardiography, and coronary computed tomography angiography. In addition, patients with MASLD would benefit from heart disease case-finding strategies in which liver stiffness measurements can play a key role. In conclusion, increased liver stiffness should be a trigger to consider a cardiac workup in metabolically compromised patients.

PNPLA3 I148M and response to treatment for hepatic steatosis: A systematic review.

BACKGROUND: It is unclear whether the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C-to-G single nucleotide polymorphism, resulting in the substitution of isoleucine to methionine at position 148 (I148M), impedes regression of hepatic steatosis when treating non-alcoholic fatty liver disease (NAFLD). OBJECTIVES: Investigate if carriage of the PNPLA3 148M allele affects the anti-steatotic efficacy of all possible anti-NAFLD interventions, identify gaps in current knowledge and provide guidance for individual treatment. METHODS: Research available in public databases was searched up to 13 November 2022. Studies were included if a treatment in NAFLD patients decreased hepatic steatosis in the pooled patient group or a PNPLA3 I148M polymorphism subgroup (II/IM/MM). The risk of bias was assessed using the Cochrane Risk-Of-Bias 2 Tool and the Newcastle-Ottawa Scale. RESULTS: Moderate evidence indicates that NAFLD patients homozygous for the PNPLA3 148M allele benefit less or not at all from omega-3 carboxylic acids to decrease liver fat, while the PNPLA3 148I allele shows moderate benefit. Low evidence suggests that interventions employing lifestyle changes are more effective to reduce liver fat in NAFLD patients homozygous for the PNPLA3 148M allele compared to patients with wild-type PNPLA3. CONCLUSIONS: NAFLD patients homozygous for the PNPLA3 148M allele might not benefit from omega-3 carboxylic acids to reduce hepatic steatosis in contrast to patients with wild-type PNPLA3. Instead, patients with two PNPLA3 148M alleles should be especially advised to adopt lifestyle changes. Genotyping for PNPLA3 I148M should be encouraged in therapeutic studies for NAFLD. REGISTRATION NUMBER (PROSPERO): CRD42022375028.

Metabolic signature of HepaRG cells exposed to ethanol and tumor necrosis factor alpha to study alcoholic steatohepatitis by LC-MS-based untargeted metabolomics.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368 mM) and tumor necrosis factor alpha (TNF-α, 50 ng/mL) for 24 h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

Metabolic Signature of Ethanol-Induced Hepatotoxicity in HepaRG Cells by Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics.

Alcoholic liver disease is highly prevalent but poorly identified and characterized, leading to knowledge gaps, which impairs early diagnosis. Excessive alcohol consumption is known to alter lipid metabolism, followed by progressive intracellular lipid accumulation, resulting in alcoholic fatty liver disease. In this study, HepaRG cells were exposed to ethanol at IC10 and 1/10 IC10 for 24 and 48 h. Metabolic alterations were investigated intra- and extracellularly with liquid chromatography-high-resolution mass spectrometry. Ion mobility was added as an extra separation dimension for untargeted lipidomics to improve annotation confidence. Distinctive patterns between exposed and control cells were consistently observed, with intracellular upregulation of di- and triglycerides, downregulation of phosphatidylcholines and phosphatidylethanolamines, sphingomyelins, and S-adenosylmethionine, among others. Several intracellular metabolic patterns could be related to changes in the extracellular environment, such as increased intracellular hydrolysis of sphingomyelins, leading to increased phosphorylcholine secretion. Carnitines showed alterations depending on the size of their carbon chain, which highlights the interplay between ß-oxidation in mitochondria and peroxisomes. Potential new biomarkers of ethanol-induced hepatotoxicity have been observed, such as ceramides with a sphingadienine backbone, octanoylcarnitine, creatine, acetylcholine, and ethoxylated phosphorylcholine. The combination of the metabolic fingerprint and footprint enabled a comprehensive investigation of the pathophysiology behind ethanol-induced hepatotoxicity.

Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code.

There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepatic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.

Human hepatic in vitro models reveal distinct anti-NASH potencies of PPAR agonists.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.

Infections at the nexus of metabolic-associated fatty liver disease.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease that affects about a quarter of the world population. MAFLD encompasses different disease stadia ranging from isolated liver steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Although MAFLD is considered as the hepatic manifestation of the metabolic syndrome, multiple concomitant disease-potentiating factors can accelerate disease progression. Among these risk factors are diet, lifestyle, genetic traits, intake of steatogenic drugs, male gender and particular infections. Although infections often outweigh the development of fatty liver disease, pre-existing MAFLD could be triggered to progress towards more severe disease stadia. These combined disease cases might be underreported because of the high prevalence of both MAFLD and infectious diseases that can promote or exacerbate fatty liver disease development. In this review, we portray the molecular and cellular mechanisms by which the most relevant viral, bacterial and parasitic infections influence the progression of fatty liver disease and steatohepatitis. We focus in particular on how infectious diseases, including coronavirus disease-19, hepatitis C, acquired immunodeficiency syndrome, peptic ulcer and periodontitis, exacerbate MAFLD. We specifically underscore the synergistic effects of these infections with other MAFLD-promoting factors.

Elafibranor restricts lipogenic and inflammatory responses in a human skin stem cell-derived model of NASH.

Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular steatosis with concomitant hepatic inflammation. Despite its pandemic proportions, no anti-NASH drugs have been approved yet. This is partially because drug development is decelerated due to the lack of adequate tools to assess the efficacy of potential new drug candidates. The present study describes the development and application of a new preclinical model for NASH using hepatic cells generated from human skin-derived precursors. Exposure of these cells to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1ß, TNF-α, TGF-ß) resulted in a characteristic NASH response, as indicated by intracellular lipid accumulation, modulation of NASH-specific gene expression, increased caspase-3/7 activity and the expression and/or secretion of inflammatory markers, including CCL2, CCL5, CCL7, CCL8, CXCL5, CXCL8, IL1a, IL6 and IL11. The human relevance of the proposed NASH model was verified by transcriptomics analyses that revealed commonly modulated genes and the identification of the same gene classes between the in vitro system and patients suffering from NASH. The application potential of this in vitro model was demonstrated by testing elafibranor, a promising anti-NASH compound currently under clinical phase III trial evaluation. Elafibranor attenuated in vitro key features of NASH, and dramatically lowered lipid load as well as the expression and secretion of inflammatory chemokines, which in vivo are responsible for the recruitment of immune cells. This reduction in inflammatory response was NFκB-mediated. In summary, this human-relevant, in vitro system proved to be a sensitive testing tool for the investigation of novel anti-NASH compounds.

Technological advancements for the development of stem cell-based models for hepatotoxicity testing.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Human-based systems: Mechanistic NASH modelling just around the corner?

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by excessive triglyceride accumulation in the liver accompanied by inflammation, cell stress and apoptosis. It is the tipping point to the life-threatening stages of non-alcoholic fatty liver disease (NAFLD). Despite the high prevalence of NASH, up to five percent of the global population, there are currently no approved drugs to treat this disease. Animal models, mostly based on specific diets and genetic modifications, are often employed in anti-NASH drug development. However, due to interspecies differences and artificial pathogenic conditions, they do not represent the human situation accurately and are inadequate for testing the efficacy and safety of potential new drugs. Human-based in vitro models provide a more legitimate representation of the human NASH pathophysiology and can be used to investigate the dysregulation of cellular functions associated with the disease. Also in silico methodologies and pathway-based approaches using human datasets, may contribute to a more accurate representation of NASH, thereby facilitating the quest for new anti-NASH drugs. In this review, we describe the molecular components of NASH and how human-based tools can contribute to unraveling the pathogenesis of this disease and be used in anti-NASH drug development. We also propose a roadmap for the development and application of human-based approaches for future investigation of NASH.

A quantitative weight-of-evidence method for confidence assessment of adverse outcome pathway networks: A case study on chemical-induced liver steatosis.

The field of chemical toxicity testing is undergoing a transition to overcome the limitations of in vivo experiments. This evolution involves implementing innovative non-animal approaches to improve predictability and provide a more precise understanding of toxicity mechanisms. Adverse outcome pathway (AOP) networks are pivotal in organizing existing mechanistic knowledge related to toxicological processes. However, these AOP networks are dynamic and require regular updates to incorporate the latest data. Regulatory challenges also persist due to concerns about the reliability of the information they offer. This study introduces a generic Weight-of-Evidence (WoE) scoring method, aligned with the tailored Bradford-Hill criteria, to quantitatively assess the confidence levels in key event relationships (KERs) within AOP networks. We use the previously published AOP network on chemical-induced liver steatosis, a prevalent form of human liver injury, as a case study. Initially, the existing AOP network is optimized with the latest scientific information extracted from PubMed using the free SysRev platform for artificial intelligence (AI)-based abstract inclusion and standardized data collection. The resulting optimized AOP network, constructed using Cytoscape, visually represents confidence levels through node size (key event, KE) and edge thickness (KERs). Additionally, a Shiny application is developed to facilitate user interaction with the dataset, promoting future updates. Our analysis of 173 research papers yielded 100 unique KEs and 221 KERs among which 72 KEs and 170 KERs, respectively, have not been previously documented in the prior AOP network or AOP-wiki. Notably, modifications in de novo lipogenesis, fatty acid uptake and mitochondrial beta-oxidation, leading to lipid accumulation and liver steatosis, garnered the highest KER confidence scores. In conclusion, our study delivers a generic methodology for developing and assessing AOP networks. The quantitative WoE scoring method facilitates in determining the level of support for KERs within the optimized AOP network, offering valuable insights into its utility in both scientific research and regulatory contexts. KERs supported by robust evidence represent promising candidates for inclusion in an in vitro test battery for reliably predicting chemical-induced liver steatosis within regulatory frameworks.

Immune cells and their derived microRNA-enriched extracellular vesicles in nonalcoholic fatty liver diseases: Novel therapeutic targets.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Despite extensive research and multiple clinical trials, there are still no FDA-approved therapies to treat the most severe forms of NAFLD. This is largely due to its complicated etiology and pathogenesis, which involves visceral obesity, insulin resistance, gut dysbiosis, etc. Although inflammation is generally believed to be one of the critical factors that drive the progression of simple steatosis to nonalcoholic steatohepatitis (NASH), the exact type of inflammation and how it contributes to NASH pathogenesis remain largely unknown. Liver inflammation is accompanied by the elevation of inflammatory mediators, including cytokines and chemokines and consequently intrahepatic infiltration of multiple types of immune cells. Recent studies revealed that extracellular vesicles (EVs) derived from inflammatory cells and hepatocytes play an important role in controlling liver inflammation during NASH. In this review, we highlight the roles of innate and adaptive immune cells and their microRNA-enriched EVs during NAFLD development and discuss potential drugs that target inflammatory pathways for the treatment of NAFLD.

Human skin stem cell-derived hepatic cells as in vitro drug discovery model for insulin-driven de novo lipogenesis.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by intrahepatic triglyceride accumulation and can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. Hepatic de novo lipogenesis (DNL), activated by glucose and insulin, is a central pathway contributing to early-stage development of MASLD. The emerging global prevalence of MASLD highlights the urgent need for pharmaceutical intervention to combat this health threat. However, the identification of novel drugs that could inhibit hepatic DNL is hampered by a lack of reliable, insulin-sensitive, human, in vitro, hepatic models. Here, we report human skin stem cell-derived hepatic cells (hSKP-HPC) as a unique in vitro model to study insulin-driven DNL (iDNL), evidenced by both gene expression and lipid accumulation readouts. Insulin-sensitive hSKP-HPC showed increased sterol regulatory element-binding protein 1c (SREBP-1c) expression, a key transcription factor for DNL. Furthermore, this physiologically relevant in vitro human steatosis model allowed both inhibition and activation of the iDNL pathway using reference inhibitors and activators, respectively. Optimisation of the lipid accumulation assay to a high-throughput, 384-well format enabled the screening of a library of annotated compounds, delivering new insights on key players in the iDNL pathway and MASLD pathophysiology. Together, these results establish the value of the hSKP-HPC model in preclinical development of antisteatotic drugs to combat MASLD.

From NAFLD to MAFLD: Aligning Translational In Vitro Research to Clinical Insights.

Although most same-stage non-alcoholic fatty liver disease (NAFLD) patients exhibit similar histologic sequelae, the underlying mechanisms appear to be highly heterogeneous. Therefore, it was recently proposed to redefine NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) in which other known causes of liver disease such as alcohol consumption or viral hepatitis do not need to be excluded. Revised nomenclature envisions speeding up and facilitating anti-MAFLD drug development by means of patient stratification whereby each subgroup would benefit from distinct pharmacological interventions. As human-based in vitro research fulfils an irrefutable step in drug development, action should be taken as well in this stadium of the translational path. Indeed, most established in vitro NAFLD models rely on short-term exposure to fatty acids and use lipid accumulation as a phenotypic benchmark. This general approach to a seemingly ambiguous disease such as NAFLD therefore no longer seems applicable. Human-based in vitro models that accurately reflect distinct disease subgroups of MAFLD should thus be adopted in early preclinical disease modeling and drug testing. In this review article, we outline considerations for setting up translational in vitro experiments in the MAFLD era and allude to potential strategies to implement MAFLD heterogeneity into an in vitro setting so as to better align early drug development with future clinical trial designs.

Transcriptomics Reveals Discordant Lipid Metabolism Effects between In Vitro Models Exposed to Elafibranor and Liver Samples of NAFLD Patients after Bariatric Surgery.

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH) is a life-threatening stage of non-alcoholic fatty liver disease (NAFLD) for which no drugs have been approved. We have previously shown that human-derived hepatic in vitro models can be used to mimic key cellular mechanisms involved in the progression of NASH. In the present study, we first characterize the transcriptome of multiple in vitro NASH models. Subsequently, we investigate how elafibranor, which is a peroxisome proliferator-activated receptor (PPAR)-α/δ agonist that has recently failed a phase 3 clinical trial as a potential anti-NASH compound, modulates the transcriptome of these models. Finally, we compare the elafibranor-induced gene expression modulation to transcriptome data of patients with improved/resolved NAFLD/NASH upon bariatric surgery, which is the only proven clinical NASH therapy. METHODS: Human whole genome microarrays were used for the transcriptomics evaluation of hepatic in vitro models. Comparison to publicly available clinical datasets was conducted using multiple bioinformatic application tools. RESULTS: Primary human hepatocytes (PHH), HepaRG, and human skin stem cell-derived hepatic progenitors (hSKP-HPC) exposed to NASH-inducing triggers exhibit up to 35% overlap with datasets of liver samples from NASH patients. Exposure of the in vitro NASH models to elafibranor partially reversed the transcriptional modulations, predicting an inhibition of toll-like receptor (TLR)-2/4/9-mediated inflammatory responses, NFκB-signaling, hepatic fibrosis, and leukocyte migration. These transcriptomic changes were also observed in the datasets of liver samples of patients with resolved NASH. Peroxisome Proliferator Activated Receptor Alpha (PPARA), PPARG Coactivator 1 Alpha (PPARGC1A), and Sirtuin 1 (SIRT1) were identified as the major common upstream regulators upon exposure to elafibranor. Analysis of the downstream mechanistic networks further revealed that angiopoietin Like 4 (ANGPTL4), pyruvate dehydrogenase kinase 4 (PDK4), and perilipin 2 (PLIN2), which are involved in the promotion of hepatic lipid accumulation, were also commonly upregulated by elafibranor in all in vitro NASH models. Contrarily, these genes were not upregulated in liver samples of patients with resolved NASH. CONCLUSION: Transcriptomics comparison between in vitro NASH models exposed to elafibranor and clinical datasets of NAFLD patients after bariatric surgery reveals commonly modulated anti-inflammatory responses, but discordant modulations of key factors in lipid metabolism. This discordant adverse effect of elafibranor deserves further investigation when assessing PPAR-α/δ agonism as a potential anti-NASH therapy.

Healthcare-Associated COVID-19 across Five Pandemic Waves: Prediction Models and Genomic Analyses.

BACKGROUND: Healthcare-associated SARS-CoV-2 infections need to be explored further. Our study is an analysis of hospital-acquired infections (HAIs) and ambulatory healthcare workers (aHCWs) with SARS-CoV-2 across the pandemic in a Belgian university hospital. METHODS: We compared HAIs with community-associated infections (CAIs) to identify the factors associated with having an HAI. We then performed a genomic cluster analysis of HAIs and aHCWs. We used this alongside the European Centre for Disease Control (ECDC) case source classifications of an HAI. RESULTS: Between March 2020 and March 2022, 269 patients had an HAI. A lower BMI, a worse frailty index, lower C-reactive protein (CRP), and a higher thrombocyte count as well as death and length of stay were significantly associated with having an HAI. Using those variables to predict HAIs versus CAIs, we obtained a positive predictive value (PPV) of 83.6% and a negative predictive value (NPV) of 82.2%; the area under the ROC was 0.89. Genomic cluster analyses and representations on epicurves and minimal spanning trees delivered further insights into HAI dynamics across different pandemic waves. The genomic data were also compared with the clinical ECDC definitions for HAIs; we found that 90.0% of the 'definite', 87.8% of the 'probable', and 70.3% of the 'indeterminate' HAIs belonged to one of the twenty-two COVID-19 genomic clusters we identified. CONCLUSIONS: We propose a novel prediction model for HAIs. In addition, we show that the management of nosocomial outbreaks will benefit from genome sequencing analyses.