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Abstract The incidence of MASLD and MASH-associated fibrosis is rapidly increasing worldwide. Drug therapy is hampered by large patient variability and partial representation of human MASH fibrosis in preclinical models. Here, we investigated the mechanisms underlying patient heterogeneity using a discovery dataset and validated in distinct human transcriptomic datasets, to improve patient stratification and translation into subgroup specific patterns. Patient stratification was performed using weighted gene co-expression network analysis (WGCNA) in a large public transcriptomic discovery dataset (n = 216). Differential expression analysis was performed using DESeq2 to obtain differentially expressed genes (DEGs). Ingenuity Pathway analysis was used for functional annotation. The discovery dataset showed relevant fibrosis-related mechanisms representative of disease heterogeneity. Biological complexity embedded in genes signature was used to stratify discovery dataset into six subgroups of various sizes. Of note, subgroup-specific DEGs show differences in directionality in canonical pathways (e.g. Collagen biosynthesis, cytokine signaling) across subgroups. Finally, a multiclass classification model was trained and validated in two datasets. In summary, our work shows a potential alternative for patient population stratification based on heterogeneity in MASLD-MASH mechanisms. Future research is warranted to further characterize patient subgroups and identify protein targets for virtual screening and/or in vitro validation in preclinical models.

Abstract Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr−/−.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

An in silico target discovery pipeline was developed by including a directional and weighted molecular disease network for metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. This approach integrates text mining, network biology, and artificial intelligence/machine learning with clinical transcriptome data for optimal translational power. At the mechanistic level, the critical components influencing disease progression were identified from the disease network using in silico knockouts. The top-ranked genes were then subjected to a target efficacy analysis, following which the top-5 candidate targets were validated in vitro. Three targets, including EP300, were confirmed for their roles in liver fibrosis. EP300 gene-silencing was found to significantly reduce collagen by 37%; compound intervention studies performed in human primary hepatic stellate cells and the hepatic stellate cell line LX-2 showed significant inhibition of collagen to the extent of 81% compared to the TGFβ-stimulated control (1 μM inobrodib in LX-2 cells). The validated in silico pipeline presents a unique approach for the identification of human-disease-mechanism-relevant drug targets. The directionality of the network ensures adherence to physiologically relevant signaling cascades, while the inclusion of clinical data boosts its translational power and ensures identification of the most relevant disease pathways. In silico knockouts thus provide crucial molecular insights for successful target identification.

Concerns have been raised about whether preclinical models sufficiently mimic molecular disease processes observed in nonalcoholic steatohepatitis (NASH) patients, bringing into question their translational value in studies of therapeutic interventions in the process of NASH/fibrosis. We investigated the representation of molecular disease patterns characteristic for human NASH in high‐fat diet (HFD)‐fed Ldlr‐/‐.Leiden mice and studied the effects of obeticholic acid (OCA) on these disease profiles. Multiplatform serum metabolomic profiles and genome‐wide liver transcriptome from HFD‐fed Ldlr‐/‐.Leiden mice were compared with those of NASH patients. Mice were profiled at the stage of mild (24 weeks HFD) and severe (34 weeks HFD) fibrosis, and after OCA intervention (24‐34 weeks; 10 mg/kg/day). Effects of OCA were analyzed histologically, biochemically, by immunohistochemistry, using deuterated water technology (de novo collagen formation), and by its effect on the human‐based transcriptomics and metabolomics signatures. The transcriptomics and metabolomics profile of Ldlr‐/‐.Leiden mice largely reflected the molecular signature of NASH patients. OCA modulated the expression of these molecular profiles and quenched specific proinflammatory‐profibrotic pathways. OCA attenuated specific facets of cellular inflammation in liver (F4/80‐positive cells) and reduced crown‐like structures in adipose tissue. OCA reduced de novo collagen formation and attenuated further progression of liver fibrosis, but did not reduce fibrosis below the level before intervention. Conclusion: HFD‐fed Ldlr‐/‐.Leiden mice recapitulate molecular transcriptomic and metabolomic profiles of NASH patients, and these signatures are modulated by OCA. Intervention with OCA in developing fibrosis reduces collagen deposition and de novo synthesis but does not resolve already manifest fibrosis in the period studied. These data show that human molecular signatures can be used to evaluate the translational character of preclinical models for NASH.

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-β signaling pathways.

Introduction: It is generally accepted that metabolic inflammation in the liver is an important driver of disease progression in NASH and associated matrix remodeling/fibrosis. However, the exact molecular inflammatory mechanisms are poorly defined in human studies. Investigation of key pathogenic mechanisms requires the use of pre-clinical models, for instance for time-resolved studies. Such models must reflect molecular disease processes of importance in patients. Herein we characterized inflammation in NASH patients on the molecular level by transcriptomics and investigated whether key human disease pathways can be recapitulated experimentally in Ldlr−/−.Leiden mice, an established pre-clinical model of NASH.Methods: Human molecular inflammatory processes were defined using a publicly available NASH gene expression profiling dataset (GSE48452) allowing the comparison of biopsy-confirmed NASH patients with normal controls. Gene profiling data from high-fat diet (HFD)-fed Ldlr−/−.Leiden mice (GSE109345) were used for assessment of the translational value of these mice.Results: In human NASH livers, we observed regulation of 65 canonical pathways of which the majority was involved in inflammation (32%), lipid metabolism (16%), and extracellular matrix/remodeling (12%). A similar distribution of pathways across these categories, inflammation (36%), lipid metabolism (24%) and extracellular matrix/remodeling (8%) was observed in HFD-fed Ldlr−/−.Leiden mice. Detailed evaluation of these pathways revealed that a substantial proportion (11 out of 13) of human NASH inflammatory pathways was recapitulated in Ldlr−/−.Leiden mice. Furthermore, the activation state of identified master regulators of inflammation (i.e., specific transcription factors, cytokines, and growth factors) in human NASH was largely reflected in Ldlr−/−.Leiden mice, further substantiating its translational value.Conclusion: Human NASH is characterized by upregulation of specific inflammatory processes (e.g., “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes,” “PI3K signaling in B Lymphocytes”) and master regulators (e.g., TNF, CSF2, TGFB1). The majority of these processes and regulators are modulated in the same direction in Ldlr−/−.Leiden mice fed HFD with a human-like macronutrient composition, thus demonstrating that specific experimental conditions recapitulate human disease on the molecular level of disease pathways and upstream/master regulators.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. Methods: A time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. Results: High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. Conclusions: An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames.

Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

To assess collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane–like interface tissue around aseptically loosened hip replacement implants.The medical indication for the primary total hip replacement was osteoarthritis in all study patients. Samples from the study patients were compared with control synovial membranes obtained from trauma (hip fracture) patients. Proteoglycans were extracted with 4M guanidinium chloride. Denatured collagen in the remaining matrix was solubilized with α‐chymotrypsin. Nonsoluble matrix and supernatant fractions were acid hydrolyzed before measurement of hydroxyproline. The proportion of soluble (in vivo–degraded) collagen of the total sample collagen content was calculated. Proteinases were stained using the avidin–biotin–peroxidase complex method.Collagen in the interface membrane from the implants was highly degraded (mean ± SEM 20 ± 3%) compared with that in the control synovial membranes (12 ± 1%; P = 0.007). In controls, the degree of collagen degradation did not correlate with levels of matrix metalloproteinase 1 (MMP‐1), MMP‐13, or cathepsin K, although MMP‐1 approached statistical significance. In interface membranes, the correlations were r = 0.88 (P = 0.002), r = 0.92 (P = 0.001), and r = 0.98 (P < 0.0001) for MMP‐1, MMP‐13, and cathepsin K, respectively.In normal synovial membrane, collagen matrix remodeling may be mainly an intracellular process. In contrast, pathologic tissue destruction in the interface membrane from prosthetic hip joints is associated with a shift toward MMP‐13 and cathepsin K, which become activated and overcome their endogenous inhibitors (tissue inhibitors of metalloproteinases and cystatin C). The highly significant correlation between collagen degradation and cathepsin K indicates an extracellular role of this acidic endoproteinase, consistent with previous observations concerning the acidity of the interface membrane. [ABSTRACT FROM AUTHOR]