Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, and fibrosis stage is the strongest predictor of liver-related outcomes. Point shear wave elastography (pSWE), based on acoustic radiation force impulse technology, is widely available in routine ultrasound systems; however, histologically validated data in well-characterized MASLD cohorts remain limited. This study aimed to evaluate the diagnostic performance of pSWE for the detection of advanced fibrosis (≥ F3) using histology as the reference standard. In this prospective single-center study, adult patients with MASLD undergoing clinically indicated liver biopsy were included. Liver stiffness was assessed using pSWE on a Siemens ultrasound system. Histological fibrosis staging (F0–F4) was performed by three blinded liver pathologists. Diagnostic performance for advanced fibrosis (≥ F3) was evaluated using receiver operating characteristic (ROC) analysis. Clinically applicable rule-in and rule-out thresholds were determined using predefined sensitivity- and specificity-based criteria. A total of 59 patients with MASLD and numerically evaluable pSWE measurements were included. Advanced fibrosis (≥ F3) was present in 18/59 patients (30.5

Liver fibrosis and portal hypertension (PH) determine prognosis in chronic liver disease. In experimental models, stimulation of NO–sGC–cGMP signaling improved fibrosis, PH and inflammation. Since fibrosis and PH improve slowly after injury cessation, we investigated whether stimulating sGC activity accelerates their regression. Liver fibrosis was induced in C57BL/6 J mice by either carbon tetrachloride (CCl4; 2 µl/g, gavage 3x/week) for 12 weeks or thioacetamide (TAA; 150 mg/kg, intraperitoneal injections 3x/week) for 12 weeks, followed by 1 (R1) or 2 (R2) weeks of regression. Animals received the sGC stimulator Riociguat (RIO; 3 mg/kg, gavage 2x/day) during regression. Disease severity was assessed by portal pressure (PP), collagen proportionate area (CPA) and whole liver transcriptomics. PH and fibrosis area peaked in the TAA model at 7.71 ± 0.57 mmHg PP and 3.87 ± 0.19

BACKGROUND:Metabolic dysfunction-associated steatotic liver disease (MASLD) progresses to metabolic dysfunction-associated steatohepatitis (MASH) and is a major cause of liver cirrhosis. Although liver inflammation is the hallmark feature of MASH versus MASLD, the involvement of the peripheral immune cell compartments in disease progression is poorly understood, and single-cell profiles of peripheral immune cells in MASLD/MASH are not known. METHODS:Patients with MASLD/MASH and healthy volunteers have been prospectively enrolled in a cross-sectional study. Patients have been histologically stratified and further characterized by liver bulk RNA sequencing (RNA-Seq). Peripheral immune cells from patients and control blood samples have been comprehensively profiled using bulk and single RNA-Seq. RESULTS:Twenty-two patients with fibrosis stage less than F3 have been histologically stratified into patients with low, medium, and high disease activity scores (NAFLD activity score [NAS]). In contrast to fibrosis, the NAS group correlated with noninvasive imaging readouts and blood biomarkers of liver damage and inflammation (ALT, AST). The prevalence of type 2 diabetes and obesity increased with the NAS stage. Bulk RNA-seq profiling of patient liver biopsies revealed gene signatures that were positively and negatively associated with NAS. Known marker genes for liver fibrosis where upregulated on RNA level. Blood bulk RNA-seq showed only moderate differences in patients versus healthy controls. In contrast, single-cell analysis of white blood cells revealed multiple alterations of immune (sub-)populations, including an increased abundance of immature B cells and myeloid suppressor cells in patients with MASLD/MASH as compared to healthy controls. CONCLUSIONS:The study gives new insights into the pathophysiology of MASLD/MASH already manifesting relatively early in peripheral immune cell compartments. This opens new avenues for the development of new biomarker diagnostics and disease therapies.

Tissues, organs, and entire organisms are composed of diverse cell populations, which are characterized by cell-type-specific gene activities. Bulk RNA-seq represents a robust, cost-effective, scalable method to measure gene activity at the bulk tissue level. However, pathomolecular processes lead to divergent changes in tissue composition and cell-type-specific gene deregulations, which cannot be resolved at the tissue bulk level without information on either change in cell-type proportion or expression at the single-cell level. Accordingly, methods have been developed that constrain bulk deconvolution by information from single-cell expression or cell-type proportion. In parallel, convolution methods have been developed to project single-cell expression to bulk tissue level (pseudobulk simulation). In the present review, we provide an overview of existing convolution and deconvolution methods, their interconnectivity, and benchmarking. Our unique approach lies in the joint consideration of both directions in a "holistic transcriptome model." Through analysis of published (de)convolution studies and benchmarks, we identified the reduced availability of suitable datasets and the use of inaccurate convolution-like methods for (de)convolution model assessment and training as key bottlenecks in the field. On that basis, we conclude with a holistic transcriptome model envisioning that a more integral approach to convolution and deconvolution is needed. With our suggestions for a unified framework we aim to spark collaborative efforts to enable major leaps forward in the field of (de)convolution.

Given their capability to differentiate into each cell type of the human body, human pluripotent stem cells (hPSCs) provide a unique platform for developmental studies. In the current study, we employed this cell system to understand the role of pancreatic progenitor differentiation and proliferation factor (PPDPF), a protein that has been little explored so far. While the zebrafish orthologue exdpf is essential for exocrine pancreas specification, its importance for mammalian and human development has not been studied yet. We implemented a four times CRISPR/Cas9 nicking approach to knockout PPDPF in human embryonic stem cells (hESCs) and differentiated PPDPFKO/KO and PPDPFWT/WT cells towards the pancreatic lineage. In contrast to data obtained from zebrafish, a very modest effect of the knockout was observed in the development of pancreatic progenitors in vitro, not affecting lineage specification upon orthotopic transplantation in vivo. The modest effect is in line with the finding that genetic variants near PPDPF are associated with random glucose levels in humans, but not with type 2 diabetes risk, supporting that dysregulation of this gene may only result in minor alterations of glycaemic balance in humans. In addition, PPDPF is less organ- and cell type specifically expressed in higher vertebrates and its so far reported functions appear highly context-dependent.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Persistent liver injury triggers a fibrogenic program that causes pathologic remodelling of the hepatic microenvironment (i.e., liver fibrosis) and portal hypertension. The dynamics of gene regulation during liver disease progression and regression remain understudied. Here, we generated hepatic transcriptome profiles in two well-established liver disease models at peak fibrosis and during spontaneous regression after the removal of the inducing agents. We linked the dynamics of key liver disease readouts, such as portal pressure, collagen proportionate area, and transaminase serum levels, to most differentially expressed genes, enabling the identification of transcriptomic signatures of progressive vs. regressive liver fibrosis and portal hypertension. These candidate biomarkers (e.g., Scube1, Tcf4, Src, Hmga1, Trem2, Mafk, Mmp7) were also validated in RNA-seq datasets of patients with cirrhosis and portal hypertension. Finally, deconvolution analysis identified major cell types and suggested an association of macrophage and portal hepatocyte signatures with portal hypertension and fibrosis area in both models.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-β1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

The translation of findings from animal models to human disease is a fundamental part in the field of drug development. However, only a small proportion of promising preclinical results in animals translate to human pathophysiology. This underscores the necessity for novel data analysis strategies to accurately evaluate the most suitable animal model for a specific purpose, ensuring cross-species translatability. To address this need, we present In Silico Treatment (IST), a computational method to assess translation of disease-related molecular expression patterns between animal models and humans. By simulating changes observed in animals onto humans, IST provides a holistic picture of how well animal models recapitulate key aspects of human disease, or how treatments transform pathogenic expression patterns to healthy ones. Furthermore, IST highlights particular genes that influence molecular features of pathogenesis or drug mode of action. We demonstrate the potential of IST with three applications using bulk transcriptomics data. First, we assessed two mouse models for idiopathic pulmonary fibrosis (IPF): one involving injury with intra-tubular Bleomycin exposure, and the other Adeno-associated-virus-induced, TGFβ1-mediated tissue transformation (AAV6.2-TGFβ1). Both models exhibited gene expression patterns resembling extracellular matrix derangement in human IPF, whereas differences in VEGF-driven vascularization were observed. Second, we confirmed known features of non-alcoholic steatohepatitis (NASH) mouse models, including choline-deficient, l-amino acid-defined diet (CDAA), carbon tetrachloride hepatotoxicity injury (CCl4) and bile duct ligation surgery (BDL). Overall, the three mouse models recapitulated expression changes related to fibrosis in human NASH, whereas model-specific differences were found in lipid metabolism, inflammation, and apoptosis. Third, we reproduced the strong anti-fibrotic signature and induction of the PPARα signaling observed in the Elafibranor experimental treatment for NASH in the CDAA model. We validated the contribution of known disease-related genes to the findings made with IST in the IPF and NASH applications. The complete data integration IST framework, including an interactive app to integrate and compare datasets, is made available as an open-source R package. Author summary Preclinical testing plays a pivotal role in the drug development process, serving as a crucial evaluation phase before a new drug can be tested on humans in clinical trials. The drug must undergo a rigorous evaluation in in vivo and in vitro preclinical studies to assess its safety and efficacy. However, positive outcomes in preclinical animal models do not always translate to positive results in humans, mainly due to biological differences. Therefore, selecting an animal model that closely mirrors human disease traits and detecting and accounting for model limitations is of paramount importance. Over the last decade, the availability of gene expression data in both animals and humans has substantially increased. Gene expression states and perturbations are routinely employed as a proxy to predict and understand changes in disease states. Here, we developed In Silico Treatment, a computational method designed to overlay the gene expression changes observed in animals onto humans, quantifying the change in human disease status. We applied this method to mouse models for idiopathic pulmonary fibrosis and non-alcoholic steatohepatitis, two severe fibrotic diseases. We successfully identified known features of the disease models and provide a granular gene-level rationale behind our predictions. Consequently, our method shows promise as an effective approach to improve animal model selection and thus clinical translation. ### Competing Interest Statement All the authors were paid employees by Boehringer Ingelheim Pharma GmbH & Co.KG

Glucagon (GCG) and glucagon-like peptide 1 (GLP-1) are peptide hormones that pharmacologically regulate bodyweight by increasing energy expenditure and reducing energy intake, respectively. Several dual (GCG receptor [GCGR]/GLP-1 receptor [GLP-1R]) agonistic peptides are in clinical development for obesity. We describe preclinical data leading to selection of BI 456906 as a dual agonist. Functional potencies of the compounds were determined in CHO-K1 cells stably expressing human GCGR and GLP-1R. EC50 for GCGR mediated cAMP increase were 0.52 nM for BI 456906, 0.92 nM for BI 456908 and 0.44 nM for BI 456987. EC50 for GLP-1R engagement were 0.33 nM, 0.61 nM and 1.43 nM, respectively. Upon acute, single dosing to lean mice, engagement of the GCGR and GLP-1R was determined by testing for improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma FGF-21 and liver nicotinamide N-methyltransferase mRNA expression (100 nmol/kg), respectively. Specificity of the biomarkers was confirmed using the selective GLP-1R agonist semaglutide and GLP-1R knockout mice. Bodyweight and glucose lowering (HbA1c) efficacies were investigated in subchronic dosing studies in diet-induced obese (DIO) and diabetic (db/db) mice, respectively. In DIO mice, BI 456906 (30 nmol/kg qd), BI 456908 (30 nmol/kg qd) and BI 456897 (10 nmol/kg qd) achieved bodyweight lowering from baseline of 25%, 27%, and 26%, respectively. In db/db mice, BI 456906 and BI 456908 (10 and 20 nmol/kg qd) substantially lowered HbA1c (0.4-0.6%); no clear effect was observed for BI 456897 (3 and 7 nmol/kg qd). As a balanced GCGR/GLP-1R agonist with robust in vivo efficacy BI 456906 (30 nmol/kg qd) was further characterized, and superior bodyweight lowering (32% vs 27%; p<0.05) was seen versus a maximally effective semaglutide dose (100 nmol/kg qd). This was attributed to an increase in energy expenditure. The selected candidate, BI 456906, is currently in clinical development in people with obesity and NASH. Disclosure R.Augustin: None. L.Thomas: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. T.Zimmermann: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. E.Simon: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. W.Rist: None. I.Uphues: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. W.Reindl: None. T.Klein: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. H.Neubauer: None. Funding Zealand Pharma A/S; Boehringer Ingelheim

The current obesity epidemic and high prevalence of metabolic diseases necessitate efficacious and safe treatments. Brown adipose tissue in this context is a promising target with the potential to increase energy expenditure, however no pharmacological treatments activating brown adipose tissue are currently available. Here, we identify AXL receptor tyrosine kinase as a regulator of adipose function. Pharmacological and genetic inhibition of AXL enhance thermogenic capacity of brown and white adipocytes, in vitro and in vivo. Mechanistically, these effects are mediated through inhibition of PI3K/AKT/PDE signaling pathway, resulting in induction of nuclear FOXO1 localization and increased intracellular cAMP levels via PDE3/4 inhibition and subsequent stimulation of the PKA-ATF2 pathway. In line with this, both constitutive Axl deletion as well as inducible adipocyte-specific Axl deletion protect animals from diet-induced obesity concomitant with increases in energy expenditure. Based on these data, we propose AXL receptor as a target for the treatment of obesity.

Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses.

Glucagon (GCG) and glucagon-like peptide 1 (GLP-1) are peptide hormones that pharmacologically regulate bodyweight by increasing energy expenditure and reducing energy intake, respectively. Several dual (GCG receptor [GCGR]/GLP-1 receptor [GLP-1R]) agonistic peptides are in clinical development for obesity. We describe preclinical data leading to selection of BI 456906 as a dual agonist. Functional potencies of the compounds were determined in CHO-K1 cells stably expressing human GCGR and GLP-1R. EC50 for GCGR mediated cAMP increase were 0.52 nM for BI 456906, 0.92 nM for BI 456908 and 0.44 nM for BI 456987. EC50 for GLP-1R engagement were 0.33 nM, 0.61 nM and 1.43 nM, respectively. Upon acute, single dosing to lean mice, engagement of the GCGR and GLP-1R was determined by testing for improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma FGF-21 and liver nicotinamide N-methyltransferase mRNA expression (100 nmol/kg), respectively. Specificity of the biomarkers was confirmed using the selective GLP-1R agonist semaglutide and GLP-1R knockout mice. Bodyweight and glucose lowering (HbA1c) efficacies were investigated in subchronic dosing studies in diet-induced obese (DIO) and diabetic (db/db) mice, respectively. In DIO mice, BI 456906 (30 nmol/kg qd), BI 456908 (30 nmol/kg qd) and BI 456897 (10 nmol/kg qd) achieved bodyweight lowering from baseline of 25%, 27%, and 26%, respectively. In db/db mice, BI 456906 and BI 456908 (10 and 20 nmol/kg qd) substantially lowered HbA1c (0.4-0.6%); no clear effect was observed for BI 456897 (3 and 7 nmol/kg qd). As a balanced GCGR/GLP-1R agonist with robust in vivo efficacy BI 456906 (30 nmol/kg qd) was further characterized, and superior bodyweight lowering (32% vs 27%; p<0.05) was seen versus a maximally effective semaglutide dose (100 nmol/kg qd). This was attributed to an increase in energy expenditure. The selected candidate, BI 456906, is currently in clinical development in people with obesity and NASH. Disclosure R.Augustin: None. L.Thomas: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. T.Zimmermann: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. E.Simon: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. W.Rist: None. I.Uphues: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. W.Reindl: None. T.Klein: Employee; Boehringer Ingelheim Pharma GmbH&Co.KG. H.Neubauer: None. Funding Zealand Pharma A/S; Boehringer Ingelheim

There is a high need for predictive human ex vivo models for non-alcoholic fatty liver disease (NAFLD). About a decade ago, precision-cut liver slices (PCLSs) have been established as an ex vivo assay for humans and other organisms. In the present study, we use transcriptomics by RNASeq to profile a new human and mouse PCLSs based assay for steatosis in NAFLD. Steatosis as quantified by an increase of triglycerides after 48 h in culture, is induced by incremental supplementation of sugars (glucose and fructose), insulin, and fatty acids (palmitate, oleate). We mirrored the experimental design for human vs. mouse liver organ derived PCLSs and profiled each organ at eight different nutrient conditions after 24 h and 48 h time in culture. Thus, the provided data allows a comprehensive analysis of the donor, species, time, and nutrient factor specific regulation of gene expression in steatosis, despite the heterogeneity of the human tissue samples. Exemplified this is demonstrated by ranking homologous gene pairs by convergent or divergent expression pattern across nutrient conditions.

Collapsing glomerulopathy (CG) is a form of podocytopathy that is challenging to manage. CG can be idiopathic or associated with other conditions including autoimmune connective tissue diseases. In the setting of autoimmune connective tissue diseases, there are no current guidelines to guide therapy. Here we report a unique and challenging case of CG with mixed connective tissue disease (MCTD) that responded to steroids followed by mycophenolate. In PubMed, we identified three previously reported cases of CG with MCTD in addition to other forms of autoimmune diseases, including Sjogren syndrome, adult-onset still's disease, and vasculitis, etc. We are providing a literature review of collapsing glomerulopathy cases in the setting of autoimmune connective tissue diseases and with MCTD. CG in the setting of autoimmune connective tissue diseases is more common in females and black patients. Response to therapy was inconsistent. Many patients progressed to dialysis despite use of various treatment modalities.

Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology.

Non‐alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence‐based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra‐Amylin NASH (GAN) diet‐induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO‐NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28–88 weeks. GAN DIO‐NASH mice with biopsy‐confirmed NASH and fibrosis received low‐caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within‐subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning‐based image analysis. GAN DIO‐NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO‐NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO‐NASH mice in preclinical drug development.