Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model.

Atypical chemokine receptor 3 (ACKR3, formerly CXC chemokine receptor 7) is a G protein-coupled receptor that recruits ß-arrestins, but is devoid of functional G protein signaling after receptor stimulation. In preclinical models of liver and lung fibrosis, ACKR3 was previously shown to be upregulated after acute injury in liver sinusoidal and pulmonary capillary endothelial cells, respectively. This upregulation was linked with a pro-regenerative and anti-fibrotic role for ACKR3. A recently described ACKR3-targeting small molecule agonist protected mice from isoproterenol-induced cardiac fibrosis. Here, we aimed to evaluate its protective role in preclinical models of liver and lung fibrosis. After confirming its in vitro pharmacological activity (i.e., ACKR3-mediated ß-arrestin recruitment and receptor binding), in vivo administration of this ACKR3 agonist led to increased mouse CXCL12 plasma levels, indicating in vivo interaction of the agonist with ACKR3. Whereas twice daily in vivo administration of the ACKR3 agonist lacked inhibitory effect on bleomycin-induced lung fibrosis, it had a modest, but significant anti-fibrotic effect in the carbon tetrachloride (CCl4)-induced liver fibrosis model. In the latter model, ACKR3 stimulation affected the expression of several fibrosis-related genes and led to reduced collagen content as determined by picro-sirius red staining and hydroxyproline quantification. These data confirm that ACKR3 agonism, at least to some extent, attenuates fibrosis, although this effect is rather modest and heterogeneous across various tissue types. Stimulating ACKR3 alone without intervening in other signaling pathways involved in the multicellular crosstalk leading to fibrosis will, therefore, most likely not be sufficient to deliver a satisfactory clinical outcome.

Atorvastatin Attenuates Diet-Induced Non-Alcoholic Steatohepatitis in APOE*3-Leiden Mice by Reducing Hepatic Inflammation.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1ß (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Semaglutide Has Beneficial Effects on Non-Alcoholic Steatohepatitis in Ldlr-/-.Leiden Mice.

Semaglutide, a glucagon-like peptide-1 receptor agonist, is an antidiabetic medication that has recently been approved for the treatment of obesity as well. Semaglutide is postulated to be a promising candidate for the treatment of non-alcoholic steatohepatitis (NASH). Here, Ldlr-/-.Leiden mice received a fast-food diet (FFD) for 25 weeks, followed by another 12 weeks on FFD with daily subcutaneous injections of semaglutide or vehicle (control). Plasma parameters were evaluated, livers and hearts were examined, and hepatic transcriptome analysis was performed. In the liver, semaglutide significantly reduced macrovesicular steatosis (-74%, p < 0.001) and inflammation (-73%, p < 0.001) and completely abolished microvesicular steatosis (-100%, p < 0.001). Histological and biochemical assessment of hepatic fibrosis showed no significant effects of semaglutide. However, digital pathology revealed significant improvements in the degree of collagen fiber reticulation (-12%, p < 0.001). Semaglutide did not affect atherosclerosis relative to controls. Additionally, we compared the transcriptome profile of FFD-fed Ldlr-/-.Leiden mice with a human gene set that differentiates human NASH patients with severe fibrosis from those with mild fibrosis. In FFD-fed Ldlr-/-.Leiden control mice, this gene set was upregulated as well, while semaglutide predominantly reversed this gene expression. Using a translational model with advanced NASH, we demonstrated that semaglutide is a promising candidate with particular potential for the treatment of hepatic steatosis and inflammation, while for the reversal of advanced fibrosis, combinations with other NASH agents may be necessary.

Unraveling the Transcriptional Dynamics of NASH Pathogenesis Affecting Atherosclerosis.

The prevalence of non-alcoholic steatohepatitis (NASH) is rapidly increasing and associated with cardiovascular disease (CVD), the major cause of mortality in NASH patients. Although sharing common risk factors, the mechanisms by which NASH may directly contribute to the development to CVD remain poorly understood. The aim of this study is to gain insight into key molecular processes of NASH that drive atherosclerosis development. Thereto, a time-course study was performed in Ldlr-/-.Leiden mice fed a high-fat diet to induce NASH and atherosclerosis. The effects on NASH and atherosclerosis were assessed and transcriptome analysis was performed. Ldlr-/-.Leiden mice developed obesity, hyperlipidemia and insulin resistance, with steatosis and hepatic inflammation preceding atherosclerosis development. Transcriptome analysis revealed a time-dependent increase in pathways related to NASH and fibrosis followed by an increase in pro-atherogenic processes in the aorta. Gene regulatory network analysis identified specific liver regulators related to lipid metabolism (SC5D, LCAT and HMGCR), inflammation (IL1A) and fibrosis (PDGF, COL3A1), linked to a set of aorta target genes related to vascular inflammation (TNFA) and atherosclerosis signaling (CCL2 and FDFT1). The present study reveals pathogenic liver processes that precede atherosclerosis development and identifies hepatic key regulators driving the atherogenic pathways and regulators in the aorta.

Therapeutic Intervention with Anti-Complement Component 5 Antibody Does Not Reduce NASH but Does Attenuate Atherosclerosis and MIF Concentrations in Ldlr-/-.Leiden Mice.

BACKGROUND: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. METHODS: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. RESULTS: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). CONCLUSION: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression.

Intramuscular short-chain acylcarnitines in elderly people are decreased in (pre-)frail females, but not in males.

This study tested the hypothesis that in human aging, a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance, and altered mitochondrial function. We used a cross-sectional study design with well-matched fit and (pre-)frail old males and females, using young males and females as healthy controls. Frailty was assessed according to the Fried criteria and physical performance was determined by 400 m walk test, short physical performance battery and handgrip strength. Muscle and plasma acylcarnitine status, and muscle mitochondrial gene expression was analyzed. Results showed that intramuscular total carnitine levels and short-chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular short-chain acylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. It is, therefore, concluded that in (pre-)frail old females, intramuscular total carnitine levels and short-chain acylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in aging research.

Dual targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative.

BACKGROUND & AIMS: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively. METHODS: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis. RESULTS: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development. CONCLUSIONS: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients.

A network-based approach for identifying suitable biomarkers for oral immunotherapy of food allergy.

BACKGROUND: Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, concerns with regards to safety and long-term efficacy of OIT remain. There is a need to identify biomarkers that predict, monitor and/or evaluate the effects of OIT. Here we present a method to select candidate biomarkers for efficacy and safety assessment of OIT using the computational approaches Bayesian networks (BN) and Topological Data Analysis (TDA). RESULTS: Data were used from fructo-oligosaccharide diet-supported OIT experiments performed in 3 independent cow's milk allergy (CMA) and 2 independent peanut allergy (PNA) experiments in mice. Bioinformatical approaches were used to understand the data structure. The BN predicted the efficacy of OIT in the CMA with 86% and indicated a clear effect of scFOS/lcFOS on allergy parameters. For the PNA model, this BN (trained on CMA data) predicted an efficacy of OIT with 76% accuracy and shows similar effects of the allergen, treatment and diet as compared to the CMA model. The TDA identified clusters of biomarkers closely linked to biologically relevant clinical symptoms and also unrelated and redundant parameters within the network. CONCLUSIONS: Here we provide a promising application of computational approaches to a) compare mechanistic features of two different food allergies during OIT b) determine the biological relevance of candidate biomarkers c) generate new hypotheses to explain why CMA has a different disease pattern than PNA and d) select relevant biomarkers for future studies.

The Neglectable Impact of Delayed Graft Function on Long-term Graft Survival in Kidneys Donated After Circulatory Death Associates With Superior Organ Resilience.

OBJECTIVE: To explore putative different impacts of delayed graft function (DGF) on long-term graft survival in kidneys donated after brain death (DBD) and circulatory death (DCD). BACKGROUND: Despite a 3-fold higher incidence of DGF in DCD grafts, large studies show equivalent long-term graft survival for DBD and DCD grafts. This observation implies a differential impact of DGF on DBD and DCD graft survival. The contrasting impact is remarkable and yet unexplained. METHODS: The impact of DGF on DBD and DCD graft survival was evaluated in 6635 kidney transplants performed in The Netherlands. DGF severity and functional recovery dynamics were assessed for 599 kidney transplants performed at the Leiden Transplant Center. Immunohistochemical staining, gene expression profiling, and Ingenuity Pathway Analysis were used to identify differentially activated pathways in DBD and DCD grafts. RESULTS: While DGF severely impacted 10-year graft survival in DBD grafts (HR 1.67; P < 0.001), DGF did not impact graft survival in DCD grafts (HR 1.08; P = 0.63). Shorter dialysis periods and superior posttransplant eGFRs in DBD grafts show that the differential impact was not caused by a more severe DGF phenotype in DBD grafts. Immunohistochemical evaluation indicates that pathways associated with tissue resilience are present in kidney grafts. Molecular evaluation showed selective activation of resilience-associated pathways in DCD grafts. CONCLUSIONS: This study shows an absent impact of DGF on long-term graft survival in DCD kidneys. Molecular evaluation suggests that the differential impact of DGF between DBD and DCD grafts relates to donor-type specific activation of resilience pathways in DCD grafts.

Evaluation of chitotriosidase as a biomarker for adipose tissue inflammation in overweight individuals and type 2 diabetic patients.

BACKGROUND: Overweight and obesity can lead to adipose tissue inflammation, which causes insulin resistance and on the long-term type 2 diabetes mellitus (T2D). The inflammatory changes of obese-adipose tissue are characterized by macrophage infiltration and activation, but validated circulating biomarkers for adipose tissue inflammation for clinical use are still lacking. One of the most secreted enzymes by activated macrophages is chitotriosidase (CHIT1). OBJECTIVE: To test whether circulating CHIT1 enzymatic activity levels reflect adipose tissue inflammation. METHODS: Plasma and adipose tissue samples of 105 subjects (35 lean, 37 overweight, and 33 T2D patients) were investigated. CHIT1 mRNA levels were determined in adipose tissue-resident innate immune cells. CHIT1 mRNA levels, protein abundance, and plasma enzymatic activity were subsequently measured in adipose tissue biopsies and plasma of control subjects with varying levels of obesity and adipose tissue inflammation as well as in T2D patients. RESULTS: In adipose tissue, CHIT1 mRNA levels were higher in stromal vascular cells compared to adipocytes, and higher in adipose tissue-residing macrophages compared to circulating monocytes (p < 0.001). CHIT1 mRNA levels in adipose tissue were enhanced in overweightcompared to lean subjects and even more in T2D patients (p < 0.05). In contrast, plasma CHIT1 enzymatic activity did not differ between lean, overweight subjects and T2D patients. A mutation of the CHIT1 gene decreases plasma CHIT1 activity. CONCLUSIONS: CHIT1 is expressed by adipose tissue macrophages and expression is higher in overweight subjects and T2D patients, indicating its potential as tissue biomarker for adipose tissue inflammation. However, these differences do not translate into different plasma CHIT1 activity levels. Moreover, a common CHIT1 gene mutation causing loss of plasma CHIT1 activity interferes with its use as a biomarker of adipose tissue inflammation. These results indicate that plasma CHIT1 activity is of limited value as a circulating biomarker for adipose tissue inflammation in human subjects.

The future of protein biomarker research in type 2 diabetes mellitus.

INTRODUCTION: The onset of type 2 diabetes mellitus (T2DM) is strongly associated with obesity and subsequent perturbations in immuno-metabolic responses. To understand the complexity of these systemic changes and better monitor the health status of people at risk, validated clinical biomarkers are needed. Omics technologies are increasingly applied to measure the interplay of genes, proteins and metabolites in biological systems, which is imperative in understanding molecular mechanisms of disease and selecting the best possible molecular biomarkers for clinical use. Areas covered: This review describes the complex onset of T2DM, the contribution of obesity and adipose tissue inflammation to the T2DM disease mechanism, and the output of current biomarker strategies. A new biomarker approach is described that combines published and new self-generated data to merge multiple -omes (i.e. genome, proteome, metabolome etc.) toward understanding of mechanism of disease on the individual level and design multiparameter biomarker panels that drive significant impacts on personalized healthcare. Expert commentary: We here propose an approach to use cross-omics analyses to contextualize published biomarker data and better understand molecular mechanisms of health and disease. This will improve the current and future innovation gaps in translation of discovered putative biomarkers to clinically applicable biomarker tests.

Combined Treatment with L-Carnitine and Nicotinamide Riboside Improves Hepatic Metabolism and Attenuates Obesity and Liver Steatosis.

Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for ß-oxidation and the TCA cycle, respectively. Ldlr -/-.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% w/w), nicotinamide riboside (NR; 0.3% w/w) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (-17%) and hepatic steatosis (-22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.

Diet-Independent Correlations between Bacteria and Dysfunction of Gut, Adipose Tissue, and Liver: A Comprehensive Microbiota Analysis in Feces and Mucosa of the Ileum and Colon in Obese Mice with NAFLD.

Development of non-alcoholic fatty liver disease (NAFLD) is linked to obesity, adipose tissue inflammation, and gut dysfunction, all of which depend on diet. So far, studies have mainly focused on diet-related fecal microbiota changes, but other compartments may be more informative on host health. We present a first systematic analysis of microbiota changes in the ileum and colon using multiple diets and investigating both fecal and mucosal samples. Ldlr-/-.Leiden mice received one of three different energy-dense (ED)-diets (n = 15/group) for 15 weeks. All of the ED diets induced obesity and metabolic risk factors, altered short-chain fatty acids (SCFA), and increased gut permeability and NAFLD to various extents. ED diets reduced the diversity of high-abundant bacteria and increased the diversity of low-abundant bacteria in all of the gut compartments. The ED groups showed highly variable, partially overlapping microbiota compositions that differed significantly from chow. Correlation analyses demonstrated that (1) specific groups of bacteria correlate with metabolic risk factors, organ dysfunction, and NAFLD endpoints, (2) colon mucosa had greater predictive value than other compartments, (3) correlating bacteria differed per compartment, and (4) some bacteria correlated with plasma SCFA levels. In conclusion, this comprehensive microbiota analysis demonstrates correlations between the microbiota and dysfunctions of gut, adipose tissue, and liver, independent of a specific disease-inducing diet.

Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.

Delayed graft function (DGF) following kidney transplantation affects long-term graft function and survival and is considered a manifestation of ischemia reperfusion injury. Preclinical studies characterize metabolic defects resulting from mitochondrial damage as primary driver of ischemia reperfusion injury. In a comprehensive approach that included sequential establishment of postreperfusion arteriovenous concentration differences over the human graft, metabolomic and genomic analysis in tissue biopsies taken before and after reperfusion, we tested whether the preclinical observations translate to the context of clinical DGF. This report is based on sequential studies of 66 eligible patients of which 22 experienced DGF. Grafts with no DGF immediately recovered aerobic respiration as indicated by prompt cessation of lactate release following reperfusion. In contrast, grafts with DGF failed to recover aerobic respiration and showed persistent adenosine triphosphate catabolism indicated by a significant persistently low post reperfusion tissue glucose-lactate ratio and continued significant post-reperfusion lactate and hypoxanthine release (net arteriovenous difference for lactate and hypoxanthine at 30 minutes). The metabolic data for the group with DGF point to a persistent post reperfusion mitochondrial defect, confirmed by functional (respirometry) and morphological analyses. The archetypical mitochondrial stabilizing peptide SS-31 significantly preserved mitochondrial function in human kidney biopsies following simulated ischemia reperfusion. Thus, development of DGF is preceded by a profound post-reperfusion metabolic deficit resulting from severe mitochondrial damage. Strategies aimed at preventing DGF should be focused on safeguarding a minimally required post-reperfusion metabolic competence.

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9.

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores -2.56 and -2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (-28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (-47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [(14)C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.

Mirtoselect, an anthocyanin-rich bilberry extract, attenuates non-alcoholic steatohepatitis and associated fibrosis in ApoE(∗)3Leiden mice.

BACKGROUND & AIMS: Anthocyanins may have beneficial effects on lipid metabolism and inflammation and are demonstrated to have hepatoprotective properties in models of restraint-stress- and chemically-induced liver damage. However, their potential to protect against non-alcoholic steatohepatitis (NASH) under conditions relevant for human pathogenesis remains unclear. Therefore, we studied the effects of the standardised anthocyanin-rich extract Mirtoselect on diet-induced NASH in a translational model of disease. METHODS: ApoE(∗)3Leiden mice were fed a Western-type cholesterol-containing diet without (HC) or with 0.1% (w/w) Mirtoselect (HCM) for 20weeks to study the effects on diet-induced NASH. RESULTS: Mirtoselect attenuated HC-induced hepatic steatosis, as observed by decreased macro- and microvesicular hepatocellular lipid accumulation and reduced hepatic cholesteryl ester content. This anti-steatotic effect was accompanied by local anti-inflammatory effects in liver, as demonstrated by reduced inflammatory cell clusters and reduced neutrophil infiltration in HCM. On a molecular level, HC diet significantly induced hepatic expression of pro-inflammatory genes Tnf, Emr1, Ccl2, Mpo, Cxcl1, and Cxcl2 while this induction was less pronounced or significantly decreased in HCM. A similar quenching effect was observed for HC-induced pro-fibrotic genes, Acta2 and Col1a1 and this anti-fibrotic effect of Mirtoselect was confirmed histologically. Many of the pro-inflammatory and pro-fibrotic parameters positively correlated with intrahepatic free cholesterol levels. Mirtoselect significantly reduced accumulation and crystallisation of intrahepatic free cholesterol, providing a possible mechanism for the observed hepatoprotective effects. CONCLUSIONS: Mirtoselect attenuates development of NASH, reducing hepatic lipid accumulation, inflammation and fibrosis, possibly mediated by local anti-inflammatory effects associated with reduced accumulation and crystallisation of intrahepatic free cholesterol.

Seven robust and easy to obtain biomarkers to measure acute stress.

With the purpose of identifying a sensitive, robust, and easy-to-measure set of biomarkers to assess stress reactivity, we here study a large set of relatively easy to obtain markers reflecting subjective, autonomic nervous system (ANS), endocrine, and inflammatory responses to acute social stress (n = 101). A subset of the participants was exposed to another social stressor the next day (n = 48) while being measured in the same way. Acute social stress was induced following standardized procedures. The markers investigated were self-reported positive and negative affect, heart rate, electrodermal activity, salivary cortisol, and ten inflammatory markers both in capillary plasma and salivary samples, including IL-22 which has not been studied in response to acute stress in humans before. Robust effects (significant effect in the same direction for both days) were found for self-reported negative affect, heart rate, electrodermal activity, plasma IL-5, plasma IL-22, salivary IL-8 and salivary IL-10. Of these seven markers, the participants' IL-22 responses on the first day were positively correlated to those on the second day. We found no correlations between salivary and capillary plasma stress responses for any of the ten cytokines and somewhat unexpectedly, cytokine responses in saliva seemed more pronounced and more in line with previous literature than cytokines in capillary plasma. In sum, seven robust and easy to obtain biomarkers to measure acute stress response were identified and should be used in future stress research to detect and examine stress reactivity. This includes IL-22 in plasma as a promising novel marker.

Translatability of mouse muscle-aging for humans: the role of sex.

Muscle-aging drives sarcopenia and is a major public health issue. Mice are frequently used as a model for human muscle-aging, however, research investigating their translational value is limited. In addition, mechanisms underlying muscle-aging may have sex-specific features in humans, but it is not yet assessed whether these are recapitulated in mice. Here, we studied the effects of aging on a functional, histological and transcriptional level at multiple timepoints in male and female mice (4, 17, 21 and 25 months), with particular emphasis on sex-differences. The effects of natural aging on the transcriptome of quadriceps muscle were compared to humans on pathway level. Significant loss of muscle mass occurred late, at 25 months, in both male (-17%, quadriceps) and female mice (-10%, quadriceps) compared to young control mice. Concomitantly, we found in female, but not male mice, a slower movement speed in the aged groups compared to the young mice (P < 0.001). Consistently, weighted gene co-expression network analysis revealed a stronger association between the aging-related reduction of movement and aging-related changes in muscle transcriptome of female compared to male mice (P < 0.001). In male, but not female mice, major distinctive aging-related changes occurred in the last age group (25 months), which highlights the necessity for careful selection of age using mice as a muscle-aging model. Furthermore, contrasting to humans, more aging-related changes were found in the muscle transcriptome of male mice compared to female mice (4090 vs. 2285 differentially expressed genes at 25 months, respectively). Subsequently, male mice recapitulated more muscle-aging related pathways characteristic for both male and female humans. In conclusion, our data show that sex has a critical effect on the mouse muscle-aging trajectory, although these do not necessarily reflect sex differences observed in the human muscle-aging trajectory.

Supplementation of Seaweed Extracts to the Diet Reduces Symptoms of Alzheimer's Disease in the APPswePS1ΔE9 Mouse Model.

We previously demonstrated that diet supplementation with seaweed Sargassum fusiforme (S. fusiforme) prevented AD-related pathology in a mouse model of Alzheimer's Disease (AD). Here, we tested a lipid extract of seaweed Himanthalia elongata (H. elongata) and a supercritical fluid (SCF) extract of S. fusiforme that is free of excess inorganic arsenic. Diet supplementation with H. elongata extract prevented cognitive deterioration in APPswePS1ΔE9 mice. Similar trends were observed for the S. fusiforme SCF extract. The cerebral amyloid-ß plaque load remained unaffected. However, IHC analysis revealed that both extracts lowered glial markers in the brains of APPswePS1ΔE9 mice. While cerebellar cholesterol concentrations remained unaffected, both extracts increased desmosterol, an endogenous LXR agonist with anti-inflammatory properties. Both extracts increased cholesterol efflux, and particularly, H. elongata extract decreased the production of pro-inflammatory cytokines in LPS-stimulated THP-1-derived macrophages. Additionally, our findings suggest a reduction of AD-associated phosphorylated tau and promotion of early oligodendrocyte differentiation by H. elongata. RNA sequencing on the hippocampus of one-week-treated APPswePS1ΔE9 mice revealed effects of H. elongata on, amongst others, acetylcholine and synaptogenesis signaling pathways. In conclusion, extracts of H. elongata and S. fusiforme show potential to reduce AD-related pathology in APPswePS1ΔE9 mice. Increasing desmosterol concentrations may contribute to these effects by dampening neuroinflammation.

Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

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.