Novel traceable CRISPR-Cas9 engineered human embryonic stem cell line (E1C3 + hSEAP + 2xKO + pCD47), has potential to evade immune detection in pigs.

Here we present the generation of a human embryonic stem cell line with the potential to escape immune rejection upon transplantation to an alternate species, in this case sus scrofa. For in vivo detection the cells were modified by CRISPR-Cas9 to express human secreted alkaline phosphatase. To avoid immune recognition and subsequent rejection by host, genes encoding hB2M and hCIITA were knocked out and the porcine gene for CD47 was introduced. Upon editing and subsequent culture, cells maintained molecular and phenotypic pluripotent charactaristics and a normal karyotype supporting viability and functionality of the engineered cell line.

Validation of computed tomography as a diagnostic tool in guinea pigs with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) and subsequent steatohepatitis (NASH) is the most common cause of liver disease and liver transplantation in humans. Affecting millions of patients worldwide, diagnosis relies on a biopsy, not without risk to the patient, and emphasises the need for improved diagnostic measures to determine and monitor disease progression. Despite intensive research, approved pharmacological treatment modalities are few, underlining that animal models with increased translational validity are important to advance preclinical drug development. This study validates the applicability of computed tomography (CT) as a non-invasive diagnostic tool for the assessment of liver steatosis in a guinea pig model of NAFLD/NASH. Guinea pigs with induced NAFLD or NASH were compared to healthy controls at two separate time points: week 16, serving as baseline measure, and week 25 to monitor disease progression over time. The animals were subsequently euthanised, and samples were collected to confirm disease stage. The data showed a strong negative correlation between liver triglycerides and Hounsfield unit (HU) values (R2 = 0.8157; p < 0.0001). A significant difference in histopathological scoring and HU values between grade 0 and more advanced stages of steatosis was recorded (p < 0.001), although the degree of liver fibrosis could not be accurately evaluated by differences in HU. In conclusion, the present study validates CT scanning for the determination of hepatic steatosis in guinea pigs, and it strongly supports the technique as a relevant non-invasive diagnostic tool in this species.

Dietary Long-Chain Fatty Acids Accelerate Metabolic Dysfunction in Guinea Pigs with Non-Alcoholic Steatohepatitis.

The composition of dietary fatty acids may be important for the development and progression of metabolic syndrome and non-alcoholic steatohepatitis (NASH). This study investigated the effect of two high-fat diets based on coconut oil, containing predominantly medium-chain fatty acids (MCFA), or cocoa butter, containing mainly long-chain fatty acids (LCFA), on glucose homeostasis and NASH in guinea pigs following 16 and 32 weeks of diet. At week 16, glucose intolerance was increased in the LCFA animals compared to the MCFA animals (p < 0.001), with both groups differing from the controls by week 32 (p < 0.0001), supported by increased hemoglobin A1c (p < 0.05). NASH was present in both high-fat groups from week 16, with advancing fibrosis appearing more progressive in the LCFA animals at week 16. In agreement, gene expression showed overall increased expression of NASH target genes in the LCFA animals compared to the MCFA animals at weeks 16 and 32 (p < 0.05 and p < 0.0001, respectively). The LCFA animals also displayed increased plasma uric acid at both time points (p < 0.05), a phenomenon linked to NASH in humans. In conclusion, this study reports that a diet high in LCFA promotes metabolic imbalance and may accelerate NASH-associated hepatic fibrosis. This highlights the importance of a critical evaluation of fatty acid composition when investigating NASH-associated endpoints.

Vitamin C Deficiency Exacerbates Dysfunction of Atherosclerotic Coronary Arteries in Guinea Pigs Fed a High-Fat Diet.

Vitamin C (vitC) deficiency has been associated with an increased risk of cardiovascular disease; while several putative mechanistic links have been proposed, functional evidence supporting a causal relationship is scarce. In this study, we investigated how vitC deficiency affects coronary artery vasomotor function and the development of coronary atherosclerotic plaques in guinea pigs subjected to chronic dyslipidemia by a high-fat diet regime. Female Hartley guinea pigs were fed either a control (low-fat diet and sufficient vitC) (N = 8) or a high-fat diet with either sufficient (N = 8) or deficient (N = 10) vitC for 32 weeks. Guinea pigs subjected to the high-fat diet developed significant atherosclerotic plaques in their coronary arteries, with no quantitative effect of vitC deficiency. In isolated coronary arteries, vasomotor responses to potassium, carbachol, nitric oxide, or bradykinin were studied in a wire myograph. Carbachol, bradykinin, and nitric oxide mediated relaxation in the coronary arteries of the control group. While vasorelaxation to carbachol and nitric oxide was preserved in the two high-fat diet groups, bradykinin-induced vasorelaxation was abolished. Interestingly, bradykinin induced a significant contraction in coronary arteries from vitC-deficient guinea pigs (p < 0.05). The bradykinin-induced contraction was unaffected by L-NAME but significantly inhibited by both indomethacin and vitC, suggesting that, during vitC deficiency, increased release of arachidonic acid metabolites and vascular oxidative stress are involved in the constrictor effects mediated by bradykinin. In conclusion, the present study shows supporting evidence that poor vitC status negatively affects coronary artery function.

Efficacy of fibroblast growth factor 21 in non-alcoholic fatty liver disease in guinea pigs.

Fibroblast growth factor 21 (FGF21) agonists have shown promising effects in preclinical models of non-alcoholic fatty liver disease (NAFLD) as well as in short-term clinical trials in patients with non-alcoholic steatohepatitis (NASH). Comparing drug formulation, dose, administration route and age, this exploratory study investigated effects of FGF21 on NAFLD-associated measures in a validated guinea pig model. In three separate studies, female guinea pigs received a high-fat diet prior to intervention with escalating doses of either recombinant native human FGF21 or a human FGF21 human recombinant analogue (FGF21/19 chimer) with an extended half-life. While no significant effects of native FGF21 on the investigated endpoints were observed, the long-acting FGF21/19 chimer significantly altered the levels of circulating lipids, increasing plasma concentrations of cholesterol (TC, LDLc and HDLc) in young guinea pigs (p < 0.01 for all three parameters). Relative liver weights were reduced in FGF21/19-treated young animals (p < 0.05) compared to mature animals, whereas FGF21/19 reduced body weights in both age groups (p < 0.001). The FGF21/19 chimer effects on dyslipidemia, body and liver weights particularly in young animals, support an age-associated difference in the FGF21 response. The limited effects of the native human FGF21 highlights potential species-associated differences of this compound.

Modelling Nonalcoholic Steatohepatitis In Vivo-A Close Transcriptomic Similarity Supports the Guinea Pig Disease Model.

The successful development of effective treatments against nonalcoholic steatohepatitis (NASH) is significantly set back by the limited availability of predictive preclinical models, thereby delaying and reducing patient recovery. Uniquely, the guinea pig NASH model develops hepatic histopathology and fibrosis resembling that of human patients, supported by similarities in selected cellular pathways. The high-throughput sequencing of guinea pig livers with fibrotic NASH (n = 6) and matched controls (n = 6) showed a clear separation of the transcriptomic profile between NASH and control animals. A comparison to NASH patients with mild disease (GSE126848) revealed a 45.2% overlap in differentially expressed genes, while pathway analysis showed a 34% match between the top 50 enriched pathways in patients with advanced NASH (GSE49541) and guinea pigs. Gene set enrichment analysis highlighted the similarity to human patients (GSE49541), also when compared to three murine models (GSE52748, GSE38141, GSE67680), and leading edge genes THRSP, CCL20 and CD44 were highly expressed in both guinea pigs and NASH patients. Nine candidate genes were identified as highly correlated with hepatic fibrosis (correlation coefficient > 0.8), and showed a similar expression pattern in NASH patients. Of these, two candidate genes (VWF and SERPINB9) encode secreted factors, warranting further investigations as potential biomarkers of human NASH progression. This study demonstrates key similarities in guinea pig and human NASH, supporting increased predictability when translating research findings to human patients.

Differential Effects of Dietary Components on Glucose Intolerance and Non-Alcoholic Steatohepatitis.

Pharmacological treatment modalities for non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are scarce, and discoveries are challenged by lack of predictive animal models adequately reflecting severe human disease stages and co-morbidities such as obesity and type 2 diabetes. To mimic human NAFLD/NASH etiology, many preclinical models rely on specific dietary components, though metabolism may differ considerably between species, potentially affecting outcomes and limiting comparability between studies. Consequently, understanding the physiological effects of dietary components is critical for high translational validity. This study investigated the effects of high fat, cholesterol, and carbohydrate sources on NASH development and metabolic outcomes in guinea pigs. Diet groups (n = 8/group) included: low-fat low-starch (LF-LSt), low-fat high-starch (LF-HSt), high-fat (HF) or HF with 4.2%, or 8.4% sugar water supplementation. The results showed that caloric compensation in HF animals supplied with sugar water led to reduced feed intake and a milder NASH phenotype compared to HF. The HF group displayed advanced NASH, weight gain and glucose intolerance compared to LF-LSt animals, but not LF-HSt, indicating an undesirable effect of starch in the control diet. Our findings support the HF guinea pig as a model of advanced NASH and highlights the importance in considering carbohydrate sources in preclinical studies of NAFLD.

Vitamin C Deficiency in the Young Brain-Findings from Experimental Animal Models.

Severe and long-term vitamin C deficiency can lead to fatal scurvy, which is fortunately considered rare today. However, a moderate state of vitamin C (vitC) deficiency (hypovitaminosis C)-defined as a plasma concentration below 23 µM-is estimated to affect up to 10% of the population in the Western world, albeit clinical hallmarks in addition to scurvy have not been linked to vitC deficiency. The brain maintains a high vitC content and uniquely high levels during deficiency, supporting vitC's importance in the brain. Actions include both antioxidant and co-factor functions, rendering vitamin C deficiency likely to affect several targets in the brain, and it could be particularly significant during development where a high cellular metabolism and an immature antioxidant system might increase sensitivity. However, investigations of a non-scorbutic state of vitC deficiency and effects on the developing young brain are scarce. This narrative review provides a comprehensive overview of the complex mechanisms that regulate vitC homeostasis in vivo and in the brain in particular. Functions of vitC in the brain and the potential consequences of deficiency during brain development are highlighted, based primarily on findings from experimental animal models. Perspectives for future investigations of vitC are outlined.

Hepatic Stellate Cell Activation and Inactivation in NASH-Fibrosis-Roles as Putative Treatment Targets?

Hepatic fibrosis is the primary predictor of mortality in patients with non-alcoholic steatohepatitis (NASH). In this process, the activated hepatic stellate cells (HSCs) constitute the principal cells responsible for the deposition of a fibrous extracellular matrix, thereby driving the hepatic scarring. HSC activation, migration, and proliferation are controlled by a complex signaling network involving growth factors, lipotoxicity, inflammation, and cellular stress. Conversely, the clearance of activated HSCs is a prerequisite for the resolution of the extracellular fibrosis. Hence, pathways regulating the fate of the HSCs may represent attractive therapeutic targets for the treatment and prevention of NASH-associated hepatic fibrosis. However, the development of anti-fibrotic drugs for NASH patients has not yet resulted in clinically approved therapeutics, underscoring the complex biology and challenges involved when targeting the intricate cellular signaling mechanisms. This narrative review investigated the mechanisms of activation and inactivation of HSCs with a focus on NASH-associated hepatic fibrosis. Presenting an updated overview, this review highlights key cellular pathways with potential value for the development of future treatment modalities.

The development of nonalcoholic steatohepatitis is subjected to breeder dependent variation in guinea pigs.

Variability in disease development due to differences in strains and breeders constitutes a substantial challenge in preclinical research. However, the impact of the breeder on non-alcoholic steatohepatitis (NASH) is not yet fully elucidated. This retrospective study investigates NASH development in guinea pigs from Charles River or Envigo fed a high fat diet (20% fat, 15% sucrose, 0.35% cholesterol) for 16 or 24/25 weeks. Charles River animals displayed more severe NASH, with higher steatosis (p < 0.05 at week 16), inflammation (p < 0.05 at both week), fibrosis (p < 0.05 at week 16) and disease activity (p < 0.05 at both weeks). Accordingly, alanine and aspartate aminotransferase were increased at week 24/25 (p < 0.01). Hepatic expression of inflammatory (Ccl2, Cxcl8) and fibrotic (Pdgf, Serpine1, Col1a1) genes was also increased (p < 0.05). Differences were observed in healthy chow (4% fat, 0% sucrose, 0% cholesterol) fed animals: Envigo animals displayed higher relative liver weights (p < 0.01 at both weeks), liver cholesterol (p < 0.0001 at week 24/25) and aspartate aminotransferase (p < 0.05 at week 16), but lower levels of alkaline phosphatase (p < 0.0001 at week 24/25). These findings accentuates the importance of the breeder and its effect on NASH development and severity. Consequently, this may affect reproducibility, study comparison and limit the potential of developing novel therapies.

Decreased expression of the GLP-1 receptor after segmental artery injury in mice.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in the renal vasculature and known to be downregulated under hypertensive conditions in rats and humans. However, little is known about the regulation in other types of renal pathology involving vascular changes. This study investigates the expression of the GLP1R in renal vasculature after glomerular injury in the nephrotoxic nephritis mouse model, high cholesterol, and atherosclerosis in the Ldlr-/- mouse on Western diet, and ex vivo injury in an organ culture model. The immunohistochemical signal of the GLP1R was significantly decreased in arteries from mice with nephrotoxic nephritis after 42 days compared to 7 days and saline control (P < 0.05). Histological evaluation of kidneys from Ldlr-/- mice on Western diet showed a decreased GLP1R specific immunohistochemical signal (P < 0.05). The dilatory response to liraglutide was decreased in Western diet fed Ldlr-/- mice compared to C57Bl/6J controls (P < 0.05). Organ culture significantly decreased the immunohistochemical signal of the GLP1R (P <0.05) and the expression of Glp1r mRNA (P < 0.005) compared to fresh. Organ cultured vessels showed vascular smooth muscle cell remodelling as Acta2 expression was decreased (P < 0.005) and Ednrb was increased (P < 0.05). In conclusion, nephrotoxic nephritis and hypercholesterolaemia led to decreased GLP1R specific immunohistochemical signal. Ex vivo vascular injury in the organ culture model leads to a decrease in expression of GLP1R expressionand contractile VSMC specific markers and increase in expression of dedifferentiation markers suggestive of an inverse relationship between phenotypic switch of the VSMC and the expression of the GLP1R; however, the causal relationship remains elusive.

Extracellular Vesicles as Drivers of Non-Alcoholic Fatty Liver Disease: Small Particles with Big Impact.

Nonalcoholic fatty liver disease (NAFLD) is becoming the leading chronic liver disease, negatively affecting the lives of millions of patients worldwide. The complex pathogenesis involves crosstalk between multiple cellular networks, but how the intricate communication between these cells drives disease progression remains to be further elucidated. Furthermore, the disease is not limited to the liver and includes the reprogramming of distant cell populations in different organs. Extracellular vesicles (EVs) have gained increased attention as mediators of cellular communication. EVs carry specific cargos that can act as disease-specific signals both locally and systemically. Focusing on NAFLD advancing to steatohepatitis (NASH), this review provides an update on current experimental and clinical findings of the potential role of EVs in hepatic inflammation and fibrosis, the main contributors to progressive NASH. Particular attention is placed on the characteristics of EV cargos and potential specificity to disease stages, with putative value as disease markers and treatment targets for future investigations.

Vitamin C Deficiency May Delay Diet-Induced NASH Regression in the Guinea Pig.

Oxidative stress is directly linked to non-alcoholic fatty liver disease (NAFLD) and the progression to steaotohepatitis (NASH). Thus, a beneficial role of antioxidants in delaying disease progression and/or accelerating recovery may be expected, as corroborated by recommendations of, e.g., vitamin E supplementation to patients. This study investigated the effect of vitamin C deficiency-often resulting from poor diets low in fruits and vegetables and high in fat-combined with/without a change to a low fat diet on NAFLD/NASH phenotype and hepatic transcriptome in the guinea pig NASH model. Vitamin C deficiency per se did not accelerate disease induction. However, the results showed an effect of the diet change on the resolution of hepatic histopathological hallmarks (steatosis, inflammation, and ballooning) (p < 0.05 or less) and indicated a positive effect of a high vitamin C intake when combined with a low fat diet. Our data show that a diet change is important in NASH regression and suggest that a poor vitamin C status delays the reversion towards a healthy hepatic transcriptome and phenotype. In conclusion, the findings support a beneficial role of adequate vitamin C intake in the regression of NASH and may indicate that vitamin C supplementation in addition to lifestyle modifications could accelerate recovery in NASH patients with poor vitamin C status.

A Long-Term Energy-Rich Diet Increases Prefrontal BDNF in Sprague-Dawley Rats.

Findings of the effect of high-fat feeding including "Cafeteria Diets" (CAF) on brain-derived neurotrophic factor (BDNF) in the hippocampus (HIP) and prefrontal cortex (PFC) in rodents are conflicting. CAF is a non-standardized, highly palatable energy-rich diet composed by everyday food items for human consumption and is known to induce metabolic syndrome and obesity in rats. However, the highly palatable nature of CAF may counteract a negative effect of chronic stress on anticipatory behavior and synaptic plasticity in the hippocampus, hence represent a confounding factor (e.g., when evaluating functional effects on the brain). This study investigated the effects of a chronic, restricted access to CAF on BDNF, monoamine neurotransmitters, and redox imbalance in HIP and PFC in male rats. Our results show that CAF induced BDNF and its receptor TrkB in PFC compared to the controls (p < 0.0005). No differences in monoamine neurotransmitters were detected in either PFC or HIP. CAF increased dehydroascorbic acid and decreased malondialdehyde in PFC (p < 0.05), suggesting an early redox imbalance insufficient to induce lipid peroxidation. This study supports that a chronic CAF on a restricted schedule increases BDNF levels in the PFC of rats, highlighting that this may be a suboptimal feeding regime when investigating the effects of diet-induced obesity in the brain and emphasizing this as a point of attention when comparing the findings.

Liraglutide treatment improves endothelial function in the Ldlr-/- mouse model of atherosclerosis and affects genes involved in vascular remodelling and inflammation.

Recent clinical intervention studies have shown that the GLP1 analogue liraglutide lowers cardiovascular risk, but the underlying mechanism has not yet been fully elucidated. This study investigated the effects of liraglutide on endothelial function in the Ldlr-/- mouse model. Mice (n = 12/group) were fed Western diet (WD) or chow for 12 weeks followed by 4 weeks of treatment with liraglutide (1 mg/kg/day) or vehicle subcutaneously. Weight loss, blood lipid content, plaque burden, vasomotor function of the aorta and gene expression pattern in aorta and brachiocephalic artery were monitored. Liraglutide treatment significantly induced weight loss (P < .0001), decreased blood triglycerides (P < .0001) and total cholesterol (P < .0001) in WD-fed mice but did not decrease plaque burden. Liraglutide also improved endothelium-mediated dilation of the distal thoracis aorta (P = .0067), but it did not affect phenylephrine or sodium nitroprusside responses. Fluidigm analyses of 96 genes showed significantly altered expression of seven genes related to inflammation, vascular smooth muscle cells and extracellular matrix composition in liraglutide-treated animals. We conclude that treatment with liraglutide decreased endothelial dysfunction and that this could be linked to decreased inflammation or regulation of vascular remodelling.

The effect of acetylsalicylic acid and pentoxifylline in guinea pigs with non-alcoholic steatohepatitis.

Therapeutic options are urgently needed for non-alcoholic fatty liver disease (NAFLD), but development is time-consuming and costly. In contrast, drug repurposing offers the advantages of re-applying compounds that are already approved, thereby reducing cost. Acetylsalicylic acid (ASA) and pentoxifylline (PTX) have shown promise for treatment of NAFLD, but have not yet been tested in combination. Guinea pigs were fed a high-fat diet for 16 weeks and then continued on the diet while being treated with ASA, PTX or ASA+PTX for 8 weeks. Chow-fed animals served as healthy controls. Guinea pigs were CT scanned before intervention start and at intervention end. Animals without steatosis (ie NAFLD) at week 16 were excluded from the data analysis. ASA and PTX alone or in combination did not improve hepatic steatosis, ballooning, inflammation or fibrosis nor did the treatments affect liver enzymes (aminotransferases and alkaline phosphatase) or circulating lipids. Liver triglyceride levels, relative liver weight and hepatic mRNA expression of monocyte chemoattractant protein 1, interleukin 8 and platelet-derived growth factor b were nominally decreased. Thus, in the current study, treatment with ASA and PTX alone or in combination for 8 weeks did not ameliorate NASH or hepatic fibrosis in guinea pigs.

Animal Models of Fibrosis in Nonalcoholic Steatohepatitis: Do They Reflect Human Disease?

Nonalcoholic steatohepatitis (NASH) is one of the most common chronic liver diseases in the world, yet no pharmacotherapies are available. The lack of translational animal models is a major barrier impeding elucidation of disease mechanisms and drug development. Multiple preclinical models of NASH have been proposed and can broadly be characterized as diet-induced, deficiency-induced, toxin-induced, genetically induced, or a combination of these. However, very few models develop advanced fibrosis while still reflecting human disease etiology or pathology, which is problematic since fibrosis stage is considered the best prognostic marker in patients and an important endpoint in clinical trials of NASH. While mice and rats predominate the NASH research, several other species have emerged as promising models. This review critically evaluates animal models of NASH, focusing on their ability to develop advanced fibrosis while maintaining their relevance to the human condition.