Novel insights into metabolic-associated steatotic liver disease preclinical models.

Metabolic-associated steatotic liver disease (MASLD) encompasses a wide spectrum of metabolic conditions associated with an excess of fat accumulation in the liver, ranging from simple hepatic steatosis to cirrhosis and hepatocellular carcinoma. Finding appropriate tools to study its development and progression is essential to address essential unmet therapeutic and staging needs. This review discusses advantages and shortcomings of different dietary, chemical and genetic factors that can be used to mimic this disease and its progression in mice from a hepatic and metabolic point of view. Also, this review will highlight some additional factors and considerations that could have a strong impact on the outcomes of our model to end up providing recommendations and a checklist to facilitate the selection of the appropriate MASLD preclinical model based on clinical aims.

Role of EpCAM+ CD133+ extracellular vesicles in steatosis to steatohepatitis transition in NAFLD.

BACKGROUND AND AIMS: Extracellular vesicles (EVs) have emerged as a potential source of circulating biomarkers in liver disease. We evaluated circulating AV+ EpCAM+ CD133+ EVs as a potential biomarker of the transition from simple steatosis to steatohepatitis. METHODS: EpCAM and CD133 liver proteins and EpCAM+ CD133+ EVs levels were analysed in 31 C57BL/6J mice fed with a chow or high fat, high cholesterol and carbohydrates diet (HFHCC) for 52 weeks. The hepatic origin of MVs was addressed using AlbCrexmT/mG mice fed a Western (WD) or Dual diet for 23 weeks. Besides, we assessed plasma MVs in 130 biopsy-proven NAFLD patients. RESULTS: Hepatic expression of EpCAM and CD133 and EpCAM+ CD133+ EVs increased during disease progression in HFHCC mice. GFP+ MVs were higher in AlbCrexmT/mG mice fed a WD (5.2% vs 12.1%) or a Dual diet (0.5% vs 7.3%). Most GFP+ MVs were also positive for EpCAM and CD133 (98.3% and 92.9% respectively), suggesting their hepatic origin. In 71 biopsy-proven NAFLD patients, EpCAM+ CD133+ EVs were significantly higher in those with steatohepatitis compare to those with simple steatosis (286.4 ± 61.9 vs 758.4 ± 82.3; p < 0.001). Patients with ballooning 367 ± 40.6 vs 532.0 ± 45.1; p = 0.01 and lobular inflammation (321.1 ± 74.1 vs 721.4 ± 80.1; p = 0.001), showed higher levels of these EVs. These findings were replicated in an independent cohort. CONCLUSIONS: Circulating levels of EpCAM+ CD133+ MVs in clinical and experimental NAFLD were increased in the presence of steatohepatitis, showing high potential as a non-invasive biomarker for the evaluation and management of these patients.

Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function.

BACKGROUND & AIMS: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown. METHODS: By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice. RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action. CONCLUSIONS: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis. LAY SUMMARY: We show that autophagy-related gene 3 (ATG3) contributes to the progression of non-alcoholic fatty liver disease in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD by stimulating mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.

Long non-coding RNA H19 as a biomarker for hepatocellular carcinoma.

BACKGROUND AND AIMS: Liver cancer stem cells (CSCs) could be involved in the carcinogenesis, recurrence, metastasis and chemoresistance of hepatocellular carcinoma (HCC). The aim of this study was to explore the role of lncRNA-H19 as a biomarker for liver cancer. METHODS: LncRNA-H19 expression levels and the functional assays were conducted in EpCAM+ CD133+ CSCs and C57BL/6J mice fed with a high-fat high-cholesterol carbohydrate (HFHCC) or standard diet for 52 weeks. Liver tissue and plasma samples from patients with cirrhosis, with or without HCC, were used for the analyses of gene expression and circulating lncRNA-H19 levels in an estimation and validation cohort. RESULTS: EpCAM+ CD133+ cells showed a stem cell-like phenotype, self-renewal capacity, upregulation of pluripotent gene expression and overexpressed lncRNA-H19 (p < .001). Suppression of lncRNA-H19 by antisense oligonucleotide treatment significantly reduced the self-renewal capacity (p < .001). EpCAM, CD133 and lncRNA-h19 expression increased accordingly with disease progression in HFHCC-fed mice (p < .05) and also in liver tissue from HCC patients (p = .0082). Circulating lncRNA-H19 levels were significantly increased in HCC patients in both cohorts (p = .013; p < .0001). In addition, lncRNA-H19 levels increased accordingly with BCLC staging (p < .0001) and decreased after a partial and complete therapeutic response (p < .05). In addition, patients with cirrhosis who developed HCC during follow-up showed higher lncRNA-H19 levels (p = .0025). CONCLUSION: LncRNA-H19 expression was increased in CSCs, in liver tissue and plasma of patients with HCC and decreased after partial/complete therapeutic response. Those patients who developed HCC during the follow-up showed higher levels of lncRNA-H19. LncRNA-H19 could constitute a new biomarker of HCC.

Impact of a Loss-of-Function Variant in HSD17B13 on Hepatic Decompensation and Mortality in Cirrhotic Patients.

A common splice variant in HSD17B13 (rs72613567:TA) was recently found to be associated with a reduced risk of developing chronic liver disease in NAFLD patients and a reduced risk of progression to advanced fibrosis and cirrhosis. In this study, we aimed to evaluate the prognosis of cirrhotic patients harboring this variant. We performed a retrospective analysis on 483 prospectively recruited patients from four different hospitals in Spain, followed-up for at least 5 years. We collected clinical, demographic, and biochemical data, and we performed a genotyping analysis for common variants previously associated with liver disease risk (HSD17B13 rs72613567:TA and PNPLA3 rs738409). Patients homozygous for the TA allele showed a higher MELD score (p = 0.047), Child−Turcotte−Pugh score (p = 0.014), and INR levels (p = 0.046), as well as decreased albumin (p = 0.004) at baseline. After multivariate analysis, patients with the "protective" variant indeed had an increased risk of hepatic decompensation [aHR 2.37 (1.09−5.06); p = 0.029] and liver-related mortality [aHR 2.32 (1.20−4.46); p = 0.012]. Specifically, these patients had an increased risk of developing ascites (Log-R 11.6; p < 0.001), hepatic encephalopathy (Log-R 10.2; p < 0.01), and higher mortality (Log-R 14.1; p < 0.001) at 5 years of follow-up. Interactions with the etiology of the cirrhosis and with the variant rs738409 in PNPLA3 are also described. These findings suggest that the variant rs72613567:TA in HSD17B13 has no protective effect, but indeed increases the risk of decompensation and death in patients with advanced chronic liver disease.

Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models.

INTRODUCTION: non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. MATERIAL AND METHODS: this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. RESULTS: HF-HFD animals had an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. CONCLUSION: in conclusion, the comparison of these models aided in the selection of a long-term, more physiological model for physiopathology studies or a more rapid NASH model for novel molecule testing.

miRNAs in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis.

BACKGROUND & AIMS: microRNAs (miRNAs) are deregulated in non-alcoholic fatty liver disease (NAFLD) and have been proposed as useful markers for the diagnosis and stratification of disease severity. We conducted a meta-analysis to identify the potential usefulness of miRNA biomarkers in the diagnosis and stratification of NAFLD severity. METHODS: After a systematic review, circulating miRNA expression consistency and mean fold-changes were analysed using a vote-counting strategy. The sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio and area under the curve (AUC) for the diagnosis of NAFLD or non-alcoholic steatohepatitis (NASH) were pooled using a bivariate meta-analysis. Deeks' funnel plot was used to assess the publication bias. RESULTS: Thirty-seven studies of miRNA expression profiles and six studies of diagnostic accuracy were ultimately included in the quantitative analysis. miRNA-122 and miRNA-192 showed consistent upregulation. miRNA-122 was upregulated in every scenario used to distinguish NAFLD severity. The miRNA expression correlation between the serum and liver tissue was inconsistent across studies. miRNA-122 distinguished NAFLD from healthy controls with an AUC of 0.82 (95% CI 0.75-0.89), and miRNA-34a distinguished non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver (NAFL) with an AUC of 0.78 (95% CI 0.67-0.88). CONCLUSION: miRNA-34a, miRNA-122 and miRNA-192 were identified as potential diagnostic markers to segregate NAFL from NASH. Both miRNA-122, in distinguishing NAFLD from healthy controls, and miRNA-34a, in distinguishing NASH from NAFL, showed moderate diagnostic accuracy. miRNA-122 was upregulated in every scenario of NAFL, NASH and fibrosis. LAY SUMMARY: microRNAs are deregulated in non-alcoholic fatty liver disease. The microRNAs, miRNA-34a, miRNA-122 and miRNA-192, were identified as potential biomarkers of non-alcoholic fatty liver and non-alcoholic steatohepatitis, at different stages of disease severity. The correlation between miRNA expression in the serum and in liver tissue was inconsistent, or even inverse.

Navigating the Link Between Non-alcoholic Fatty Liver Disease/Non-alcoholic Steatohepatitis and Cardiometabolic Syndrome.

The global prevalence of non-alcoholic fatty liver disease (NAFLD) is nearly 25% and is increasing rapidly. The spectrum of liver damage in NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis, characterised by the presence of lobular inflammation and hepatocyte ballooning degeneration, with or without fibrosis, which can further develop into cirrhosis and hepatocellular carcinoma. Not only is NAFLD a progressive liver disease, but numerous pieces of evidence also point to extrahepatic consequences. Accumulating evidence suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); in fact, CVDs are the most common cause of mortality in patients with NAFLD. Obesity, type 2 diabetes and higher levels of LDL are common risk factors in both NAFLD and CVD; however, how NAFLD affects the development and progression of CVD remains elusive. In this review, we comprehensively summarise current data on the key extrahepatic manifestations of NAFLD, emphasising the possible link between NAFLD and CVD, including the role of proprotein convertase substilisin/kenin type 9, extracellular vesicles, microbiota, and genetic factors.

Fibroblast growth factor 21 is a hepatokine involved in MASLD progression.

AIM: We aimed to assess the role of FGF21 in metabolic dysfunction-associated steatotic liver disease (MASLD) at a multi-scale level. METHODS: We used human MASLD pathology samples for FGF21 gene expression analyses (qPCR and RNAseq), serum to measure circulating FGF21 levels and DNA for genotyping the FGF21 rs838133 variant in both estimation and validation cohorts. A hepatocyte-derived cell line was exposed to free fatty acids at different timepoints. Finally, C57BL/6J mice were fed a high-fat and choline-deficient diet (CDA-HFD) for 16 weeks to assess hepatic FGF21 protein expression and FGF21 levels by ELISA. RESULTS: A significant upregulation in FGF21 mRNA expression was observed in the liver analysed by both qPCR (fold change 5.32 ± 5.25 vs. 0.59 ± 0.66; p = 0.017) and RNA-Seq (3.5 fold; FDR: 0.006; p < 0.0001) in MASLD patients vs. controls. Circulating levels of FGF21 were increased in patients with steatohepatitis vs. bland steatosis (386.6 ± 328.9 vs. 297.9 ± 231.5 pg/mL; p = 0.009). Besides, sex, age, A-allele from FGF21, GG genotype from PNPLA3, ALT, type 2 diabetes mellitus and BMI were independently associated with MASH and significant fibrosis in both estimation and validation cohorts. In vitro exposure of Huh7.5 cells to high concentrations of free fatty acids (FFAs) resulted in overexpression of FGF21 (p < 0.001). Finally, Circulating FGF21 levels and hepatic FGF21 expression were found to be significantly increased (p < 0.001) in animals under CDA-HFD. CONCLUSIONS: Hepatic and circulating FGF21 expression was increased in MASH patients, in Huh7.5 cells under FFAs and in CDA-HFD animals. The A-allele from the rs838133 variant was also associated with an increased risk of steatohepatitis and significant and advanced fibrosis in MASLD patients.

Circulating bone morphogenetic protein 8A is a novel biomarker to predict advanced liver fibrosis.

BACKGROUND & AIMS: Advanced hepatic fibrosis is the main risk factor of liver-related morbidity and mortality in patients with chronic liver disease. In this study, we assessed the potential role of bone morphogenetic protein 8A (BMP8A) as a novel target involved in liver fibrosis progression. METHODS: Histological assessment and BMP8A expression were determined in different murine models of hepatic fibrosis. Furthermore, serum BMP8A was measured in mice with bile duct ligation (BDL), in 36 subjects with histologically normal liver (NL) and in 85 patients with biopsy-proven non-alcoholic steatohepatitis (NASH): 52 with non- or mild fibrosis (F0-F2) and 33 with advanced fibrosis (F3-F4). BMP8A expression and secretion was also determined in cultured human hepatocyte-derived (Huh7) and human hepatic stellate (LX2) cells stimulated with transforming growth factor ꞵ (TGFꞵ). RESULTS: Bmp8a mRNA levels were significantly upregulated in livers from fibrotic mice compared to control animals. Notably, serum BMP8A levels were also elevated in BDL mice. In addition, in vitro experiments showed increased expression and secretion to the culture supernatant of BMP8A in both Huh7 and LX2 cells treated with TGFꞵ. Noteworthy, we found that serum BMP8A levels were significantly higher in NASH patients with advanced fibrosis than in those with non- or mild fibrosis. In fact, the AUROC of circulating BMP8A concentrations to identify patients with advanced fibrosis (F3-F4) was 0.74 (p˂0.0001). Moreover, we developed an algorithm based on serum BMP8A levels that showed an AUROC of 0.818 (p˂0.0001) to predict advanced fibrosis in NASH patients. CONCLUSION: This study provides experimental and clinical evidence indicating that BMP8A is a novel molecular target linked to liver fibrosis and introduces an efficient algorithm based on serum BMP8A levels to screen patients at risk for advanced hepatic fibrosis.

Bone morphogenetic protein 2 is a new molecular target linked to non-alcoholic fatty liver disease with potential value as non-invasive screening tool.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the commonest cause of chronic liver disease worldwide, being non-alcoholic steatohepatitis (NASH) its most clinically relevant form. Given the risks associated with taking a liver biopsy, the design of accurate non-invasive methods to identify NASH patients is of upmost importance. BMP2 plays a key role in metabolic homeostasis; however, little is known about its involvement in NAFLD onset and progression. This study aimed to elucidate the impact of BMP2 in NAFLD pathophysiology. METHODS: Hepatic and circulating levels of BMP2 were quantified in serum and liver specimens from 115 biopsy-proven NAFLD patients and 75 subjects with histologically normal liver (NL). In addition, BMP2 content and release was determined in cultured human hepatocytes upon palmitic acid (PA) overload. RESULTS: We found that BMP2 expression was abnormally increased in livers from NAFLD patients than in subjects with NL and this was reflected in higher serum BMP2 levels. Notably, we observed that PA upregulated BMP2 expression and secretion by human hepatocytes. An algorithm based on serum BMP2 levels and clinically relevant variables to NAFLD showed an AUROC of 0.886 (95%CI, 0.83-0.94) to discriminate NASH. We used this algorithm to develop SAN (Screening Algorithm for NASH): a SAN < 0.2 implied a low risk and a SAN ≥ 0.6 indicated high risk of NASH diagnosis. CONCLUSION: This proof-of-concept study shows BMP2 as a new molecular target linked to NAFLD and introduces SAN as a simple and efficient algorithm to screen individuals at risk for NASH.

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation.

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr -/-). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m2), diabetes mellitus and ALT were found to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.

Analysis of Common Pathways and Markers From Non-Alcoholic Fatty Liver Disease to Immune-Mediated Diseases.

Metabolic associated fatty liver disease (MAFLD) is the most prevalent form of liver disease worldwide, accounting for a high liver-related mortality and morbidity with extensive multi-organ involvement. This entity has displaced viral hepatitis as the main cause of severe forms of hepatic diseases, although the onset and transition of MAFLD stages still remains unclear. Nevertheless, innate and adaptive immune responses seem to play an essential role in the establishment and further progression of this disease. The immune system is responsible of safeguard and preserves organs and systems function, and might be altered under different stimuli. Thus, the liver suffers from metabolic and immune changes leading to different injuries and loss of function. It has been stablished that cell-cell crosstalk is a key process in the hepatic homeostasis maintenance. There is mounting evidence suggesting that MAFLD pathogenesis is determined by a complex interaction of environmental, genetic and host factors that leads to a full plethora of outcomes. Therefore, herein we will revisit and discuss the interplay between immune mechanisms and MAFLD, highlighting the potential role of immunological markers in an attempt to clarify its relationship.

Hepatitis C Virus Clearance by Direct-Acting Antivirals Agents Improves Endothelial Dysfunction and Subclinical Atherosclerosis: HEPCAR Study.

INTRODUCTION: Hepatitis C virus (HCV) infection has been related to increased cardiovascular (CV) risk. The aim of this study was to analyze the impact of sustained virological response (SVR) on endothelial dysfunction and subclinical atherosclerosis in patients with hepatitis C virus treated with direct-acting antiviral agents. METHODS: A total of 114 patients were prospectively recruited and underwent CV risk assessment including (i) endothelial dysfunction determined through laser Doppler flowmetry and (ii) subclinical atherosclerosis, elucidated by the ankle-brachial index (ABI). Atherogenic lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides); markers of oxidative stress (oxidized low-density lipoprotein antibodies [OLAbs]), soluble markers of adhesion (vascular cell adhesion molecule [VCAM], e-selectin, and soluble markers of angiogenesis; and vascular endothelial growth factor, endothelial [EMPs] and platelet [PMPs] apoptotic microparticles, and cell-free DNA [cfDNA]) were measured. All determinations were performed at baseline, 12 weeks (SVR time), and 1 year after treatment. RESULTS: In patients with endothelial dysfunction, area of hyperemia improved after virus clearance (P = 0.013) and was related to significant decrease in VCAM, e-selectin (P < 0.001), and cfDNA (P = 0.017) and to increased OLAb levels (P = 0.001). In patients with subclinical atherosclerosis at baseline, a significantly improved ABI was seen after HCV clearance (P < 0.001). Levels of both EMPs and PMPs also decreased after SVR and at follow-up (P = 0.006 and P = 0.002, respectively). DISCUSSION: HCV clearance improved not only liver function but also endothelial dysfunction and subclinical atherosclerosis promoted by decrease in levels of VCAM, e-selectin, cfDNA, and PMPs and EMPs.

Metabolic characterization of two different non-alcoholic fatty liver disease pre-clinical mouse models

Introduction: non-alcoholic fatty liver disease is one of the most prevalent liver disorders in the developed world. Currently, there is no approved pharmacological therapy except for lifestyle intervention. Therefore, there is a need to increase the knowledge of preclinical models in order to boost novel discoveries that could lead to a better therapeutic management. Material and methods: this study characterized the effects of two different diets, a long-term high-fat high-fructose diet (HF-HFD) and a choline-deficient, methionine supplemented high-fat diet (CDA-HFD) in C57BL/6J mice for 52 weeks or 16 weeks, respectively. Body weight, lipid and hepatic profile were analyzed and liver histology was subsequently evaluated. Results: HF-HFD animals had an increased body weight and total cholesterol levels, whereas the opposite occurred in CDA-HFD. Both HF-HFD and CDA-HFD animals had higher ALT and AST levels. With regard to histology findings, HF-HFD and CDA-HFD diets induced an increased collagen deposit and intrahepatic steatosis accumulation. Conclusion: in conclusion, the comparison of these models aided in the selection of a long-term, more physiological model for physiopathology studies or a more rapid NASH model for novel molecule testing

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