Drug repurposing in MASLD and MASH-cirrhosis: Targets and treatment approaches based on pathways analysis.

Designing and predicting novel drug targets to accelerate drug discovery for treating metabolic dysfunction-associated steatohepatitis (MASH)-cirrhosis is a challenging task. The presence of superimposed (nested) and co-occurring clinical and histological phenotypes, namely MASH and cirrhosis, may partly explain this. Thus, in this scenario, each sub-phenotype has its own set of pathophysiological mechanisms, triggers, and processes. Here, we used gene/protein and set enrichment analysis to predict druggable pathways for the treatment of MASH-cirrhosis. Our findings indicate that the pathogenesis of MASH-cirrhosis can be explained by perturbations in multiple, simultaneous, and overlapping molecular processes. In this scenario, each sub-phenotype has its own set of pathophysiological mechanisms, triggers, and processes. Therefore, we used systems biology modeling to provide evidence that MASH and cirrhosis paradoxically present unique and distinct as well as common disease mechanisms, including a network of molecular targets. More importantly, pathway analysis revealed straightforward results consistent with modulation of the immune response, cell cycle control, and epigenetic regulation. In conclusion, the selection of potential therapies for MASH-cirrhosis should be guided by a better understanding of the underlying biological processes and molecular perturbations that progressively damage liver tissue and its underlying structure. Therapeutic options for patients with MASH may not necessarily be of choice for MASH cirrhosis. Therefore, the biology of the disease and the processes associated with its natural history must be at the forefront of the decision-making process.

Valproate decreases transgenerationally blood pressure by affecting thyrotropin-releasing hormone promoter DNA methylation and gene expression in spontaneously hypertensive rat.

Central TRH, a neuropeptide, is involved in cardiovascular regulation. We demonstrated that the overexpression of diencephalic TRH (dTRH) in SHR rats can be prevented by antisense treatment, normalizing blood pressure (BP). Valproate (VPA) is an inhibitor of histone deacetylases (HDAC) which modulates gene expression through epigenetic modifications such as DNA methylation. AIMS: Study the role of HDAC inhibition in the regulation of dTRH gene expression and its effect on the pathogenesis of hypertension. MAIN METHODS: We treated 7-weeks-old male and female SHR and WKY rats with VPA for 10 weeks and evaluated BP, dTRH mRNA and methylation gene status. KEY FINDINGS: VPA attenuated the elevated BP and dTRH mRNA expression characteristic of SHR. Indeed, we found a significant 62% reduction in dTRH mRNA expression in the SHR + VPA group compared to control SHR. The decrease TRH mRNA expression induced by VPA was confirmed "in vitro" in a primary neuron culture using trichostatin A. With methylation specific PCR we demonstrated a significant increase in TRH promoter DNA methylation level in SHR + VPA group compared to control SHR. After 2 weeks of treatment interruption, rats were mated. Although they did not receive any treatment, the offspring born from VPA-treated SHR parents showed similar changes in BP, dTRH expression and methylation status, implying a transgenerational inheritance. Our findings suggest that dTRH modulation by epigenetics mechanism affects BP and could be inherited by the next generation in SHR rats.

SARS-CoV-2 targets the liver and manipulates glucose metabolism.

A recent publication by Barreto and colleagues showed that SARS-CoV-2 directly triggers hyperglycemia by infecting hepatocytes and inducing phosphoenolpyruvate carboxykinase (PEPCK)-dependent gluconeogenesis. Here, we discuss the biological importance of these findings, including the hepatic tropism of SARS-CoV-2. We also comment on the clinical implications of the bidirectional connection between COVID-19 and noncommunicable diseases.

Non-alcoholic fatty liver disease mediates the effect of obesity on arterial hypertension.

BACKGROUND: It has been consistently shown that obesity contributes directly to arterial hypertension and cardiovascular disease (CVD), independently of other risk factors. Likewise, non-alcoholic fatty liver disease (NAFLD) is acknowledged as a contributor and a risk enhancer for CVD. OBJECTIVES: We tested the hypothesis of a causal role of NAFLD in the effect of obesity on arterial hypertension. METHODS: Using causal mediation analysis, we quantified the magnitude of the body mass index (BMI) effect on arterial hypertension and CV-traits mediated by NAFLD. First, we analysed data from 1348 young adults in the Bogalusa Heart Study (BHS), a cohort aimed at assessing the natural history of CVD. Then, we used data from 3359 participants of the National Health and Nutrition Examination Survey (2017-2018 cycle, NHANES) to replicate the findings. RESULTS: We found that roughly 92% of the effects of BMI on arterial hypertension in the BHS and 51% in the NHANES population are mediated by NAFLD. In addition, indirect effects of BMI on systolic (SBP) and diastolic (DBP) blood pressure, and heart rate (HR) through NAFLD explained up to 91%, 93%, and 100% of the total effect, respectively, in the BHS. In the NHANES survey, indirect effects of BMI through NAFLD on CV traits explain a significant proportion of the total effects (SBP = 60.4%, HR = 100%, and pulse pressure = 88%). CONCLUSION: NAFLD mediates a substantial proportion of the effect of obesity on the presence of hypertension and CV-parameters independently of relevant covariates. This conclusion has implications for clinical management.

Advances in our understanding of the molecular heterogeneity of fatty liver disease: toward informed treatment decision making.

INTRODUCTION: nonalcoholic fatty liver disease (NAFLD) is a complex disorder resulting from intricate relationships with diverse cardiometabolic risk factors and environmental factors. NAFLD may result in severe chronic liver damage and potentially declining liver function. AREAS COVERED: Accumulated knowledge over the last decade indicates that the disease trajectory presents substantial heterogeneity. In addition, overlapping features with the diseases of the metabolic syndrome, combined with heterogeneity in disease mechanisms, further complicates NAFLD diagnosis and prognosis, and hampers progress in biomarker and pharmacological discoveries. Here, we explore solving the heterogeneous clinical landscape of NAFLD by cluster analysis of molecular signatures that serve as a proxy for disease stratification into molecular sub-types. First, we collected information on NAFLD and metabolic syndrome-associated protein-coding genes by data mining the literature. Next, we performed pathways enrichment and cluster analyses to decipher and dissect the different patterns of phenotypic heterogeneity. Our approach showed unique biological pathways for every clinical subtype/group, namely NAFLD + obesity, NAFLD + arterial hypertension, NAFLD + dyslipidemia, and NAFLD + type 2 diabetes. EXPERT OPINION: Patients with NAFLD may be benefited by a better understanding of the disease biology, which involves 'dissection' of the molecular sub-phenotypes that drive the disease progression.

Hypothyroidism-associated immunosuppression involves induction of galectin-1-producing regulatory T cells.

Hypothyroidism exerts deleterious effects on immunity, but the precise role of the hypothalamic-pituitary-thyroid (HPT) axis in immunoregulatory and tolerogenic programs is barely understood. Here, we investigated the mechanisms underlying hypothyroid-related immunosuppression by examining the regulatory role of components of the HPT axis. We first analyzed lymphocyte activity in mice overexpressing the TRH gene (Tg-Trh). T cells from Tg-Trh showed increased proliferation than wild-type (WT) euthyroid mice in response to polyclonal activation. The release of Th1 pro-inflammatory cytokines was also increased in Tg-Trh and TSH levels correlated with T-cell proliferation. To gain further mechanistic insights into hypothyroidism-related immunosuppression, we evaluated T-cell subpopulations in lymphoid tissues of hypothyroid and control mice. No differences were observed in CD3/CD19 or CD4/CD8 ratios between these strains. However, the frequency of regulatory T cells (Tregs) was significantly increased in hypothyroid mice, and not in Tg-Trh mice. Accordingly, in vitro Tregs differentiation was more pronounced in naïve T cells isolated from hypothyroid mice. Since Tregs overexpress galectin-1 (Gal-1) and mice lacking this lectin (Lgals1-/- ) show reduced Treg function, we investigated the involvement of this immunoregulatory lectin in the control of Tregs in settings of hypothyroidism. Increased T lymphocyte reactivity and reduced frequency of Tregs were found in hypothyroid Lgals1-/- mice when compared to hypothyroid WT animals. This effect was rescued by the addition of recombinant Gal-1. Finally, increased expression of Gal-1 was found in Tregs purified from hypothyroid WT mice compared with their euthyroid counterpart. Thus, a substantial increase in the frequency and activity of Gal-1-expressing Tregs underlies immunosuppression associated with hypothyroid conditions, with critical implications in immunopathology, metabolic disorders, and cancer.

COVID-19 and non-alcoholic fatty liver disease: Biological insights from multi-omics data.

We explored the shared pathophysiological mechanisms between COVID-19 and non-alcoholic fatty liver disease (NAFLD) by integrating multi-omics data. We studied common genetic risk factors and underlying biological processes using functional enrichment analysis. To understand the sex-specific pathways involved in the clinical course of SARS-CoV-2 infection, we processed sex-stratified data from COVID-19 genome-wide association datasets. We further explored the transcriptional signature of the liver cells in healthy and COVID-19 tissue specimens. We also integrated genetic and metabolomic information. We found that COVID-19 and NAFLD share biological disease mechanisms, including pathways that regulate the inflammatory and lipopolysaccharide response. Single-cell transcriptomics revealed enrichment of complement-related pathways in Kupffer cells, syndecan-mediated signalling in plasma cells, and epithelial-to-mesenchymal transition in hepatic stellate cells. The strategy of pathway-level analysis of genomic and metabolomic data uncovered l-lactic acid, Krebs cycle intermediate compounds, arachidonic acid and cortisol among the most prominent shared metabolites.

The serum uric acid/creatinine ratio is associated with nonalcoholic fatty liver disease in the general population.

Serum uric acid-to-creatinine ratio (sUA/CrR) may be associated with metabolic syndrome components, but limited evidence exists on a relationship between sUA/Cr and NAFLD. Here, we investigated the association between sUA/CrR and NAFLD.We performed a cross-sectional analysis in 3359 subjects who participated in the NHANES 2017-2018 survey and consumed less than 30 and 20 g alcohol (men and women, respectively), with no positive tests of viral hepatitis. Liver steatosis was defined by controlled attenuation parameter and fibrosis by stiffness measurements obtained via transient elastography. We modeled the relationship between NAFLD and relevant demographic, anthropometric, and biochemical variables.sUA/CrR was significantly higher in participants with NAFLD than those without NAFLD. LASSO logit regression showed that only logarithmized age (p = 1.2e-3), waist circumference (WC) (p = 1.8e-5), triglycerides (p = 5e-6), and sUA/CrR (p = 3e-5) were retained in the model. Multivariate logistic analysis demonstrated a significant association between sUA/CrR and NAFLD; the OR for NAFLD of one log(sUA/CrR) increase was 2.61 (95% CI: 1.86-3.68, p < 3e-8) after adjusting for relevant covariables, including aminotransaminase levels and the effect of sUA/CrR remained significant for highest WC quintiles. The model's predictive power with vs. without sUA/CrR was slightly but significantly better (Auroc: 0.859 ± 0.006 vs. 0.855 ± 0.007, p < 1.1e-2). Mediation analysis showed that SUA/CrR modestly mediates the effect of WC and insulin resistance but not glycohemoglobin on NAFLD.In conclusion, elevated sUA/CrR was significantly associated with NAFLD in the general population. Therefore, kidney function should be closely monitored in NAFLD patients.

Genetics in non-alcoholic fatty liver disease: The role of risk alleles through the lens of immune response.

The knowledge on the genetic component of non-alcoholic fatty liver disease (NAFLD) has grown exponentially over the last 10 to 15 years. This review summarizes the current evidence and the latest developments in the genetics of NAFLD and non-alcoholic steatohepatitis (NASH) from the immune system's perspective. Activation of innate and or adaptive immune response is an essential driver of NAFLD disease severity and progression. Lipid and immune pathways are crucial in the pathophysiology of NAFLD and NASH. Here, we highlight novel applications of genomic techniques, including single-cell sequencing and the genetics of gene expression, to elucidate the potential involvement of NAFLD/NASH-risk alleles in modulating immune system cells. Together, our focus is to provide an overview of the potential involvement of the NAFLD/NASH-related risk variants in mediating the immune-driven liver disease severity and diverse systemic pleiotropic effects.

Alcohol consumption and metabolic syndrome: Clinical and epidemiological impact on liver disease.

Alcohol use and metabolic syndrome are highly prevalent in the population and frequently co-exist. Both are implicated in a large range of health problems, including chronic liver disease, hepatocellular carcinoma, and liver-related outcomes (i.e. decompensation or liver transplantation). Studies have yielded mixed results regarding the effects of mild-moderate alcohol consumption on the risk of metabolic syndrome and fatty liver disease, possibly due to methodological differences. The few available prospective studies have indicated that mild-moderate alcohol use is associated with an increase in liver-related outcomes. This conclusion was substantiated by systems biology analyses suggesting that alcohol and metabolic syndrome may play a similar role in fatty liver disease, potentiating an already existing dysregulation of common vital homeostatic pathways. Alcohol and metabolic factors are independently and jointly associated with liver-related outcomes. Indeed, metabolic syndrome increases the risk of liver-related outcomes, regardless of alcohol intake. Moreover, the components of metabolic syndrome appear to have additive effects when it comes to the risk of liver-related outcomes. A number of population studies have implied that measures of central/abdominal obesity, such as the waist-to-hip ratio, can predict liver-related outcomes more accurately than BMI, including in individuals who consume harmful quantities of alcohol. Many studies even point to synergistic interactions between harmful alcohol use and many metabolic components. This accumulating evidence showing independent, combined, and modifying effects of alcohol and metabolic factors on the onset and progression of chronic liver disease highlights the multifactorial background of liver disease in the population. The available evidence suggests that more holistic approaches could be useful for risk prediction, diagnostics and treatment planning.

Single cell gene expression profiling of nasal ciliated cells reveals distinctive biological processes related to epigenetic mechanisms in patients with severe COVID-19.

OBJECTIVE: To explore the molecular processes associated with cellular regulatory programs in patients with COVID-19, including gene activation or repression mediated by epigenetic mechanisms. We hypothesized that a comprehensive gene expression profiling of nasopharyngeal epithelial cells might expand our understanding of the pathogenic mechanisms of severe COVID-19. METHODS: We used single-cell RNA sequencing (scRNAseq) profiling of ciliated cells (n = 12,725) from healthy controls (SARS-CoV-2 negative n = 13) and patients with mild/moderate (n = 13) and severe (n = 14) COVID-19. ScRNAseq data at the patient level were used to perform gene set and pathway enrichment analyses. We prioritized candidate miRNA-target interactions and epigenetic mechanisms. RESULTS: We found that mild/moderate COVID-19 compared to healthy controls had upregulation of gene expression signatures associated with mitochondrial function, misfolded proteins, and membrane permeability. In addition, we found that compared to mild/moderate disease, severe COVID-19 had downregulation of epigenetic mechanisms, including DNA and histone H3K4 methylation and chromatin remodelling regulation. Furthermore, we found 11-ranked miRNAs that may explain miRNA-dependent regulation of histone methylation, some of which share seed sequences with SARS-CoV-2 miRNAs. CONCLUSION: Our results may provide novel insights into the epigenetic mechanisms mediating the clinical course of SARS-CoV-2 infection.

MicroRNAs as messengers of liver diseases: has the message finally been decrypted?

MicroRNAs (miRNAs), which are regarded as crucial regulators of gene expression and diverse aspects of cell biology, can be present in various body fluids as highly stable molecules. It is also known that miRNAs exert tissue-specific regulation of gene transcription. Large amount of clinical and experimental evidence provided the rationale for raising the intriguing question of whether miRNAs can mediate cell-cell communication. For those reasons, miRNAs have been considered as the 'Holy Grail' of biomarkers allowing non-invasive diagnostic screening and early detection of a variety of diseases, including solid and non-solid cancers. In a study published in Clin. Sci. (Lond.) (2011) 120(5):183-193 (https://doi.org/10.1042/CS20100297), Gui et al. investigated the hypothesis that circulating miRNAs could be used to identify patients with liver pathologies. Specifically, the authors profiled circulating miRNAs in patients with hepatocellular carcinoma (HCC), liver cirrhosis (LC), and healthy controls and found that serum miR-885-5p levels were significantly higher in samples of patients with HCC (6.5-fold increase) and LC (8.8-fold increase). In this commentary, we highlight biological aspects associated with mir-122-the 'liver-specific' miRNA, which has been associated with a diverse range of liver pathologies. In addition, we discuss the relevance of mir-885-5p as potential biomarker for detecting human cancers. Finally, we provide some clues about how presumably unrelated miRNAs such as miR-122 and miR-885-5p may act in similar biological processes (BPs), making the miRNA regulatory networks more complex than anticipated.

PNPLA3 rs738409 and risk of fibrosis in NAFLD: Exploring mediation pathways through intermediate histological features.

BACKGROUND AND AIMS: It is unclear whether rs738409 (p.I148M) missense variant in patatin-like phospholipase domain-containing 3 rs738409 promotes fibrosis development by triggering specific fibrogenic pathways or by creating an unfavorable microenvironment by promoting steatosis, inflammation, and ultimately fibrosis. We tested the hypothesis that intermediate histologic traits, including steatosis, lobular and portal inflammation, and ballooning may determine the effect of rs738409 on liver fibrosis among individuals with biopsy-proven NAFLD. APPROACH AND RESULTS: Causal mediation models including multiple mediators in parallel or sequentially were performed to examine the effect of rs738409, by decomposing its total effect on fibrosis severity into direct and indirect effects, mediated by histology traits in 1153 non-Hispanic White patients. Total effect of rs738409 on fibrosis was ß = 0.19 (95% CI: 0.09-0.29). The direct effect of rs738409 on fibrosis after removing mediators' effects was ß = 0.09 (95% CI: 0.01-0.17) and the indirect effect of rs738409 on fibrosis through all mediators' effects were ß = 0.010 (95% CI: 0.04-0.15). Among all mediators, the greatest estimated effect size was displayed by portal inflammation (ß = 0.09, 95% CI: 0.05-0.12). Among different sequential combinations of histology traits, the path including lobular inflammation followed by ballooning degeneration displayed the most significant indirect effect (ß = 0.023, 95% CI: 0.011-0.037). Mediation analysis in a separate group of 404 individuals with biopsy-proven NAFLD from other races and ethnicity showed similar results. CONCLUSIONS: In NAFLD, nearly half of the total effect of the rs738409 G allele on fibrosis severity could be explained by a direct pathway, suggesting that rs738409 may promote fibrosis development by activating specific fibrogenic pathways. A large proportion of the indirect effect of rs738409 on fibrosis severity is mediated through portal inflammation.

Computational Pipeline for Next-Generation Sequencing (NGS) Studies in Genetics of NASH.

High-throughput sequencing (HTS) technologies have contributed to expand current knowledge of the biology of complex diseases, including nonalcoholic fatty liver disease (NAFLD). Genome-wide association studies, whole exome sequencing, and sequencing of entire genes are used to identify variants and/or mutations that predispose to the disease pathogenesis. Here, we present a tutorial that may guide readers to manage high volume of genetics data in the context of Next-Generation Sequencing (NGS) studies.

Metabolic dysfunction-associated fatty liver disease: advances in genetic and epigenetic implications.

PURPOSE OF REVIEW: Fatty liver associated with metabolic dysfunction, also known under the acronym NAFLD (nonalcoholic fatty liver disease) is the leading global cause of chronic liver disease. In this review, we address the state of research on genetics and epigenetics of NAFLD with focus on key discoveries and conceptual advances over the past 2 years. RECENT FINDINGS: The analysis of NAFLD-associated genetic variant effects on the whole-transcriptome, including quantitative trait loci (QTL) associated with gene expression (eQTL) or splicing (sQTL) may explain pleiotropic effects. Functional experiments on NAFLD-epigenetics, including profiling of liver chromatin accessibility quantitative trait loci (caQTL) show co-localization with numerous genome-wide association study signals linked to metabolic and cardiovascular traits. Novel studies provide insights into the modulation of the hepatic transcriptome and epigenome by tissue microbiotas. Genetic variation of components of the liver cellular respirasome may result in broad cellular and metabolic effects. Mitochondrial noncoding RNAs may regulate liver inflammation and fibrogenesis. RNA modifications as N6-methyladenosine may explain sex-specific differences in liver gene transcription linked to lipid traits. SUMMARY: The latest developments in the field of NAFLD-genomics can be leveraged for identifying novel disease mechanisms and therapeutic targets that may prevent the morbidity and mortality associated with disease progression. VIDEO ABSTRACT: http://links.lww.com/COL/A23.

The Protection Conferred by HSD17B13 rs72613567 Polymorphism on Risk of Steatohepatitis and Fibrosis May Be Limited to Selected Subgroups of Patients With NAFLD.

INTRODUCTION: Our study aimed to explore how PNPLA3 rs738409 or phenotypic risk factors may moderate the relationship between HSD17B13 rs72613567 and risk of steatohepatitis and fibrosis. METHODS: This analysis consisted of 1,153 non-Hispanic whites with biopsy-proven nonalcoholic fatty liver disease enrolled in the nonalcoholic steatohepatitis Clinical Research Network studies. Nonalcoholic fatty liver disease severity was determined by liver histology scored centrally according to the nonalcoholic steatohepatitis Clinical Research Network criteria. Moderation and logistic regression analyses were performed to identify the influence of moderators (PNPLA3 rs738409, age, sex, body mass index, and diabetes) on the relationship between HSD17B13 rs72613567 and risk of steatohepatitis and fibrosis. RESULTS: HSD17B13 rs72613567 genotype frequency was as follows: (-/-), 64%; (-/A), 30%; (A/A), 6%. Moderation analysis showed that the protective effect of HSD17B13 rs72613567 A-allele on risk of steatohepatitis remained only significant among patients with PNPLA3 rs738409 genotype CC (ß coeff: -0.19, P = 0.019), women (ß coeff: -0.18, P < 0.001), patients of age ≥ 45 years (ß coeff: -0.18, P < 0.001), patients with body mass index ≥ 35 kg/m2 (ß coeff: -0.17, P < 0.001), and patients with diabetes (ß coeff: -0.18, P = 0.020). Among women, the protective effect of HSD17B131 rs72613567 A-allele on risk of steatohepatitis was stronger in those aged ≥ 51 years. Logistic regression-based sensitivity analysis including various important subgroups confirmed our observations. DISCUSSION: The protection conferred by HSD17B13 rs72613567 A-allele on risk of steatohepatitis and fibrosis may be limited to selected subgroups of individuals who are aged ≥ 45 years, women and have class ≥ 2 obesity or diabetes, and those with PNPLA3 rs738409 CC genotype.