Liver endothelial cells in NAFLD and transition to NASH and HCC.

Non-alcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome, which is characterised by obesity, insulin resistance, hypercholesterolemia and hypertension. NAFLD is the most frequent liver disease worldwide and more than 10% of NAFLD patients progress to the inflammatory and fibrotic stage of non-alcoholic steatohepatitis (NASH), which can lead to end-stage liver disease including hepatocellular carcinoma (HCC), the most frequent primary malignant liver tumor. Liver sinusoidal endothelial cells (LSEC) are strategically positioned at the interface between blood and hepatic parenchyma. LSECs are highly specialized cells, characterised by the presence of transcellular pores, called fenestrae, and exhibit anti-inflammatory and anti-fibrotic characteristics under physiological conditions. However, during NAFLD development they undergo capillarisation and acquire a phenotype similar to vascular endothelial cells, actively promoting all pathophysiological aspects of NAFLD, including steatosis, inflammation, and fibrosis. LSEC dysfunction is critical for the progression to NASH and HCC while restoring LSEC homeostasis appears to be a promising approach to prevent NAFLD progression and its complications and even reverse tissue damage. In this review we present current information on the role of LSEC throughout the progressive phases of NAFLD, summarising in vitro and in vivo experimental evidence and data from human studies.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating the development of novel therapies against disease progression.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Analysis of the Role of Stellate Cell VCAM-1 in NASH Models in Mice.

Non-alcoholic fatty liver disease (NAFLD) can progress to non-alcoholic steatohepatitis (NASH), characterized by inflammation and fibrosis. Fibrosis is mediated by hepatic stellate cells (HSC) and their differentiation into activated myofibroblasts; the latter process is also promoted by inflammation. Here we studied the role of the pro-inflammatory adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) in HSCs in NASH. VCAM-1 expression was upregulated in the liver upon NASH induction, and VCAM-1 was found to be present on activated HSCs. We therefore utilized HSC-specific VCAM-1-deficient and appropriate control mice to explore the role of VCAM-1 on HSCs in NASH. However, HSC-specific VCAM-1-deficient mice, as compared to control mice, did not show a difference with regards to steatosis, inflammation and fibrosis in two different models of NASH. Hence, VCAM-1 on HSCs is dispensable for NASH development and progression in mice.

Integrated omics analysis for characterization of the contribution of high fructose corn syrup to non-alcoholic fatty liver disease in obesity.

BACKGROUND: High-Fructose Corn Syrup (HFCS), a sweetener rich in glucose and fructose, is nowadays widely used in beverages and processed foods; its consumption has been correlated to the emergence and progression of Non-Alcoholic Fatty Liver Disease (NAFLD). Nevertheless, the molecular mechanisms by which HFCS impacts hepatic metabolism remain scarce, especially in the context of obesity. Besides, the majority of current studies focuses either on the detrimental role of fructose in hepatic steatosis or compare separately the additive impact of fructose versus glucose in high fat diet-induced NAFLD. AIM: By engaging combined omics approaches, we sought to characterize the role of HFCS in obesity-associated NAFLD and reveal molecular processes, which mediate the exaggeration of steatosis under these conditions. METHODS: Herein, C57BL/6 mice were fed a normal-fat-diet (ND), a high-fat-diet (HFD) or a HFD supplemented with HFCS (HFD-HFCS) and upon examination of their metabolic and NAFLD phenotype, proteomic, lipidomic and metabolomic analyses were conducted to identify HFCS-related molecular alterations of the hepatic metabolic landscape in obesity. RESULTS: Although HFD and HFD-HFCS mice displayed comparable obesity, HFD-HFCS mice showed aggravation of hepatic steatosis, as analysis of the lipid droplet area in liver sections revealed (12,15 % of total section area in HFD vs 22,35 % in HFD-HFCS), increased NAFLD activity score (3,29 in HFD vs 4,86 in HFD-HFCS) and deteriorated hepatic insulin resistance, as compared to the HFD mice. Besides, the hepatic proteome of HFD-HFCS mice was characterized by a marked upregulation of 5 core proteins implicated in de novo lipogenesis (DNL), while an increased phosphatidyl-cholines(PC)/phosphatidyl-ethanolamines(PE) ratio (2.01 in HFD vs 3.04 in HFD-HFCS) was observed in the livers of HFD-HFCS versus HFD mice. Integrated analysis of the omics datasets indicated that Tricarboxylic Acid (TCA) cycle overactivation is likely contributing towards the intensification of steatosis during HFD-HFCS-induced NAFLD. CONCLUSION: Our results imply that HFCS significantly contributes to steatosis aggravation during obesity-related NAFLD, likely deriving from DNL upregulation, accompanied by TCA cycle overactivation and deteriorated hepatic insulin resistance.

Significance of Circulating Cell-Free DNA Biomarkers in HBeAg-Negative Chronic Hepatitis B Virus Infection and Their Changes after Treatment Initiation.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a common chronic liver disease that is closely associated with increased morbidity and mortality. Circulating cell-free DNA (cf-DNA) and global DNA methylation, expressed as circulating levels of 5-methyl-2'-deoxycytidine, are increasingly used to monitor chronic inflammatory diseases of several etiologies. This study attempts to investigate the serum levels of circulating cf-DNA and 5-methyl-2'-deoxycytidine in HBeAg-negative patients with chronic infection (carriers) and chronic hepatitis B (CHB), as well as their changes after treatment initiation in CHB. METHODS: Serum samples from a total of 61 HBeAg-negative patients (30 carriers and 31 CHB patients) were included in order to quantify the levels of circulating cf-DNA and 5-methyl-2'-deoxycytidine. In addition, serum samples from 17 CHB patients in complete virological and biochemical remission after initiation of treatment with a nucleos(t)ide analogue were included. RESULTS: Circulating cf-DNA concentration was significantly increased after the initiation of treatment (15 vs. 10 ng/mL, p = 0.022). There was a trend in higher mean levels of circulating 5-methyl-2'-deoxycytidine in carriers compared to CHB patients (211.02 vs. 175.66 ng/mL, p = 0.089), as well as a trend in increasing 5-methyl-2'-deoxycytidine levels after treatment initiation in CHB patients compared to pre-treatment levels (215 vs. 173 ng/mL, p = 0.079). CONCLUSIONS: Both circulating levels of cf-DNA and 5-methyl-2'-deoxycytidine might be useful biomarkers in order to monitor liver disease activity and response to antiviral treatment in HBeAg-negative chronic HBV patients, but further studies are essential in order to validate these intriguing findings.

Hepatocyte Mitochondrial Dynamics and Bioenergetics in Obesity-Related Non-Alcoholic Fatty Liver Disease.

PURPOSE OF THE REVIEW: Mitochondrial dysfunction has long been proposed to play a crucial role in the pathogenesis of a considerable number of disorders, such as neurodegeneration, cancer, cardiovascular, and metabolic disorders, including obesity-related insulin resistance and non-alcoholic fatty liver disease (NAFLD). Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify their formation through biogenesis and the opposite processes of fission and fusion, the fragmentation, and connection of mitochondrial network areas respectively. Herein, we review and discuss the current literature on the significance of mitochondrial adaptations in obesity and metabolic dysregulation, emphasizing on the role of hepatocyte mitochondrial flexibility in obesity and NAFLD. RECENT FINDINGS: Accumulating evidence suggests the involvement of mitochondrial morphology and bioenergetics dysregulations to the emergence of NAFLD and its progress to non-alcoholic steatohepatitis (NASH). Most relevant data suggests that changes in liver mitochondrial dynamics and bioenergetics hold a key role in the pathogenesis of NAFLD. During obesity and NAFLD, oxidative stress occurs due to the excessive production of ROS, leading to mitochondrial dysfunction. As a result, mitochondria become incompetent and uncoupled from respiratory chain activities, further promoting hepatic fat accumulation, while leading to liver inflammation, insulin resistance, and disease's deterioration. Elucidation of the mechanisms leading to dysfunctional mitochondrial activity of the hepatocytes during NAFLD is of predominant importance for the development of novel therapeutic approaches towards the treatment of this metabolic disorder.

PCSK9/LDLR System and Rheumatoid Arthritis-Related Atherosclerosis.

Background/Aims: Rheumatoid arthritis (RA) is associated with the emergence of cardiovascular disease, while chronic inflammation is considered a common denominator for their parallel progression. The Proprotein convertase subtilisin/kexin type 9 (PCSK9)/LDL-Receptor (LDLR) system is of high importance during atherogenesis, via regulating the clearance of LDL from the circulation; nevertheless the role of this molecular mechanism during RA-related atheromatosis is not known. Methods: Herein, high-resolution ultrasound measurements for arterial hypertrophy, atheromatosis and arterial stiffness as well as comprehensive biochemical profiling were performed in 85 RA patients. The circulating levels of PCSK9 and LDLR were measured and their potential associations as well as of the PCSK9/LDLR ratio with patients' characteristics and the degree of atherosclerosis were investigated. Results: Increased LDLR levels and decreased PCSK9/LDLR ratio were found in RA patients with at least 2 atheromatic plaques as compared to the ones without any plaques. In addition the levels of both PCSK9 and LDLR were positively correlated with the presence of atheromatic plaques as an age- and gender- adjusted multivariate analysis revealed. Conclusions: Our data imply that the PCSK9/LDLR system plays a significant role during RA-related atherosclerosis and may therefore be used as a screening tool for disease progression in the future.

Hepatic Senescence Accompanies the Development of NAFLD in Non-Aged Mice Independently of Obesity.

Senescence is considered to be a cardinal player in several chronic inflammatory and metabolic pathologies. The two dominant mechanisms of senescence include replicative senescence, predominantly depending on age-induced telomere shortening, and stress-induced senescence, triggered by external or intracellular harmful stimuli. Recent data indicate that hepatocyte senescence is involved in the development of nonalcoholic fatty liver disease (NAFLD). However, previous studies have mainly focused on age-related senescence during NAFLD, in the presence or absence of obesity, while information about whether the phenomenon is characterized by replicative or stress-induced senescence, especially in non-aged organisms, is scarce. Herein, we subjected young mice to two different diet-induced NAFLD models which differed in the presence of obesity. In both models, liver fat accumulation and increased hepatic mRNA expression of steatosis-related genes were accompanied by hepatic senescence, indicated by the increased expression of senescence-associated genes and the presence of a robust hybrid histo-/immunochemical senescence-specific staining in the liver. Surprisingly, telomere length and global DNA methylation did not differ between the steatotic and the control livers, while malondialdehyde, a marker of oxidative stress, was upregulated in the mouse NAFLD livers. These findings suggest that senescence accompanies NAFLD emergence, even in non-aged organisms, and highlight the role of stress-induced senescence during steatosis development independently of obesity.

Novel genetic risk variants for pediatric celiac disease.

BACKGROUND: Celiac disease is a complex chronic immune-mediated disorder of the small intestine. Today, the pathobiology of the disease is unclear, perplexing differential diagnosis, patient stratification, and decision-making in the clinic. METHODS: Herein, we adopted a next-generation sequencing approach in a celiac disease trio of Greek descent to identify all genomic variants with the potential of celiac disease predisposition. RESULTS: Analysis revealed six genomic variants of prime interest: SLC9A4 c.1919G>A, KIAA1109 c.2933T>C and c.4268_4269delCCinsTA, HoxB6 c.668C>A, HoxD12 c.418G>A, and NCK2 c.745_746delAAinsG, from which NCK2 c.745_746delAAinsG is novel. Data validation in pediatric celiac disease patients of Greek (n = 109) and Serbian (n = 73) descent and their healthy counterparts (n = 111 and n = 32, respectively) indicated that HoxD12 c.418G>A is more prevalent in celiac disease patients in the Serbian population (P < 0.01), while NCK2 c.745_746delAAinsG is less prevalent in celiac disease patients rather than healthy individuals of Greek descent (P = 0.03). SLC9A4 c.1919G>A and KIAA1109 c.2933T>C and c.4268_4269delCCinsTA were more abundant in patients; nevertheless, they failed to show statistical significance. CONCLUSIONS: The next-generation sequencing-based family genomics approach described herein may serve as a paradigm towards the identification of novel functional variants with the aim of understanding complex disease pathobiology.