The Mechanisms and Physiological Consequences of Diurnal Hepatic Cell Size Fluctuations: A Brief Review.

Liver size in mammals fluctuates throughout the day and correlates with changes in hepatocyte size. However, the role of these daily changes in liver and hepatocyte size and the underlying molecular mechanisms remain largely unknown. In this review, we highlight the view that hepatocyte size, and thus, overall organ size, is subject to regulation by the circadian clock and feeding/fasting cycles. To that end, we provide an overview of the current literature dealing with this phenomenon and elaborate the role of feeding and nutrients in this process. We will discuss the role of hepatic protein content and synthesis, which are both subject to diurnal regulation, in daily hepatocyte and liver size fluctuations. Although there is evidence that changes in hepatocyte and liver size are associated with daily variations in macromolecule content, there is also evidence that these changes in size may be actively regulated by modifications of the cells' osmotic environment. Future research will need to examine the intriguing possibility that hepatocyte and liver size fluctuations may be required for normal liver function and to reveal the underlying molecular mechanisms behind this process.

Bcl-3 promotes TNF-induced hepatocyte apoptosis by regulating the deubiquitination of RIP1

Tumor necrosis factor-α (TNF) is described as a main regulator of cell survival and apoptosis in multiple types of cells, including hepatocytes. Dysregulation in TNF-induced apoptosis is associated with many autoimmune diseases and various liver diseases. Here, we demonstrated a crucial role of Bcl-3, an IκB family member, in regulating TNF-induced hepatic cell death. Specifically, we found that the presence of Bcl-3 promoted TNF-induced cell death in the liver, while Bcl-3 deficiency protected mice against TNF/D-GalN induced hepatoxicity and lethality. Consistently, Bcl-3-depleted hepatic cells exhibited decreased sensitivity to TNF-induced apoptosis when stimulated with TNF/CHX. Mechanistically, the in vitro results showed that Bcl-3 interacted with the deubiquitinase CYLD to synergistically switch the ubiquitination status of RIP1 and facilitate the formation of death-inducing Complex II. This complex further resulted in activation of the caspase cascade to induce apoptosis. By revealing this novel role of Bcl-3 in regulating TNF-induced hepatic cell death, this study provides a potential therapeutic target for liver diseases caused by TNF-related apoptosis.

463 New era in hypercholesterolemia treatment, inclisiran: early and sustained LDL-C reduction with a twice per year administration

Inclisiran is a synthetic small-interfering RNA (siRNA) that works with the RNA interference (RNAi) mechanism. SiRNA binds its target mRNA, leading to silencing the protein synthesis by the related mRNA degradation. Inclisiran is designed to bind solely PCSK9 mRNA, decreasing PCSK9 expression, thus leading to lower LDL-C level. Several chemical modifications were added to obtain a stable compound delivering a rapid effect and generally well tolerated [Khvorova A. Oligonucleotide therapeutics—a new class of cholesterol-lowering drugs. N Engl J Med 2017; 376 4–7]. High cholesterol levels and prolonged time of exposure enhance risk of new or recurrent CV events, therefore also timing became crucial for atherosclerotic cardiovascular disease (ASCVD) patients [Ference BA, Graham I, Tokgozoglu L, et al. Impact of lipids on cardiovascular health: JACC health promotion series. J Am Coll Cardiol 2018; 72 1141–1156]. Therefore, an early and effective LDL-C lowering effect is positively correlated with CV risk reduction, together with the life-long LDL-C reduction that will impact definitively on the global CV risk [Cohen JC, Boerwinkle E, Mosley TH Jr, et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006; 354 1264–1272]. The siRNA conjugation with a triantennary GalNAC leads to a specific targeted hepatic delivery therefore, the 284 mg inclisiran dose is undetectable in blood stream after 24–48 h from the subcutaneous injection [Wright RS, Collins MG, StoekenbroekRM, et al. Effects of renal impairment on the pharmacokinetics, efficacy, and safety of inclisiran: an analysis of the ORION-7 and ORION-1 studies. Mayo Clin Proc 2020; 95 77–89]. The LDL-C lowering effect starts early upon the hepatic cell entry (24–48 h) and the LDL-C level drop is already significant at 14 days post injection, and by Day 30 the mean reduction is about 50%, as shown in the ORION-1 phase II trial [Ray KK, Landmesser U, Leiter LA, et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N Engl J Med 2017; 376 1430–1440]. Other chemical modifications at the siRNA back-bone level, protect inclisiran from degradation by liver nucleases, which may occur upon the hepatic cell uptake. In the cytoplasm, RNAi mechanism occurs by the siRNA—RISC protein complex coupling. Physiologically, this bond last for long and the inclisiran back-bone modifications further enhance the complex stability [Khvorova A. Oligonucleotide therapeutics—a new class of cholesterol-lowering drugs. N Engl J Med 2017; 376 4–7]. Moreover, one siRNA-RISC complex has an effect on multiple PCSK9 mRNA units, allowing inclisiran administration twice per year (after initial dose at baseline and 3 months), granting an early, sustained and effective LDL-C level reduction that lasts for 6 months. A pooled analysis of the 3 phase III trials (ORION-9/10/11) shows a time averaged (18 months) LDL-C reduction of 50.5% on top of therapy with statins±ezetimibe [Wright RS, Ray KK, Raal FJ, et al. Pooled patient-level analysis of inclisiran trials in patients with familial hypercholesterolemia or atherosclerosis. J Am Coll Cardiol 2021; 77 1182–1193]. Inclisiran provides effective evidence-based results on lowering LDL-C levels in different high CV risk populations (HeFH/established ASCVD/ASCVD-risk equivalent), which is demonstrated to be crucial for the reduction of patients’ CV risk. Furthermore, the twice per year administrations may positively improve adherence, thereby simplifying patient management and control during follow-up. Based on these findings, we are stepping into a new era of biologic therapeutics, where inclisiran represents the new, effective and safe therapeutic candidate for lowering LDL-C levels.

Anticholesterolemic Activity of Three Vegetal Extracts (Artichoke, Caigua, and Fenugreek) and Their Unique Blend

Hepatic-related diseases, in particular hyperlipidemia and hypercholesterolemia, are a thorn on the side of the national health institutes around the globe. Indeed, liver lipid and cholesterol dysregulation could lead to atherosclerotic plaque formation and cardiovascular diseases. Currently, statin administration and monacolin K consumption are the main therapies proposed to counter this alarming connection, but relevant side effects are known. To overcome this issue, safe nutraceutical formulations and/or vegetal extracts, endowed with anticholesterolemic activity, could be instrumental in hypercholesterolemia prevention and treatment. In the present work, the anticholesterolemic efficacy of three vegetal extracts used in traditional medicine (artichoke, caigua, and fenugreek), their unique blend (ACFB), and the monacolin K-containing red yeast extract (RYR), was investigated with an in vitro approach based on hepatic cell line HepG2. The impact on cholesterol of the three extracts, their blend, and RYR were investigated by determining hepatocyte total and free cholesterol and bile acids biosynthesis. According to our results, the anticholesterolemic activity of the vegetal extracts was confirmed, and a novel choleretic activity of caigua extract was evidenced. ACFB showed to be safer than RYR while showing a similar effect on total and free cholesterol and bile acids synthesis compared to it. The anticholesterolemic activity of the blend was obtained with lower vegetal extract concentrations compared with the single vegetal extract, potentially indicating an additive effect between the extracts. In conclusion, the vegetal extracts and their blend, ACFB, are safe and are endowed with anticholesterolemic activity, potentially providing complementary therapies to the statin-based ones for hyperlipidemia and hypercholesterolemia-related complications.

Cytotoxicity and Mitochondrial Effects of Phenolic and Quinone-Based Mitochondria-Targeted and Untargeted Antioxidants on Human Neuronal and Hepatic Cell Lines: A Comparative Analysis

Mitochondriotropic antioxidants (MC3, MC6.2, MC4 and MC7.2) based on dietary antioxidants and analogs (caffeic, hydrocaffeic, trihydroxyphenylpropanoic and trihydroxycinnamic acids) were developed. In this study, we evaluate and compare the cytotoxicity profile of novel mitochondria-targeted molecules (generally known as MitoCINs) on human HepG2 and differentiated SH-SY5Y cells with the quinone-based mitochondria-targeted antioxidants MitoQ and SkQ1 and with two non-targeted antioxidants, resveratrol and coenzyme Q10 (CoQ10). We further evaluate their effects on mitochondrial membrane potential, cellular oxygen consumption and extracellular acidification rates. Overall, MitoCINs derivatives reduced cell viability at concentrations about six times higher than those observed with MitoQ and SkQ1. A toxicity ranking for both cell lines was produced: MC4 < MC7.2 < MC3 < MC6.2. These results suggest that C-6 carbon linker and the presence of a pyrogallol group result in lower cytotoxicity. MC3 and MC6.2 affected the mitochondrial function more significantly relative to MitoQ, SkQ1, resveratrol and CoQ10, while MC4 and MC7.2 displayed around 100–1000× less cytotoxicity than SkQ1 and MitoQ. Based on the mitochondrial and cytotoxicity cellular data, MC4 and MC7.2 are proposed as leads that can be optimized to develop safe drug candidates with therapeutic application in mitochondrial oxidative stress-related diseases.

Cyclin-dependent kinase 19 upregulation correlates with an unfavorable prognosis in hepatocellular carcinoma

Objectives Cyclin-dependent kinase 19 (CDK19) is a component of the mediator coactivator complex, which is required for transcriptional activation. In this study, we utilized public databases and wet-bench hepatic cell line experiments to elucidate the potential roles of CDK19 in hepatocellular cancer (HCC). Materials and methods We studied the relationships between CDK19 expression and several clinical features related to HCC via the Oncomine and UALCAN databases. The prognostic value of CDK19 was tested using the Kaplan–Meier Plotter database. We presented the mutations of CDK19 and addressed the relation of CDK19 expression with immune cell infiltration by means of the cBioPortal, Catalogue of Somatic Mutations in Cancer (COSMIC) and Tumor IMmune Estimation Resource (TIMER) databases. Hub genes were obtained and further analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. To test the in silico findings, we knocked down CDK19 with short hairpin RNA (shRNA) technology in two hepatic cell lines and conducted several functional characterization experiments. Results Marked CDK19 upregulation was found in HCC tissues versus normal liver tissues, and CDK19 mRNA expression had high diagnostic value in HCC patients. Subgroup analysis showed that CDK19 overexpression was associated with sex, tumor stage and TP53 mutation status. The prognostic value of CDK19 upregulation for overall survival (OS) was significant in patients with stage 2–3, stage 3–4, and grade 2 disease. One percent of the patients had CDK19 mutations, but no relationship between CDK19 mutation and prognosis was observed. CDK19 was positively correlated with the abundances of CD4 + T cells, macrophages and dendritic cells. We identified 10 genes correlated with CDK19, 8 of which presented excellent prognostic value in HCC. These hub genes were directly involved in cell division and regulation of the G2/M cell cycle transition. Protein–protein interaction (PPI) and pathway predictions indicated that CDK19 is highly likely to be involved in several cellular functions, such as proliferation, migration, and invasion. These functions were strongly interfered from two independent hepatic cell lines after CDK19 knockdown. Conclusions CDK19 could be a prognostic marker in HCC, and its therapeutic potential in HCC needs further study.

Autoantibodies directed against apolipoprotein A-1 as a potential contributor to non-alcoholic fatty liver disease

Background Non-Alcoholic Fatty Liver Disease (NAFLD) represents an increasing cause of liver disease worldwide. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in these patients. Although NAFLD pathophysiology is not fully understood alterations in fat metabolism seem to play a role. Autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) are a novel cardiovascular risk factor to which have been recently attributed a metabolic role in addition to a well-established macrophage-mediated inflammatory effect and have a function as a disruptor of the cholesterol pathway. Purpose This study aims at evaluating a possible role of anti-apoA-1 IgG in NAFLD. Methods Serum from 137 NAFLD patients were tested for anti-apoA-1 IgG prevalence. In vitro, SREBP1, SREBP2 expressions were assessed in the human hepatic cell line HepaRG by western blot analysis and bodipy staining was used to evaluate the lipid droplet content. Mescoscale technology platform was used to measure TNF-α, IL-6 and IL-8 cytokines/chemokines in HepaRG supernatants. Oil Red O staining was used to detect lipid accumulation in liver sections from ApoE−/− mice. Results Elevated anti-apoA-1 IgG seropositivity was found in patients with NAFLD (46%). In vitro, anti-apoA-1 IgG and not control IgG induced lipid accumulation in hepatic cells (5.9 vs 2.5, P=0.0008) and this lipid overload was associated with a high SREBP1 but not SREBP2 expression. Furthermore, anti-apoA-1 IgG and not control antibodies caused a significant large increase of the proinflammatory cytokines IL-6 (680 vs. 163 pg/mL, P=0.03) and TNF-α (391 vs 266 pg/mL, P=0.04) as well as of the chemokine IL-8 (174.1 vs. 72.6 ng/mL, P=0.03) detected in the hepatic cell supernatants. In vivo, anti-apoA-1 IgG and not control IgG also induced higher lipid accumulation in the livers of ApoE−/− mice (1.23 vs 0.53, P=0.03). Conclusion Anti-apoA-1 IgG are frequent in NAFLD, cause a strong inflammatory response and promote lipid accumulation through SREBP1 activation in human hepatic cells. We hypothesize that anti-apoA1 IgG may be a potential contributor in the development of NAFLD. FUNDunding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Geneva University Hospital

Human Three-Dimensional Hepatic Models: Cell Type Variety and Corresponding Applications

Owing to retained hepatic phenotypes and functions, human three-dimensional (3D) hepatic models established with diverse hepatic cell types are thought to recoup the gaps in drug development and disease modeling limited by a conventional two-dimensional (2D) cell culture system and species-specific variability in drug metabolizing enzymes and transporters. Primary human hepatocytes, human hepatic cancer cell lines, and human stem cell–derived hepatocyte-like cells are three main hepatic cell types used in current models and exhibit divergent hepatic phenotypes. Primary human hepatocytes derived from healthy hepatic parenchyma resemble in vivo–like genetic and metabolic profiling. Human hepatic cancer cell lines are unlimitedly reproducible and tumorigenic. Stem cell–derived hepatocyte-like cells derived from patients are promising to retain the donor’s genetic background. It has been suggested in some studies that unique properties of cell types endue them with benefits in different research fields of in vitro 3D modeling paradigm. For instance, the primary human hepatocyte was thought to be the gold standard for hepatotoxicity study, and stem cell–derived hepatocyte-like cells have taken a main role in personalized medicine and regenerative medicine. However, the comprehensive review focuses on the hepatic cell type variety, and corresponding applications in 3D models are sparse. Therefore, this review summarizes the characteristics of different cell types and discusses opportunities of different cell types in drug development, liver disease modeling, and liver transplantation.