Reply to Abenavoli et al. Comment on "Clayton-Chubb et al. Understanding NAFLD: From Case Identification to Interventions, Outcomes, and Future Perspectives. Nutrients 2023, 15, 687".

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Understanding NAFLD: From Case Identification to Interventions, Outcomes, and Future Perspectives.

While non-alcoholic fatty liver disease (NAFLD) is a prevalent and frequent cause of liver-related morbidity and mortality, it is also strongly associated with cardiovascular disease-related morbidity and mortality, likely driven by its associations with insulin resistance and other manifestations of metabolic dysregulation. However, few satisfactory pharmacological treatments are available for NAFLD due in part to its complex pathophysiology, and challenges remain in stratifying individual patient's risk for liver and cardiovascular disease related outcomes. In this review, we describe the development and progression of NAFLD, including its pathophysiology and outcomes. We also describe different tools for identifying patients with NAFLD who are most at risk of liver-related and cardiovascular-related complications, as well as current and emerging treatment options, and future directions for research.

Addressing the liver progenitor cell response and hepatic oxidative stress in experimental non-alcoholic fatty liver disease/non-alcoholic steatohepatitis using amniotic epithelial cells.

BACKGROUND: Non-alcoholic fatty liver disease is the most common liver disease globally and in its inflammatory form, non-alcoholic steatohepatitis (NASH), can progress to cirrhosis and hepatocellular carcinoma (HCC). Currently, patient education and lifestyle changes are the major tools to prevent the continued progression of NASH. Emerging therapies in NASH target known pathological processes involved in the progression of the disease including inflammation, fibrosis, oxidative stress and hepatocyte apoptosis. Human amniotic epithelial cells (hAECs) were previously shown to be beneficial in experimental models of chronic liver injury, reducing hepatic inflammation and fibrosis. Previous studies have shown that liver progenitor cells (LPCs) response plays a significant role in the development of fibrosis and HCC in mouse models of fatty liver disease. In this study, we examined the effect hAECs have on the LPC response and hepatic oxidative stress in an experimental model of NASH. METHODS: Experimental NASH was induced in C57BL/6 J male mice using a high-fat, high fructose diet for 42 weeks. Mice received either a single intraperitoneal injection of 2 × 106 hAECs at week 34 or an additional hAEC dose at week 38. Changes to the LPC response and oxidative stress regulators were measured. RESULTS: hAEC administration significantly reduced the expansion of LPCs and their mitogens, IL-6, IFNγ and TWEAK. hAEC administration also reduced neutrophil infiltration and myeloperoxidase production with a concurrent increase in heme oxygenase-1 production. These observations were accompanied by a significant increase in total levels of anti-fibrotic IFNß in mice treated with a single dose of hAECs, which appeared to be independent of c-GAS-STING activation. CONCLUSIONS: Expansion of liver progenitor cells, hepatic inflammation and oxidative stress associated with experimental NASH were attenuated by hAEC administration. Given that repeated doses did not significantly increase efficacy, future studies assessing the impact of dose escalation and/or timing of dose may provide insights into clinical translation.

Dynamic Regulation of Caveolin-1 Phosphorylation and Caveolae Formation by Mammalian Target of Rapamycin Complex 2 in Bladder Cancer Cells.

The mammalian target of rapamycin (mTOR) and associated phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway is commonly up-regulated in cancer, including bladder cancer. mTOR complex 2 (mTORC2) is a major regulator of bladder cancer cell migration and invasion, but the mechanisms by which mTORC2 regulates these processes are unclear. A discovery mass spectrometry and reverse-phase protein array-based proteomics dual approach was used to identify novel mTORC2 phosphoprotein targets in actively invading cancer cells. mTORC2 targets included focal adhesion kinase, proto-oncogene tyrosine-protein kinase Src, and caveolin-1 (Cav-1), among others. Functional testing shows that mTORC2 regulates Cav-1 localization and dynamic phosphorylation of Cav-1 on Y14. Regulation of Cav-1 activity by mTORC2 also alters the abundance of caveolae, which are specialized lipid raft invaginations of the plasma membrane associated with cell signaling and membrane compartmentalization. Our results demonstrate a unique role for mTORC2-mediated regulation of caveolae formation in actively migrating cancer cells.

An exploratory study examining how nano-liquid chromatography-mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis. METHODS: Sixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed. RESULTS: Phosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen. CONCLUSIONS: Simultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.

Antibody Validation by Western Blotting.

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

Reverse Phase Protein Microarrays.

While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

Pacemaker interactions induce reentrant wave dynamics in engineered cardiac culture.

Pacemaker interactions can lead to complex wave dynamics seen in certain types of cardiac arrhythmias. We use experimental and mathematical models of pacemakers in heterogeneous excitable media to investigate how pacemaker interactions can be a mechanism for wave break and reentrant wave dynamics. Embryonic chick ventricular cells are cultured in vitro so as to create a dominant central pacemaker site that entrains other pacemakers in the medium. Exposure of those cultures to a potassium channel blocker, E-4031, leads to emergence of peripheral pacemakers that compete with each other and with the central pacemaker. Waves emitted by faster pacemakers break up over the slower pacemaker to form reentrant waves. Similar dynamics are observed in a modified FitzHugh-Nagumo model of heterogeneous excitable media with two distinct sites of pacemaking. These findings elucidate a mechanism of pacemaker-induced reentry in excitable media.

Chaotic dynamics in cardiac aggregates induced by potassium channel block.

Chaotic rhythms in deterministic models can arise as a consequence of changes in model parameters. We carried out experimental studies in which we induced a variety of complex rhythms in aggregates of embryonic chick cardiac cells using E-4031 (1.0-2.5 µM), a drug that blocks the hERG potassium channel. Following the addition of the drug, the regular rhythm evolved to display a spectrum of complex dynamics: irregular rhythms, bursting oscillations, doublets, and accelerated rhythms. The interbeat intervals of the irregular rhythms can be described by one-dimensional return maps consistent with chaotic dynamics. A Hodgkin-Huxley-style cardiac ionic model captured the different types of complex dynamics following blockage of the hERG mediated potassium current.

Stochastic and spatial influences on drug-induced bifurcations in cardiac tissue culture.

The addition of a drug that specifically blocks a potassium channel in spontaneously beating aggregates of chick heart cells leads to complex bifurcations over time. A stochastic partial differential equation model based on discrete ionic currents recorded in these cells demonstrates that drug diffusion and noise can induce the coupled beats and bursting rhythms observed. These results provide further evidence that stochastic events at a subcellular level are needed to understand complex cardiac arrhythmias and play an important role in the onset of these arrhythmias.

Beyond insulin resistance in NASH: TNF-alpha or adiponectin?

Adiponectin has antilipogenic and anti-inflammatory effects, while tumor necrosis factor alpha (TNF-alpha) reduces insulin sensitivity and has proinflammatory effects. We examined (1) the extent to which hypoadiponectinemia and TNF-alpha activation are features of nonalcoholic steatohepatitis (NASH) and (2) whether serum levels of these markers correlate with the severity of histological changes in 109 subjects with nonalcoholic fatty liver disease (NAFLD), including 80 with NASH and 29 with simple steatosis. By multivariate analysis, subjects with NASH had reduced adiponectin level and increased TNF-alpha and soluble TNF receptor 2 (sTNFR2)-but not leptin levels, compared with controls matched by age, sex, and body mass index; these differences were independent of the increased insulin resistance (by homeostasis model [HOMA-IR]) in NASH. When compared with simple steatosis, NASH was associated with lower adiponectin levels and higher HOMA-IR, but there were no significant differences in the levels of TNF-alpha and sTNFR2. The majority of subjects with steatohepatitis (77%) had adiponectin levels less than 10 microg/mL and HOMA-IR greater than 3 units, but only 33% of those with pure steatosis had these findings. HOMA-IR and low serum adiponectin were also independently associated with increased grades of hepatic necroinflammation. In conclusion, hypoadiponectinemia is a feature of NASH independent of insulin resistance. Reduced adiponectin level is associated with more extensive necroinflammation and may contribute to the development of necroinflammatory forms of NAFLD.