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Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Primary sclerosing cholangitis (PSC) is an idiopathic and heterogenous cholestatic liver disease characterized by chronic inflammation and fibrosis of the biliary tree. Currently, no effective therapies are available for this condition, whose incidence is rising. At present, specificity and sensitivity of current serum markers used to diagnose PSC are limited and often unreliable. In this study, we characterize circulating extracellular vesicles and provide supporting data on their potential use as novel surrogate biomarkers for PSC. EVs are membrane surrounded structures, 100–1000 nm in size, released by cells under various conditions and which carry a variety of bioactive molecules, including small non-coding RNAs, lipids and proteins. In recent years, a large body of evidence has pointed to diagnostic implications of EVs and relative cargo in various human diseases. We isolated EVs from serum of well-characterized patients with PSC or control subjects by differential centrifugation and size-exclusion chromatography. A complete characterization identified elevated levels of circulating EVs in PSC patients compared to healthy control subjects (2000 vs. 500 Calcein-FITC + EVs/μL). Tissue and cell specificity of circulating EVs was assessed by identification of liver-specific markers and cholangiocyte marker CK-19. Further molecular characterization identified 282 proteins that were differentially regulated in PSC-derived compared to healthy control-EVs. Among those, IL-13Ra1 was the most significantly and differentially expressed protein in PSC-derived EVs and correlated with the degree of liver fibrosis. In addition to protein profiling, we performed a miRNA-sequencing analysis which identified 11 among established, liver-specific (e.g., miR-122 and miR-192) and novel miRNAs. One of the newly identified miRNAs, miR-4645-3p, was significantly up-regulated fourfold in PSC-derived EVs compared to circulating EVs isolated from healthy controls. This study provides supporting evidence of the potential role of circulating EVs and associated protein and miRNA cargo as surrogate noninvasive and reliable biomarker for PSC.

The interplay between intracellular and intravascular lipolysis is crucial for maintaining circulating lipid levels and systemic energy homeostasis. Adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL), the primary triglyceride (TG) lipases responsible for these two spatially separate processes, are highly expressed in adipose tissue. Yet the mechanisms underlying their coordinated regulation remain undetermined. Here, we demonstrate that genetic ablation of G0S2, a specific inhibitory protein of ATGL, completely abolished diet-induced hypertriglyceridemia and significantly attenuated atherogenesis in mice. These effects were attributable to enhanced whole-body TG clearance, not altered hepatic TG secretion. Specifically, G0S2 deletion increased circulating LPL concentration and activity, predominantly through LPL production from white adipose tissue (WAT). Strikingly, transplantation of G0S2-deficient WAT normalized plasma TG levels in mice with hypertriglyceridemia. In conjunction with improved insulin sensitivity and decreased ANGPTL4 expression, the absence of G0S2 enhanced the stability of LPL protein in adipocytes, a phenomenon that could be reversed upon ATGL inhibition. Collectively, these findings highlight the pivotal role of adipocyte G0S2 in regulating both intracellular and intravascular lipolysis, and the possibility of targeting G0S2 as a viable pharmacological approach to reducing levels of circulating TGs.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical \"find-me\" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel \"find me\" signals that contributes to macrophage infiltration associated with obesity.

Therapy for nonalcoholic steatohepatitis (NASH) is limited. Andrographolide (ANDRO), a botanical compound, has a potent anti-inflammatory activity due to its ability to inhibit NF-κB. ANDRO has been also shown to inhibit the NLRP3 inflammasome, a relevant pathway in NASH. Our aim was to evaluate the effects of ANDRO in NASH and its influence on inflammasome activation in this setting. Thus, mice were fed a choline-deficient-amino-acid–defined (CDAA) diet with/without concomitant ANDRO administration (1 mg/kg, 3-times/week). Also, we assessed serum levels of alanine-aminotransferase (ALT), liver histology, hepatic triglyceride content (HTC) and hepatic expression of pro-inflammatory, pro-fibrotic and inflammasome genes. Inflammasome activation was also evaluated in fat-laden HepG2 cells. Our results showed that ANDRO administration decreased HTC and attenuated hepatic inflammation and fibrosis in CDAA-fed mice. ANDRO treatment determined a strong reduction in hepatic macrophage infiltration and reduced hepatic mRNA levels of both pro-inflammatory and pro-fibrotic genes. In addition, mice treated with ANDRO showed reduced expression of inflammasome genes. Finally, ANDRO inhibited LPS-induced interleukin-1β expression through NF-κB inhibition in fat-laden HepG2 cells and inflammasome disassembly. In conclusion, ANDRO administration reduces inflammation and fibrosis in experimental NASH. Inflammasome modulation by a NF-κB-dependent mechanism may be involved in the therapeutic effects of ANDRO.

Background/Aims Hepatocyte cell death is a key feature of nonalcoholic steatohepatitis (NASH). As the contribution of specific caspases remains unclear, our aim was to ascertain the effect of caspase 3 suppression on liver injury and fibrogenesis. Methods C57BL/6 wild-type (WT) and caspase 3 knock out ( Casp3 − / − ) mice were placed on a methionine- and choline-deficient (MCD) diet for 6 weeks to induce steatohepatitis and liver fibrosis. Thereafter, liver injury, liver fibrosis and hepatocellular apoptosis were quantified in liver sections. Additionally, expression of proteins associated with liver inflammation and fibrogenesis was analyzed. Results WT mice fed MCD diet showed marked activation of caspase 3 in hepatocytes, in conjunction with steatohepatitis and increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3 − / − mice fed the MCD diet showed similar serum aminotransferase levels and NAFLD activity scores (NAS) compared with WT MCD-fed mice. However, Casp3 − / − mice on the MCD diet showed a marked reduction in expression of transcripts for profibrogenic genes, which translated into reduced hepatic collagen deposition. These changes were associated with decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling. Casp3 − / − mice on the MCD showed a reduction in expression of chemokine receptor 2 (CCR2) leading to ameliorated infiltration of inflammatory lymphocyte antigen 6 complex, locus C1 (Ly6c) positive monocytes. Conclusion These findings support a prominent role for hepatocyte caspase 3 activation in NASH-related apoptosis, fibrogenesis and fibrosis which in part is mediated via CCR2-dependent infiltration of Ly6c positive monocytes.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in adults and children worldwide. NAFLD has become a severe health issue and it can progress towards a more severe form of the disease, the non-alcoholic steatohepatitis (NASH). A combination of environmental factors, host genetics, and gut microbiota leads to excessive accumulation of lipids in the liver (steatosis), which may result in lipotoxicity and trigger hepatocyte cell death, liver inflammation, fibrosis, and pathological angiogenesis. NASH can further progress towards liver cirrhosis and cancer. Over the last few years, cell-derived extracellular vesicles (EVs) have been identified as effective cell-to-cell messengers that transfer several bioactive molecules in target cells, modulating the pathogenesis and progression of NASH. In this review, we focused on recently highlighted aspects of molecular pathogenesis of NASH, mediated by EVs via their bioactive components. The studies included in this review summarize the state of art regarding the role of EVs during the progression of NASH and bring novel insight about the potential use of EVs for diagnosis and therapeutic strategies for patients with this disease.

Nonalcoholic fatty liver disease (NAFLD) is currently one of most common forms of chronic liver disease globally. NAFLD represents a wide spectrum of liver involvement from nonprogressive isolated steatosis to nonalcoholic steatohepatitis (NASH), characterized by liver necroinflammation and fibrosis and currently one of the top causes of end‐stage liver disease and hepatocellular carcinoma. At present, there is a lack of effective treatments, and a central barrier to the development of therapies is the requirement for an invasive liver biopsy for diagnosis of NASH. Discovery of reliable, noninvasive biomarkers are urgently needed. In this study, we tested whether circulating extracellular vesicles (EVs), cell‐derived small membrane‐surrounded structures with a rich cargo of bioactive molecules, may serve as reliable noninvasive “liquid biopsies” for NASH diagnosis and assessment of disease severity. Total circulating EVs and hepatocyte‐derived EVs were isolated by differential centrifugation and size‐exclusion chromatography from serum samples of healthy individuals, patients with precirrhotic NASH, and patients with cirrhotic NASH. EVs were further characterized by flow cytometry, electron microscopy, western blotting, and dynamic light scattering assays before performing a proteomics analysis. Our findings suggest that levels of total and hepatocyte‐derived EVs correlate with NASH clinical characteristics and disease severity. Additionally, using proteomics data, we developed understandable, powerful, and unique EV‐based proteomic signatures for potential diagnosis of advanced NASH. Conclusion: Our study shows that the quantity and protein constituents of circulating EVs provide strong evidence for EV protein–based liquid biopsies for NAFLD/NASH diagnosis. Circulating liver‐derived extracellular vesicles (EVs) are released in patients with advanced nonalcoholic steatohepatitis (NASH) and correlate with disease outcomes. Analysis of EV protein cargo identified potential multiprotein signatures that can be used as noninvasive biomarkers as an alternative to costly and invasive liver biopsy.

The NLRP3 inflammasome is an intracellular, multiprotein complex that promotes the auto-catalytic activation of caspase-1 and the subsequent maturation and secretion of the pro-inflammatory cytokines, IL-1β and IL-18. Persistent activation of the NLRP3 inflammasome has been implicated in the pathophysiology of a number of inflammatory and autoimmune diseases, including neuroinflammation, cardiovascular disease, non-alcoholic steatohepatitis, lupus nephritis and severe asthma. Here we describe the preclinical profile of JT002, a novel small molecule inhibitor of the NLRP3 inflammasome. JT002 potently reduced NLRP3-dependent proinflammatory cytokine production across a number of cellular assays and prevented pyroptosis, an inflammatory form of cell death triggered by active caspase-1. JT002 demonstrated in vivo target engagement at therapeutically relevant concentrations when orally dosed in mice and prevented body weight loss and improved inflammatory and fibrotic endpoints in a model of Muckle–Wells syndrome (MWS). In two distinct models of neutrophilic airway inflammation, JT002 treatment significantly reduced airway hyperresponsiveness and airway neutrophilia. These results provide a rationale for the therapeutic targeting of the NLRP3 inflammasome in severe asthma and point to the use of JT002 in a variety of inflammatory disorders.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.