A PROTEIN COMPLEX OF LIVER ORIGIN ACTIVATES A PRO-INFLAMMATORY PROGRAM THAT DRIVES HEPATIC AND INTESTINAL INJURY IN ALCOHOL-ASSOCIATED LIVER DISEASE.

BACKGROUND AND AIMS: There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction. RESULTS: Alcohol-fed RageΔMye mice exhibited decreased NFκB signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. Additionally, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, as it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induces a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis. CONCLUSION: We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.

Ablation of secreted phosphoprotein-1 in hepatocytes increases fatty acid oxidation and ameliorates alcohol-associated liver disease.

BACKGROUND: Previously, we demonstrated that Spp1-/- mice exhibit a greater susceptibility to alcohol-induced liver injury than wild-type (WT) mice. Notably, alcohol triggers the expression of osteopontin (encoded by SPP1) in hepatocytes. However, the specific role of hepatocyte-derived SPP1 in either mitigating or exacerbating alcohol-associated liver disease (AALD) has yet to be elucidated. We hypothesized that hepatocyte-derived SPP1 plays a role in AALD by modulating the regulation of steatosis. METHODS: We analyzed hepatic SPP1 expression using four publicly available datasets from patients with alcoholic hepatitis (AH). Additionally, we examined SPP1 expression in the livers of WT mice subjected to either a control or ethanol Lieber-DeCarli (LDC) diet for 6 weeks. We compared the relationship between SPP1 expression and significantly dysregulated genes in AH with controls using correlation and enrichment analyses. To investigate the specific impact of hepatocyte-derived SPP1, we generated hepatocyte-specific Spp1 knock-out (Spp1ΔHep) mice and subjected them to either a control or ethanol Lieber-DeCarli diet for 6 weeks. RESULTS: Alcohol induced hepatic SPP1 expression in both humans and mice. Our analysis, focusing on genes correlated with SPP1, revealed an enrichment of fatty acid oxidation (FAO) in three datasets, and peroxisome proliferator-activated receptor signaling in one dataset. Notably, FAO genes correlating with SPP1 were downregulated in patients with AH. Ethanol-fed WT mice exhibited higher serum-free fatty acids (FFAs), adipose tissue lipolysis, and hepatic fatty acid (FA) transporters. In contrast, ethanol-fed Spp1ΔHep mice displayed lower liver triglycerides, FFAs, and serum alanine transaminase and greater FAO gene expression than WT mice, indicating a protective effect against AALD. Primary hepatocytes from Spp1∆Hep mice exhibited heightened expression of genes encoding proteins involved in FAO. CONCLUSIONS: Alcohol induces the expression of SPP1 in hepatocytes, leading to impaired FAO and contributing to the development of AALD.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

BACKGROUND AND AIMS: Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. APPROACH AND RESULTS: We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. CONCLUSIONS: Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

Macrophage-derived Osteopontin (SPP1) Protects From Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, lobular inflammation, hepatocyte ballooning degeneration, and fibrosis, all of which increase the risk of progression to end-stage liver disease. Osteopontin (OPN, SPP1) plays an important role in macrophage (MF) biology, but whether MF-derived OPN affects NASH progression is unknown. METHODS: We analyzed publicly available transcriptomic datasets from patients with NASH, and used mice with conditional overexpression or ablation of Spp1 in myeloid cells and liver MFs, and fed them a high-fat, fructose, and cholesterol diet mimicking the Western diet, to induce NASH. RESULTS: This study demonstrated that MFs with high expression of SPP1 are enriched in patients and mice with nonalcoholic fatty liver disease (NAFLD), and show metabolic but not pro-inflammatory properties. Conditional knockin of Spp1 in myeloid cells (Spp1KI Mye) or in hepatic macrophages (Spp1KI LvMF) conferred protection, whereas conditional knockout of Spp1 in myeloid cells (Spp1ΔMye) worsened NASH. The protective effect was mediated by induction of arginase-2 (ARG2), which enhanced fatty acid oxidation (FAO) in hepatocytes. Induction of ARG2 stemmed from enhanced production of oncostatin-M (OSM) in MFs from Spp1KI Mye mice. OSM activated STAT3 signaling, which upregulated ARG2. In addition to hepatic effects, Spp1KI Mye also protected through sex-specific extrahepatic mechanisms. CONCLUSION: MF-derived OPN protects from NASH, by upregulating OSM, which increases ARG2 through STAT3 signaling. Further, the ARG2-mediated increase in FAO reduces steatosis. Therefore, enhancing the OPN-OSM-ARG2 crosstalk between MFs and hepatocytes may be beneficial for patients with NASH.

The PTP1B selective inhibitor MSI-1436 mitigates Tunicamycin-induced ER stress in human hepatocarcinoma cell line through XBP1 splicing modulation.

Protein tyrosine phosphatase PTP1B is considered as a key metabolic enzyme that has been reported to be associated with insulin resistance onset, and underlying cellular metabolic malfunctions, including ER stress and mitochondrial failure. In this study, effects of selective PTP1B inhibition using MSI-1436 on cellular apoptosis, oxidative stress, mitochondrial dysfunction and ER stress have been assessed using an in vitro model of Tunicamycin induced ER stress in HepG2 cell line. Inhibition of PTP1B using MSI-1436 significantly increased cell viability and reduced the number of apoptotic cells as well as the expression of key apoptosis initiators and effectors. MSI-1436 further mitigated ER stress, by downregulating the expression of IRE1, ATF6 and PERK transcripts, all being key ER stress sensors. Interestingly, MSI-1436 inhibited the XBP1 splicing, and thus its UPR-associated transcriptional activity. PTP1B inhibition further enabled to restore proper mitochondrial biogenesis, by improving transmembrane potential, and diminishing intracellular ROS while restoring of endogenous antioxidant enzymes genes expression. PTP1B inhibition using MSI-1436 could improve cellular apoptosis and metabolic integrity through the mitigation of ER and mitochondrial stress signalling pathways, and excessive ROS accumulation. This strategy may be useful for the treatment of metabolic disorders including IR, NAFLD and diabetes.

Ablation of high-mobility group box-1 in the liver reduces hepatocellular carcinoma but causes hyperbilirubinemia in Hippo signaling-deficient mice.

Silencing the Hippo kinases mammalian sterile 20-like 1 and 2 (MST1/2) activates the transcriptional coactivator yes-associated protein (YAP) in human hepatocellular carcinoma (HCC). Hepatocyte-derived high-mobility group box-1 (HMGB1) regulates YAP expression; however, its contribution to HCC in the context of deregulated Hippo signaling is unknown. Here, we hypothesized that HMGB1 is required for hepatocarcinogenesis by activating YAP in Hippo signaling-deficient (Mst1/2ΔHep ) mice. Mst1/2ΔHep mice developed HCC within 3.5 months of age and had increased hepatic expression of HMGB1 and elevated YAP activity compared to controls. To understand the contribution of HMGB1, we generated Mst1/2&Hmgb1ΔHep mice. They exhibited decreased YAP activity, cell proliferation, inflammation, fibrosis, atypical ductal cell expansion, and HCC burden at 3.5 months compared to Mst1/2∆Hep mice. However, Mst1/2&Hmgb1ΔHep mice were smaller, developed hyperbilirubinemia, had more liver injury with intrahepatic biliary defects, and had reduced hemoglobin compared to Mst1/2ΔHep mice. Conclusion: Hepatic HMGB1 promotes hepatocarcinogenesis by regulation of YAP activity; nevertheless, it maintains intrahepatic bile duct physiology under Hippo signaling deficiency.

Maternal Transmission of Human OGG1 Protects Mice Against Genetically- and Diet-Induced Obesity Through Increased Tissue Mitochondrial Content.

Obesity and related metabolic disorders are pressing public health concerns, raising the risk for a multitude of chronic diseases. Obesity is multi-factorial disease, with both diet and lifestyle, as well as genetic and developmental factors leading to alterations in energy balance. In this regard, a novel role for DNA repair glycosylases in modulating risk for obesity has been previously established. Global deletion of either of two different glycosylases with varying substrate specificities, Nei-like endonuclease 1 (NEIL1) or 8-oxoguanine DNA glycosylase-1 (OGG1), both predispose mice to diet-induced obesity (DIO). Conversely, enhanced expression of the human OGG1 gene renders mice resistant to obesity and adiposity. This resistance to DIO is mediated through increases in whole body energy expenditure and increased respiration in adipose tissue. Here, we report that hOGG1 expression also confers resistance to genetically-induced obesity. While Agouti obese (Ay/a) mice are hyperphagic and consequently develop obesity on a chow diet, hOGG1 expression in Ay/a mice (Ay/aTg ) prevents increased body weight, without reducing food intake. Instead, obesity resistance in Ay/aTg mice is accompanied by increased whole body energy expenditure and tissue mitochondrial content. We also report for the first time that OGG1-mediated obesity resistance in both the Ay/a model and DIO model requires maternal transmission of the hOGG1 transgene. Maternal, but not paternal, transmission of the hOGG1 transgene is associated with obesity resistance and increased mitochondrial content in adipose tissue. These data demonstrate a critical role for OGG1 in modulating energy balance through changes in adipose tissue function. They also demonstrate the importance of OGG1 in modulating developmental programming of mitochondrial content and quality, thereby determining metabolic outcomes in offspring.

A Novel Role for the DNA Repair Enzyme 8-Oxoguanine DNA Glycosylase in Adipogenesis.

Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1-/-) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1-/- mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.

The DNA Repair Protein OGG1 Protects Against Obesity by Altering Mitochondrial Energetics in White Adipose Tissue.

Obesity and related metabolic pathologies represent a significant public health concern. Obesity is associated with increased oxidative stress that damages genomic and mitochondrial DNA. Oxidatively-induced lesions in both DNA pools are repaired via the base-excision repair pathway, initiated by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). Global deletion of OGG1 and common OGG1 polymorphisms render mice and humans susceptible to metabolic disease. However, the relative contribution of mitochondrial OGG1 to this metabolic phenotype is unknown. Here, we demonstrate that transgenic targeting of OGG1 to mitochondria confers significant protection from diet-induced obesity, insulin resistance, and adipose tissue inflammation. These favorable metabolic phenotypes are mediated by an increase in whole body energy expenditure driven by specific metabolic adaptations, including increased mitochondrial respiration in white adipose tissue of OGG1 transgenic (Ogg1Tg) animals. These data demonstrate a critical role for a DNA repair protein in modulating mitochondrial energetics and whole-body energy balance.

Cognitive effects of vanillic acid against streptozotocin-induced neurodegeneration in mice.

CONTEXT: Vanillic acid (VA), a flavoring agent used in food and drug products, obtained naturally from the plant Angelica sinensis (Oliv.) Diels (Apiaceae), used in the traditional Chinese medicine. It is reported to possess strong antioxidant, anti-inflammatory, and neuroprotective effects. However, the pharmacological effects on oxidative stress-induced neurodegeneration are not well investigated. OBJECTIVE: This study investigates the neuroprotective effect of VA on streptozotocin (STZ)-induced neurodegeneration in mice through behavioral and biochemical parameters. MATERIALS AND METHODS: The behavioral effects were determined using the Y-maze and open-field habituation memory. In biochemical parameters, acetylcholinesterase (AChE), corticosterone, tumor necrosis factor (TNF)-α, and antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase) were measured. Five groups of animals used were of control, negative control, and three separate groups treated with 25, 50, and 100 mg/kg of VA, respectively, for 28 d. Intracerebroventricular (ICV) injections of STZ were performed for all groups except control on 14th and 16th of 28 d of VA treatment. RESULTS: VA improved spatial learning and memory retention by preventing oxidative stress compared with control animals. VA at 50 and 100 mg/kg dose significantly (p < 0.001) improved the habituation memory, decreased the AChE, corticosterone, TNF-α, and increased the antioxidants (p < 0.001). VA (100 mg/kg) exhibited dose-dependent effect in all parameters with p < 0.001 except antioxidants in which VA showed the significance of p < 0.01. DISCUSSION AND CONCLUSION: VA exhibited reduction in AChE, TNF-α, and corticosterone with improved antioxidants to contribute neuroprotection and could be an effective therapeutic agent for treating neurodegenerative disorders.

Assessment of injectable and cohesive nanohydroxyapatite composites for biological functions.

Pressing need for utilization of injectables/fillers in various forms of orthopaedic treatments/surgeries commands an equal demand for better graft material. Injectable bone graft material based on biomimetically synthesized nanohydroxyapatite was developed and subjected to ball milling for different times; three materials thus produced were evaluated for their biological properties. The three composites tested were found to have some difference in proliferation and differentiation on mesenchymal stem cells in cultures. In vivo studies were performed by implanting the graft materials with or without cells in the bone drill hole injury created in the femur of Wistar rats. Our studies show that the composites lead to well-healed injury site with normal histology without inflammation or fibrous tissue formation and bone deformity. This material needs to be tested on large animals for further ascertaining its applicability in clinical use.