Nonalcoholic Steatohepatitis Fibrosis Research Articles

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

Thrombospondin1 as a potential therapeutic target for human nonalcoholic fatty liver disease.

AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

Clinical profile of non-alcoholic fatty liver disease in nonobese patients.

Non-alcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome. Worryingly, it has been increasingly reported among nonobese patients. This study aims to analyse patient characteristics of biopsy-proven NAFLD in an Asian cohort and explore differences stratified by body mass index (BMI). Clinical, laboratory, and histological data were collected from 263 adults with biopsy-proven NAFLD. Patients with and without obesity (BMI cut-off 25) were compared. The ability to predict advanced liver fibrosis with three non-invasive scores, the NAFLD Fibrosis score (NFS), Fibrosis-4 (FIB4), and the aspartate aminotransferase to platelet ratio index (APRI), was compared. Obese subjects had a lower mean age (49.5±12.5 vs 54.0±12.9years, P=0.017), a higher prevalence of diabetes (52.4% vs 36.8%, P=0.037), and a higher waist circumference (113.9±16.0cm vs 87.0±18.4cm, P=0.022). The prevalence of dyslipidaemia (68.0% vs 61.4%, P=0.353) and hypertension (61.7% vs 49.1%, P=0.190) was comparable between the two groups. The distribution of non-alcoholic steatohepatitis (NASH) (63.1% versus 61.4%, P=0.710) and advanced fibrosis (31.6% versus 26.3%, P=0.447) were also similar in both groups. All three non-invasive scores (NFS, FIB4, and APRI) performed poorly in predicting advanced fibrosis in nonobese patients with NAFLD. The FIB4 was the most accurate non-invasive score in predicting advanced fibrosis in the obese group. Obese and nonobese patients are equally at risk of NASH and advanced fibrosis. While the FIB4 is the most accurate non-invasive score in predicting advanced fibrosis among obese individuals, further research is warranted to develop a nonobese specific score to correctly identify nonobese NAFLD patients with advanced fibrosis.

GPx7 ameliorates non-alcoholic steatohepatitis by regulating oxidative stress

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases. NAFLD can further progress to irreversible liver failure such as non-alcoholic steatohepatitis (NASH) fibrosis and cirrhosis. However, specific regulator of NASH- fibrosis has yet to be established. Here, we found that glutathione peroxidase 7 (GPx7) was markedly expressed in NASH fibrosis. Although GPx7 is an antioxidant enzyme protecting other organs, whether GPx7 plays a role in NASH fibrosis has yet to be studied. We found that knockdown of GPx7 in transforming growth factor-β (TGF-β) and free fatty acids (FFA)- treated LX-2 cells elevated the expression of pro-fibrotic and pro-inflammatory genes and collagen synthesis. Consistently, GPx7 overexpression in LX-2 cells led to the suppression of ROS production and reduced the expression of pro-fibrotic and pro-inflammatory genes. Further, NASH fibrosis induced by choline-deficient amino acid defined, high fat diet (CDAHFD) feeding was significantly accelerated by knockdown of GPx7, as evidenced by up-regulated liver fibrosis and inflammation compared with CDAHFD control mice. Collectively, these results suggest that GPx7 might be a novel therapeutic target to prevent the progression and development of NAFLD.

Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells

BackgroundThe transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression. AimTo evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2). MethodsHepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH. ResultsChemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs. ConclusionChemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.

1850-P: High Prevalence of Fatigue and Pruritus in Patients with Advanced Fibrosis Due to Nonalcoholic Steatohepatitis (NASH): The Impact on Patient-Reported Outcomes (PROs)

Background: PROs of NASH patients have not been fully assessed. We assessed the impact of fatigue and pruritus on PROs of patients with advanced NASH. Methods: Baseline PROs including fatigue and pruritis (SF-36, CLDQ-NASH, EQ-5D, and WPAI instruments) were collected from patients with biopsy-proven NASH and advanced fibrosis (stage≥F3). Results: Fatigue (33%) and pruritus (27%) were reported among NASH patients (n=1,669; mean age 58±9 years, 48% F3, 42% psychiatric comorbidities). Fatigued patients were younger, female, cirrhotic, diabetic with higher BMI and more comorbidities (all p<0.05) than those without fatigue. Fatigued patients also had lower albumin, higher alkaline phosphatase (ALP), bile acids, glucose, HbA1c, C-reactive protein (CRP), GGT, ELF, Fibrotest, NFS, and liver stiffness (p<0.05). PROs of fatigued patients were significantly impaired (mean up to -31.4% of a PRO range). In multivariate analysis, predictors of fatigue were female gender [OR=1.47 (1.15-1.89)], history of depression [2.18 (1.67-2.83)], lower serum albumin, younger age, non-Asian race, diabetes, other nervous system or psychiatric comorbidities and some laboratory tests (p<0.05). NASH patients with pruritus had similar demographic profile to fatigued patients but had more skin-related comorbidities than patients without pruritus and a lower hemoglobin and serum albumin, higher ALP, fasting glucose, HOMA, HbA1c, CRP, GGT, higher liver stiffness, ELF, and NFS (all p<0.05). Pruritus patients had impairment in all PROs (all p<0.01). Female gender [odds ratio (OR)=1.33 [1.05-1.68)], history of depression (1.53 (1.19-1.96), nervous [1.40 (1.11-1.77)] and skin-related [1.59 (1.25-2.01)] comorbidities and lower serum albumin were associated with a greater risk of pruritus (p<0.05). Conclusion: One-third of patients with advanced NASH have fatigue and pruritus which negatively impact their PROs. Disclosure Z. Younossi: Consultant; Self; AbbVie Inc., Bristol-Myers Squibb, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Novo Nordisk Inc., Terns and Viking. Q. Anstee: Consultant; Self; 89Bio, Abbott Laboratories, Acuitas Medical, Allergan/Tobira, AstraZeneca, Axcella, Blade, BNN Cardio, Celgene, Cirius, CymaBay, EcoR1, E3Bio, Eli Lilly & Company Ltd., Galmed, Genentech, Genfit S, Intercept Pharma Europe Ltd., Inventiva, IQVIA, Janssen, Madrigal, MedImmune, Metacrine, NewGene, NGMBio, North Sea Therapeutics, Novartis, Novo Nordisk A/S, Pfizer Ltd., Poxel, ProSciento, Raptor Pha. Research Support; Self; Abbvie, Allergan/Tobira, AstraZeneca, GlaxoSmithKline, Glympse Bio, Novartis Pharma AG, Pfizer Ltd., Vertex. Speaker’s Bureau; Self; Abbott Laboratories, Allergan/Tobira, BMS, Clinical Care Options, Falk, Fishawack, Genfit SA, Gilead, Integritas Communications, Kenes, MedScape. V.W.S. Wong: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Gilead Sciences, Inc. Consultant; Self; AbbVie Inc., Allergan plc., Echosens, Intercept Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Pfizer Inc. M. Trauner: Advisory Panel; Self; Gilead Sciences, Inc. Research Support; Self; Gilead Sciences, Inc. Speaker’s Bureau; Self; Gilead Sciences, Inc. Other Relationship; Self; Gilead Sciences, Inc. M. Camargo: Employee; Self; Gilead Sciences, Inc. Z. Goodman: None. L. Henry: None. M. Stepanova: None. M. Romero-Gomez: Consultant; Self; Kaleido Biosciences, Merck & Co., Inc., Novo Nordisk Inc., Siemens Corporation, Zydus Pharmaceuticals, Inc. R. Myers: None. E. Lawitz: None. Funding Gilead Sciences Foundation

1821-P: Foz Foz Mice with a Mutation in the Alms1 Gene Model the Metabolic Syndrome with Advanced Stages of Nonalcoholic Steatohepatitis and Fibrosis

Nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH) is highly prevalent in modern societies frequently consuming diets rich in fats and carbohydrates. NASH is characterized by liver steatosis, inflammation and increased risk for liver fibrosis in human. There is no single animal model available that encompasses the defining features of NASH; therefore, it is of importance to thoroughly characterize available rodent models for NASH research and new drug discovery. Foz/foz mice have a mutation in the Alms1 gene, a ubiquitous protein essential for proper primary cilium function. They have similar defects such as metabolic syndrome, NASH and liver fibrosis to human with the same gene mutation. To better characterize the model, foz/foz mice were fed with western diet (WD, D12079B) for various duration. Compared with WT mice, foz/foz mice on WD had (1) increased BW gain driven by hyperphagia; (2) insulin/leptin resistance with decreased adiponectin levels and hyperlipidemia; (3) increased fat mass and hepatomegaly; (4) increased liver enzymes and elevated TIMP1 in plasma; (5) Liver gene expressions profiles of foz/foz mice showed up-regulation of fibrogenic genes and Lipogenesis, inflammation genes. Histopathology assessment showed foz/foz mice developed severe hepatic steatosis and increased marker of macrophage populations (F4/80 IHC staining). liver collagen content was significantly increased at 16 and 24 wks post WD feeding (PSR staining) compared to wild type mice fed the same diet. Our results suggest that the foz/foz mouse model has robust hallmark features of NASH such as metabolic syndrome, steatohepatitis, and fibrosis. In addition, our data suggest the presence of metabolic complications in other organs as well, as foz/foz mice develop proteinuria over time. The WD-induced foz/foz model exhibits several clinically relevant features and serves as a translational model of NASH. Disclosure J. Liu: None. S. Wang: None. K.E. D’Aquino: Employee; Self; Janssen Pharmaceuticals, Inc. Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. N.H. Wallace: None. M.M. Rankin: Employee; Self; Janssen Pharmaceuticals, Inc. Stock/Shareholder; Self; Johnson & Johnson. F. Du: None. S.A. Hinke: Employee; Self; Janssen Pharmaceuticals, Inc. Stock/Shareholder; Self; Johnson & Johnson. K.A. Albarazanji: None. A.R. Nawrocki: Employee; Self; Janssen Pharmaceuticals, Inc.

1449-P: The Enhanced Liver Fibrosis (ELF) Score Predicts Adverse Clinical and Patient-Reported Outcomes in Patients with Advanced Fibrosis Due to Nonalcoholic Steatohepatitis (NASH)

Background: ELF score is a non-invasive assessment for NASH fibrosis. We assessed associations between ELF, clinical outcomes and PROs in advanced NASH. Methods: NASH patients with advanced fibrosis (Stage ≥F3) were enrolled in 4 clinical trials of simtuzumab and selonsertib - neither showed efficacy. Liver biopsies, ELF scores, and PROs were collected at baseline and week 48. Disease progression (histologic progression to cirrhosis, hepatic decompensation, listed for or liver transplanted, death) was monitored over time (median 16 months; IQR 14-20 months). Results: Patients (n=2155) with biopsy-proven advanced NASH [ 52.5% F4, 40% male, 78% white, 72% type 2 diabetes, mean baseline ELF 10.4±1.1 (F4: 11.4±1.2 vs. F3: 10.3±1.0, p<0.0001)] were included;16.7% F3-NASH had disease progression (96% progressed to cirrhosis or clinical symptoms); 7.3% F4-NASH experienced clinical events (39% ascites, 24% hepatic encephalopathy). Those who progressed had higher baseline ELF: 10.6 ± 1.0 vs. 9.9 ± 0.9 (F3) and 11.5 ± 1.2 vs. 10.6 ± 1.0 (F4) (both p<0.0001). In F3 and F4 patients, baseline ELF was independently associated with disease progression: adjusted hazard ratio (aHR) = 2.48 (2.11-2.93) and 2.83 (2.20-3.63) per 1 ELF point (p<0.0001). After adjustment for baseline ELF, increases in ELF were associated with disease progression: aHR (F3)=1.47 (1.20-1.80) and (F4)=1.68 (1.21-2.33) per +1 additional ELF point (both p<0.002). ELF scores were associated with PRO impairments- ELF cutoff of 10.43 described 18/23 PRO scores (p<0.05) including all domains of CLDQ-NASH. The average magnitude of PRO impairment was -6.1% of a PRO range in NASH with ELF≥10.43 vs. ELF<10.43. Patients with a decrease in ELF of at least 0.83 points had improvement in 11/23 PROs (all domains of CLDQ-NASH, up to +9.8%, p≤0.05). Conclusions: ELF at baseline and its changes over time are good predictors of adverse clinical and PROs in advanced NASH. Disclosure Z. Younossi: Consultant; Self; AbbVie Inc., Bristol-Myers Squibb, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Novo Nordisk Inc., Terns and Viking. Q. Anstee: Consultant; Self; 89Bio, Abbott Laboratories, Acuitas Medical, Allergan/Tobira, AstraZeneca, Axcella, Blade, BNN Cardio, Celgene, Cirius, CymaBay, EcoR1, E3Bio, Eli Lilly & Company Ltd., Galmed, Genentech, Genfit S, Intercept Pharma Europe Ltd., Inventiva, IQVIA, Janssen, Madrigal, MedImmune, Metacrine, NewGene, NGMBio, North Sea Therapeutics, Novartis, Novo Nordisk A/S, Pfizer Ltd., Poxel, ProSciento, Raptor Pha. Research Support; Self; Abbvie, Allergan/Tobira, AstraZeneca, GlaxoSmithKline, Glympse Bio, Novartis Pharma AG, Pfizer Ltd., Vertex. Speaker’s Bureau; Self; Abbott Laboratories, Allergan/Tobira, BMS, Clinical Care Options, Falk, Fishawack, Genfit SA, Gilead, Integritas Communications, Kenes, MedScape. M. Trauner: Advisory Panel; Self; Gilead Sciences, Inc. Research Support; Self; Gilead Sciences, Inc. Speaker’s Bureau; Self; Gilead Sciences, Inc. Other Relationship; Self; Gilead Sciences, Inc. V.W.S. Wong: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Gilead Sciences, Inc. Consultant; Self; AbbVie Inc., Allergan plc., Echosens, Intercept Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Pfizer Inc. M. Camargo: Employee; Self; Gilead Sciences, Inc. L. Henry: None. M. Stepanova: None. M. Romero-Gomez: Consultant; Self; Kaleido Biosciences, Merck & Co., Inc., Novo Nordisk Inc., Siemens Corporation, Zydus Pharmaceuticals, Inc. E. Lawitz: None. R. Myers: None. Funding Gilead Sciences Foundation

Targeted Delivery of Notch Inhibitor Attenuates Obesity-Induced Glucose Intolerance and Liver Fibrosis

As the prevalence of obesity-induced type 2 diabetes mellitus (T2DM) and nonalcoholic steatohepatitis (NASH) continue to increase, the need for pharmacologic therapies becomes urgent. However, endeavors to identify and develop novel therapeutic strategies for these chronic conditions are balanced by the need for safety, impeding clinical translation. One shared pathology of these two diseases is a maladaptive reactivation of the Notch signaling pathway in liver. Notch antagonism with γ-secretase inhibitors effectively suppresses hepatic glucose production and reduces liver fibrosis in NASH, but its extrahepatic side effects, particularly goblet cell metaplasia, limit therapeutic utility. To overcome this barrier, we developed a nanoparticle-mediated delivery system to target γ-secretase inhibitor to liver (GSI NPs). GSI NP application reduced hepatic glucose production in diet-induced obese mice and reduced hepatic fibrosis and inflammation in mice fed a NASH-provoking diet, without apparent gastrointestinal toxicity. By changing the delivery method, these results provide proof-of-concept for the repurposing of a previously intolerable medication to address unmet needs in the clinical landscape for obesity-induced T2DM and NASH.

Non-invasive diagnosis of non-alcoholic steatohepatitis and advanced fibrosis in Japan: A targeted literature review.

Despite the invasive nature of liver biopsy, it remains the current standard for diagnosing non-alcoholic steatohepatitis (NASH) and fibrosis staging. Given the rising prevalence of non-alcoholic fatty liver disease (NAFLD) in Japan, there is a need for reliable non-invasive tests to accurately and efficiently identify NASH and advanced (F3/F4) fibrosis. A review of published works from English and Japanese sources was undertaken in PubMed, Embase, and Ichushi Web to identify studies reporting diagnostic characteristics of NITs in biopsy-proven Japanese NAFLD/NASH patients including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic curve. The performance of non-invasive tests for two diagnostic questions were assessed, namely: (i) identifying NASH cases among NAFLD; and (ii) distinguishing advanced fibrosis (F3-4) from milder fibrosis (F0-2). Twenty-five studies reported outcomes for serum biomarkers, imaging, scoring systems, and novel complex techniques (based on multivariable regression models) for both diagnostic questions. Serum biomarkers were the most commonly assessed method for NASH identification, whereas scoring systems and imaging techniques were most commonly studied for fibrosis staging. In general, tests for NASH identification showed higher PPVs than NPVs, suggesting their usefulness in identifying probable NASH cases. The reverse was observed for fibrosis staging, with higher NPVs than PPVs, suggesting their use in excluding patients at low risk of F3/F4 disease rather than identifying definite F3/F4 fibrosis. In Japanese studies, simple scoring systems and imaging techniques showed particular usefulness in prediction of fibrosis staging, and combinations of serum biomarkers showed diagnostic potential for NASH screening.

Periostin antisense oligonucleotide prevents hepatic steatosis and fibrosis in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, inflammation, and hepatocellular injury with varying degrees of fibrosis. There are currently no established treatment approaches for NASH other than lifestyle interventions. Periostin, a matricellular protein required for tissue remodeling and fibrosis, plays an important role in hepatic steatosis and fibrosis and could be a potential target for NASH treatment. Advances in molecular biology and biochemical engineering have led to the development of antisense oligonucleotides (ASOs) that can inhibit target genes with no significant toxic effects. Herein, we investigated the therapeutic effects of periostin-targeting ASO (PNASO) in NASH. C57BL/6J mice were fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with or without intraperitoneal injection of mouse PNASO. To explore the role of periostin in hepatocellular steatosis, Hc3716 cells, an immortalized human hepatocyte line, were treated with recombinant periostin in vitro. The induced periostin expression in the liver of CDAHFD-fed mice was significantly suppressed by PNASO. The deletion of hepatic periostin by PNASO significantly ameliorated hepatic steatosis while restoring the expression levels of peroxisome proliferator-activated receptor-alpha (PPAR-α) and its target genes. PNASO also inhibited hepatic fibrosis, reflected by the reduction of alpha-smooth muscle actin, collagen type I, and other fibrotic markers. In vitro experiments demonstrated that treatment with recombinant periostin increased cellular lipid accumulation in Hc3716 cells accompanied with the downregulation of PPAR-α. Periostin-targeting ASO is a potential therapeutic approach for the efficient treatment of hepatic steatosis and fibrosis in NASH.

MON-644 Prevalence of Non-Alcoholic Fatty Liver Disease and Liver Fibrosis in Patients with Type 2 Diabetes Mellitus

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. The more severe form is non-alcoholic steatohepatitis (NASH) which can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). NASH is more common in patients with type 2 diabetes mellitus (T2DM). However, its true prevalence in unselected patients with T2DM in the United States remains unknown. In 2019, the American Diabetes Association recommended screening for NASH and liver fibrosis in all patients with T2DM with steatosis and/or elevated ALT. Screening focuses on liver fibrosis as associated with increased risk of cirrhosis and HCC. Still, a liver biopsy remains the gold standard to accurately assess the severity of liver disease. The aim of this study was to determine the prevalence of liver fibrosis in unselected patients with T2DM presenting to primary care or endocrinology clinics at a university hospital in the US. Secondary outcomes were to assess the prevalence of steatosis controlled attenuation parameter (CAP) and performance of vibration-controlled transient elastography (VCTE) as a non-invasive tool to identify patients with significant liver fibrosis. Patients with T2DM between ages of 21-79 and without a history of alcohol intake or other causes secondary causes of NAFLD were recruited for the study. Participants underwent screening for NAFLD at the time of their clinic visit by means of point-of-care CAP and VCTE. Initial evaluation also included obtaining patient demographics, routine chemistries, and fasting samples (on visit #2 if not fasting initially) for metabolic measurements and fibrosis biomarkers. Liver biopsies were offered to patients with a liver stiffness measurement (LSM) ≥8.0 kPa (i.e., highly likely to have moderate-to-severe fibrosis or ≥F2), or those with ≥7 kPa if AST ≥20 and had an APRI and/or FIB-4 score suggestive of being at high-risk of liver fibrosis (i.e., at least mild-to-moderate fibrosis or ≥F1). A total of 469 patients were recruited (age 59±12; 56% females; 60% non-Hispanic whites, 30% African Americans, 4% Asian; BMI 33±6 Kg/m2; A1c 7.5±1.7%; FPG 143±60 mg/dL; AST 22±11 U/L; ALT 24±17 U/L; triglycerides 156±151 mg/dL; LDL-C 88±37 mg/dL; HDL-C 47±13 mg/dL). The prevalence of NAFLD by CAP (≥280) was 67% with a mean CAP of 305±3. The prevalence of any fibrosis was 24% patients. Among those with fibrosis, 15% had moderate-to-severe fibrosis or ≥F2. In those that underwent a liver biopsy, 61% had moderate-to-severe fibrosis (F2-3). Our ongoing study demonstrates the high prevalence of liver steatosis and fibrosis in patients with T2DM. NASH is a common but under-recognized complication of T2DM that requires greater awareness among clinicians taking care of patients with diabetes. While the optimal screening strategy remains unclear, an approach based on plasma biomarkers and CAP/VCTE deserves further exploration moving forward.

Novel Combinatorial Regimen of Garcinol and Curcuminoids for Non-alcoholic Steatohepatitis (NASH) in Mice

Non-alcoholic steatohepatitis (NASH) is a progressive form of Non-alcoholic fatty liver disease (NAFLD), a chronic liver disease with a significant unmet clinical need. In this study, we examined the protective effects of Garcinia indica extract standardized to contain 20% w/w of Garcinol (GIE) and 95% Curcuminoids w/w from Curcuma longa (Curcuminoids) in a Stelic animal model (STAM) of NASH. The STAM mice developed steatosis, hepatocyte ballooning, and inflammation, which were significantly reduced by the combination of GIE and Curcuminoids, resulting in a lower NAFLD activity score. The treatment reduced fibrosis as observed by Sirius red staining, liver hydroxyproline content and mRNA levels of TGF- β and collagen in the liver. Immunostaining with alpha-smooth muscle actin (α SMA) revealed a significant reduction in hepatic stellate cells. Intriguingly, the combination regimen markedly decreased the mRNA levels of MCP1 and CRP and both mRNA and protein levels of TNF-α. NF-kB, reduced the hepatic and circulating FGF21 levels and altered the nonenzymatic (glutathione) and enzymatic antioxidant markers (Glutathione peroxidase, and superoxide dismutase). Our results suggest that the combination of GIE and Curcuminoids can reduce the severity of NASH by reducing steatosis, fibrosis, oxidative stress, and inflammation. The results suggest that the combinatorial regimen could be an effective supplement to prevent the progression of liver steatosis to inflammation and fibrosis in NASH.

Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid β-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported. A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. Inhibition of ACC reduced the activation of TGF-β-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation). In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells - the primary cell responsible for generating fibrotic scar in the liver - and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.

334 OBETICHOLIC ACID IMPROVES EXPERIMENTAL NON-INVASIVE MARKERS OF NON-ALCOHOLIC STEATOHEPATITIS AND ADVANCED FIBROSIS; RESULTS OF A SECONDARY ANALYSIS FROM THE MONTH-18 INTERIM ANALYSIS OF THE REGENERATE STUDY

In the REGENERATE 18-month interim analysis, obeticholic acid (OCA) improved surrogate endpoints of liver fibrosis in patients with non-alcoholic steatohepatitis (NASH). New biomarker indices are being developed, including FibroMeter (FM), which is designed to predict fibrosis stage ≥2 using age, gender, alpha-2-macroglobulin, international normalized ratio, platelets, urea, and gamma-glutamyltransferase. FM Vibration-Controlled Transient Elastography (VCTE) uses the same biomarkers (excluding urea) with liver stiffness (LS). The FibroScan AST (FAST™) score uses LS by VCTE, Controlled Attenuation Parameter score, and aspartate aminotransferase to identify patients with NASH and NAFLD Activity Score (NAS) ≥4 and fibrosis stage ≥2. NASH patients with fibrosis stages 2 and 3 were randomized (1:1:1) to placebo (N=311), OCA 10 mg (N=312), or OCA 25 mg (N=308) once daily. Changes in FM (N=604), FM VCTE (N=604), and FAST (N=391) were analyzed using a mixed-effect repeated measures model (MRMM), with factors of treatment, baseline, visit, visit-by-treatment interaction, and stratification. Least square means and p-values were based on MMRM. At baseline, no significant differences were observed in scores across treatment groups (figure 1). Patients with stage 3 fibrosis at baseline had higher scores than those with stage 2 fibrosis. OCA-treated patients experienced improvements in FM, FM VCTE, and FAST at Month 6 through Month 18. No improvements were observed with placebo (figure 1). OCA treatment resulted in early and sustained improvements in non-invasive assessments of fibrosis in NASH. Improvements in FM and FM VCTE are consistent with OCA’s anti-fibrotic effect, while improvements in FAST are consistent with amelioration of NASH inflammation and fibrosis.

Intragastric Balloon Placement Induces Significant Metabolic and Histologic Improvement in Patients With Nonalcoholic Steatohepatitis

Obese patients with nonalcoholic steatohepatitis (NASH) are at risk for cirrhosis if significant weight loss is not achieved. The single fluid-filled intragastric balloon (IGB) induces meaningful weight loss and might be used in NASH treatment. We performed an open-label prospective study to evaluate the effects of IGB placement on metabolic and histologic features of NASH. Twenty-one patients with early hepatic fibrosis (81% female; mean age, 54 years; average body mass index, 44 kg/m2) underwent magnetic resonance elastography (MRE) and endoscopic ultrasound with core liver biopsy collection at time IGB placement and removal at a single center from October 2016 through March 2018. The primary outcome measure was the changes in liver histology parameters after IGB, including change in nonalcoholic fatty liver disease activity score (NAS) and fibrosis score. We also evaluated changes in weight, body mass index, waist to hip ratio, aminotransaminases, fasting levels of lipids, fasting glucose, glycosylated hemoglobin, and MRE-detected liver stiffness. Six months after IGB, patients' mean total body weight loss was 11.7% ± 7.7%, with significant reductions in HbA1c (1.3% ± 0.5%) (P = .02). Waist circumference decreased by 14.4 ± 2.2 cm (P = .001). NAS improved in 18 of 20 patients (90%), with a median decrease of 3 points (range, 1-4 points); 16 of 20 patients (80%) had improvements of 2 points or more. Fibrosis improved by 1.17 stages in 15% of patients, and MRE-detected fibrosis improved by 1.5 stages in 10 of 20 patients (50%). Half of patients reached endpoints approved by the Food and Drug Administration of for NASH resolution and fibrosis improvement. Percent total body weight loss did not correlate with reductions in NAS or fibrosis. Other than post-procedural pain (in 5% of patients), no serious adverse events were reported. In a prospective study, IGB facilitated significant metabolic and histologic improvements in NASH. IGB appears to be safe and effective for NASH management when combined with a prescribed diet and exercise program. ClinicalTrials.gov no: NCT02880189.

TGF-β1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis

Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-β1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH). Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (•OH) and oxidative DNA damage were measured using an •OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. In culture, TGF-β1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H2O2-triggered •OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct •OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions +2-+13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-β1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA+pSMAD2+- and αSMA+NOX4+-positive hepatic stellate cells from patients with NASH and advanced fibrosis. Downregulation of CYGB by the TGF-β1/pSMAD2/SP3-M1 pathway brings about •OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH. Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-β1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

African Americans have a distinct clinical and histologic profile with lower prevalence of NASH and advanced fibrosis relative to Caucasians.

Racial/ethnic disparities have been reported in the prevalence of nonalcoholic fatty liver disease (NAFLD). Thus, we aimed to understand the inter-ethnic clinical, biochemical, and histological differences in a large cohort of Caucasians and African-Americans (AA). Laboratory and liver biopsy data of 942 NAFLD patients were retrospectively analyzed. Nine hundred seven patients were included in the analysis: 677 (74.6%) Caucasians and 230 (25.3%) AA. AA had higher mean BMI compared to Caucasians (42.6 ± 9.5 vs. 39 ± 8.6 kg/m2). The prevalence of nonalcoholic steatohepatitis (NASH), defined by NAFLD activity score (NAS . 5), was higher in the Caucasians (n = 67) compared to AA (n = 7) (9.8% vs. 3%, P = 0.0007). One hundred fifteen patients (12.8%) had advanced fibrosis: 109 (16.2%) Caucasians and six (2.6%) AA. No AA patients had stage 4 fibrosis or cirrhosis. Multivariate logistic regression analysis revealed advanced fibrosis was significantly associated with age at liver biopsy (OR 1.03, 95% CI 1.0.1.1, P = 0.017, lower platelet count (OR 0.99, 95% CI 0.98.0.99, P = <0.0001), AST/ALT ratio (OR 5.19, 95% CI 2.9.9.2, P <0.0001) and Caucasian race (OR 7.49, 95% CI 2.53.22.2, P = 0.0003). Advanced fibrosis in AA was predicted by lower platelet count and AST/ALT ratio. Whereas Advanced fibrosis in Caucasians was predicted by age at biopsy, lower platelet count and AST/ALT ratio. The AA have a distinct clinical and histologic phenotype. Caucasians have a significantly greater proportion of NASH and are eight times more likely to develop advanced fibrosis than AA.

Polymorphisms in Interleukin 13 Signaling and Interacting Genes Predict Advanced Fibrosis and Hepatocellular Carcinoma Development in Non-Alcoholic Steatohepatitis.

Background: non-alcoholic steatohepatitis (NASH) recently headlined hepatocellular carcinoma (HCC) worldwide. This study aims to unveil the role of some unaddressed critical players that might aid in understanding, predicting, and targeting NASH and NASH-HCC. Methods: Serum interleukin 13 (IL-13) levels and single nucleotide polymorphisms (SNPs) within interleukin (IL)-13 rs20541, IL-13 receptors (IL-13R1) rs2248841, (IL-13R2) rs5946040, signal transducer activator of transcription 6 (STAT6) rs167769, yes-associated protein (YAP1) rs11225163, programmed death-ligand 1 (PD-L1) rs2282055, and programmed death-ligand 2 (PD-L2) rs7854413 genes were analyzed by qRT-PCR. Multiple stepwise regression analysis was performed on a cohort of 134 Egyptian male patients diagnosed with NASH and NASH-HCC. RESULTS: higher serum alpha-fetoprotein (AFP) and higher serum IL-13 levels were directly associated with HCC development in NASH (odds ratio (OR) 19.6 and 1.9 p < 0.01). Reversibly, the presence of the C/C genotype in STAT6 rs167769 and the C allele carrier YAP1 rs11225163 were inversely associated with HCC in NASH patients (OR 0.015 and 0.047 p < 0.01). A predictive model was formulated with 97.5% specificity, 90.9% sensitivity, and 94.8% accuracy. Moreover, higher serum IL-13 levels and the presence of PD-L2 rs7854413 C allele carriers were associated with advanced fibrosis progression in NASH patients (OR 1.432 and 3.797 p < 0.01). Serum levels of IL-13 and C/C genotype in STAT6 rs167769 significantly increased the predictive capacity of serum AFP to predict HCC in F1–F2 and in F3–F4 fibrosis grades NASH patients. Conclusion: association between serum IL-13 and PD-L2 rs7854413 polymorphism successfully predict advanced fibrosis in NASH. However, HCC development in NASH is associated with higher serum AFP, IL-13 levels, and STAT6 rs167769, YAP1 rs11225163 polymorphisms.

Mast Cell Concentrations Correlate with Hepatic Fibrosis in Cancer Patients

BackgroundThe pathogenesis of liver fibrosis in non‐alcoholic steatohepatitis (NASH) has been studied in animal models and humans, with some researchers finding a direct correlation between the concentration of mast cells and the stage of fibrosis in NASH. This suggests that mast cells play a key role in the progression of fibrosis in NASH patients.This study investigates patients with liver fibrosis in a more varied clinical setting with exposure to hepatotoxic chemotherapy regimens and checkpoint inhibitor therapy with various underlying causes of hepatic fibrosis including alcohol use, chronic hepatitis C or Hepatitis B infection, and NASH. The goal is to determine if correlations between mast cell concentration (MCC) and fibrosis remain in patients with multifactorial liver injury.DesignHepatic tissue from seventy‐seven patients (13 resections and 64 biopsies), was retrospectively selected from the database of a large cancer center, and reviewed. Of the cases selected, 38 were male and 39 were female. The gender breakdown by stage of fibrosis is detailed in figure 1. Patients ranged in age from 28–87. The stage of fibrosis was determined using the Batts‐Ludwig criteria. Potential injury‐inducing factors including chemotherapy regimen, checkpoint inhibitor therapy use, and any background liver disease were noted. Six of the patients had multiple primary cancers, 47 had cancer plus underlying liver disease, 18 had liver disease alone, 11 were taking checkpoint inhibitors, 30 were on chemotherapy, 7 were taking hormone therapy, and 6 had one primary cancer with no liver involvement or underlying liver disease.A tryptase immunohistochemical stain (clone AA1, Dako) was performed on a representative section from each case to evaluate MCC. Mast cells in five 40× fields (1 mm2) in both portal tracts and lobular areas were counted and separately recorded. MCC was then compared to the stage of fibrosis using an independent student’s t‐test calculation (Graphpad Prism statistics software).ResultsMCC in portal areas significantly correlated with each histologic stage of fibrosis (p‐values &lt; 0.05, Fig 1). Lobular MCC showed a statistically significant correlation between stage 0 and stage 1–2 (p‐value 0.0043) but did not correlate with fibrosis in later stages (Fig. 1). Though a positive trend is seen in MCC compared to the stage of fibrosis in both portal tracts and lobular areas, a stronger association is apparent in the portal tracts (Fig 2).ConclusionIn medically complex patients from a large cancer center with exposure to various chemotherapy types and checkpoint inhibitor therapy, mast cell concentrations within portal areas directly correlate with the stage of hepatic fibrosis. This finding further supports the role of mast cells in the development of hepatic fibrosis.Support or Funding InformationMD Anderson Department Chair Research Fund for FellowsFigure 1Figure 2