Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

Outcomes of Onyx® Embolization of Type II Endoleaks After Endovascular Repair of Abdominal Aortic Aneurysms.

BACKGROUND: Type II endoleaks (T2ELs) are common following endovascular repair of abdominal aortic aneurysms (EVAR). Embolization with ethylene vinyl alcohol copolymer (Onyx) may present an effective treatment alternative for T2ELs. Due to limited data supporting its use, we sought to analyze outcomes of Onyx embolization for T2ELs. METHODS: Retrospective review of consecutive patients treated for T2ELs utilizing Onyx embolization agent from 2009-2018. All pre- and post-Onyx intervention CT scans were analyzed for diameter and volume changes with 3D reconstruction software. The primary outcomes were change in maximum AAA diameter and volume. Secondary outcomes included additional interventions, rupture, and mortality. A subset analysis was performed with patients with isolated T2ELs (no other types of endoleaks present). RESULTS: We identified 85 patients (73 males, mean age 77.6 ± 7.6 years) who underwent 112 Onyx interventions. Average time to first Onyx intervention after index EVAR was 3.3 ± 2.6 years and average sac growth was 6.3 ± 6.7 mm. Patients underwent mean 1.3 Onyx interventions using a mean of 4.9 ± 4.7 ml for treatment. Three complications occurred (Onyx extravasation, colon ischemia, and access site hematoma). Mean follow-up was 2.5 ± 2.1 years after initial Onyx treatment. At the most recent follow-up, sac diameter stabilization was seen in 47% and reduction >5 mm was seen in 19%. Sac growth of >5 mm was seen in 34% of patients following the first Onyx intervention. In our subset of isolated T2EL, 72% had sac stabilization or reduction >5 mm. Four patients experienced a ruptured aneurysm (3 had active type 1 endoleaks). Rupture-free survival was 95% at 5 years, and overall survival was 54% at 5 years. Notably, increasing Onyx interventions were not associated with sac stabilization or reduction (OR 0.6, P = 0.1). On multivariable analysis, AAA sac diameter stabilization or reduction was independently associated with BMI >30 kg/m2 (OR 4.2, P = 0.01) and having only 1 Onyx intervention (OR 3.8, P = 0.02). CONCLUSIONS: Onyx for embolization of T2ELs resulted in AAA sac diameter stabilization or reduction in 66% of patients, and up to 72% in isolated T2ELs. Further, increasing Onyx interventions were not associated with either aneurysm sac stabilization or reduction. Given its similar outcomes to other embolization strategies in the literature, Onyx embolization for management of T2ELs needs to be judiciously considered, particularly for T2ELs persisting after an initial Onyx embolization intervention.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

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.