Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure

Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2a (eIf2a) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-kappa B inhibitor I kappa Ba. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent

Pharmacogene expression during progression of metabolic dysfunction-associated steatotic liver disease: Studies on mRNA and protein levels and their relevance to drug treatment

\ua9 2024. Metabolic dysfunction-associated steatotic liver disease (MASLD) is common worldwide. Genes and proteins contributing to drug disposition may show altered expression as MASLD progresses. To assess this further, we undertook transcriptomic and proteomic analysis of 137 pharmacogenes in liver biopsies from a large MASLD cohort. We performed sequencing on RNA from 216 liver biopsies (206 MASLD and 10 controls). Untargeted mass spectrometry proteomics was performed on a 103 biopsy subgroup. Selected RNA sequencing signals were replicated with an additional 187 biopsies. Comparison of advanced MASLD (fibrosis score 3/4) with milder disease (fibrosis score 0–2) by RNA sequencing showed significant alterations in expression of certain phase I, phase II and ABC transporters. For cytochromes P450, CYP2C19 showed the most significant decreased expression (30 % of that in mild disease) but significant decreased expression of other CYPs (including CYP2C8 and CYP2E1) also occurred. CYP2C19 also showed a significant decrease comparing the inflammatory form of MASLD (MASH) with non-MASH biopsies. Findings for CYP2C19 were confirmed in the replication cohort. Proteomics on the original discovery cohort confirmed decreased levels of several CYPs as MASLD advanced but this decrease was greatest for CYP2C19 where levels fell to 40 % control. This decrease may result in decreased CYP2C19 activity that could be problematic for prescription of drugs activated or metabolized by CYP2C19 as MASLD advances. More limited decreases for other P450s suggest fewer issues with non-CYP2C19 drug substrates. Negative correlations at RNA level between CYP2C19 and several cytokine genes provided initial insights into the mechanism underlying decreased expression

The PDGFRα-laminin B1-keratin 19 cascade drives tumor progression at the invasive front of human hepatocellular carcinoma

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor a (PDGFR alpha) and laminin beta 1 (LAMB1) expression. PDGFR alpha has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFR alpha-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFR alpha expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFR alpha expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFR alpha-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of alpha 2 beta 1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFR alpha signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFR alpha-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFR alpha-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression

Serum levels of fibrogenesis biomarkers reveal distinct endotypes predictive of response to weight loss in advanced nonalcoholic fatty liver disease

\ua9 2023 Lippincott Williams and Wilkins. All rights reserved.Background: NAFLD is associated with activation of fibroblasts and hepatic fibrosis. Substantial patient heterogeneity exists, so it remains challenging to risk-stratify patients. We hypothesized that the amount of fibroblast activity, as assessed by circulating biomarkers of collagen formation, can define a "high-risk, high-fibrogenesis" patient endotype that exhibits greater fibroblast activity and potentially more progressive disease, and this endotype may be more amendable to dietary intervention. Methods: Patients with clinically confirmed advanced NAFLD were prescribed a very low-calorie diet (VLCD) intervention (800 kcal/d) to induce weight loss, achieved using total diet replacement. Serum markers of type III (PRO-C3) and IV collagen (PRO-C4) fibrogenesis were assessed at baseline every second week until the end of the VLCD, and 4 weeks post-VLCD and at 9 months follow-up. Results: Twenty-six subjects had a mean weight loss of 9.7% with VLCD. This was associated with significant improvements in liver biochemistry. When stratified by baseline PRO-C3 and PRO-C4 into distinct fibrosis endotypes, these predicted substantial differences in collagen fibrogenesis marker dynamics in response to VLCD. Patients in the high activity group (PRO-C3 11.4 ng/mL and/or PRO-C4 236.5 ng/mL) exhibited a marked reduction of collagen fibrogenesis, ranging from a 40%-55% decrease in PRO-C3 and PRO-C4, while fibrogenesis remained unchanged in the low activity group. The biochemical response to weight loss was substantially greater in patients a priori exhibiting a high fibroblast activity endotype in contrast to patients with low activity. Conclusions: Thus, the likelihood of treatment response may be predicted at baseline by quantification of fibrogenesis biomarkers

Investigation of Performance and Cavitation Treatment in a Kaplan Hydro Turbine

Cavitation is a phenomenon that occurs in various turbomachinery applications causing drawbacks on the. Some of these downsides are damaging the components of the system, generating noise and vibration, and loss of the turbine efficiency over time. Thus, it is imperative to address issue of cavitation to increase the life span of the equipment in addition to improve the system performance. This thesis introduces a method used to mitigate the cavitation phenomenon in a 3-inch Kaplan hydro turbine via injecting air at the leading edge of the rotor blades. The study is based on modeling the turbine using Computational Fluid Dynamics (CFD) software as well as carrying out experimental tests. The simulations were conducted at different air injection pressures over a spectrum of rotational speeds using Large Eddy Simulation (LES) for turbulence and volume of fluid for multiphase interactions: water, vapor water and air. The cavitation behavior was observed first without aeration, then followed by air injection simulations to investigate the effect of aeration. Each case was simulated for 12 cycles at rotational speeds of 1000, 2000, 3000, 4000, and 5000 rpm. The Vapor Volume Fraction (VVF) and the output mechanical power were monitored throughout the simulations. The data acquired from the simulations were compared to the experimental results for verifications. It was observed that the cavitation was mitigated in both the computer simulations and the experiment testing reaching up to 49.7% as an average reduction, while the output power was reduced by 6.6%

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal

Background: The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objectives: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Study design: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. Results: Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. Main limitations: The relatively low number of equine oocytes and embryo transfer procedures performed. Conclusions: For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Background &amp; Aims: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. Methods: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. Results: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. Conclusions: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice

NASH limits anti-tumour surveillance in immunotherapy-treated HCC

Hepatocellular carcinoma (HCC) can have viral or non-viral causes(1-5). Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need(6,7). Here we report the progressive accumulation of exhausted, unconventionally activated CD8(+)PD1(+) T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8(+)PD1(+) T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8(+)PD1(+)CXCR6(+), TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8(+) T cells or TNF neutralization, suggesting that CD8(+) T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8(+)PD1(+) T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment