Hepatocellular carcinoma in patients with metabolic dysfunction-associated fatty liver disease: Can we stratify at-risk populations?

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a new nomenclature recently proposed by a panel of international experts so that the entity is defined based on positive criteria and linked to pathogenesis, replacing the traditional non-alcoholic fatty liver disease (NAFLD), a definition based on exclusion criteria. NAFLD/MAFLD is currently the most common form of chronic liver disease worldwide and is a growing risk factor for development of hepatocellular carcinoma (HCC). It is estimated than 25% of the global population have NAFLD and is projected to increase in the next years. Major Scientific Societies agree that surveillance for HCC should be indicated in patients with NAFLD/ MAFLD and cirrhosis but differ in non-cirrhotic patients (including those with advanced fibrosis). Several studies have shown that the annual incidence rate of HCC in NAFLD-cirrhosis is greater than 1%, thus surveillance for HCC is cost-effective. Risk factors that increase HCC incidence in these patients are male gender, older age, presence of diabetes and any degree of alcohol consumption. In non-cirrhotic patients, the incidence of HCC is much lower and variable, being a great challenge to stratify the risk of HCC in this group. Furthermore, large epidemiological studies based on the general population have shown that diabetes and obesity significantly increase risk of HCC. Some genetic variants may also play a role modifying the HCC occurrence among patients with NAFLD. The purpose of this review is to discuss the epidemiology, clinical and genetic risk factors that may influence the risk of HCC in NAFLD/MAFLD patients and propose screening strategy to translate into better patient care.

[Diagnosis and treatment of non-alcoholic fatty liver disease. Argentine Association for the Study of Liver Diseases, year 2019]. / Guía de diagnóstico y tratamiento del hígado graso no alcohólico. Asociación Argentina para el estudio de las enfermedades del hígado, año 2019.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease worldwide, with an estimated global prevalence of approximately 25%, that is much higher in patients with overweight, obesity and type 2 diabetes. NAFLD is considered as the hepatic manifestation of metabolic syndrome. It has a wide spectrum, from simple steatosis to steatohepatitis, fibrosis, cirrhosis and its complications, such as hepatocellular carcinoma. Most of the affected patients will not evolve to advanced fibrosis or cirrhosis. Despite this, it has been described that the hepatic disease is the third cause of death among patients with nonalcoholic fatty liver, after cardiovascular and malignant diseases. Among the huge number of patients affected, the main challenge is to identify those who are at risk of developing cirrhosis or its complications and to recognize the diagnostic and treatment options. In this Guideline, endorsed by the Argentine Association for the Study of Liver Diseases, the definitions, epidemiological aspects, natural history and a practical approach to possible algorithms to estimate the severity of liver disease in the individual patient are reviewed; in addition to analyzing advances in treatment and proposing recommendations for follow-up. It is important to note that no data on the incidence or prevalence of the disease have been published in the general population of Argentina, and it is encouraged to carry them out.

El hígado graso no alcohólico (HGNA) es la enfermedad hepática crónica más frecuente en todo el mundo, con una prevalencia aproximada de 25% a nivel global. Su prevalencia es mucho mayor en pacientes con sobrepeso, obesidad y diabetes tipo 2 y es considerada como la manifestación hepática del síndrome metabólico. El espectro de la enfermedad hepática es muy amplio, desde la esteatosis simple a la esteatohepatitis, fibrosis, cirrosis y sus complicaciones, como el hepatocarcinoma. La mayoría de los pacientes afectados no progresará a la fibrosis avanzada/cirrosis. A pesar de esto, se ha descripto que la hepatopatía es la tercera causa de muerte entre los pacientes con HGNA, luego de las enfermedades cardiovasculares y las malignas. Entre la enorme cantidad de afectados, lo más importante es identificar a los que están en riesgo de evolución a la cirrosis o sus complicaciones y conocer las opciones de diagnóstico y tratamiento. En esta Guía organizada por la Asociación Argentina para el Estudio de las Enfermedades del Hígado se revisan las definiciones, los aspectos epidemiológicos, la historia natural y un enfoque práctico sobre algoritmos posibles para estimar la gravedad de la hepatopatía en cada caso, además de analizar los avances en el tratamiento y recomendaciones para el seguimiento. Es importante señalar que no se han publicado datos sobre incidencia o prevalencia de la enfermedad en población general de Argentina, y se alienta a la realización de los mismos.

Hepatocellular carcinoma in South America: Evaluation of risk factors, demographics and therapy.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Most studies addressing the epidemiology of HCC originate from developed countries. This study reports the preliminary findings of a multinational approach to characterize HCC in South America. METHODS: We evaluated 1336 HCC patients seen at 14 centres in six South American countries using a retrospective study design with participating centres completing a template chart of patient characteristics. The diagnosis of HCC was made radiographically or histologically for all cases according to institutional standards. Methodology of surveillance for each centre was following AASLD or EASL recommendations. RESULTS: Sixty-eight percent of individuals were male with a median age of 64 years at time of diagnosis. The most common risk factor for HCC was hepatitis C infection (HCV, 48%), followed by alcoholic cirrhosis (22%), Hepatitis B infection (HBV, 14%) and NAFLD (9%). We found that among individuals with HBV-related HCC, 38% were diagnosed before age 50. The most commonly provided therapy was transarterial chemoembolization (35% of HCCs) with few individuals being considered for liver transplant (<20%). Only 47% of HCCs were diagnosed during surveillance, and there was no difference in age of diagnosis between those diagnosed incidentally vs by surveillance. Nonetheless, being diagnosed during surveillance was associated with improved overall survival (P = .01). CONCLUSIONS: Our study represents the largest cohort to date reporting characteristics and outcomes of HCC across South America. We found an important number of HCCs diagnosed outside of surveillance programmes, with associated increased mortality in those patients.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.

Noxa mediates hepatic stellate cell apoptosis by proteasome inhibition.

AIM: Induction of hepatic stellate cell (HSC) apoptosis is a viable therapeutic strategy to reduce liver fibrogenesis. Although BH3-only proteins of the Bcl-2 family trigger pro-apoptotic pathways, the BH3-only proteins mediating HSC apoptosis have not been well defined. Our aim, using proteasome inhibition as a model to induce HSC apoptosis, was to examine the BH3-only proteins contributing to cell death of this key liver cell subtype. METHODS: Apoptosis was induced by treating LX-2 cells, an immortalized human hepatic stellate cell line, and primary rat stellate cells with the proteasome inhibitor MG-132. RESULTS: Treatment with proteasome inhibitors increased expression of Noxa both at the mRNA (16-fold) and protein (22-fold) levels indicating that both transcriptional and post-translational mechanisms contributed to the increase in cellular Noxa levels. Knockdown of Noxa by siRNA significantly attenuated cell death, mechanistically implicating Noxa as a key apoptotic mediator of proteasome inhibitor-induced cell death. Given the pivotal role for the anti-apoptotic Bcl-2 protein A1 in activated HSC survival, we determined if Noxa bound to this survival protein. Noxa was shown to physically bind the anti-apoptotic Bcl-2 protein A1 by co-immunoprecipitation. CONCLUSIONS: Noxa contributes to proteasome inhibitor-induced apoptosis of stellate cells likely by binding A1. Strategies to therapeutically increase Noxa expression may be useful for inducing HSC apoptosis.

[Why are there few publications by the Argentine gastroenterology? Considerations on a bibliometric analysis of Argentine publications on gastroenterology]. / Por qué publica poco la gastroenterología Argentina? Consideraciones sobre un análisis bibliométrico de publicaciones Argentinas en gastroenterología.

The publication of scientific findings is the main way to communicate advances. Our aim was to perform a bibliometric and comparative analysis of the Argentinean gastroenterological research output. We analyzed Argentinean gastroenterological publications selectively retrieved from LILACS (between years 1982-2006) and EMBASE (1996-2007) databases by means of specially constructed filter based on author address and subject headings. The global Argentinean scientific research output is far below that of developed countries and has been affected in direct manner by economic, political and social disturbances in the country. The gastroenterological research output from Argentina represent about 6% of national biomedical research. While 54% belongs to gastroenterology and 46% to hepatology, 65% are based on clinical research and 67% were originally contributions. Only 11% have been published in high impact factor journals. The comparative analysis within countries with health indicators similarities has shown a low biomedical and gastroenterology research output, however, the rate of acceptance at the 18 top gastroenterological journals is acceptable (15%). The contributions of registered specialists were lower for gastroenterologists compared with those from hepatologists (8.7% and 16.4% respectively). The research projects at public hospital funded by the pharmaceutical industry overcome those funded independently. Indeed, it seems that the independent research is being progressively replaced by that supported by the industry due to economic benefits for researchers even when there is a very low participation rate in publications (3%) by Argentinean researchers. We conclude that the Argentinean biomedical and gastroenterological research output is scanty compared with developed countries and countries with comparable health indicators. Our analysis suggests that efforts must be taken to attain objectives directed to develop and improve the Argentinean biomedical and gastroenterological scientific work and publication.

Overexpression of mcl-1 attenuates liver injury and fibrosis in the bile duct-ligated mouse.

Hepatocyte apoptosis contributes to liver injury and fibrosis after cholestatic injury. Our aim was to ascertain if the anti-apoptotic protein Mcl-1 alters liver injury or fibrosis in the bile duct-ligated mouse. Markers of apoptosis and fibrosis were compared in wild-type and transgenic mice expressing human Mcl-1 after bile duct ligation. Compared to hMcl-1 transgenic animals, ligated wild-type mice displayed a significant increase in TUNEL-positive cells and in caspase 3/7-positive hepatocytes. Consistent with apoptotic injury, the pro-apoptotic protein Bak underwent a conformational change to an activated form upon cholestatic injury, a change mitigated by hMcl-1 overexpression. Likewise, liver histology, number of bile infarcts, serum ALT values, markers of hepatic fibrosis, and animal survival were improved in bile duct-ligated mice transgenic for hMcl-1 as compared to wild-type mice. In conclusion, increased Mcl-1 expression plays a role in hepatoprotection upon cholestatic liver injury.

TRAIL mediates liver injury by the innate immune system in the bile duct-ligated mouse.

UNLABELLED: The contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand expressed by cells of the innate immune system, to cholestatic liver injury has not been explored. Our aim was to ascertain if TRAIL contributes to liver injury in the bile duct-ligated (BDL) mouse. C57/BL6 wild-type (wt), TRAIL heterozygote (TRAIL(+/-)), and TRAIL knockout (TRAIL(-/-)) mice were used for these studies. Liver injury and fibrosis were examined 7 and 14 days after BDL, respectively. Hepatic TRAIL messenger RNA (mRNA) was 6-fold greater in BDL animals versus sham-operated wt animals (P < 0.01). The increased hepatic TRAIL expression was accompanied by an increase in liver accumulation of natural killer 1.1 (NK 1.1)-positive NK and natural killer T (NKT) cells, the predominant cell types expressing TRAIL. Depletion of NK 1.1-positive cells reduced hepatic TRAIL mRNA expression and serum alanine aminotransferase (ALT) values. Consistent with a role for NK/NKT cells in this model of liver injury, stress ligands necessary for their recognition of target cells were also up-regulated in hepatocytes following BDL. Compared to sham-operated wt mice, BDL mice displayed a 13-fold increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and an 11-fold increase in caspase 3/7-positive hepatocytes (P < 0.01). The number of TUNEL and caspase 3/7-positive cells was reduced by >80% in BDL TRAIL knockout animals (P < 0.05). Likewise, liver histology, number of bile infarcts, serum ALT values, hepatic fibrosis, and animal survival were also improved in BDL TRAIL(-/-) animals as compared to wt animals. CONCLUSION: These observations support a pivotal role for TRAIL in cholestatic liver injury mediated by NK 1.1-positive NK/NKT cells.

Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis.

Hepatocyte lipoapoptosis, a critical feature of nonalcoholic steatohepatitis, can be replicated in vitro by incubating hepatocytes with saturated free fatty acids (FFA). These toxic FFA induce Bim expression, which is requisite for their cytotoxicity. Because the FoxO3a transcription factor has been implicated in Bim expression, our aim was to determine if FFA induce Bim by a FoxO3a-dependent mechanism. In Huh-7 cells, the saturated FFA, palmitic and stearic acid, increased Bim mRNA 16-fold. Treatment of cells with the saturated FFA induced FoxO3a dephosphorylation (activation) and nuclear translocation and stimulated a FoxO luciferase-based reporter assay; direct binding of FoxO3a to the Bim promoter was also confirmed by a chromatin immunoprecipitation assay. A small interfering RNA-targeted knockdown of FoxO3a abrogated FFA-mediated Bim induction and apoptosis. FoxO3a was activated by a phosphatase 2A-dependent mechanism, since okadaic acid- and small interfering RNA-targeted knockdown of this phosphatase blocked FoxO3a dephosphorylation, Bim expression, and apoptosis. Consistent with these data, phosphatase 2A activity was also stimulated 3-fold by saturated FFA. Immunoprecpitation studies revealed an FFA-dependent association between FoxO3a and protein phosphatase 2A. FFA-mediated FoxO3a activation by protein phosphatase 2A was also observed in HepG2 cells and murine hepatocytes. In conclusion, saturated FFA stimulate protein phosphatase 2A activity, which activates FoxO3a, inducing expression of the intracellular death mediator Bim.

Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity.

BACKGROUND: Elevated circulating free fatty acids (FFA) contribute to the development of hepatic steatosis and promote hepatocyte apoptosis by incompletely defined mechanisms. Although the death ligand TRAIL has been implicated in a variety of pathological liver diseases, the role of TRAIL in mediating apoptosis of FFA induced steatotic hepatocytes is unknown. AIM: We examined TRAIL cytotoxicity in an in vitro model of hepatocyte steatosis induced by FFA. METHODS: Hepatocytes (Huh 7 cells, HepG2 cells, and primary rat hepatocytes) were rendered steatotic by incubation with oleic acid. Apoptosis was assessed morphologically and biochemically by caspase activity. TRAIL receptor regulation was examined using immunoblot analysis and siRNA for targeted knockdown. c-jun N-terminal kinase (JNK) inhibition was attained with SP600125. RESULTS: Oleic acid sensitised the cells to TRAIL but not TNF-alpha cytotoxicity. FFA sensitisation to TRAIL occurred at much lower concentrations than required for FFA mediated sensitisation to Fas, or FFA induced lipoapoptosis. Oleic acid treatment led to upregulation of the cognate TRAIL receptor death receptor 5 (DR5) but not death receptor 4 (DR4). The upregulation of DR5 was JNK dependent. siRNA targeted knockdown of either DR5 or DR4 demonstrated that DR5 was responsible for FFA sensitisation to TRAIL killing. DR5 expression was enhanced in steatotic human liver samples. CONCLUSION: Our results suggest that FFA induced hepatocyte steatosis sensitises to TRAIL by a DR5 mediated JNK dependent mechanism.