Fernando J. Barreyro

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-α 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.

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.

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

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. (HEPATOLOGY 2008.)

Proton pump inhibitor therapy does not increase the incidence of spontaneous bacterial peritonitis in cirrhosis: A multicenter prospective study

BACKGROUND & AIM Retrospective studies show an association between proton pump inhibitor (PPI) therapy and spontaneous bacterial peritonitis (SBP). We investigate the relationship between PPI and SBP in decompensated cirrhotic patients in a large nationwide prospective study. METHODS Seven hundred seventy patients with a diagnosis of decompensated cirrhosis were admitted consecutively in 23 hospitals in Argentina from March 2011 to April 2012; the patients were carefully investigated for PPI consumption in the previous 3 months. In total, 251 patients were excluded because of active gastrointestinal hemorrhage, antibiotic use during the preceding weeks, HIV-positive status and immunosuppressive therapy. RESULTS Two hundred twenty-six out of 519 patients (43.5%) had received PPI therapy within the last 3 months. In 135 patients, PPIs were administered for longer than 2 weeks. A bacterial infection was shown in 255 patients (49.1%). SBP was diagnosed in 95 patients out of 394 patients with ascites (24.7%). There was no significant difference in the rate of PPI consumption between the infected and the non-infected patients (44.3% vs. 42.8%) or between the SBP patients and the patients with ascites without SBP (46% vs. 42%). In the SBP patients, the duration of PPI administration did not influence the rate of SBP occurrence. The type of bacteria and the origin of SBP infection were similar in the patients with and without PPI. CONCLUSION In the current large, multicenter, prospective study, PPI therapy, specifically evaluated at admission of consecutive cirrhotic patients, was not associated with a higher risk of SBP.

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.

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.

PIB: A Score to Select Sorafenib Treatment Candidates for Hepatocellular Carcinoma in Resource-Limited Settings

James S Leathers1,*, Domingo Balderramo2, Jhon Prieto3, Fernando Diehl2, Esteban Gonzalez-Ballerga4, Melina R. Ferreiro4, Enrique Carrera5, Fernando Barreyro6, Javier DiazFerrer7, Dupinder Singh8, Angelo Z. Mattos9, Flair Carrilho10, and Jose D. Debes8,** 1School of Medicine, Vanderbilt University, Nashville, USA 2Department of Gastroenterology, Hospital Privado Universitario de Córdoba, Instituto Universitario de Ciencias Biomédicas de Córdoba, Córdoba, Argentina