Ali Canbay

Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis

BACKGROUND & AIMSnThe pathogenesis of nonalcoholic steatohepatitis (NASH) remains poorly understood. Although apoptosis is a common mechanism of liver injury, the extent and clinical significance of apoptosis in NASH has not been examined. Thus, the aims of this study were to quantify hepatocyte apoptosis in NASH, correlate it with disease severity, and identify possible mechanisms of apoptosis induction.nnnMETHODSnHepatocyte apoptosis was assessed in NASH, simple steatosis, alcoholic hepatitis, and controls without liver disease using the TUNEL assay and immunohistochemistry for activated caspases 3 and 7. Liver specimens were also graded according to the magnitude of inflammation and fibrosis.nnnRESULTSnTUNEL-positive cells were significantly increased in liver biopsy specimens from patients with NASH compared with simple steatosis and controls. Unexpectedly, TUNEL-positive cells were also greater in NASH vs. alcoholic hepatitis. Immunohistochemistry demonstrated active caspases 3 and 7 in NASH specimens, confirming the occurrence of apoptosis in this disease. A positive correlation was observed between hepatocyte apoptosis and hepatic fibrosis and inflammatory activity, respectively. The Fas receptor was strongly expressed in hepatocytes in liver specimens from NASH patients as compared with controls.nnnCONCLUSIONSnHepatocyte apoptosis is significantly increased in patients with NASH and correlates with disease severity, suggesting that antiapoptotic therapy may be useful in this syndrome.

Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway

Nonalcoholic fatty liver disease (NAFLD) is a serious health problem. Although NAFLD represents a form of lipotoxicity, its pathogenesis remains poorly understood. The aim of this study was to examine the cellular mechanisms involved in free fatty acid (FFA)‐mediated hepatic lipotoxicity. FFA treatment of liver cells resulted in Bax translocation to lysosomes and lysosomal destabilization with release of cathepsin B (ctsb), a lysosomal cysteine protease, into the cytosol. This process was also partially dependent on ctsb. Lysosomal destabilization resulted in nuclear factor κB–dependent tumor necrosis factor α expression. Release of ctsb into the cytoplasm was also observed in humans with NAFLD and correlated with disease severity. In a dietary murine model of NAFLD, either genetic or pharmacological inactivation of ctsb protected against development of hepatic steatosis, liver injury, and insulin resistance with its associated “dysmetabolic syndrome.” In conclusion, these data support a lipotoxic model of FFA‐mediated lysosomal destabilization. Supplemental material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:185–194.)

Apoptosis: the nexus of liver injury and fibrosis.

Apoptosis associated with liver disease is increasingly viewed as a nexus through which many key pathways converge. Apoptotic responses incorporate soluble stimuli, inflammatory cells, resident parenchymal cells, and, it now appears, fibrogenic cells as well. Cellular apoptosis is the first cellular response to many toxic events, and accompanies viral hepatitis, alcohol-induced liver disease, nonalcoholic fatty liver disease, cholestatic liver diseases, and ischemia/reperfusion injury.1–5 Moreover, hepatocyte apoptosis is significantly increased in patients with alcoholic hepatitis and nonalcoholic steatohepatitis, and correlates with disease severity and hepatic fibrosis.1,5 Despite their pervasive concurrence in liver damage, the relationship between apoptosis and either hepatic inflammation or fibrosis has not been fully explored, in part because of the prevailing—and overstated—dogma that cell death by apoptosis is “innocuous.” This presumption has been based primarily on analyses of organ development, where physiologic apoptosis is tightly restricted to discrete subsets of cells both spatially and temporally. In contrast, “pathologic” apoptosis in adult tissues induces large numbers of cells, is non-selective, injures large aggregates of cells, and may be sustained over decades. In contrast to physiologic apoptosis, pathologic apoptosis in liver may not only result from inflammation and fibrosis, but may in turn amplify these responses. In particular, hepatic stellate cells (HSC), the key fibrogenic cell type in liver, contribute to apoptosis and inflammation. Given these emerging and sometimes contradictory observations, a review of the relationship between apoptosis, inflammation, and fibrosis is timely. Critical insights into these complex relationsmay help promote rationallybased therapies for patients with chronic liver diseases. Furthermore, we anticipate that these concepts will help stimulate further investigation into mechanisms connecting cell death to inflammation and fibrosis.

Apoptotic body engulfment by a human stellate cell line is profibrogenic.

Hepatocyte apoptosis and stellate cell activation are both features of chronic liver diseases, but a relationship between these events has not been explored. In macrophages, engulfment of apoptotic bodies induces expression of transforming growth factor-β (TGF-β), a profibrogenic cytokine. We examined whether a similar response occurs in stellate cells. Fluorescently labeled hepatocyte apoptotic bodies were added to cultures of primary and immortalized human stellate cells. Stellate cells, but not hepatocytes, readily engulfed apoptotic bodies in a time-dependent manner as assessed by confocal microscopy. The activation of primary and immortalized human stellate cells after incubation with apoptotic bodies, as well as their fibrogenic activity, was indicated by an increase in α-smooth muscle actin (primary cells), TGF-β1, and collagen α1(I) mRNA (primary and immortalized cells). The profibrogenic response was dependent upon apoptotic body engulfment, because nocodazole, a microtubule-inhibiting agent, blocked both the engulfment and the increase of TGF-β1 and collagen α1(I) mRNA. As described in primary rodent stellate cells, up-regulation of collagen α1(I) mRNA was inhibited by a PI-3K inhibitor (LY294002) and a p38 mitogen-activated protein kinase inhibitor (SB203580) in LX-1 cells. In conclusion, these data support a model in which engulfment of hepatocyte apoptotic bodies by stellate cells leads to a fibrogenic response by eliciting a kinase-signaling pathway.

Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice

BACKGROUND/AIMSnHepatic steatosis sensitizes the liver to injury and inflammation by unclear mechanisms. Because Fas has been linked to liver injury and inflammation, Fas expression and sensitization to Fas signaling was examined in models of hepatic steatosis.nnnMETHODSnMice were fed a carbohydrate diet while control animals received standard chow. Sensitization to Fas was examined following administration of Jo2 antibody. For the in vitro experiments, HepG2 cells were incubated with or without a mixture of long chain fatty acids (2:1 oleate:palmitate). Sensitization of the cells to Fas was examined using the CH11 antibody.nnnRESULTSnMice fed a high caloric diet developed hepatic steatosis, hyperlipidemia, insulin resistance, and hyperleptinemia, all features of the human syndrome. Fas expression in hepatocytes was increased as compared to lean animals and was coupled to cytotoxic signaling. Indeed, hepatocyte apoptosis, liver injury and chemokine generation were all accentuated in obese animals following administration of Jo-2, a Fas agonist. Hep G2 cells cultured in the presence of free fatty acids also developed cellular steatosis, upregulated Fas expression and were more sensitive to apoptosis by a Fas agonist.nnnCONCLUSIONSnCollectively, these data implicate Fas as a link between obesity associated fatty liver and increased susceptibility to liver damage.

Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis.

Although a lysosomal, cathepsin B-dependent (Ctsb-dependent) pathway of apoptosis has been described, the contribution of this pathway to tissue damage remains unclear. Our aim was to ascertain if Ctsb inactivation attenuates liver injury, inflammation, and fibrogenesis after bile duct ligation (BDL). In 3-day BDL mice, hepatocyte apoptosis, mitochondrial cytochrome c release, and serum alanine aminotransferase (ALT) values were reduced in Ctsb-/- versus Ctsb+/+ animals. Likewise, R-3032 (a Ctsb inhibitor) also reduced these parameters in BDL WT mice. Both genetic and pharmacologic inhibition of Ctsb in the BDL mouse reduced (a). hepatic inflammation, as assessed by transcripts for CXC chemokines and neutrophil infiltration, and (b). fibrogenesis, as assessed by transcripts for stellate cell activation and sirius red staining for hepatic collagen deposition. These differences could not be ascribed to alterations in cholestasis. These findings support a prominent role for the lysosomal pathway of apoptosis in tissue injury and link apoptosis to inflammation and fibrogenesis. Ctsb inhibition may be therapeutic in liver diseases.

Bile Acids Up-regulate Death Receptor 5/TRAIL-receptor 2 Expression via a c-Jun N-terminal Kinase-dependent Pathway Involving Sp1

Bile acids up-regulate death receptor 5 (DR5)/TRAIL-receptor 2 (TRAIL-R2) expression thereby sensitizing hepatocytes to TRAIL-mediated apoptosis. However, the precise mechanism by which bile acids enhance DR5/TRAIL-R2 expression is unknown. Although several bile acids enhanced DR5/TRAIL-R2 expression, deoxycholic acid (DCA) was the most potent. DCA stimulated JNK activation and the JNK inhibitor SP600125 blocked DCA-induced DR5/TRAIL-R2 mRNA and protein expression. Reporter gene analysis identified a 5′-flanking region containing two Sp1 binding sites within the DR5/TRAIL-R2 promoter as bile acid responsive. Sp1 binding to one of the two sites was enhanced by DCA treatment as evaluated by electrophoretic mobility shift assays and chromatin immunoprecipitation studies. JNK inhibition with SP600125 also blocked binding of Sp1 to the DR5/TRAIL-R2 promoter. Finally, point mutations of the Sp1 binding site attenuated promoter activity. In conclusion, Sp1 is a bile acid-responsive transcription factor that mediates DR5/TRAIL-R2 gene expression downstream of JNK.

Oxysterols induce cyclooxygenase-2 expression in cholangiocytes: implications for biliary tract carcinogenesis.

Cyclooxygenase‐2 (COX‐2), which is expressed by cholangiocytes in biliary tract disorders, has recently been implicated in biliary tract carcinogenesis. The mechanisms responsible for this COX‐2 expression remain unclear. In human diseases, bile contains oxygenated derivatives of cholesterol (oxysterols) which possess diverse biological properties. Therefore, we determined if oxysterols modulate COX‐2 expression. The effect of an oxysterol (22(R)‐hydroxycholesterol, 22‐HC) on COX‐2 expression in KMBC cells, a human cholangiocarcinoma cell line, was examined. 22‐HC enhanced COX‐2 protein expression. This oxysterol activated p42/44 and p38 MAPK, but not JNK 1/2. A p42/44 MAPK inhibitor did not block COX‐2 induction, while p38 MAPK inhibitor effectively attenuated COX‐2 induction. Although COX‐2 mRNA levels were increased by 22‐HC, this increase was not transcriptionally regulated, as 22‐OH did not increase activity in a COX‐2 promoter gene assay. In contrast, COX‐2 mRNA stability was augmented by 22‐HC treatment, and this effect was reversed by a p38 MAPK inhibitor. In conclusion, the results demonstrate that the oxysterol 22‐HC stabilizes COX‐2 mRNA via a p38 MAPK‐dependent mechanism. This enhanced COX‐2 protein expression by oxysterols may participate in the genesis and progression of cholangiocarcinoma. (HEPATOLOGY 2004;39:732–738.)

22 The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse

Liver injury is characterized by hepatocyte apoptosis and collagen-producing activated hepatic stellate cells (HSC). Hepatocyte apoptosis promotes liver injury and fibrosis, whereas activated HSC apoptosis limits hepatic fibrosis. Pharmacological inhibition of liver cell apoptosis may potentially attenuate liver injury and fibrosis by blocking hepatocyte apoptosis or promote fibrosis by permitting accumulation of activated HSCs. To ascertain the net effect of inhibiting liver cell apoptosis on liver injury, inflammation, and hepatic fibrogenesis, we examined the effect of a pancaspase inhibition IDN-6556 on these parameters in the bile duct ligated (BDL) mouse. Hepatocyte apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and immunofluorescence for active caspases 3/7, and liver injury by histopathology and serum alanine aminotransferase (ALT) determinations. Real-time polymerase chain reaction was used to measure mRNA transcripts for markers of hepatic inflammation, HSC activation, and fibrosis. Immunohistochemistry for alpha-smooth muscle actin was performed to identify HSC activation. Collagen deposition was quantitated by Sirius red staining and digital imaging techniques. Hepatocyte apoptosis and liver injury (bile infarcts and serum ALT values) were reduced in IDN-6556-treated versus saline-treated 3-day BDL mice. Markers for liver inflammation [chemokine (C-X-C) ligand 1 and macrophage inflammatory protein-2 chemokine expression] and hepatic fibrogenesis (transforming growth factor-beta and collagen I expression) were also attenuated. Consistent with these data, HSC activation as assessed by alpha-smooth muscle actin mRNA expression and immunohistochemistry was markedly reduced in both 3- and 10-day BDL animals. Collectively, these data suggest hepatocyte apoptosis initiates cascades culminating in liver injury and fibrosis. The pan-caspase inhibitor IDN-6556 is a promising agent for cholestatic liver injury.