Matthias Froh

Reduced Early Alcohol-Induced Liver Injury in CD14-Deficient Mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 ± 0.3%) was increased significantly by ethanol (7.3 ± 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 ± 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 ± 0.3, p < 0.05). Additionally, NF-κB, TGF-β, and TNF-α were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Role of Lipopolysaccharide-Binding Protein in Early Alcohol-Induced Liver Injury in Mice

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-α participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 ± 31 U/L) over high-fat controls (24 ± 7 U/L), effects which were blunted significantly in LBP knockout mice (60 ± 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 ± 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 ± 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-α mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.

Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF‐κB activation and cytokine production

Kupffer cells play a significant role in the pathogenesis of several liver diseases; therefore, a potential therapeutic strategy would be to inactivate the Kupffer cell with a gene‐delivery system. Although recombinant adenovirus provides robust, transgene expression in parenchymal cells, whether adenovirus transduces Kupffer cells is unclear. Thus, the purpose of this study was to evaluate this possibility. In animals infected with adenovirus, Kupffer cells were identified positively to express adenoviral transgenes by immunohistochemical techniques and Western blot analysis, indicating that Kupffer cells are transduced in vivo. Indeed, isolated Kupffer cells were transduced in vitro with recombinant adenovirus in a dose‐dependent manner. Moreover, adenoviral transduction of Kupffer cells was blocked by inhibitors of αVβ5 integrin, the co‐receptor for adenovirus binding, supporting the hypothesis that adenovirus transduces Kupffer cells via an αVβ5 integrin‐dependent mechanism. Indeed, it is shown here that Kupffer cells express αVβ5 integrins. In a functional assay, infection of isolated Kupffer cells with adenovirus containing superoxide dismutase or IκBα super‐repressor blunted LPS‐induced nuclear transcription factor kappa B (NF‐κB) activation and tumor necrosis factor α (TNF‐α) production but not IL‐10 production. Moreover, superoxide production was blocked by expression of superoxide dismutase. These data support the hypothesis that LPS‐induced NF‐κB activation and TNF‐α production in Kupffer cells are oxidant‐dependent. These findings suggest that Kupffer cell‐targeted approaches may be a potential therapeutic strategy against many inflammatory diseases including early alcohol‐induced liver injury.

TNFα is required for cholestasis-induced liver fibrosis in the mouse

TNFalpha, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFalpha knockout mice (TNFalpha-/-). Survival after BDL was 60% in wild-type mice (TNFalpha+/+) and 90% in TNFalpha-/- mice. Body weight loss and liver to body weight ratios were reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFalpha+/+ mice (268.6+/-28.2U/L) compared to TNFalpha-/- mice (105.9U/L+/-24.4). TNFalpha-/- mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, alpha-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-beta mRNA, a profibrogenic cytokine, were markedly reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Thus, our data indicate that TNFalpha induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury

The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress.

Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat.

Background. Oxygen‐derived free radicals play a central role in ischemia/reperfusion injury after organ transplantation and are degraded by endogenous radical scavengers such as superoxide dismutase (SOD). Overexpression of SOD by delivery of the cytosolic SOD gene with an adenovirus (Ad.SOD1) decreases organ injury and increases survival in a rat model of liver transplantation. However, it is unclear which of the three isoforms of SOD provides the most protective effect. The purpose of this study was to identify the isoform with the highest effectiveness against ischemia/reperfusion injury after transplantation of fatty livers, which are particularly susceptible. Methods. Donor rats were given ethanol by gavage before harvest to induce steatotic livers. Some of the donors were infected with adenoviruses expressing either the gene lacZ encoding bacterial &bgr;‐galactosidase (Ad.lacZ), Ad.SOD1, Ad.SOD2 (mitochondrial isoform), or Ad.SOD3 (extracellular isoform). After transplantation, SOD activity in liver, survival, histopathology, transaminases, and activation of nuclear factor (NF)‐&kgr;B, I&kgr;B kinase, Jun‐N‐terminal kinase (JNK), and tumor necrosis factor (TNF)‐&agr; were evaluated. Results. Ad.SOD1 treatment increased survival, blunted transaminase release, and reduced necrosis, whereas Ad.SOD3 had no protective effect. Ad.SOD2 was not as protective as Ad.SOD1. Ad.SOD1 reduced the activation of NF‐&kgr;B, blunted JNK activity, and reduced TNF‐&agr; activity. Ad.SOD2 treatment resulted in lower kinase, TNF‐&agr;, and NF‐&kgr;B activities but was not as effective as Ad.SOD1. I&kgr;B kinase activity was not affected. Conclusion. This study demonstrates that cytosolic SOD represents the most effective isoform of SOD to protect transplanted livers from failure; this may be related to lowered NF‐&kgr;B and JNK activities because of reduced oxygen‐derived radical production.

Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation

Abstract Background and aims. Sclerosing cholangitis in critically ill patients (SC-CIP) with sepsis and acute respiratory distress syndrome (ARDS) is a cholestatic liver disease with a rapid progression to liver cirrhosis and hepatic failure. Data on outcome of these patients after liver transplantation (LT) are sparse. Patients and methods. Eleven patients (46 ± 12 years; mean labMELD-score: 27 ± 7) with SC-CIP underwent LT. Six patients had severe polytrauma with multiple bone fractures, sepsis and ARDS. Five non-traumatic patients acquired SC-CIP during long-term intensive-care-unit stays due to sepsis and ARDS. Time to diagnosis, the microbiologic results and the survival rates after LT were evaluated. Results. SC-CIP was diagnosed by endoscopic retrograde cholangiopancreatography (ERCP) within 3 ± 1 months after manifestation of cholestasis and histologically confirmed in explanted livers. The predominant microorganisms isolated in bile were: Enterococcus and Candida albicans. Mean follow-up after LT was 28 ± 20 months. One female patient (non-traumatic) died due to sepsis 26 days after LT. All other patients left the hospital alive, but two (non-traumatic) patients died from sepsis, and one (traumatic) patient died in a hemorrhagic shock, thereafter. Seven of 11 patients (5 with polytrauma) are still alive and have a good quality of life. The survival of the SC-CIP patients after LT was comparable with that of patients transplanted due to alcoholic liver cirrhosis. Conclusion. SC-CIP develops rapidly within several months. Enterococcus and C. albicans were the main isolated microorganisms in the bile. Sepsis was the main cause of death after LT. Overall, SC-CIP is a good indication for LT in selected patients.

TNF α‐induced ras activation due to ethanol promotes hepatocyte proliferation independently of liver injury in the mouse

Tumor necrosis factor α (TNFα) has been shown to be both proapoptotic and mitogenic for hepatocytes and necessary for alcohol‐induced liver injury. Ras, a known proto‐oncogene, is very important in the regulation of cellular responses to TNFα. Therefore, the purpose of this study was to investigate the role of Ras in alcohol‐induced pathogenesis. Male C57Bl/6 mice were fed ethanol or high‐fat control diet via intragastric cannulation for 4 weeks. Ras activity was increased significantly after 4 weeks of ethanol and correlated with an increase in pathologic features. However, in mice deficient in the receptor‐type 1 for TNFα (TNFR1‐/‐), ethanol‐induced liver injury and the increase in Ras activity were significantly blunted compared with wild‐type mice. Furthermore, it was demonstrated that H‐, K‐, and R‐Ras isoforms were increased after ethanol exposure in wild‐type mice. In TNFR1‐/‐ mice, R‐Ras activity remained elevated by ethanol, whereas H‐Ras and K‐Ras activity was blunted significantly under these conditions. Interestingly, hepatocellular proliferation, which was elevated approximately fivefold after 4 weeks of chronic ethanol in wild‐type mice, was also blunted in TNFR1‐/‐ mice given ethanol. Inhibition of Ras with adenovirus containing a dominant‐negative Ras had no effect on ethanol‐induced liver injury, but significantly blunted ethanol‐induced hepatocyte proliferation by more than 50%. Overexpression of mitochondrial superoxide dismutase using recombinant adenovirus blunted lipid peroxidation and attenuated hepatic injury resulting from ethanol, but had no effect on Ras activation and hepatocyte proliferation caused by ethanol. In conclusion, these data support the hypotheses that hepatocellular oxidative stress leads to cell death and that TNFα‐induced Ras activation is important in hepatic proliferation in response to ethanol‐induced liver injury. (HEPATOLOGY 2004;39:721–731.)

M1567 CpG Motifs Induce Proinflammatory Events in Hepatic Stellate Cells and Are Involved in Bile Duct Ligation-Induced Hepatic Fibrosis In Vivo

periphery of the fibrotic bands. Control animals, for each group had normal liver histology and SA-βgal was negative. The expression of p16, p21, and p53, was positive since early stages, with a significant increase of p16 from 15% in-group 1 to 70% in-group 3 that declined after withdrawal of TAA to 16%. No significant changes were noted in p21 and p53 labeling indexes. Conclusions: This study provides evidence that cellular senescence applies to liver cirrhosis and carries a risk of cell cycle checkpoints alterations that may affect liver regeneration at the cirrhotic stage. The selective loss of p16 after withdrawal of TAA may allow clonal expansion of hepatocytes, which may be relevant to understanding hepatocarcinogenesis This work was supported in part by grants UNAM, PAPIIT IN-208107, and CONACyT Salud 2004-CO1-175.