A case report on a large, peduncular intra-abdominal hepatocellular carcinoma extending into the retroperitoneum.

Here, we discuss a relatively uncommon presentation of a hepatocellular carcinoma and discuss its preoperative planning and surgical intervention required to reach complete resection.

Electrodiagnostic and Advanced Neuroimaging Characterization for Successful Treatment of Spinal Extradural Arachnoid Cyst.

BACKGROUND: Spinal extradural arachnoid cysts are relatively uncommon. Rarely, large cysts presented with spinal cord compression requiring surgical intervention. CASE DESCRIPTION: We report a patient with a progressively enlarging spinal extradural arachnoid cyst causing worsening right S1 radiculopathy and gastrocnemius muscle atrophy. Electromyography and nerve conduction studies revealed an S1 motor radiculopathy. Serial magnetic resonance imaging findings confirmed enlargement of the small cyst originating from the sacral thecal sac on the right while 2 smaller cysts on the left remained stable. Dynamic computed tomography myelogram revealed connection to the thecal sac behind the right S1 nerve root. We performed a right hemilaminectomy from L5 to S2, exposed the cyst pedicle ligated it, and marsupialized the cyst. After surgery, the patient showed clinical and electrodiagnostic improvement. CONCLUSION: This case illustrates the principles of timely surgical intervention after advanced diagnostic imaging and electrodiagnostic testing to improve neurologic function and minimize complications.

Cannabidiol attenuates alcohol-induced liver steatosis, metabolic dysregulation, inflammation and neutrophil-mediated injury.

Cannabidiol (CBD) is a non-psychoactive component of marijuana, which has anti-inflammatory effects. It has also been approved by FDA for various orphan diseases for exploratory trials. Herein, we investigated the effects of CBD on liver injury induced by chronic plus binge alcohol feeding in mice. CBD or vehicle was administered daily throughout the alcohol feeding study. At the conclusion of the feeding protocol, serums samples, livers or isolated neutrophils were utilized for molecular biology, biochemistry and pathology analysis. CBD significantly attenuated the alcohol feeding-induced serum transaminase elevations, hepatic inflammation (mRNA expressions of TNFα, MCP1, IL1ß, MIP2 and E-Selectin, and neutrophil accumulation), oxidative/nitrative stress (lipid peroxidation, 3-nitrotyrosine formation, and expression of reactive oxygen species generating enzyme NOX2). CBD treatment also attenuated the respiratory burst of neutrophils isolated from chronic plus binge alcohol fed mice or from human blood, and decreased the alcohol-induced increased liver triglyceride and fat droplet accumulation. Furthermore, CBD improved alcohol-induced hepatic metabolic dysregulation and steatosis by restoring changes in hepatic mRNA or protein expression of ACC-1, FASN, PPARα, MCAD, ADIPOR-1, and mCPT-1. Thus, CBD may have therapeutic potential in the treatment of alcoholic liver diseases associated with inflammation, oxidative stress and steatosis, which deserves exploration in human trials.

Pepcan-12 (RVD-hemopressin) is a CB2 receptor positive allosteric modulator constitutively secreted by adrenals and in liver upon tissue damage.

Pepcan-12 (RVD-hemopressin; RVDPVNFKLLSH) is the major peptide of a family of endogenous peptide endocannabinoids (pepcans) shown to act as negative allosteric modulators (NAM) of cannabinoid CB1 receptors. Noradrenergic neurons have been identified to be a specific site of pepcan production. However, it remains unknown whether pepcans occur in the periphery and interact with peripheral CB2 cannabinoid receptors. Here, it is shown that pepcan-12 acts as a potent (K i value ~50 nM) hCB2 receptor positive allosteric modulator (PAM). It significantly potentiated the effects of CB2 receptor agonists, including the endocannabinoid 2-arachidonoyl glycerol (2-AG), for [35S]GTPγS binding and cAMP inhibition (5-10 fold). In mice, the putative precursor pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH) was identified with pepcan-12 in brain, liver and kidney. Pepcan-12 was increased upon endotoxemia and ischemia reperfusion damage where CB2 receptors play a protective role. The adrenals are a major endocrine site of production/secretion of constitutive pepcan-12, as shown by its marked loss after adrenalectomy. However, upon I/R damage pepcan-12 was strongly increased in the liver (from ~100 pmol/g to ~500 pmol/g) independent of adrenals. The wide occurrence of this endogenous hormone-like CB2 receptor PAM, with unforeseen opposite allosteric effects on cannabinoid receptors, suggests its potential role in peripheral pathophysiological processes.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis.

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvß6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.

The novel, orally available and peripherally restricted selective cannabinoid CB2 receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity.

BACKGROUND AND PURPOSE: Here, we have characterized 3-cyclopropyl-1-(4-(6-((1,1-dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazolidine-2,4-dione hydrochloride (LEI-101) as a novel, peripherally restricted cannabinoid CB2 receptor agonist, using both in vitro and in vivo models. EXPERIMENTAL APPROACH: We investigated the effects of LEI-101 on binding and functional activity. We assessed its in vitro and in vivo selectivity. Efficacy of LEI-101 was determined in a mouse model of cisplatin-induced nephrotoxicity. KEY RESULTS: LEI-101 behaved as a partial agonist at CB2 receptors using ß-arrestin and GTPγS assays and was ~100-fold selective in CB2 /CB1 receptor-binding assays. It did not display any activity on endocannabinoid hydrolases and nor did it react with serine hydrolases in an activity-based protein profiling assay. In mice, LEI-101 had excellent oral bioavailability reaching high concentrations in the kidney and liver with minimal penetration into the brain. LEI-101 up to a dose of 60 mg·kg(-1) (p.o.) did not exert any CNS-mediated effects in the tetrad assay, in mice. LEI-101 (p.o. or i.p.) at 3 or 10 mg·kg(-1) dose-dependently prevented kidney dysfunction and/or morphological damage induced by cisplatin in mice. These protective effects were associated with improved renal histopathology, attenuated oxidative stress and inflammation in the kidney. These effects were absent in CB2 receptor knockout mice. CONCLUSION AND IMPLICATIONS: These results indicate that LEI-101 is a selective, largely peripherally restricted, orally available CB2 receptor agonist with therapeutic potential in diseases that are associated with inflammation and/or oxidative stress, including kidney disease.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis.

UNLABELLED: Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. CONCLUSION: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.

Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice.

BACKGROUND & AIMS: The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults. METHODS: We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type 2(-/-) (CB2-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses. RESULTS: Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB2 and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4. CONCLUSIONS: MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2 h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6 h of reperfusion and peaking at 24 h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.

Interferon-γ induces autophagy with growth inhibition and cell death in human hepatocellular carcinoma (HCC) cells through interferon-regulatory factor-1 (IRF-1).

Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, anti-viral, and anti-proliferative effects. In this study, we examined the effects of IFN-γ on autophagy and cell growth in human hepatocellular carcinoma (HCC) cells. IFN-γ inhibited cell growth of Huh7 cells with non-apoptotic cell death. IFN-γ induced autophagosome formation and conversion/turnover of microtubule associated protein 1 light chain 3 (LC3) protein. Furthermore, overexpression of IRF-1 also induced autophagy in Huh7 cells. Silencing IRF-1 expression with target small hairpin RNA blocked autophagy induced by IFN-γ. Silencing of the autophagy signals Beclin-1 or Atg5 attenuated the inhibitory effect of IFN-γ on Huh7 cells with decreased cell death. Additionally, IFN-γ activated autophagy in freshly cultured human HCC cells. Together, these findings show that IFN-γ induces autophagy through IRF-1 signaling pathway and the induction of autophagy contributes to the growth-inhibitory effect of IFN-γ with cell death in human liver cancer cells.

Mast cells play a critical role in the systemic inflammatory response and end-organ injury resulting from trauma.

BACKGROUND: Much of the morbidity after trauma results from excessive activation of the innate immune system. This is manifested as a systemic inflammatory response and associated end-organ damage. Although mast cells are known to be important in many immune responses, their role in the systemic response to severe trauma is unknown. STUDY DESIGN: C57BL/6J-KitW-sh/BsmJ (mast cell deficient) and wild type mice were subjected to 1.5 hours of hemorrhagic shock plus bilateral femur fracture and soft tissue injury (HS/T), followed by resuscitation at 4.5 hours. Blood withdrawal volumes, mean arterial pressures, circulating cytokine, chemokine, high mobility group box-1 (HMGB-1), double strain DNA (dsDNA), transaminase levels, and histology in liver and lung were compared between groups. RESULTS: Mast cell deficient mice exhibited greater hemodynamic stability than wild type mice. At baseline, the mast cell deficient mice exhibited no difference in any of the organ injury or inflammatory markers measured. As expected, wild type mice subjected to HS/T exhibited end-organ damage manifested by marked increases in circulating alanine transaminase, aspartate aminotransferase, and dsDNA levels, as well as histologic evidence of tissue necrosis. In clear contrast, mast cell deficient mice exhibited almost no tissue damage. Similarly, the magnitude of increased circulating cytokine and chemokine induced by HS/T was much less in the mast cell deficient mice than in the wild type group. CONCLUSIONS: Mast cell deficiency resulted in a damped systemic inflammatory response, greatly attenuated multiple organ injury, and more stable hemodynamics in HS/T. So mast cells appear to be a critical component of the initial host response to severe injury.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

A critical role for IFN regulatory factor 1 in NKT cell-mediated liver injury induced by alpha-galactosylceramide.

NKT cells are remarkably abundant in mouse liver. Compelling experimental evidence has suggested that NKT cells are involved in the pathogenesis of many liver diseases. Activation of NKT cells with alpha-galactosylceramide (alpha-GalCer) causes liver injury through mechanisms that are not well understood. We undertook studies to characterize the key pathways involved in alpha-GalCer-induced liver injury. We found that expression of the transcription factor IFN regulatory factor 1 (IRF-1) in mouse liver was dramatically upregulated by alpha-GalCer treatment. Neutralization of either TNF-alpha or IFN-gamma inhibited alpha-GalCer-mediated IRF-1 upregulation. alpha-GalCer-induced liver injury was significantly suppressed in IRF-1 knockout mice or in wild-type C56BL/6 mice that received a microRNA specifically targeting IRF-1. In contrast, overexpression of IRF-1 greatly potentiated alpha-GalCer-induced liver injury. alpha-GalCer injection also induced a marked increase in hepatic inducible NO synthase expression in C56BL/6 mice, but not in IRF-1 knockout mice. Inducible NO synthase knockout mice exhibited significantly reduced liver injury following alpha-GalCer treatment. Finally, we demonstrated that both NKT cells and hepatocytes expressed IRF-1 in response to alpha-GalCer. However, it appeared that the hepatocyte-derived IRF-1 was mainly responsible for alpha-GalCer-induced liver injury, based on the observation that inhibition of IRF-1 by RNA interference did not affect alpha-GalCer-induced NKT cell activation. Our findings revealed a novel mechanism of NKT cell-mediated liver injury in mice, which has implications in the development of human liver diseases.

Donor graft interferon regulatory factor-1 gene transfer worsens liver transplant ischemia/reperfusion injury.

BACKGROUND: Liver ischemia and reperfusion (IR) injury is a phenomenon that leads to graft dysfunction after liver transplantation. Understanding the molecular mechanisms behind this process is crucial to developing strategies to prevent short- and long-term graft dysfunction. The purpose of this study was to explore the role of the transcription factor interferon regulatory factor-1 (IRF-1) in a model of orthotopic rat liver transplantation. METHODS: Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 3 hours preservation in cold University of Wisconsin solution. Adenovirus-expressing IRF-1 (AdIRF-1) or control gene vector (Adnull) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as controls. Recipients were humanely killed 1-24 hours post-transplantation. RESULTS: Rats that underwent OLT with long-term preserved grafts (18 hours) displayed increased hepatic nuclear expression of IRF-1 protein at 1 and 3 hours. Rats pretreated with AdIRF-1 before transplantation had elevated alanine aminotransferase levels and increased expression of interferon (IFN)-beta, IFN-gamma, interleukin-12, and inducible nitric oxide synthase in the short-term period (3 hours) when compared with donor livers pretreated with Adnull. AdIRF-1 pretreated donor livers also exhibited increased susceptibility to early apoptosis in the transplanted grafts as shown by increased terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and expression of cleaved caspase-3. Additionally, AdIRF-1 pretreated donor livers had increased activation of the MAP kinase Jun N-terminal kinase as compared with Adnull pretreated donor livers. CONCLUSION: IRF-1 is an important regulator of IR injury after OLT in rats. Targeting of IRF-1 may be a potential strategy to ameliorate ischemic liver injury after transplantation to minimize organ dysfunction.

Preactivation of NKT cells with alpha-GalCer protects against hepatic ischemia-reperfusion injury in mouse by a mechanism involving IL-13 and adenosine A2A receptor.

Hepatic preconditioning has emerged as a promising strategy of activating natural pathways to augment tolerance to liver ischemia-reperfusion (IR) injury. Liver-resident natural killer T (NKT) cells play an important role in modulating the local immune and inflammatory responses. This work was aimed to investigate whether preactivation of NKT cells could provide a beneficial "preconditioning" effect to ameliorate the subsequent hepatic IR injury. To selectively activate NKT cells, C57BL/6 mice were treated intraperitoneally with the glycolipid antigen alpha-galactosylceramide (alpha-GalCer) 1 h prior to hepatic ischemia. Significantly reduced liver IR injury was observed in mice pretreated with alpha- GalCer, and this protective effect was specifically abrogated by a CD1d blocking antibody. Serum TNF-alpha, IFN-gamma, and IL-13 levels were markedly increased shortly after alpha-GalCer injection. Pretreatment with a neutralizing antibody against TNF-alpha or IFN-gamma did not influence the protective effect of alpha-GalCer preconditioning, whereas preadministration of an IL-13 neutralizing antibody completely abolished the effect. Treatment with alpha-GalCer also led to an increased expression of adenosine A2A receptor (A2AR) in the liver, and blockade of A2AR by SH58261 diminished alpha-GalCer pretreatment-mediated attenuation of liver IR injury. In contrast, administration of the selective A2AR agonist CGS21680 reversed the counteracting effect of the IL-13 neutralizing antibody on alpha-GalCer preconditioning. Additionally, alpha-GalCer pretreatment was associated with a decreased neutrophil accumulation in the ischemic liver. These findings provide the first evidence that hepatic preconditioning by preactivation of NKT cells with alpha-GalCer protects the liver from IR injury via an IL-13 and adenosine A2AR-dependent mechanism.

Wnt/beta-catenin signaling regulates cytokine-induced human inducible nitric oxide synthase expression by inhibiting nuclear factor-kappaB activation in cancer cells.

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor kappaB (NF-kappaB) and has recently been shown to be a target of the Wnt/beta-catenin pathway. In this study, we tested the hypothesis that Wnt/beta-catenin signaling might regulate cytokine- or tumor necrosis factor alpha (TNFalpha)-induced hiNOS expression through interaction with NF-kappaB. A cytokine mixture of TNFalpha + interleukin (IL)-1beta + IFNgamma induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of beta-catenin produced a dose-dependent decrease in NF-kappaB reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNFalpha-induced hiNOS NF-kappaB activation showed decreased p50 binding and decreased NF-kappaB reporter activity in the beta-catenin-mutant HAbeta18 cells. Conversely, enhanced p50 binding and increased NF-kappaB reporter activity were seen in HAbeta85 cells, which lack beta-catenin signaling. Coimmunoprecipitation confirmed that beta-catenin complexed with both p65 and p50 NF-kappaB proteins. NF-kappaB-dependent Traf1 protein expression also inversely correlated with the level of beta-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased beta-catenin protein and increased TNFalpha-induced p65 NF-kappaB binding as well as iNOS and Traf1 expression. Finally, beta-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that beta-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-kappaB-dependent gene expression. These findings underscore the complex role of Wnt/beta-catenin, NF-kappaB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis.

Glucose up-regulates HIF-1 alpha expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status.

It has been suggested that hypoxia-inducible factor 1 (HIF-1), a key regulator in cell's adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up-regulated the expression of HIF-1alpha, the oxygen-dependent subunit of HIF-1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose-regulated HIF-1alpha expression, we investigated the relationships between HIF-1alpha expression, reactive oxygen species (ROS), and redox status. Low levels of HIF-1alpha protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa. The glutathione (GSH) precursor, N-acetyl cysteine, induced HIF-1alpha protein expression in hypoxic neurons while the GSH synthesis inhibitor, l-buthionine sulfoximine, inhibited the expression. Moreover, (-)-epicatechin gallate, a ROS scavenger, elevated HIF-1alpha expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF-1alpha expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF-1alpha stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF-1alpha and that a reducing environment is required to stabilize HIF-1alpha in neurons exposed to hypoxia.

HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling.

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.