l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.

Background & Aims-Improving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver.

A number of laboratories have sought to elucidate the role of nitric oxide (NO) in both acute and chronic inflammatory diseases. It is now well appreciated that NO can influence many aspects of the inflammatory cascade ranging from its own expression to recruitment of leucocytes to the effected tissue. With the advent of mice selectively deficient in the various isoforms of nitric oxide synthase (NOS), the role that NO may play in various disease states can now be examined in vivo. One such syndrome that has gained much attention in recent years is ischaemia and reperfusion-induced tissue injury. Ischaemia-reperfusion (I/R) injury is an important clinical consideration in situations such as transplantation, trauma, liver or bowel resection and haemorrhagic shock. A hallmark of I/R is the production of reactive oxygen species (ROS) during the reperfusion phase and it is thought that the production of ROS mediate much of the post-ischaemic tissue injury. This review will examine the current state of knowledge regarding the regulatory mechanisms by which NO can influence various aspects of the inflammatory cascade as well as its role in a model of I/R injury in vivo.

The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle ␣-actin (␣SMA) promoter was generated (AdSMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced. Conclusion: Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis. (HEPATOLOGY 2009;50:1512-1523

The mouse model of liver ischemia and reperfusion injury has proven to be valuable for our understanding of the role that reactive oxygen and nitrogen metabolites play in postischemic tissue injury. This methods paper provides a detailed protocol for inducing partial liver ischemia followed by reperfusion. Liver ischemia is induced in anesthetized mice by cross-clamping the hepatic artery and portal vein for varying lengths of time resulting in deprivation of blood flow to approximately of 70% of the liver. Restoration of blood flow to the ischemic lobes enhances superoxide production concomitant with a rapid and marked decrease in the bioavailability of nitric oxide resulting in alterations in the redox state of the liver in favor of a more oxidative environment. This hepatocellular oxidative stress induces the activation of oxidant-sensitive transcription factors followed by the upregulation of pro-inflammatory cytokines and mediators that ultimately lead to liver injury. This model can be induced in any strain or sex of mouse and requires 1-2 months of practice to become proficient in the surgery and animal manipulation. The role of different reactive metabolites of oxygen and nitrogen may be evaluated using genetically-engineered mice as well as selective molecular, cellular and/or pharmacological agents.

Ethanol consumption is known to cause significant acute liver damage resulting in hepatic fibrosis and eventual cirrhosis when consumed chronically. The mechanism(s) by which ethanol exerts its damaging effects on the liver are not well understood; however, recent scientific investigation has begun to delineate the earliest events in alcoholic liver disease. From these studies, it is apparent that components of the innate immune system and, in particular, Kupffer cells, play a significant role in this process. It is also becoming clear that other parts of the immune system including T cells may also be responsible for mediating the devastating effects of chronic alcohol consumption on the liver. This review will highlight recent experiments demonstrating a role for the innate immune response in the initiation and progression of alcohol-induced liver hepatitis and subsequent organ damage.

Background Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNFα) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic NKT cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Methods Male C57Bl/6 wild type (Wt) and IL-12-deficient (IL12−/−) mice were fed a choline deficient diet (CDD) for 0, 10 or 20 weeks. Findings CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function and increased hepatic expression of the Th1-type cytokines IL-12, interferon gamma (IFNγ) and TNFα in wt mice. Absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFNγ and TNFα expression. Treatment of CDD fed mice with lipopolysaccharide led to a significant increase in hepatic IL12 expression and Kupffer cell (KC)_depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD fed mice failed to produce increased quantities of IL12 upon activation in vitro when compared to similarly treated KCs from control fed mice suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT and NK cells. Conclusions Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC and IL-12-dependent manner. Our results suggest a pivotal and multi-functional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process which is likely active within human non-alcoholic fatty liver disease.

Although it is clear that bile acid accumulation is the major initiator of fibrosis caused by cholestatic liver disease, endotoxemia is a common side effect. However, the depletion of hepatic macrophages with gadolinium chloride blunts hepatic fibrosis. Because endotoxin is a key activator of hepatic macrophages, this study was designed to test the hypothesis that LPS signaling through CD14 contributes to hepatic fibrosis caused by experimental cholestasis. Wild-type mice and CD14 knockout mice (CD14(-/-)) underwent sham operation or bile duct ligation and were killed 3 wk later. Measures of liver injury, such as focal necrosis, biliary cell proliferation, and inflammatory cell influx, were not significantly different among the strains 3 wk after bile duct ligation. Markers of liver fibrosis such as Sirius red staining, liver hydroxyproline, and alpha-smooth muscle actin expression were blunted in CD14(-/-) mice compared with wild-type mice after bile duct ligation. Despite no difference in lymphocyte infiltration, the macrophage/monocyte activation marker OX42 (CD11b) and the oxidative stress/lipid peroxidation marker 4-hydroxynonenal were significantly upregulated in wild-type mice after bile duct ligation but not in CD14(-/-) mice. Increased profibrogenic cytokine mRNA expression in the liver after bile duct ligation was significantly blunted in CD14(-/-) mice compared with the wild type. The hypothesis that LPS was involved in experimental cholestatic liver fibrosis was tested using mice deficient in LPS-binding protein (LBP(-/-)). LBP(-/-) mice had less liver injury and fibrosis (Siruis red staining and hydroxyproline content) compared with wild-type mice after bile duct ligation. In conclusion, these data demonstrate that endotoxin in a CD14-dependent manner exacerbates hepatic fibrogenesis and macrophage activation to produce oxidants and cytokines after bile duct ligation.