Svetlana Radaeva

Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity

Endogenous cannabinoids acting at CB(1) receptors stimulate appetite, and CB(1) antagonists show promise in the treatment of obesity. CB(1) (-/-) mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB(1) in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB(1) agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB(1). High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB(1) density, and basal rates of fatty acid synthesis, and the latter is reduced by CB(1) blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB(1) ligands. We conclude that anandamide acting at hepatic CB(1) contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation.

Interleukin 22 (IL‐22) plays a protective role in T cell‐mediated murine hepatitis: IL‐22 is a survival factor for hepatocytes via STAT3 activation

The central role of T cell activation in hepatocellular injury has been well documented. In this article, we provide evidence suggesting that T cells may also play a protective role in liver disease by releasing interleukin‐22 (IL‐22), a recently identified T cell‐derived cytokine whose biological significance is unclear. IL‐22 messenger RNA and protein expression are significantly elevated in T cell‐mediated hepatitis induced by concanavalin A (ConA) but are less extensively elevated in the carbon tetrachloride‐induced liver injury model. Activated CD3+ T cells are likely responsible for the production of IL‐22 in the liver after injection of ConA. The IL‐22 receptor is normally expressed at high levels by hepatocytes and further induced after ConA injection. IL‐22 blockade with a neutralizing antibody reduces signal transducer and activator of transcription factor 3 (STAT3) activation and worsens liver injury in T cell‐mediated hepatitis, whereas injection of recombinant IL‐22 attenuates such injury. In vitro treatment with recombinant IL‐22 or overexpression of IL‐22 promotes cell growth and survival in human hepatocellular carcinoma HepG2 cells. Stable overexpression of IL‐22 in HepG2 cells constitutively activates STAT3 and induces expression of a variety of antiapoptotic (e.g., Bcl‐2, Bcl‐xL, Mcl‐1) and mitogenic (e.g., c‐myc, cyclin D1, Rb2, CDK4) proteins. Blocking STAT3 activation abolishes the antiapoptotic and mitogenic actions of IL‐22 in hepatic cells. In conclusion, the T cell‐derived cytokine IL‐22 is a survival factor for hepatocytes; this suggests that T cell activation may also prevent and repair liver injury by releasing hepatoprotective cytokine IL‐22 in addition to its previously documented central role in hepatocellular injury. (HEPATOLOGY 2004;39:1332–1342.)

Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases

Hepatic lymphocytes are enriched in NK and NKT cells that play important roles in antiviral and antitumor defenses and in the pathogenesis of chronic liver disease. In this review, we discuss the differential distribution of NK and NKT cells in mouse, rat, and human livers, the ultrastructural similarities and differences between liver NK and NKT cells, and the regulation of liver NK and NKT cells in a variety of murine liver injury models. We also summarize recent findings about the role of NK and NKT cells in liver injury, fibrosis, and repair. In general, NK and NKT cells accelerate liver injury by producing proinflammatory cytokines and killing hepatocytes. NK cells inhibit liver fibrosis via killing early–activated and senescent–activated stellate cells and producing IFN–γ. In regulating liver fibrosis, NKT cells appear to be less important than NK cells as a result of hepatic NKT cell tolerance. NK cells inhibit liver regeneration by producing IFN–γ and killing hepatocytes; however, the role of NK cells on the proliferation of liver progenitor cells and the role of NKT cells in liver regeneration have been controversial. The emerging roles of NK/NKT cells in chronic human liver disease will also be discussed. Understanding the role of NK and NKT cells in the pathogenesis of chronic liver disease may help us design better therapies to treat patients with this disease.

Endocannabinoids Acting at Cannabinoid-1 Receptors Regulate Cardiovascular Function in Hypertension

Background—Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension. Methods and Results—In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB1) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats. Conclusions—We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB1-mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Opposing roles of STAT1 and STAT3 in T cell–mediated hepatitis: regulation by SOCS

T cell-mediated fulminant hepatitis is a life-threatening event for which the underlying mechanism is not fully understood. Injection of concanavalin A (Con A) into mice recapitulates the histological and pathological sequelae of T cell-mediated hepatitis. In this model, both signal transducer and activator of transcription factor 1 (STAT1) and STAT3 are activated in the liver. Disruption of the STAT1 gene by way of genetic knockout attenuates liver injury, suppresses CD4(+) and NK T cell activation, and downregulates expression of proapoptotic interferon regulatory factor-1 protein and suppressor of cytokine signaling-1 (SOCS1), but enhances STAT3 activation and STAT3-controlled antiapoptotic signals. Studies from IFN-gamma-deficient mice indicate that IFN-gamma not only is the major cytokine responsible for STAT1 activation but also partially accounts for STAT3 activation. Moreover, downregulation of STAT3 activation in IL-6-deficient mice is associated with decreased STAT3-controlled antiapoptotic signals and expression of SOCS3, but upregulation of STAT1 activation and STAT1-induced proapoptotic signals and exacerbation of liver injury. Taken together, these findings suggest that STAT1 plays a harmful role in Con A-mediated hepatitis by activation of CD4(+) and NK T cells and directly inducing hepatocyte death, whereas STAT3 protects against liver injury by suppression of IFN-gamma signaling and induction of antiapoptotic protein Bcl-X(L). STAT1 and STAT3 in hepatocytes also negatively regulate one another through the induction of SOCS.

STAT1 Inhibits Liver Fibrosis in Mice by Inhibiting Stellate Cell Proliferation and Stimulating NK Cell Cytotoxicity

Liver fibrosis, a common scarring response to chronic liver injury, is a precursor to cirrhosis and liver cancer. Here, we identified signal transducer and activator of transcription 1 (STAT1) as an important negative regulator in liver fibrosis. Our findings show that disruption of the STAT1 gene accelerated liver fibrosis and hepatic stellate cell (HSC) proliferation in an in vivo model of carbon tetrachloride (CCl4)‐induced liver fibrosis. In vitro treatment with IFN‐γ inhibited proliferation and activation of wild‐type HSCs, but not STAT1−/− HSCs. Moreover, compared to wild‐type cells, cellular proliferation stimulated by serum or platelet‐derived growth factor (PDGF) was enhanced and accelerated in STAT1−/− HSCs, which was partially mediated via elevated PDGF receptor β expression on such cells. Polyinosinic‐polycytidylic acid (poly I:C) or IFN‐γ treatment inhibited liver fibrosis in wild‐type mice but not in STAT1−/− mice. Induction of NK cell killing of activated HSCs by poly I:C was attenuated in STAT1−/− mice compared to wild‐type mice, which was likely due to reduced NKG2D and TRAIL expression on STAT1−/− NK cells. Finally, activation of TGF‐β/Smad3 signaling pathway was accelerated, whereas induction of Smad7 was diminished in the liver of STAT1−/− mice after CCl4 administration compared to wild‐type mice. In conclusion, activation of STAT1 attenuates liver fibrosis through inhibition of HSC proliferation, attenuation of TGF‐β signaling, and stimulation of NK cell killing of activated HSCs. STAT1 could be a new therapeutic target for treating liver fibrosis. (HEPATOLOGY 2006;44:1441–1451.)

Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease

Fatty liver, formerly associated predominantly with excessive alcohol intake, is now also recognized as a complication of obesity and an important precursor state to more severe forms of liver pathology including ischemia/reperfusion injury. No standard protocol for treating fatty liver exists at this time. We therefore examined the effects of 10 days of interleukin 6 (IL‐6) injection in 3 murine models of fatty liver: leptin deficient ob/ob mice, ethanol‐fed mice, and mice fed a high‐fat diet. In all 3 models, IL‐6 injection decreased steatosis and normalized serum aminotransferase. The beneficial effects of IL‐6 treatment in vivo resulted in part from an increase in mitochondrial β oxidation of fatty acid and an increase in hepatic export of triglyceride and cholesterol. However, administration of IL‐6 to isolated cultured steatotic hepatocytes failed to decrease lipid contents, suggesting that the beneficial effects of IL‐6 in vivo do not result from its effects on hepatocytes alone. IL‐6 treatment increased hepatic peroxisome proliferator‐activated receptor (PPAR) α and decreased liver and serum tumor necrosis factor (TNF) α. Finally, 10 days of treatment with IL‐6 prevented the susceptibility of fatty livers to warm ischemia/reperfusion injury. In conclusion, long‐term IL‐6 administration ameliorates fatty livers and protects against warm ischemia/reperfusion fatty liver injury, suggesting the therapeutic potential of IL‐6 in treating human fatty liver disease. Supplementary material for this article can be found on the Hepatology website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:933–941.)

Host factors and failure of interferon‐α treatment in hepatitis C virus

Failure of interferon‐α (IFN‐α) treatment in patients with chronic hepatitis C virus (HCV) infection is a challenging obstacle for clinical and experimental hepatology. Both viral and host factors have been implicated in reducing responsiveness to IFN‐α therapy. The role of viral factors has been studied extensively and has been summarized in several review articles; however, much less attention has been paid to host factors. In this paper, we review evidence of host factor involvement in IFN‐α treatment failure. We discuss possible underlying mechanisms responsible for these effects. Potential therapeutic strategies to enhance the effectiveness of IFN‐α therapy for HCV are also proposed. (HEPATOLOGY 2004;39:880ndash;890.)

Crucial Role of IL-4/STAT6 in T Cell-Mediated Hepatitis: Up-Regulating Eotaxins and IL-5 and Recruiting Leukocytes

T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-γ/STAT1, IL-6/STAT3, or TNF-α/NF-κB signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 −/− and STAT6−/− mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6−/− mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4−/− and STAT6−/− mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.

Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice

Cholangiocellular carcinoma (CC), the second most common primary liver cancer, is associated with a poor prognosis. It has been shown that CCs harbor alterations of a number of tumor-suppressor genes and oncogenes, yet key regulators for tumorigenesis remain unknown. Here we have generated a mouse model that develops CC with high penetrance using liver-specific targeted disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from bile ducts of mutant mice at 2 months of age and continue to grow, leading to tumor formation in all animals at 4-7 months of age. We show that CC formation follows a multistep progression of histopathological changes that are associated with significant alterations, including increased levels of phosphorylated AKT, FOXO1, GSK-3beta, mTOR, and ERK and increased nuclear levels of cyclin D1. We further demonstrate that SMAD4 and PTEN regulate each other through a novel feedback mechanism to maintain an expression balance and synergistically repress CC formation. Finally, our analysis of human CC detected PTEN inactivation in a majority of p-AKT-positive CCs, while about half also lost SMAD4 expression. These findings elucidate the relationship between SMAD4 and PTEN and extend our understanding of CC formation.