Chandrashekhar R. Gandhi

Inhibition of T‐cell responses by hepatic stellate cells via B7‐H1–mediated T‐cell apoptosis in mice

In the injured liver, hepatic stellate cells (HSCs) secrete many different cytokines, recruit lymphocytes, and thus participate actively in the pathogenesis of liver disease. Little is known of the role of HSCs in immune responses. In this study, HSCs isolated from C57BL/10 (H2b) mice were found to express scant key surface molecules in the quiescent stage. Activated HSCs express major histocompatibility complex class I, costimulatory molecules, and produce a variety of cytokines. Stimulation by interferon γ (IFN‐γ) or activated T cells enhanced expression of these molecules. Interestingly, addition of the activated (but not quiescent) HSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti‐CD3–mediated T‐cell receptor ligation in a dose‐dependent manner. High cytokine production by the T cells suggests that the inhibition was probably not a result of suppression of their activation. T‐cell division was also found to be normal in a CFSE dilution assay. The HSC‐induced T‐cell hyporesponsiveness was associated with enhanced T‐cell apoptosis. Activation of HSCs was associated with markedly enhanced expression of B7‐H1. Blockade of B7‐H1/PD‐1 ligation significantly reduced HSC immunomodulatory activity, suggesting an important role of B7‐H1. In conclusion, the bidirectional interactions between HSCs and immune cells may contribute to hepatic immune tolerance. (HEPATOLOGY 2004;40:1312–1321.)

Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats

Endothelin-1 (ET-1), the most powerful agent to cause constriction of the hepatic vasculature, is synthesized in the liver by sinusoidal endothelial cells. Circulating ET-1 levels have been shown to increase in liver cirrhosis. As liver could be a major source of increased plasma ET-1 as well as a target for its pathologic actions, this study was designed to determine hepatic ET-1 and ET receptor(s) in experimental liver cirrhosis. Cirrhosis was induced in rats by intraperitoneal administration of carbon tetrachloride for 8 weeks. Hepatic ET-1 was measured by radioimmunoassay and ET receptors were determined by radioligand competition binding procedure. A four fold increase in ET-1 concentration accompanied by a 65% increase in ET-receptor density was observed in the cirrhotic liver. There was no change in the ET receptor affinity. The capacity of the liver to metabolize ET-1 was reduced significantly in cirrhosis. Interestingly, transforming growth factor-beta, hepatic levels of which increase in cirrhosis, stimulated ET-1 synthesis in cultured Ito cells. It has been shown that ET-1 is a potent constrictor of Ito cells that proliferate and transform into highly contractile myofibroblasts in liver cirrhosis. Thus, interactions between ET-1 and Ito cells may have significant implications in the pathogenesis and complications of liver cirrhosis.

Wnt'er in liver: Expression of Wnt and frizzled genes in mouse

The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing β‐catenin nuclear translocation and activation. Although β‐catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled‐related protein‐1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. Conclusion: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways. HEPATOLOGY 2007;45:195–204.)

Mechanisms of endotoxin‐induced NO, IL‐6, and TNF‐α production in activated rat hepatic stellate cells: Role of p38 MAPK

Compelling experimental evidence indicates that the interactions between endotoxin and hepatic stellate cells (HSCs) can play a significant role in the pathogenesis of liver disease. Endotoxin‐induced release of a multifunctional mediator NO (via inducible NO synthase) and the proinflammatory cytokines tumor necrosis factor α (TNF‐α) and interleukin (IL)‐6 by HSCs could be an important mechanism of pathological changes in the liver. However, the signaling mechanisms of these effects are poorly understood. In this study, we found that endotoxin causes activation of mitogen‐activated protein kinases (MAPKs) (extracellular signal‐regulated protein kinase [ERK] 1 and 2, p38, and c‐Jun NH2‐terminal kinase [JNK]) and nuclear factor κB (NF‐κB) and production of H2O2 in culture‐activated HSCs. However, only p38 and NF‐κB were found to be responsible for the synthesis of NO, IL‐6, and TNF‐α. Exogenous H2O2 caused modest stimulation of TNF‐α synthesis, did not affect the synthesis of NO or IL‐6, and did not activate NF‐κB or MAPKs. Inhibition of p38 and NF‐κB activation by SB203580 and pyrrolidine dithiocarbamate, respectively, blocked endotoxin‐induced H2O2, NO, TNF‐α, and IL‐6 synthesis. Inhibition of ERK1/2 and JNK phosphorylation did not alter these effects of endotoxin. Whereas SB203580 inhibited endotoxin‐induced NF‐κB activation, pyrrolidine dithiocarbamate did not affect p38 phosphorylation in endotoxin‐stimulated cells. In conclusion, endotoxin‐induced synthesis of NO, TNF‐α, and IL‐6 in HSCs is mediated by p38 and NF‐κB, with involvement of H2O2 in TNF‐α production. (HEPATOLOGY 2006;44:389–398.)

Augmenter of liver regeneration: An important intracellular survival factor for hepatocytes

BACKGROUND/AIMS Augmenter of liver regeneration (ALR), a protein synthesized and stored in hepatocytes, is associated with mitochondria, and possesses sulfhydryl oxidase and cytochrome c reductase activities. We sought to determine the effects of ALR depletion in hepatocytes by antisense oligonucleotide transfection. METHODS Rat hepatocytes in primary culture were transfected with antisense oligonucleotide for ALR mRNA (ALR-AS) or scrambled oligonucleotide. Various analyses were performed at times up to 24h after transfection. RESULTS Treatment with ALR-AS caused a decrease in ALR mRNA, cellular depletion of ALR protein primarily from mitochondria, and decreased viability. Flow cytometric analysis of ALR-AS-transfected hepatocytes stained with annexin-Vcy3 and 7-aminoactinomycin D revealed apoptosis as the predominant cause of death up to 6h; incubation beyond this time resulted in necrosis in addition to apoptosis. ALR-AS-transfection caused release of mitochondrial cytochrome c, activation of caspase-3, profound reduction in the ATP content, and cellular release of LDH. Inhibition of caspase-3 inhibited the early phase of ALR-AS-induced death but not the late phase that included ALR and LDH release. CONCLUSIONS These results suggest that ALR is critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis.

Inhibition of Kupffer cell‐mediated early proinflammatory response with carbon monoxide in transplant‐induced hepatic ischemia/reperfusion injury in rats

Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down‐regulate them. Using wild‐type (WT) to enhanced green fluorescent protein (EGFP)‐transgenic rat LTx with 18‐hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP+ host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45+ leukocytes (myeloid, T, B, and natural killer cells) and EGFP+ platelet‐like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT‐PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury‐induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF‐α), interleukin‐6 (IL‐6), and inducible nitric oxide synthase (iNOS)] were not up‐regulated in purified CD45+ cells of donor or host origin. Instead, TNF‐α and IL‐6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68+ cells, whereas iNOS mRNA up‐regulation was seen in hepatocytes. Nearly all CD68+ cells at 1 hour after LTx were EGFP− donor Kupffer cells, and CO efficiently inhibited TNF‐α and IL‐6 up‐regulation in the CD68+ Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down‐regulation of lipopolysaccharide (LPS)‐induced inflammatory responses by CO. Conclusion: These results indicate that CO ameliorates hepatic I/R injury by down‐regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up‐regulating distinctive sets of mediators in the acute phase of hepatic I/R injury. (HEPATOLOGY 2008;48:1608–1620.)

Enhanced synthesis and reduced metabolism of endothelin-1 (ET-1) by hepatocytes - an important mechanism of increased endogenous levels of ET-1 in liver cirrhosis

BACKGROUND/AIMS Hepatic concentration of endothelin-1 (ET-1) is increased in human and experimental liver cirrhosis. Because of its potent actions in the liver, ET-1 has been suggested to play an important role in the pathophysiology of cirrhosis. Since hepatocytes are the major cell type to metabolize ET-1, we investigated whether their reduced capacity to degrade ET-1 is a mechanism of its elevated levels in cirrhosis. METHODS The expression of ET-1 receptors, ET-1 and endothelin converting enzyme (ECE), and metabolism of ET-1 and ECE activity were compared in hepatocytes isolated from control and carbon tetrachloride-induced cirrhotic rats. RESULTS ET-1 receptor density and receptor-mediated internalization of ET-1 were significantly increased in cirrhotic hepatocytes relative to the control cells. However, compared to control hepatocytes, metabolism of ET-1 by the cirrhotic cells was reduced significantly. Interestingly, hepatocytes were found to contain preproET-1 mRNA, ECE-1 mRNA and ET-1. PreproET-1 mRNA and ET-1 levels were increased in cirrhotic hepatocytes but their ECE mRNA and ECE activity were not altered. CONCLUSIONS These results provide the first evidence that hepatocytes have the ability to synthesize ET-1 and demonstrate that decreased metabolism and enhanced synthesis, of ET-1 in hepatocytes are an important mechanism of its elevated levels in cirrhosis.

Endothelin stimulates platelet‐activating factor synthesis by cultured rat Kupffer cells

Endothelins are potent peptide mediators that elicit glycogenolytic and vasoconstrictor actions in the liver. Endothelins were found to stimulate the synthesis and release of the lipid mediator platelet-activating factor in cultured rat Kupffer cells. Endothelin-mediated synthesis of platelet-activating factor required extracellular calcium in that the calcium chelator, EGTA and nifedipine, a calcium ion channel blocker, inhibited platelet-activating factor synthesis. The phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited endothelin-induced platelet activating factor synthesis. Endothelin-stimulated platelet activating factor synthesis was inhibited after treatment of Kupffer cells with cholera toxin, whereas pertussis toxin inhibited only this response to endothelin-1. Agents that elevate intracellular cyclic AMP levels were found to inhibit endothelin-induced platelet-activating factor synthesis in Kupffer cells. Staurosporine, a protein kinase C inhibitor minimized endothelin-induced platelet-activating factor synthesis but phorbol myristate acetate, an activator of protein kinase C, did not affect endothelin-induced platelet activating factor synthesis. Thus, the current study demonstrates that activation of an endothelin receptor in cultured rat Kupffer cells results in the synthesis and release of platelet-activating factor. The importance of endothelin-mediated platelet-activating factor synthesis relates to the mechanism of intercellular signaling occurring between endothelial cells (i.e., the site of endothelin synthesis) and Kupffer cells (i.e., the site of formation of secondary mediators such as platelet-activating factor and eicosanoids) within the rat liver exposed to various types of pathophysiological episodes.

Increased hepatic platelet activating factor (PAF) and PAF receptors in carbon tetrachloride induced liver cirrhosis

Background and aims: The liver is a major site for the synthesis and actions of platelet activating factor (PAF), a potent hepatic vasoconstrictor and systemic vasodilator. As PAF is implicated in portal hypertension and hyperdynamic circulation associated with liver cirrhosis, we characterised changes in the hepatic PAF system in experimental cirrhosis. Methods: In rats made cirrhotic by carbon tetrachloride (CCl4) administration for eight weeks, we determined hepatic levels of PAF and its cognate receptor, and the effects of PAF and PAF antagonist (BN52021) on portal and arterial pressure. Results: Compared with control rats, cirrhotic rats had higher hepatic PAF levels, higher apparent hepatic efflux of PAF, and higher PAF levels in arterial blood (p<0.01, p<0.01, p<0.05, respectively). Relative to controls, cirrhotic livers had elevated hepatic PAF receptors (by mRNA and protein levels and [3H]PAF binding), higher (p<0.01) baseline hepatic portal pressure, and an augmented (p = 0.03) portal pressure response to PAF infusion (1 μg/kg). Portal infusion of BN52021 (5 mg/kg) showed that elevated endogenous PAF was responsible for 23% of the cirrhotic portal pressure increase but made no contribution to systemic hypotension. Finally, increased PAF receptor density was observed in the contractile perisinusoidal stellate cells isolated from cirrhotic livers relative to those from control livers. Conclusions: In cirrhosis, increased hepatic release of PAF elevates systemic PAF; in combination with upregulated hepatic PAF receptors in stellate cells, this contributes to portal hypertension.

A novel mouse model of depletion of stellate cells clarifies their role in ischemia/reperfusion- and endotoxin-induced acute liver injury

BACKGROUND & AIMS Hepatic stellate cells (HSCs) that express glial fibrillary acidic protein (GFAP) are located between the sinusoidal endothelial cells and hepatocytes. HSCs are activated during liver injury and cause hepatic fibrosis by producing excessive extracellular matrix. HSCs also produce many growth factors, chemokines and cytokines, and thus may play an important role in acute liver injury. However, this function has not been clarified due to unavailability of a model, in which HSCs are depleted from the normal liver. METHODS We treated mice expressing HSV-thymidine kinase under the GFAP promoter (GFAP-Tg) with 3 consecutive (3 days apart) CCl4 (0.16 μl/g; ip) injections to stimulate HSCs to enter the cell cycle and proliferate. This was followed by 10-day ganciclovir (40 μg/g/day; ip) treatment, which is expected to eliminate actively proliferating HSCs. Mice were then subjected to hepatic ischemia/reperfusion (I/R) or endotoxin treatment. RESULTS CCl4/ganciclovir treatment caused depletion of the majority of HSCs (about 64-72%), while the liver recovered from the initial CCl4-induced injury (confirmed by histology, serum ALT and neutrophil infiltration). The magnitude of hepatic injury due to I/R or endotoxemia (determined by histopathology and serum ALT) was lower in HSC-depleted mice. Their hepatic expression of TNF-α, neutrophil chemoattractant CXCL1 and endothelin-A receptor also was significantly lower than the control mice. CONCLUSIONS HSCs play an important role both in I/R- and endotoxin-induced acute hepatocyte injury, with TNF-α and endothelin-1 as important mediators of these effects.