N. Navari

Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells.

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.

Modulation of Liver Fibrosis by Adipokines

Hepatic fibrosis is an integrated process triggered by chronic liver damage, leading to the accumulation of extracellular matrix. In patients with chronic liver disease, this process is favored by the presence of obesity or overweight, which are also relevant risk factors for the progression of nonalcoholic steatohepatitis. In this paper, we review the available evidence indicating the modulation of the fibrogenic process by adipokines, a group of cytokines secreted primarily by adipose tissue. In particular, we discuss in detail the role of leptin and adiponectin, which favor and limit the fibrogenic process, respectively. The possible involvement of other recently identified adipokines is also briefly outlined.

The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma

Objective The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in hepatocellular carcinoma (HCC), but its biological role has not been clarified. In this study, we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. Design ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacological inhibitor, XMD8-92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. Results In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue compared with normal liver tissue. Inhibition of ERK5 decreased HCC cell proliferation and increased the proportion of cells in G0/G1 phase. These effects were associated with increased expression of p27 and p15 and decreased CCND1. Treatment with XMD8-92 or ERK5 silencing prevented cell migration induced by epidermal growth factor or hypoxia and caused cytoskeletal remodelling. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 was silenced for ERK5. Moreover, systemic treatment with XMD8-92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. Conclusions ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC.

n-3 polyunsaturated fatty acids worsen inflammation and fibrosis in experimental nonalcoholic steatohepatitis.

n‐3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n‐3 PUFA on the development and progression of experimental steatohepatitis.

1256 DIETARY SUPPLEMENTATION WITH OLIVE OIL WITH OR WITHOUT N-3 PUFA DIFFERENTIALLY AFFECTS INFLAMMATION AND PROGRESSION OF EXPERIMENTAL NONALCOHOLIC STEATOHEPATITIS

Background/aims: While it is established that excessive calorie intake plays a role in the pathogenesis of nonalcoholic steatohepatitis, the significance of individual dietary factors is still elusive. Supplementation with n-3 polyunsaturated fatty acids (PUFA) has been shown to ameliorate fatty liver in experimental models, and clinical studies are underway. In this study we compared the effects of supplementation with olive oil (mostly composed of oleic acid, 18:1 n-9) or olive oil and n-3 PUFA on the progression of experimental steatohepatitis. Methods: Balb/C mice (≥5 mice/group) were fed a methionine and choline deficient (MCD) diet or a control diet for 4 or 8 weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25mg together with 75mg olive oil), or olive oil alone (OO, 100mg), two times a week by intragastric gavage Results: Aminotransferase levels were not different comparing mice supplemented with n-3 or OO after 4w of MCD diet, while after 8w mice on MCD/n-3 had more severe necroinflammation compared to MCD/OO (ALT: 373±170 vs. 87±29 UI/L, P < 0.05). Liver/body weight ratio was significantly lower in MCD/n-3 mice after 8w of treatment (5.1±0.8 vs. 6.8±1.0%, P< 0.05). At 8w, the score of inflammation and fibrosis was significantly higher in mice receiving MCD/n-3 than in MCD/OO animals, with a marked increase in the number of lipogranulomas (26.4±8.4 vs. 5.1±5. per field, P < 0.001). A significant increase in intrahepatic expression of TNF-alpha and CCL2 was observed at both 4w and 8w in MCD/n-3 mice, which also had increased expression of CD11b at 4 weeks. In addition, increased transcript levels of the profibrogenic genes TIMP-1 and TGF-beta, and lower expression of PPAR-alpha was observed in MCD/n-3 mice. After 8 week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO (5.1±1.4 vs. 7.0±0.9mmHg, P < 0.05). No major differences were observed comparing n-3 and OO supplementation together with a control diet. Conclusions: In a model of steatohepatiits, supplementation with n-3 PUFA and OO is associated with more severe necroinflammation and fibrosis than in mice treated with OO only.