Background: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. Aims: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. Methods: A model of CCl 4 induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl 4 , and indices of fibrosis were assessed at eight weeks. Results: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of α-smooth muscle actin positive cells and α1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased α1(I)-procollagen mRNA expression in activated HSC. Conclusions: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.

Sodium-glucose cotransporter 2 inhibitors (SGLT2-Is) comprise a new class of antidiabetic agents that inhibit glucose reabsorption in the renal proximal tubules. Although a recent report demonstrated the potential ability of SGLT2-Is to attenuate cancer growth of SGLT2-expressing cancer cells, little is known about the effects of SGLT2-Is on hepatocellular carcinoma (HCC). Here, we investigate the anti-cancer properties of a SGLT2-I, canagliflozin, against human liver cancer cells. SGTL2 mRNA and protein expression were detected in Huh7 and HepG2 cells, although not in HLE as well as primary human hepatocytes and hepatic stellate cells. Canagliflozin exerted antiproliferative effects on SGLT2-expressing Huh7 and HepG2 cells in a dose-dependent manner by inhibiting glycolytic metabolism including glucose uptake, lactate and intracellular ATP production. This agent also induced G2/M arrest and apoptosis with inhibited phosphorylation of ERK, p38 and AKT and cleavage of caspase3. Xenograft tumor growth assay showed that oral administration of canagliflozin (10 mg/kg/day) significantly reduced subcutaneous tumor burdens in a glycemic status-independent manner, and attenuated intratumor vascularization in Huh7- and HepG2-derived xenograft tumors in BALB/c nude mice. In vitro, canagliflozin suppressed the increased human umbilical vein endothelial cell (HUVEC) proliferation and tubular formation which were observed in Huh7 or HepG2 co-cultures. By contrast, canagliflozin had no effect on tumor growth and intratumor angiogenesis in SGLT2-null HLE-derived xenograft models. These results indicate that SGLT2-I therapy is a potential new strategy for the treatment of HCC.

Alcohol is the most common cause of liver disease in the world. Chronic alcohol consumption leads to hepatocellular injury and liver inflammation. Inflammatory cytokines, such as TNF-α and IFN-γ, induce liver injury in the rat model of alcoholic liver disease (ALD). Hepatoprotective cytokines, such as IL-6, and anti-inflammatory cytokines, such as IL-10, are also associated with ALD. IL-6 improves ALD via activation of the signal transducer and activator of transcription 3 (STAT3) and the subsequent induction of a variety of hepatoprotective genes in hepatocytes. IL-10 inhibits alcoholic liver inflammation via activation of STAT3 in Kupffer cells and the subsequent inhibition of liver inflammation. Alcohol consumption promotes liver inflammation by increasing translocation of gut-derived endotoxins to the portal circulation and activating Kupffer cells through the LPS/Toll-like receptor (TLR) 4 pathways. Oxidative stress and microflora products are also associated with ALD. Interactions between pro- and anti-inflammatory cytokines and other cytokines and chemokines are likely to play important roles in the development of ALD. The present study aims to conduct a systemic review of ALD from the aspect of inflammation.

Nonalcoholic steatohepatitis (NASH) may cause fibrosis, cirrhosis, and hepatocellular carcinoma (HCC); however, the exact mechanism of disease progression is not fully understood. Angiogenesis has been shown to play an important role in the progression of chronic liver disease. The aim of this study was to elucidate the role of angiogenesis in the development of liver fibrosis and hepatocarcinogenesis in NASH. Zucker rats, which naturally develop leptin receptor mutations, and their lean littermate rats were fed a choline-deficient, amino acid-defined diet. Both Zucker and littermate rats showed marked steatohepatitis and elevation of oxidative stress markers (e.g., thiobarbital acid reactive substances and 8-hydroxydeoxyguanosine). In sharp contrast, liver fibrosis, glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions, and HCC developed in littermate rats but not in Zucker rats. Hepatic neovascularization and the expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, only increased in littermate rats, almost in parallel with fibrogenesis and carcinogenesis. The CD31-immunopositive neovessels were mainly localized either along the fibrotic septa or in the GST-P-positive lesions. Our in vitro study revealed that leptin exerted a proangiogenic activity in the presence of VEGF. In conclusion, these results suggest that leptin-mediated neovascularization coordinated with VEGF plays an important role in the development of liver fibrosis and hepatocarcinogenesis in NASH. Supplementary material for this article can be found on the HEPATOLOGY website

Background: Endotoxin plays an important role in the initiation and aggravation of alcoholic liver disease. In this study, we evaluated plasma endotoxin levels and serum concentrations of cytokines and lipopolysaccharide binding protein (LBP) during the acute and recovery phase of patients with alcoholic hepatitis; we also explored the prognostic factors associated with a fatal outcome.Methods: Fourteen patients, consisting of eight patients with alcoholic hepatitis (AH), five cirrhotics with superimposed AH (LC+AH), and one patient with severe alcoholic hepatitis (SAH), were studied. Among these, two with LC+AH died of hepatic failure.Results: Plasma endotoxin levels in the acute phase were higher in patients with AH (184.4 ± 159.4 pg/ml) and LC+AH (206.9 ± 174.9 pg/ml) than in healthy subjects (10.4 ± 5.5 pg/ml, p < 0.001). In particular, in one patient with SAH and one of two nonsurvivors, plasma endotoxin levels were markedly high relative to the other cases. In most survivors, plasma endotoxin levels decreased in the recovery phase, whereas they further increased at the terminal stage in one of two nonsurvivors. Serum interleukin (IL)‐6 and IL‐8 levels in the acute phase were significantly higher in patients with AH and LC+AH as compared with healthy subjects. These levels were especially high in nonsurvivors and in one patient with SAH. IL‐10 increased in two nonsurvivors, one patient with SAH, and one with LC+AH. In the recovery phase, these cytokine levels in survivors tended to decrease, but in nonsurvivors, IL‐6 remained high, and IL‐8 and IL‐10 further increased. Tumor necrosis factor‐α levels were below the detection limit throughout the course in all patients. Serum lipopolysaccharide binding protein (LBP) generally was elevated in the acute phase and decreased in the recovery phase in all survivors, but in one of the nonsurvivors, LBP was elevated markedly at the terminal stage. In the acute phase, plasma endotoxin levels were correlated positively with white blood cell counts, neutrophil counts, and serum IL‐8. IL‐8 was correlated positively with neutrophil counts and negatively with serum Cholinesterase, hepaplastin test, and serum albumin levels. IL‐6 was correlated positively with white blood cell and neutrophil counts, C‐reactive protein, and serum total bilirubin and negatively with hepaplastin test and serum total protein levels. Serum LBP was correlated positively with white blood cell and neutrophil counts.Conclusions: Endotoxemia and related elevation of IL‐8 may play an important role in the activation and migration of neutrophils in patients with alcoholic hepatitis. Marked elevation of inflammatory cytokines, IL‐6 and IL‐8, are related to severity and poor prognosis of alcoholic hepatitis. Serum LBP may serve as an index of inflammatory reaction in alcoholics.

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of α-smooth muscle actin-positive cells and mRNA expression of α2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-β were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and α-SMA and α2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-β, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.

DPP4-I markedly inhibits liver fibrosis development in rats via suppression of HSCs proliferation and collagen synthesis. These suppressive effects are associated with dephosphorylation of ERK1/2, p38 and Smad2/3 in the HSCs. Since DPP4-I is widely used in clinical practice, this drug may represent a potential new therapeutic strategy against liver fibrosis in the near future.

It has been suggested that the tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in spontaneous resolution of liver fibrosis. The aim of this study was to investigate whether TIMP-1 altered spontaneous resolution of liver fibrosis in conjunction with matrix metalloproteinases (MMP) inhibition and hepatic stellate cell (HSC) activation. The livers of liver-targeted TIMP-1 transgenic (TIMP-Tg) and control hybrid (Cont) mice were harvested at 0, 3, 7, and 28 days following spontaneous recovery from CCl(4)-induced liver fibrosis. The extent of fibrosis resolution, MMP expression, alpha-smooth-muscle actin (alpha-SMA) positive cells, and procollagen-(I) messenger RNA (mRNA) in the liver were assessed at the respective periods in both groups. We also examined the effect of TIMP-1 on HSC apoptosis. The TIMP-Tg mice showed significantly attenuated resolution of spontaneous liver fibrosis compared with the Cont mice. The hydroxyproline content, number of alpha-SMA positive cells, and procollagen-(I) mRNA rapidly decreased with time in the Cont mice, whereas these markers were little changed in TIMP-Tg mice. The level of the active form of metalloproteinases-2 (MMP-2) in the TIMP-Tg mice was less than that in the Cont mice. TIMP-1 markedly decreased the nonparenchyma apoptotic cells in the liver fibrosis resolution model, and it also inhibited HSC apoptosis associated with suppression of caspase-3 activity in vitro. In conclusion, TIMP-1 significantly attenuated spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC.