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Background and Aim: Recently, pemafibrate and a low-carbohydrate diet (LCD) have each been reported to improve fatty liver disease. However, it is unclear whether their combination improves fatty liver disease and is equally effective in obese and non-obese patients., Methods: In 38 metabolic-associated fatty liver disease (MAFLD) patients, classified by baseline body mass index (BMI), changes in laboratory values, magnetic resonance elastography (MRE), and magnetic resonance imaging-proton density fat fraction (MRI-PDFF) were studied after 1 year of combined pemafibrate plus mild LCD., Results: The combination treatment resulted in weight loss (P = 0.002), improvement in hepatobiliary enzymes (γ-glutamyl transferase, P = 0.027; aspartate aminotransferase, P < 0.001; alanine transaminase [ALT], P < 0.001), and improvement in liver fibrosis markers (FIB-4 index, P = 0.032; 7 s domain of type IV collagen, P = 0.002; M2BPGi, P < 0.001). Vibration-controlled transient elastography improved from 8.8 to 6.9 kPa (P < 0.001) and MRE improved from 3.1 to 2.8 kPa (P = 0.017) in the liver stiffness. MRI-PDFF improved from 16.6% to 12.3% in liver steatosis (P = 0.007). In patients with a BMI of 25 or higher, improvements of ALT (r = 0.659, P < 0.001) and MRI-PDFF (r = 0.784, P < 0.001) were significantly correlated with weight loss. However, in patients with a BMI below 25, the improvements of ALT or PDFF were not accompanied by weight loss., Conclusions: Combined treatment with pemafibrate and a low-carbohydrate diet resulted in weight loss and improvements in ALT, MRE, and MRI-PDFF in MAFLD patients. Although such improvements were associated with weight loss in obese patients, the improvements were observed irrespective of weight loss in non-obese patients, indicating this combination can be effective both in obese and non-obese MAFLD patients., (© 2023 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Human amniotic epithelial cells (hAECs) are increasingly gaining attention as novel sources for cell transplantation. In clinical practice, intraportal infusion is considered one of the leading approaches for transplantation; however, this has not yet been validated for in vivo transplantation of hAECs. Thus, this study aims to investigate the distribution of hAECs post intraportal infusion and compare this distribution with other cell administration routes., Methods: Wistar/ST rats were divided into 4 groups (n = 3 for each) based on cell administration route: group 1, intraportal; group 2, the spleen; group 3, tail veins; and group 4, penile veins. Subsequently, hAECs (1 × 10 7 ) stained with XenoLight DiR were infused into each recipient. Cell distribution was evaluated using an in vivo imaging system., Results: DiR signals were detected in the rat livers of groups 1 and 2 with those in group 2 being much weaker than those in group 1. Necrosis of small intestine was observed in 2 cases in group 2. DiR signals were detected in the lungs in groups 3 and 4 because of systemic circulation; however, all the animals died within 20 minutes of infusions., Conclusions: Intraportal infusion is potentially applicable for safe and efficient transplantation of hAECs into the liver, whereas hAECs administration via the spleen carries a risk of thrombosis in a narrow portal vein system. Our results also indicate that hAECs administration via the systemic circulation could cause pulmonary embolism in clinical settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hepatic sinusoidal obstruction syndrome (SOS)/veno-occlusive disease is a life-threatening complication after hematopoietic stem cell transplantation (HSCT). We previously reported the efficacy of the Hokkaido Ultrasonography (US)-based scoring system (HokUS-10) for US findings. To establish easier-to-use criteria, we retrospectively evaluated US findings from 441 patients, including 30 patients with SOS using the HokUS-10 scoring system. Using logistic regression analysis, we established the novel diagnostic criteria HokUS-6. In the presence of ascites, US diagnosis was made in the presence of two of the following 6 parameters: moderate amount of ascites, the appearance of a paraumbilical vein blood flow signal, gallbladder wall thickening, portal vein dilatation, portal vein velocity decrease, and hepatic artery resistive index increase. The AUC, sensitivity, and specificity of HokUS-6 were 0.974 (95% confidence interval 0.962-0.990), 95.2%, and 96.9%, respectively. The scores were significantly higher in patients with severe SOS than in those with non-severe SOS (p = 0.013). Furthermore, the scores before HSCT were significantly higher in patients who developed SOS than in controls (p = 0.001). The HokUS-6 is an easy and useful way to diagnose and identify the risk of SOS.

In this study, we investigated the effects of dietary supplementation of Citrus tumida hort. ex Tanaka on food intake, body and fat tissue weights, and metabolic profiles of plasma and liver in mice. Supplementation with 5% (w/w) of peels of immature C. tumida (PIC) for 4 weeks significantly suppressed body weight gain and decreased adipose tissue weight in epididymal, perirenal, and subcutaneous fats. Metabolome analyses showed that 2-hydroxyvaleric acid levels were reduced in the blood plasma of mice fed with PIC. PIC supplementation significantly elevated dipeptide (Thr-Asp, Ser-Glu, and Ala-Ala), glucuronic acid, and S -methylglutathione levels, and significantly reduced betaine aldehyde levels in the liver. In conclusion, PIC supplementation affects the metabolism of fatty acids, pectin, glutathione, and choline, showing potential beneficial effects for metabolic syndrome and obesity. PIC may be developed as a functional food and used in the treatment of these diseases.

Exogenously hypercholesterolemic (ExHC) rats develop diet-induced hypercholesterolemia (DIHC) when fed with dietary cholesterol. Previously, we reported that, under the high-sucrose-diet-feeding condition, a loss-of-function mutation in Smek2 results in low activity of fatty acid synthase (FAS) followed by the shortage of hepatic triacylglycerol content in ExHC rats and the onset of DIHC. However, the relationship between the Smek2 mutation and FAS dysfunction is still unclear. Here, we focused on carbohydrate metabolism, which provides substrates for FAS, and analyzed carbohydrate and lipid metabolisms in ExHC rats to clarify how the deficit of Smek2 causes DIHC. Male ExHC and SD rats were fed high-sucrose or high-starch diets containing 1% cholesterol for 2 weeks. Serum cholesterol levels of the ExHC rats were higher, regardless of the dietary carbohydrate. Hepatic triacylglycerol levels were higher in only the SD rats fed the high-sucrose diet. Moreover, the ExHC rats exhibited a diabetes-like status and accumulation of hepatic glycogen and low hepatic mRNA levels of liver-type phosphofructokinase (Pfkl), which encodes a rate-limiting enzyme for glycolysis. These results suggest that the glucose utilization, particularly glycolysis, is impaired in the liver of ExHC rats. To evaluate how the diet with extremely low glucose affect to DIHC, ExHC.BN-Dihc2BN, a congenic strain that does not develop DIHC, and ExHC rats were fed a high-fructose diet containing 1% cholesterol for 2 weeks. The serum cholesterol and hepatic triacylglycerol levels were similar in the strains. Results of water-soluble metabolite analysis with primary hepatocytes, an increase in fructose-6-phosphate and decreases in succinate, malate and aspartate in ExHC rats, support impaired glycolysis in the ExHC rats. Thus, the Smek2 mutation causes abnormal hepatic glucose utilization via downregulation of Pfkl expression. This abnormal glucose metabolism disrupts hepatic fatty acid synthesis and causes DIHC in the ExHC rats., Competing Interests: The authors have declared that no competing interests exist.

Background and Aim: Liver stiffness (LS), measured by transient elastography, has been validated as a non-invasive surrogate for liver fibrosis., Methods: We investigated the long-term predictive ability of LS for hepatocellular carcinoma (HCC) development and overall survival in 1146 patients with chronic hepatitis C by using LS value at enrollment. We also investigated chronological changes in LS based on antiviral therapy and its outcome in 752 patients., Results: During the mean follow-up period of 6.6 years, 190 patients developed HCC. Cumulative HCC incidence rates at 5 years were clearly stratified as 1.7% in the ≤ 5 kPa, 3.3% in 5.1-10 kPa, 16.7% in 10.1-15 kPa, 24.4% in 15.1-20 kPa, 36.3% in 20.1-25 kPa, and 43.7% in > 25 kPa subgroups (P < 0.001). Overall survival was also stratified: 10-year survival rates were 99.3% in the ≤ 5 kPa, 95.4% in 5.1-10 kPa, 81.4% in 10.1-15 kPa, 79.5% in 15.1-20 kPa, 66.1% in 20.1-25 kPa, and 49.1% in > 25 kPa subgroups (P < 0.001). LS decreased at a rate of 8.1% per year in those who achieved sustained virological responses, but increased at 0.1% per year in those who could not achieve sustained virological response instead of antiviral therapy, and increased at 3.7% per year in those who did not undergo antiviral therapy., Conclusions: Liver stiffness measurements can be useful in the prediction of HCC development and overall survival and in the evaluation of chronological changes in liver fibrosis grade during and after antiviral therapy., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Hydrodynamics-based gene delivery (HGD) is an efficient method for transfecting plasmid DNA into hepatocytes in vivo. However, the resulting gene expression is transient, and occurs in a non-tissue specific manner. The piggy Bac (PB) transposon system allows chromosomal integration of a transgene in vitro. This study aimed to achieve long-term in vivo expression of a transgene by performing hepatocyte-specific chromosomal integration of the transgene using PB and HGD. Using this approach, we generated a novel mouse model for a hepatic disorder. A distinct signal from the reporter plasmid DNA was discernible in the murine liver approximately two months after the administration of PB transposons carrying a reporter gene. Then, to induce the hepatic disorder, we first administered mice with a PB transposon carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain gene, and poly(A) sites), and then with a plasmid expressing the Cre recombinase under the control of a liver-specific promoter. We showed that this system can be used for in situ manipulation and analysis of hepatocyte function in vivo in non-transgenic (Tg) animals.

Accumulation of abdominal fat triggers metabolic syndrome, which is a cluster of metabolic abnormalities, such as dyslipidemia, glucose intolerance, insulin resistance or hyperinsulinemia, and hypertension, that leads to the development of diabetes and cardiovascular disease. Mushrooms have been used as a foodstuff and folk medicine worldwide. Among these mushrooms, Sparassis crispa (SC) is a relatively newly cultivated edible and medicinal mushroom, which has been reported to have anti-diabetic and anti-hypertensive properties. However, little is known about the anti-obesity and anti-hyperlipidemic properties of SC. In the present study, we investigated the effects of dietary SC on lipid metabolism and energy expenditure in Sprague-Dawley rats with diet-induced obesity and diabetes, and conducted respiratory gas analysis to determine how energy metabolism is altered by SC. After feeding periods of 3 and 7 weeks, dietary SC had significantly reduced hepatic triacylglycerol and cholesterol contents in a dose-dependent manner. These changes were attributable to suppression of fatty acid and cholesterol synthesis in the liver and increased insulin sensitivity in the body. In addition, after a feeding period of 6 weeks, dietary SC significantly increased energy expenditure through carbohydrate oxidation, reducing abdominal fat mass after 7 weeks. In conclusion, our results indicate that in addition to the previously reported anti-diabetic and anti-hypertensive activities, dietary SC exhibits anti-obesity and anti-hyperlipidemic activities that help protect against metabolic syndrome.

Bosentan is a substrate of hepatic uptake transporter organic anion-transporting polypeptides (OATPs), and undergoes extensive hepatic metabolism by cytochrome P450 (P450), namely, CYP3A4 and CYP2C9. Several clinical investigations have reported a nonlinear relationship between bosentan doses and its systemic exposure, which likely involves the saturation of OATP-mediated uptake, P450-mediated metabolism, or both in the liver. Yet, the underlying causes for the nonlinear bosentan pharmacokinetics are not fully delineated. To address this, we performed physiologically based pharmacokinetic (PBPK) modeling analyses for bosentan after its intravenous administration at different doses. As a bottom-up approach, PBPK modeling analyses were performed using in vitro kinetic parameters, other relevant parameters, and scaling factors. As top-down approaches, three different types of PBPK models that incorporate the saturation of hepatic uptake, metabolism, or both were compared. The prediction from the bottom-up approach (models 1 and 2) yielded blood bosentan concentration-time profiles and their systemic clearance values that were not in good agreement with the clinically observed data. From top-down approaches (models 3, 4, 5-1, and 5-2), the prediction accuracy was best only with the incorporation of the saturable hepatic uptake for bosentan. Taken together, the PBPK models for bosentan were successfully established, and the comparison of different PBPK models identified the saturation of the hepatic uptake process as a major contributing factor for the nonlinear pharmacokinetics of bosentan., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Egg-white protein (EWP) is known to reduce lymphatic TAG transport in rats. In this study, we investigated the effects of dietary EWP on body fat mass. Male rats, 4 weeks old, were fed diets containing either 20 % EWP or casein for 28 d. Carcass protein levels and gastrocnemius leg muscle weights in the EWP group were significantly higher than those in the casein group. In addition, carcass TAG levels and abdominal fat weights in the EWP group were significantly lower than those in the casein group; adipocyte size in abdominal fat in the EWP group was smaller than that in the casein group. To identify the involvement of dietary fat levels in the rats, one of two fat levels (5 or 10 %) was added to their diet along with the different protein sources (EWP and casein). Abdominal fat weight and serum and hepatic TAG levels were significantly lower in the EWP group than in the casein group. Moreover, significantly higher values of enzymatic activity related to β-oxidation in the liver were observed in the EWP group compared with the casein group. Finally, abdominal fat weight reduction in the EWP group with the 10 % fat diet was lower than that in the EWP group with the 5 % fat diet. In conclusion, our results indicate that, in addition to the inhibition of dietary TAG absorption reported previously, dietary EWP reduces body fat mass in rats through an increase of body protein mass and the acceleration of β-oxidation in the liver.

In vivo inoculation of cells such as tumor cells and induced pluripotent stem (iPS)/embryonic stem (ES) cells into immunocompromised mice has been considered as a powerful technique to evaluate their potential to proliferate or differentiate into various cell types originating from three germ cell layers. Subcutaneous grafting and grafting under the kidney capsule have been widely used for this purpose, but there are some demerits such as the requirement of a large number of tumor cells for inoculation and frequent failure of tumorigenesis. Therefore, grafting into other sites has been explored, including intratesticular or intramuscular grafting as well as grafting into the cochleae, liver, or salivary glands. In this study, we found that intrapancreatic parenchymal injection of cells is useful for allowing a small number of cells (~15 × 10³ cells or ~30 cell clumps μL -1 ·site -1 ) to proliferate and sometimes differentiate into various types of cells. It requires only surgical exposure of the pancreas over the dorsal skin and subsequent injection of cells towards the pancreatic parenchyma under dissecting microscope-based observation using a mouthpiece-controlled glass micropipette. We now name this technology "intrapancreatic parenchymal cell transplantation (IPPCT)", which will be useful, especially when only a small number of cells or colonies are available., Competing Interests: The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The capacity of the liver to regenerate is likely to be encoded as a plasticity of molecular networks within the liver. By applying a combination of comprehensive analyses of the epigenome, transcriptome, and proteome, we herein depict the molecular landscape of liver regeneration. We demonstrated that histone H3 Lys-4 was trimethylated at the promoter regions of many loci, among which only a fraction, including cell-cycle-related genes, were transcriptionally up-regulated. A cistrome analysis guided by the histone methylation patterns and the transcriptome identified FOXM1 as the key transcription factor promoting liver regeneration, which was confirmed in vitro using a hepatocarcinoma cell line. The promoter regions of cell-cycle-related genes and Foxm1 acquired higher levels of trimethylated histone H3 Lys-4, suggesting that epigenetic regulations of these key regulatory genes define quiescence and regeneration of the liver cells. A quantitative proteome analysis of the regenerating liver revealed that conditional protein degradation also mediated regeneration-specific protein expression. These sets of informational resources should be useful for further investigations of liver regeneration., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background and Aims: Because of the low penetration rate of transient elastography (TE) or its limitations in patients with obesity, narrow intercostal spaces, or ascites, the physical appearance of the liver as visualized using ultrasonography (US) is still thought to provide important information for the prediction of liver fibrosis. We examined the accuracy of various US signs when assessing the presence of liver cirrhosis, compared with TE., Methods: We enrolled 189 patients who had undergone both conventional US and TE examinations. We then assessed the associations between US parameters of the liver (surface, edge, and parenchymal texture) or the US score (sum of each parameter score), and the presence of liver cirrhosis as determined based on a liver stiffness measurement (LSM) of >15., Results: A significant increase in the LSM was observed according to the liver surface score (P < 0.001), liver edge score (P < 0.001), parenchymal texture score (P < 0.001), and US score (P < 0.001). The areas under the curves (AUROC) for the prediction of an LSM >15 for the liver surface, liver edge, parenchymal texture, and the US score were 0.859, 0.768, 0.837, and 0.902, respectively. The AUROC of the US score was higher than that of the APRI score (0.823) or the FIB-4 index (0.804). Using an optimal cut-off value of 3.5, the sensitivity and specificity of the US score were 0.815 and 0.858, respectively., Conclusions: The US score was clinically useful for the diagnosis of an LSM >15. The US score can be used as a substitute for TE data in patients with obesity, narrow intercostal spaces, or ascites or in hospitals where TE is unavailable.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis., (Copyright © 2016 the American Physiological Society.)

The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

Etoposide, a topoisomerase 2 (TOP2) inhibitor, is associated with the development of KMT2A (MLL)-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL) rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL)-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP) assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq) identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll)-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

We report the case of a 69-year-old woman with reactive lymphoid hyperplasia (RLH) of the liver. She underwent partial hepatectomy under a preoperative diagnosis of hepatocellular carcinoma; however, histopathological analysis revealed RLH. The liver nodule showed the imaging feature of perinodular enhancement in the arterial dominant phase on contrast-enhanced computed tomography and magnetic resonance imaging, which could be a useful clue for identifying RLH in the liver. Histologically, the perinodular enhancement was compatible with prominent sinusoidal dilatation surrounding the liver nodule.

Background & Aims: Fetuin-A (α2HS-glycoprotein), a liver secretory glycoprotein, is known as a transforming growth factor (TGF)-β1 signalling inhibitor. Serum fetuin-A concentration is associated with nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. However, the usefulness of serum fetuin-A as a predictive fibrosis biomarker in NAFLD patients remains unclear. In this study, we investigated the relationship between circulating fetuin-A levels and fibrosis-related markers [platelet count, NAFLD fibrosis score and carotid intima media thickness (IMT)] in subjects with NAFLD., Methods: A total of 295 subjects (male, 164; female, 131) who received medical health check-ups were enrolled in this study. NAFLD was diagnosed using abdominal ultrasonography. Serum fetuin-A was measured by ELISA. IMT was assessed using a high-resolution ultrasound scanner. Using recombinant human fetuin-A, we investigated the effects of fetuin-A on hepatic stellate cells, which play a pivotal role in the process of hepatic fibrosis., Results: Serum fetuin-A concentration was significantly correlated with platelet count (R = 0.19, P < 0.01), NAFLD fibrosis score (R = -0.25, P < 0.01) and mean IMT (R = -0.22, P < 0.01). Multivariate analyses revealed that the fetuin-A concentration is a significant and independent determinant of platelet count, NAFLD fibrosis score and mean IMT. Recombinant fetuin-A suppressed TGF-β1 signalling and fibrosis-related gene expression and increased the expression of TGF-β1 pseudoreceptor bone morphogenic protein and activin membrane-bound inhibitor (BAMBI)., Conclusions: Serum fetuin-A level is associated with liver/vessel fibrosis-related markers in NAFLD patients. Circulating fetuin-A could be a useful serum biomarker for predicting liver and vascular fibrosis progression in NAFLD patients., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hydrodynamics-based gene delivery (HGD) is a widely recognized technique for delivering exogenous DNA with high efficiency to murine hepatocytes. In this study, we investigated stimulation of exogenous DNA uptake and expression using a commercially available reagent for HGD. We also examined which mouse strain and mouse liver lobe would achieve the best gene delivery performance. Mice were injected with a solution containing reporter plasmid DNA or DNA and a gene delivery reagent. One day after the HGD procedure, liver samples were isolated and subjected to biochemical and histochemical analyses. The reporter plasmid DNA showed the strongest signal when the DNA was dissolved in TransIT-EE Hydrodynamic Delivery Solution (Takara Bio Inc., Shiga, Japan). Evaluation of transgene expression in each hepatic lobe in ICR, C57BL/6N, Balb/cA, and B6C3F1 mice showed that ICR mice exhibited the best gene transfer and that the right median lobe had the highest level of transgene expression. These findings suggest the importance of choice in mouse strains and liver lobes when performing gene-based manipulations of the liver.

Background: Exogenously hypercholesterolemic (ExHC) rats develop hypercholesterolemia and low hepatic triacylglycerol (TAG) levels when dietary cholesterol is loaded. The responsible gene Smek2 was identified via linkage analysis using the original strain Sprague-Dawley (SD) rats. In this study, we compared SD and ExHC rats to investigate a relationship between hypercholesterolemia and the low hepatic TAG levels observed in ExHC rats., Methods: Male 4-weeks-old ExHC and SD rats were fed a 1% cholesterol diet for 1 week. Serum and liver parameters were analyzed. Gene expression and enzyme activities related to TAG metabolism were also assessed., Results: We reproducibly observed higher serum cholesterol and lower hepatic TAG levels in ExHC rats than in SD rats. Golgi apparatus in the livers of ExHC rats secreted β-very-low-density lipoprotein (β-VLDL) that had higher cholesterol ester (CE) and lower TAG content than those in the β-VLDL secreted by SD rats. Gene expression related to fatty acid and TAG synthesis in ExHC rats was lower than that in SD rats. Enzymatic activities for fatty acid synthesis were also relatively lower in ExHC rats. Moreover, the fatty acid composition of hepatic and serum CE in ExHC rats showed that these CEs were not modified after secretion from the liver despite the similar activities of serum lecithin-cholesterol acyltransferase (LCAT) in ExHC rats to those in SD rats., Conclusions: Low production of liver TAG and secretion of CE-rich, TAG-poor β-VLDL without modification by LCAT in the circulation contributed to hypercholesterolemia induced by dietary cholesterol in ExHC rats.

Purpose: Nicotinic acid is one of the older drugs used to treat hyperlipidemia, the greatest risk factor of coronary heart disease. Nicotinic acid is also a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD). In mammals, α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) plays a key role in NAD biosynthesis from tryptophan. However, the relationship between ACMSD and cholesterol metabolism has not been clarified enough yet. The present study was performed to make clear the relationship between ACMSD and cholesterol metabolism using hypercholesterolemic rats and rat primary hepatocytes., Methods: Male Sprague-Dawley rats were fed a diet containing cholesterol for 10 days to induce hypercholesterolemia. The NAD levels in the plasma and liver and hepatic ACMSD activity were determined. In vitro study, the expression of ACMSD and the transcriptional factors that regulate cholesterol metabolism were determined using rat primary hepatocytes treated with cholesterol and 25-hydroxycholesterol or simvastatin, a statin medication, by quantitative real-time PCR analysis and Western blotting analysis., Results: The hepatic NAD level of the hypercholesterolemic group was significantly higher than the control, and the hepatic ACMSD activity of this group was significantly suppressed. There was a significant negative correlation between the hepatic ACMSD activity and liver cholesterol levels. Additionally, in primary rat hepatocytes treated with cholesterol and 25-hydroxycholesterol or simvastatin, ACMSD gene and protein expression was subjected to sterol-dependent regulation. This gene expression changed in parallel to sterol regulatory element-binding protein (SREBP)-2 expression., Conclusion: These results provide the first evidence that ACMSD is associated with cholesterol metabolism, and ACMSD gene expression may be upregulated by SREBP-2.

Background: Donor safety is a major concern in living-donor liver transplantation. However, partial grafts do not meet the functional demands of recipients and lead to small-for-size syndrome (SFSS). In a previous study, we showed that olprinone (OLP), a selective phosphodiesterase ІІІ inhibitor, up-regulates endothelial nitric oxide synthase level in the liver and attenuates shear stress, sinusoidal endothelial cell injury, and hepatocyte apoptosis after excessive liver resection in a rat model. We aimed to examine whether OLP treatment has beneficial effects on SFSS in a rat model of partial liver transplantation (PLT)., Methods: We performed experiments in a rat model of 30% PLT. In the OLP group, we inserted an osmotic pump with OLP into the peritoneal cavity 48 h before liver graft sampling. Recipient rats were not treated with OLP. We examined the liver microstructure by electron microscopy and biochemical examination, and determined the 7-d survival of recipients., Results: In the OLP group 1 h after PLT, the sinusoidal endothelial cells of the liver were well preserved and we observed few vacuolar structures in hepatocytes. The total serum bilirubin level 1 wk after PLT tended to be lower in the OLP group than in the controls, and the liver microstructures were also well preserved in the OLP group. The probability of survival in the OLP group (100%; 14 of 14 rats) was significantly higher than that in the control group (75%; 15 of 20 rats)., Conclusions: Olprinone treatment was demonstrated to have therapeutic potential to overcome SFSS., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The liver is an important organ for supporting the life of an individual. Gene transfer toward this organ has been attempted in many laboratories to date; however, there have been few reports on improved liver-targeted gene delivery by using a nonviral vector. In this study, we examined the effect of various types of gene delivery carriers on enhancing the uptake and gene expression of exogenous DNA in murine hepatocytes when a hydrodynamics-based gene delivery (HGD) is performed via tail-vein injection. Mice were singly injected with a large amount of phosphate-buffered saline containing reporter plasmid DNA and/or with a gene delivery carrier. One day after the gene delivery, the animals' livers were dissected and subjected to biochemical, histochemical, and molecular biological analyses. The strongest signal from the reporter plasmid DNA was observed when the DNA was mixed with a polyethylenimine- (PEI-) based reagent. Coinjection with pCRTEIL (a loxP-floxed reporter construct) and pTR/NCre (a liver-specific Cre expression vector) resulted in the liver-specific recombination of pCRTEIL. The combination of PEI with HGD would thus be a valuable tool for liver-specific manipulation to examine the function of a gene of interest in the liver and for creating liver disease models.

Hyperbilirubinemia in the early phase after allogeneic hematopoietic SCT (HSCT) is due to various causes. One of the most important causes of hyperbilirubinemia is veno-occlusive disease/sinusoidal obstructive syndrome (VOD/SOS). However, the prognosis of patients who are clinically diagnosed as SOS varies. We retrospectively evaluated 82 patients who underwent their first allogeneic HSCT. GVHD prophylaxis was a combination of short-term MTX and CsA (n=77) or tacrolimus (n=5). Thirty-three patients developed hyperbilirubinemia, with a bilirubin level of at least 2 mg/dL, within 20 days after HSCT. Of these patients, 24 were diagnosed as VOD/SOS using the modified Seattle criteria. Twenty-six recovered to a bilirubin level of <2 mg/dL. We focused on the serum alkaline phosphatase/total bilirubin ratio (ALP/TB) at the onset of hyperbilirubinemia and found that it significantly predicted the recovery from hyperbilirubinemia. OS was significantly higher in patients with a lower ALP/TB ratio (P=0.00056). In addition, a lower ALP/TB ratio was associated with better survival even in patients who were clinically diagnosed as SOS (P<0.001). The ALP/TB ratio at the onset of hyperbilirubinemia may be a useful predictor for the prognosis of hyperbilirubinemia and SOS early after HSCT.

To investigate the effects of dietary Grifola frondosa on cholesterol, normal mice were fed a diet containing 1% cholesterol (HC group) or 1% cholesterol and 10% freeze-dried G. frondosa powder (HC+G group) for 4 weeks and hepatic and plasma lipid levels were compared with those of a cholesterol-free diet-fed mice (N group). Hepatic total cholesterol (TC), triacylglycerol contents were considerably increased and plasma TC / phospholipid (PL) was also increased significantly in the HC group compared with the N group. However, plasma TC content decreased in the HC+G group compared with the HC group. To characterize the mechanisms responsible for lowered plasma cholesterol in G. frondosa-supplemented mice, hepatic gene expression was profiled using DNA microarray and gene ontology. Genome analyses revealed that de novo cholesterol synthesis genes were suppressed following cholesterol intake. However, expression of bile acid biosynthesis and low-density lipoprotein receptor genes showed little change. Scarb1, Abcg5, and Abcg8, involved in cholesterol transport and excretion, were slightly upregulated in the HC+G group compared with the HC group. These data indicate the plasma cholesterol-lowering effect of G. frondosa. Moreover, fatty acid (FA) β-oxidation was promoted via adipocytokine signaling pathways, and Saa, encodes serum amyloid A related to arteriosclerosis, was suppressed in the HC+G group.

Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.

MAPKs are involved in acetaminophen (APAP)-hepatotoxicity, but the regulatory mechanism remains unknown. Here, we explored the role of Spred-2 that negatively regulates Ras/ERK pathway in APAP-hepatotoxicity. Spred-2 knockout (KO) mice demonstrated exacerbated liver injury, an event that was associated with increased numbers of CD4(+) T, CD8(+) T and NK cells in the liver compared to the control. Levels of CXCL9/CXCL10 that attract and activate these cells were increased in Spred-2 KO-liver. Kupffer cells isolated from Spred-2 KO mice after APAP challenge expressed higher levels of CXCL9/CXCL10 than those from the control. Upon stimulation with APAP or IFNγ, naïve Kupffer cells from Spred-2 KO mice expressed higher levels of CXCL9/CXCL10. NK cell-depletion attenuated APAP-hepatotoxicity with lowered hepatic IFNγ and decreased numbers of not only NK cells but also CD4(+) T and CD8(+) T cells in the liver. These results suggest that Spred-2 negatively regulates APAP-hepatotoxicity under the control of Kupffer cells and NK cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Disseminated adenovirus disease after allogeneic hematopoietic stem cell transplantation (HSCT) is lethal in most cases, especially when it develops as fulminant hepatic failure. We encountered a patient who developed fulminant hepatic failure caused by adenovirus infection. She did not show manifestations of graft-versus-host disease and the results of serum tests for viral infection were all negative. Abdominal computed tomography (CT) findings were consistent with peliosis hepatitis. She died of fulminant hepatic failure, however, and pathological examinations of the liver specimen obtained after her death revealed adenovirus infection. In this report, we review the clinical characteristics and imaging findings of fulminant hepatic failure caused by adenovirus infection.

Purpose: The aim of the study was to clarify the effect of the interval between transcatheter hepatic arterial embolization (TAE) with Lipiodol plus gelatin sponge particles and radiofrequency (RF) ablation on the extent of ablation., Materials and Methods: Eight healthy swine were divided into four groups: RF ablation (ablation only), RF ablation immediately after TAE (immediate ablation), RF ablation 3 days after TAE (3-day ablation), and RF ablation 6 days after TAE (6-day ablation). Five ablated lesions were created in each swine (10 per group). A 2-cm expandable LeVeen needle electrode was used for RF ablation. Ablated lesions are composed of an outer reddish zone and an inner whitish zone., Results: The average longest length of the major, intermediate, and minor axes and the volume in the immediate ablation, 3-day ablation, and 6-day ablation groups were significant longer and greater (1.52 and 1.52, 1.46 and 1.50, and 1.37 and 1.35 times greater in the red zone and the whitish area, respectively) than those in the ablation-only group (P < 0.05/3). Accumulation of Lipiodol was still noted in the hepatic sinusoids in the 3-day and 6-day ablation groups., Conclusion: RF ablation delayed to 6 days following TAE produced larger ablation volumes than did RF ablation alone.

To compare and estimate the effects of dietary intake of three kinds of mushrooms (Pleurotus ostreatus, Grifola frondosa, and Hypsizigus marmoreus), mice were fed a diet containing 10-14% of each mushroom for 4 weeks. Triacylglycerol in the liver and plasma decreased and plasma cholesterol increased in the P. ostreatus-fed group compared with those in the control group. Cholesterol in the liver was lower in the G. frondosa-fed group than in the control group, but no changes were found in the H. marmoreus-fed group. DNA microarray analysis of the liver revealed differences of gene expression patterns among mushrooms. Ctp1a and Fabp families were upregulated in the P. ostreatus-fed group, which were considered to promote lipid transport and β-oxidation. In the G. frondosa-fed group, not only the gene involved in signal transduction of innate immunity via TLR3 and interferon but also virus resistance genes, such as Mx1, Rsad2, and Oas1, were upregulated.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver. Although the isolation methods of liver macrophages have been well-described, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1. After dissociation of the liver cells by two-step perfusion method, a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS. Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages (Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 10(6) cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks (Figure 3). The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4). The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills. This method might be applicable to other mammalian species.

Previously, we developed a simple and efficient method to isolate liver macrophages from a mixed primary culture of adult rat liver cells. To extend the applicability of this method, we isolated macrophages from mixed primary cultures of bovine liver cells. Macrophage cells proliferated on the cell sheet of mixed bovine liver cells after 8-16d of culture. These cells were detached by shaking of the culture flasks. Subsequent transfer and brief incubation in plastic dishes resulted in selective adhesion of macrophages. After rinses with PBS, attached macrophages were harvested. More than 10(6) cells could be harvested from the culture flask at intervals of 2-3d for more than three weeks. The isolated cells were strongly positive for bovine macrophage markers, such as CD68, CD172a and Iba-1. These cells exhibited functional properties of macrophages, including active phagocytosis of polystyrene microbeads, proliferative response to recombinant bovine granulocyte-macrophage colony-stimulating factor, upregulation of specific inflammatory cytokine genes upon stimulation with lipopolysaccharide, and formation of multinucleated giant cells. The shaking and attachment method provides a simple and efficient alternative to obtain bovine liver macrophages without requiring complex equipment or specialized technical skills., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and alpha-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10(6) cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills., (2010 Elsevier B.V. All rights reserved.)

Caveolin, a member of the membrane-anchoring protein family, accumulates various growth receptors in caveolae and inhibits their function. Upregulation of caveolin attenuates cellular proliferation and growth. However, the role of caveolin in regulating insulin signals remains controversial. Here, we demonstrate that caveolin potently enhances insulin receptor (IR) signaling when overexpressed in the liver in vivo. Adenovirus-mediated gene transfer was used to overexpress caveolin specifically in the liver of diabetic obese mice, which were generated with a high-fat diet. Expression of molecules involved in IR signaling, such as IR or Akt, remained unchanged after gene transfer. However, hepatic glycogen synthesis was markedly increased with a decrease in phosphoenolpyruvate carboxykinase protein expression. Insulin sensitivity was increased after caveolin gene transfer as determined by decreased blood glucose levels in response to insulin injection and fasting blood glucose levels. Glucose tolerant test performance was also improved. Similar improvements were obtained in KKA(y) genetically diabetic mice. Adenovirus-mediated overexpression of caveolin-3 in hepatic cells also enhanced IR signaling, as shown by increased phosphorylation of IR in response to insulin stimulation and higher glycogen synthesis at baseline. These effects were attributed mostly to increased insulin receptor activity and caveolin-mediated, direct inhibition of protein tyrosine phosphatase 1B, which was increased in obese mouse livers. In conclusion, our results suggest that caveolin is an important regulator of glucose metabolism that can enhance insulin signals.

Purpose: To define the effects of hepatic artery chemoembolization with cisplatin-lipiodol suspension and gelatin sponge particles on swine liver tissue and estimate the concentration of cisplatin that would have a minimal negative effect on normal liver parenchyma., Materials and Methods: Twelve pigs were divided into four groups: group A was the control group in which hepatic arteries were embolized with lipiodol and gelatin sponge particle (n = 3); group B animals were embolized with 10 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3), group C with 20 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3), and group D with 30 mg/mL cisplatin-lipiodol suspension plus gelatin sponge particle (n = 3). Pigs were euthanized 1 week after embolization, and the resected livers were cut into 10-mm-thick sections. The livers and necrotic foci were contoured in each section, and the necrosis volume ratio was calculated., Results: The necrosis volume ratios of the livers in groups A, B, C, and D were 0.832% +/- 0.334, 2.324% +/- 1.126, 8.056% +/- 3.276, and 11.82% +/- 4.921, respectively. Significant differences (P < .05) in necrosis volume ratio were found between groups A and C, groups A and D, groups B and C, and groups B and D; no significant difference was found between groups A and B., Conclusions: Hepatic artery chemoembolization with higher doses of cisplatin causes greater damage to liver tissue; 10 mg/mL cisplatin-lipiodol suspension causes minimal damage, similar to that without cisplatin, and is related to minimal negative changes in a swine model.

Purpose: To evaluate the effectiveness of transcatheter arterial embolization (TAE) on radiofrequency (RF) ablation volume and compare portal vein (PV) flow and liver parenchymal pressure before and after treatment., Materials and Methods: Eight healthy female swine were divided into four groups to be treated with RF ablation alone (RF-only group), RF ablation after TAE with gelatin sponge particles (GSPs; RF/TAE group), RF ablation after TAE with Lipiodol plus GSPs (RF/TAE/Lipiodol group), and PV embolization (PVE) with GSPs after TAE with Lipiodol plus GSPs (RF/TAE/Lipiodol/PVE group). Five ablations were created in each swine, with 10 ablations per group. A 2-cm expandable LeVeen needle electrode was used for RF ablation., Results: The greatest ablation volume (18,410.1 mm(3) +/- 3,986.4) was observed in the RF/TAE/Lipiodol/PVE group. Of the RF-only, RF/TAE, and RF/TAE/Lipiodol groups, the RF/TAE/Lipiodol group (14,835.5 mm(3) +/- 2,743.2) had a significantly larger ablation volume than the RF-only (8,002.6 mm(3) +/- 2,788.3) and RF/TAE groups (10,398.5 mm(3) +/- 2965.8; P < .05/3). PV pressures increased significantly after TAE (P < .01) compared with the pressure before TAE in the RF/TAE/Lipiodol and RF/TAE/Lipiodol/PVE groups, but not in the RF/TAE group. A marked increase in liver parenchymal pressure was seen during RF ablation; however, there were no significant differences among groups. Accumulation of Lipiodol was noted in the sinusoids in the RF/TAE/Lipiodol and RF/TAE/Lipiodol/PVE groups., Conclusions: TAE with blockade of PV flow before RF ablation was associated with greater ablation volumes. Liver parenchymal pressure showed no correlation with increased ablation volume.

To investigate changes in bile acid biosynthesis in chicken (Gallus gallus) during embryonic stages, we studied the contribution of hepatic and plasma total bile acid levels, mRNA expression of cholesterol 7 alpha-hydroxylase (CYP7A1), and the expression of its regulatory genes in two embryo models (i.e., broilers and layers) differing in lipid metabolism. Total bile acid levels in plasma and liver were low during embryonic stages, as well as expression of CYP7A1. At hatch (P0), hepatic and plasma total bile acid levels and CYP7A1 mRNA expression in liver were markedly increased in both models. The hepatic mRNA expression of liver X receptor (LXR)alpha, a regulator of CYP7A1 gene expression gradually decreased with developmental stages of both broilers and layers. The hepatic mRNA expression of farnesoid X receptor (FXR), a repressor of CYP7A1 gene expression, also decreased with embryonic development. The present results showed that the mRNA expression of CYP7A1 and synthesis of bile acids was low in embryonic stages, suggesting that FXR might be a key regulator of CYP7A1 gene expression in the chicken embryo.

Aim: To evaluate the effects of combined radiofrequency ablation and transcatheter arterial embolization with iodized oil on ablation time, maximum output, coagulation diameter, and portal angiography in a porcine liver model., Methods: Radiofrequency ablation (RFA) was applied to in vivo livers of 10 normal pigs using a 17-gauge 3.0 cm expandable LeVeen RF needle electrode with or without transcatheter arterial embolization (TAE) with iodized oil (n=5). In each animal, 2 areas in the liver were ablated. Direct portography was performed before and after RFA. Ablation was initiated at an output of 30 W, and continued with an increase of 10 W per minute until roll-off occurred. Ablation time and maximum output until roll-off, and coagulated tissue diameter were compared between the 2 groups. Angiographic changes on portography before and after ablation were also reviewed., Results: For groups with and without TAE with iodized oil, the ablation times until roll-off were 320.6 +/- 30.9 seconds and 445.1 +/- 35.9 seconds, respectively, maximum outputs were 69.0 +/- 7.38 W and 87.0 +/- 4.83 W and maximal diameters of coagulation were 41.7 +/- 3.85 mm and 33.2 +/- 2.28 mm. Significant reductions of ablation time and maximum output, and significantly larger coagulation diameter were obtained with RFA following TAE with iodized oil compared to RFA alone. Portography after RFA following TAE with iodized oil revealed more occlusion of the larger portal branches than with RFA alone., Conclusion: RFA following TAE with iodized oil can increase the volume of coagulation necrosis with lower output and shorter ablation time than RFA alone in normal pig liver tissue.

Hepatic stellate cells (HSCs; also called as vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the space between parenchymal cells and sinusoidal endothelial cells of the hepatic lobule, and store 80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. HSCs in Arctic animals such as polar bears and Arctic foxes store 20-100 times the levels of vitamin A found in human or rat. HSCs play an important role in the liver regeneration. A gradient of vitamin A-storage capacity exists among the SCs in a hepatic lobule. The gradient was expressed as a symmetrical biphasic distribution starting at the periportal zone, peaking at the middle zone, and sloping down toward the central zone in the hepatic lobule. In pathological conditions such as liver fibrosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped SCs to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation, and functions of the HSCs. Molecular mechanisms in the reversible regulation of the SCs by ECM imply cell surface integrin-binding to ECM components followed by signal transduction processes and then cytoskeleton assembly. SCs also exist in extrahepatic organs such as pancreas, lung, kidney, and intestine. Hepatic and extrahepatic SCs form the SC system.

A patient presented with hematemesis due to gastric variceal bleeding with an intratumoral arterioportal shunt. Contrast-enhanced CT revealed gastric varices and hepatocellular carcinoma with tumor thrombi in the right portal vein. Angiography and angio-CT revealed a marked intratumoral arterioportal shunt accompanied with reflux into the main portal vein and gastric varices. Balloon-occluded retrograde venography from the gastro-renal shunt showed no visualization of gastric varices due to rapid blood flow through the intratumoral arterioportal shunt. The hepatic artery was temporarily occluded with a balloon catheter to reduce the blood flow through the arterioportal shunt, and then concurrent balloon-occluded retrograde transvenous obliteration (BRTO) was achieved. Vital signs stabilized immediately thereafter, and contrast-enhanced CT revealed thrombosed gastric varices. Worsening of hepatic function was not recognized. BRTO combined with temporary occlusion of the hepatic artery is a feasible interventional procedure for ruptured high flow gastric varices with an intratumoral arterioportal shunt.

Background: Neutrophil plays an important role in hepatic ischemia-reperfusion injury. We investigated neutrophil infiltration in liver tissue, Kupffer cells' role in neutrophil accumulation, and apoptosis and regeneration of hepatocytes in liver ischemia-reperfusion injury., Methods: Vascular microclamps were placed across the pedicles of the median and left lateral lobes for 90 minutes after 30% hepatectomy with the resection of caudate, right lateral and quadrate lobes and papillary process. Gadolinium chloride (GdCl(3)) was used to destroy Kupffer cells. Neutrophil activity was inhibited with Urge-8, a monoclonal antibody against neutrophil produced in our laboratory. GdCl(3) (10 mg/kg) and Urge-8 (50 mg/kg) were given intravenously in respective groups. Ischemia control, GdCl(3) and Urge-8 groups were compared., Results: Following hepatic reperfusion, serum interleukin-8 (IL-8) levels and hepatic neutrophil counts peaked at 3 hours, and peak concentrations of alanine aminotransferase (ALT) occurred at 6 hours. Animals of the control group showed increases in neutrophil infiltration in liver tissue, liver enzyme levels, and apoptosis index of hepatocytes and decreases in overall survival rate and proliferating cell nuclear antigen (PCNA) expression of hepatocytes. The survival rates and PCNA proportion of hepatocytes were higher and the levels of hepatic neutrophil infiltration, liver enzymes, and hepatocyte apoptosis after reperfusion were lower in the GdCl(3) and Urge-8 groups than those in the ischemia control group., Conclusions: Blockades of Kupffer cells' activity and neutrophil infiltration by GdCl(3) and Urge-8 eliminate neutrophil-mediated hepatic injury and enhance subsequent hepatic regeneration during liver ischemia-reperfusion.

Hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) [EC4.1.1.45] plays a key role in regulating NAD biosynthesis from tryptophan. The aim of this study was to evaluate the ACMSD mRNA expression after pyrazinamide or peroxisome proliferators ingestion. When rats were fed a control (pyrazinamide- and clofibrate-free) diet, 1% pyrazinamide- or 0.24% clofibrate-containing diets for 8 days, hepatic ACMSD activity and mRNA in rats consuming the clofibrate-containing diet was strongly suppressed, as compared with those fed the control and pyrazinamide diet. Pyrazinamide suppressed liver and kidney ACMSD activities, but did not affect ACMSD mRNA. Blood NAD was increased in the clofibrate and pyrazinamide groups. Shifting from the control diet to a clofibrate diet suppressed ACMSD mRNA strongly at day 1 and continued through day 4. However ACMSD activity decreased gradually. In rats fed with several kinds of peroxisome-proliferator-containing diets such as phthalate ester, bezafibrate, Wy-14,643, 2-(-4-chlorophenoxy) propionic acid, or dehydroisoandrosterone for 8 days, hepatic ACMSD mRNA was drastically decreased by all the peroxisome proliferators. These results suggest that the transcription level of hepatic ACMSD is modulated by peroxisome proliferators, and the fluctuation of the hepatic ACMSD mRNA expression was followed by that of the ACMSD activity. However, pyrazinamide does not affect the transcription level of hepatic ACMSD.

Under physiological conditions, hepatic stellate cells (HSCs) within liver lobules store about 80% of the total body vitamin A in lipid droplets in their cytoplasm, and these cells show zonal heterogeneity in terms of vitamin A-storing capacity. Vitamin A is essential for the growth and differentiation of cells, and it is well known that liver cells including HSCs show a remarkable growth capacity after partial hepatectomy (PHx). However, the status of vitamin A storage in HSCs in the liver regeneration is not yet known. Therefore, we conducted the present study to examine vitamin A storage in these cells during liver regeneration. Morphometry at the electron microscopic level, fluorescence microscopy for vitamin A autofluorescence, and immunofluorescence microscopy for desmin and alpha-smooth muscle actin (alpha-SMA) were performed on sections of liver from male Wistar strain rats at various times after the animal had been subjected to 70% PHx. The mean area of vitamin A-storing lipid droplets per HSC gradually decreased toward 3 days after PHx, and then returned to normal within 14 days after it. However, the heterogeneity of vitamin A-storing lipid droplet area per HSC within the hepatic lobule disappeared after PHx and did not return to normal by 14 days thereafter, even though the liver volume had returned to normal. These results suggest that HSCs alter their vitamin A-storing capacity during liver regeneration and that the recovery of vitamin A homeostasis requires a much longer time than that for liver volume.

To investigate the mechanisms involved in the atrophy of the intestines in lampreys (Lampetra japonica) during the spawning migration stage, we examined by morphological methods their intestines with special reference to degradation of extracellular matrix (ECM) components. Stellate cells are known to be distributed not only in the liver (hepatic stellate cells) but also in other organs, such as the pancreas, intestine, lung, and kidney (extrahepatic stellate cells). Hepatic stellate cells are well known to be able to biosynthesize, secrete, and degrade ECM. Therefore, we investigated the cellular and molecular mechanisms involved in the atrophy of the intestines by focusing on these intestinal extrahepatic stellate cells. The cells were found to contain phagocytosed and degraded collagen fibrils, which are one of the ECM components. A positive reaction for trimetaphosphatase (TMPase, a cytochemical marker of lysosomes) was preferentially detected in round or elongated vesicles in the intestinal extrahepatic stellate cells and the deposits of the reaction products coexisted with the degraded collagen fibrils. However, the basement membrane of the intestine, which membrane is also an ECM component, was preserved throughout the spawning migration stage of the lamprey and accumulated as a mass of thick membrane, suggesting the existence of a special mechanism for selective digestion of ECM components. These results indicate that the intestinal extrahepatic stellate cells in Lampetra japonica during its spawning migration stage might play an important mechanistic role in the atrophy of lamprey intestines by phagocytizing collagen fibrils and digesting the phagocytized collagen fibrils in their lysosomes., (Copyright (c) 2005 Wiley-Liss, Inc.)

The role of interleukin (IL)-6 in inflammatory injury remains controversial. The present study elucidated the role of IL-6 in liver damage during severe inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS; 1 mg/kg) using IL-6 null (-/-) mice and corresponding wild-type (WT) mice. Histological study showed that LPS treatment caused more severe liver injury with centrilobular vacuolation of hepatocytes and neutrophilic infiltration in the liver of IL-6 (-/-) mice; in contrast, neutrophilic infiltration and mild vacuolar change of hepatocytes were found in the liver of LPS-treated WT mice. Protein levels of proinflammatory molecules, such as IL-1beta, macrophage inflammatory protein-1alpha, and macrophage chemoattractant protein-1, in the livers were significantly greater in IL-6 (-/-) mice than in WT mice after LPS challenge. These results directly indicate that IL-6 is protective against liver injury induced by bacterial endotoxin, at least partly, via the modulation of proinflammatory cytokines and chemokines.

Hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD; formerly termed picolinic carboxylase) [EC4.1.1.45] plays a key role in regulating NAD biosynthesis and the generation of quinolinate (quinolinic acid) from tryptophan. Quinolinate is a potent endogenous excitotoxin of neuronal cells. We previously reported that ingestion of fatty acids by rats leads to a decrease in their hepatic ACMSD activity. However, the mechanism of this phenomenon is not clarified. We previously purified ACMSD and cloned cDNA encoding rat ACMSD. Therefore, in this study, we examined the differential effect of fatty acids on ACMSD mRNA expression by Northern blot. Moreover, we measured quinolinic acid concentration in rats fed on fatty acid. When diets containing 2% level of fatty acid were given to male Sprague-Dawley rats (4 weeks old) for 8 days, long-chain saturated fatty acids and oleic acid did not affect ACMSD mRNA expression in the liver. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) strongly suppressed the liver ACMSD mRNA expression. In rats fed with high linoleic acid diet for 8 days, serum quinolinic acid was significantly increased as compared with the rats fed on a fatty acid-free diet under the condition of the approximately same calorie ingestion. These results suggest that the transcription level of ACMSD is modulated by polyunsaturated fatty acids, and suppressive potency of ACMSD mRNA is n-3 fatty acid family>linoleic acid (n-6 fatty acid)>saturated fatty acid. Moreover, this study provides the information that a high polyunsaturated fatty acid diet affects the production of quinolinic acid in serum by suppressing the ACMSD activity.

To investigate intercellular junctions between mammalian hepatic stellate cells, we examined cultured human and rat hepatic stellate cells at the ultrastructural and molecular levels. Intercellular junctions between cultured human stellate cells, which developed irrespective of the type of culture substratum, were detected by transmission electron microscopy. On the basis of their characteristic ultrastructure, these junctions were identified in cultured human hepatic stellate cells as adherens junctions but not as tight junctions, desmosomes, or gap junctions. N-cadherin, alpha-catenin and beta-catenin, and p120ctn were detected by Western blotting in rat stellate cells as molecular components of the intercellular adhesive structures. Immunofluorescence for pan-cadherin, alpha-catenin, and beta-catenin were also detected in cultured human stellate cells. Moreover, pan-cadherin and beta-catenin were co-localized at the contact regions between the cultured human stellate cells. These data suggest that the junctional adhesion between the stellate cells can be formed both in vivo and in vitro. Thus, hepatic stellate cells may participate in the structural organization of the cells in liver lobules through the formation of intercellular adherens junctions. This is the first description of the presence of cell-cell junctions between hepatic stellate cells in mammals at the fine structural and molecular levels.

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.

Hepatic stellate cells (HSCs), a mesenchymal cell type in hepatic parenchyma, have unique features with respect to their cellular origin, morphology, and function. Normal, quiescent HSCs function as major vitamin A-storing cells containing over 80% of total vitamin A in the body to maintain vitamin A homeostasis. HSCs are located between parenchymal cell plates and sinusoidal endothelial cells, and extend well-developed, long processes surrounding sinusoids in vivo as pericytes. However, HSCs are known to be 'activated' or 'transdifferentiated' to myofibroblast-like phenotype lacking cytoplasmic lipid droplets and long processes in pathological conditions such as liver fibrosis and cirrhosis, as well as merely during cell culture after isolation. HSCs are the predominant cell type producing extracellular matrix (ECM) components as well as ECM degrading metalloproteases in hepatic parenchyma, indicating that they play a pivotal role in ECM remodeling in both normal and pathological conditions. Recent findings have suggested that HSCs have a neural crest origin from their gene expression pattern similar to neural cell type and/or smooth muscle cells and myofibroblasts. The morphology and function of HSCs are regulated by ECM components as well as by cytokines and growth factors in vivo and in vitro. Liver regeneration after partial hepatectomy might be an invaluable model to clarify the HSC function in elaborate organization of liver tissue by cell-cell and cell-ECM interaction and by growth factor and cytokine regulation.

An effective way to overcome shortage of donors in liver transplantation (LTx) is to consider such from non-heart-beating donors (NHBDs). We investigated how a liver graft should be treated before and/or after procurement for successful LTx from an NHBD. Porcine LTx was performed with FR167653 (FR), a dual inhibitor of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and/or prostaglandin E(1) (PG). Animals were allocated to an FR group (n=4, donors and recipients were treated with FR), a PG group (n=4, donors and recipients were treated with PG), or an FRPG group (n=4, donors and recipients were treated with both FR and PG). No recipients in the FR group and only two of four recipients in the PG group survived, whereas all recipients in the FRPG group survived. Suppression of TNF-alpha and IL-1beta and maintenance of microcirculation are the key to successful transplantation from NHBDs.