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Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. It occurs with a prevalence of up to 25%, of which 10-20% cases progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. The histopathology of NASH is characterized by neutrophilic infiltration, and endotoxins from gram-negative rods have been postulated as a contributing factor. Elevations in endotoxin levels in the blood can be classified as intestinal and hepatic factors. In recent years, leaky gut syndrome, which is characterized by impaired intestinal barrier function, has become a significant issue. A leaky gut may prompt intestinal bacteria dysbiosis and increase the amount of endotoxin that enters the liver from the portal vein. These contribute to persistent chronic inflammation and progressive liver damage. In addition, hepatic factors suggest that liver damage can be induced by low-dose endotoxins, which does not occur in healthy individuals. In particular, increased expression of CD14, an endotoxin co-receptor in the liver, may result in leptin-induced endotoxin hyper-responsiveness in obese individuals. Thus, elevated blood endotoxin levels contribute to the progression of NASH. The current therapeutic targets for NASH treat steatosis and liver inflammation and fibrosis. While many clinical trials are underway, no studies have been performed on therapeutic agents that target the intestinal barrier. Recently, a randomized placebo-controlled trial examined the role of the intestinal barrier in patients with NAFLD. To our knowledge, this study was the first of its kind and study suggested that the intestinal barrier may be a novel target in the future treatment of NAFLD., Competing Interests: ANa receives grants and research support from Gilead, Mylan EPD, EA Pharma, Kowa, Taisho, and Biofermin. ANa is also a consultant for Gilead, Boehringer Ingelheim, Bristol Myers Squibb, Kowa, Astellas, EA Pharma, and Mylan EPD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kessoku, Kobayashi, Imajo, Tanaka, Yamamoto, Takahashi, Kasai, Ozaki, Iwaki, Nogami, Honda, Ogawa, Kato, Higurashi, Hosono, Yoneda, Okamoto, Usuda, Wada, Kobayashi, Saito and Nakajima.)

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin-angiotensin-aldosterone system (RAS).

The Fibrosis-4 (FIB4) index could indicate the liver fibrosis in patients with chronic hepatic diseases. It was calculated using the following formula: (age × aspartate aminotransferase [U/L]) / (platelet count [10 3 /μL] × √alanine aminotransferase [U/L]). However, the clinical impact of the FIB4 index in the acute phase of acute heart failure (AHF) has not been sufficiently investigated.A total 1,468 AHF patients were analyzed. The median FIB4 index was 2.71 [1.85-4.22]. The patients were divided into three groups according to the quartiles of their FIB4 index (low-FIB4 [Q1, ≤ 1.847], middle-FIB4 [Q2/Q3, 1.848-4.216], and high-FIB4 [Q4, ≥ 4.216] groups). A Kaplan-Meier curve analysis showed that the prognosis, such as all-cause mortality and HF events within 365 days, was significantly poorer in the high-FIB4 group than in the middle-FIB4 and low-FIB4 groups. A multivariate Cox regression model identified high FIB4 index as an independent predictor of 365-day all-cause death (hazard ratio (HR): 1.660, 95% CI: 1.136-2.427) and HF events (HR: 1.505, 95% CI: 1.145-1.978). The multivariate logistic regression analysis showed that the high plasma volume status (PVS) (Q4, odds ratio [OR]: 2.099, 95% CI: 1.429-3.082), low systolic blood pressure (SBP) (< 100 mmHg, OR: 3.825, 95% CI: 2.504-5.840), and low left ventricular ejection fraction (< 40%, OR: 1.321, 95% CI: 1.002-1.741) were associated with a high FIB4 index.A high FIB4 index can predict adverse outcomes in AHF patients, which indicate that congestive liver and liver hypoperfusion occur due to low cardiac output in the acute phase of AHF.

Background: The laxative drug lubiprostone improves intestinal permeability in healthy volunteers. We aimed to assess efficacy and safety of lubiprostone in patients with non-alcoholic fatty liver disease (NAFLD) with constipation via attenuation of intestinal permeability., Methods: This randomised, double-blind, placebo-controlled, phase 2a study in Yokohama City University Hospital, Japan, recruited patients (aged 20-85 years) with NAFLD and constipation, alanine aminotransferase (ALT) at least 40 U/L, liver stiffness (≤6·7 kPa), and hepatic fat fraction at least 5·2% when assessed by MRI-proton density fat fraction. Eligible patients were randomly assigned (11:10:9) by a computer-based system and stratified by age and sex to receive 24 μg lubiprostone, 12 μg lubiprostone, or placebo, orally, once per day for 12 weeks. The primary endpoint was the absolute changes in ALT at 12 weeks. Efficacy analysis was done by intention to treat. Safety was assessed in all treated patients. This trial was registered with University Hospital Medical Information Network Clinical Trials Registry (UMIN000026635)., Findings: Between March 24, 2017, and April 3, 2018, we screened 288 patients, of whom 150 (52%) were randomly assigned to treatment: 55 patients were assigned to receive 24 μg lubiprostone, 50 to receive 12 μg lubiprostone, and 45 to receive placebo. A greater decrease in the absolute ALT levels from baseline to 12 weeks was seen in the 24 μg lubiprostone group (mean -13 U/L [SD 19]) than in the placebo group (1 U/L [24]; mean difference -15 U/L [95% CI -23 to -6], p=0·0007) and in the 12 μg lubiprostone group (-12 U/L [21]) than in the placebo group (mean difference -13 U/L [-22 to -5], p=0·0023). 18 (33%) of 55 patients in the 24 μg group had at least one adverse event, as did three (6%) of 47 patients in the 12 μg group and three (7%) of 43 in the placebo group. The most common adverse event was diarrhoea (17 [31%] of patients in the 24 μg group, three [6%] in the 12 μg group, none in the placebo group). No life-threatening events or treatment-related deaths occurred., Interpretation: Lubiprostone was well tolerated and reduced the levels of liver enzymes in patients with NAFLD and constipation. Further studies are necessary to better define the efficacy and tolerability of lubiprostone in patients with NAFLD without constipation., Funding: Mylan EPD G.K., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Warm ischemia and reperfusion injury (IRI) is a prognostic factor in donation after cardiac death donor transplantation. However, a reliable method to predict IRI before transplantation has not been established. The aim of this study was to identify predictive markers of hepatic IRI by simultaneous measurement of endogenous molecules using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Rats were subjected to hepatic warm ischemia (70%) for 30 or 90 minutes and subsequent reperfusion. The livers were collected at the end of ischemia and 1 hour, 6 hours, and 24 hours after reperfusion. The liver tissue sections were applied to IMS (m/z 200-2000). Candidate molecules were identified by tandem mass spectrometry. Imaging mass spectrometry (IMS) revealed a significant increase in the taurine conjugates of dihydroxycholanoic acid (TDHCA) during ischemia and a tendency to return to the basal level after reperfusion. Notably, high-resolution measurements revealed focal accumulation of TDHCA in the intrahepatic bile duct with ischemic time. In conclusion, IMS is a useful method to detect minute changes provoked by ischemia, which are barely detectable in assays involving homogenization. Accordingly, focal accumulation of TDHCA during ischemia may be a candidate marker for predicting later IRI., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O 2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background and Purpose: We reported previously that hydrogen gas (H 2 ) reduced hepatic ischemia and reperfusion injury (IRI) after prolonged cold storage (CS) of livers retrieved from heart-beating donors. The present study was designed to assess whether H 2 reduced hepatic IRI during donation of a cardiac death (DCD) graft with subsequent CS., Methods: Rat livers were harvested after 30-min cardiac arrest and stored for 4 h in University of Wisconsin solution. The graft was reperfused with oxygenated buffer, with or without H 2 (H 2 or NT groups, respectively), at 37° for 90 min on isolated perfused rat liver apparatus., Results: In the NT group, liver enzyme leakage, apoptosis, necrosis, energy depletion, redox status, impaired microcirculation, and bile production were indicative of severe IRI, whereas in the H 2 group these impairments were significantly suppressed. The phosphorylation of cytoplasmic MKK4 and JNK were enhanced in the NT group and suppressed in the H 2 group. NFkB-p65 and c-Fos in the nucleus were unexpectedly unchanged by IRI regardless of H 2 treatment, indicating the absence of inflammation in this model., Conclusion: H 2 was observed to ameliorate IRI in the DCD liver by maintaining microcirculation, mitochondrial functions, and redox status, as well as suppressing the cytoplasmic MKK4-JNK-mediated cellular death pathway.

The contribution of caspases to hepatic ischemia/reperfusion (I/R)-induced apoptosis has not been completely understood yet. Several studies have demonstrated increased caspase activity during I/R and the protective effect of caspase inhibitors against I/R injuries. However, reports with opposing results also exist. Herein, we examined the contribution of caspases to the I/R-induced hepatic apoptosis in rats using caspase inhibitors and specific substrates of caspases. Hepatic I/R was induced via a 2-h occlusion of the portal vein and the hepatic artery, without conducting bile duct occlusion. DNA laddering and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were increased at 3 h after reperfusion. Pretreatment with caspase inhibitors (Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-cmk) 2 or 10 mg/kg intravenously (i.v.), 20 mg/kg intraperitoneally (i.p.), Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) 3 mg/kg i.v.) failed to reduce apoptosis induced by I/R. Interestingly, apoptosis induced by the portal triad (hepatic artery, portal vein, and bile duct) occlusion/reperfusion could be marginally attenuated using Z-Asp-cmk (2 mg/kg i.v.). The cleavage activity for Ac-DEVD-α-(4-methylcoumaryl-7-amide) (MCA), a caspase-3/7/8/9 substrate, was significantly increased by I/R. Conversely, the cleavage activities for Ac-DNLD-MCA and MCA-VDQVDGW[K-DNP]-NH 2 , specific substrates for caspase-3 and -7 respectively, were decreased by I/R. Protein expression of the cellular inhibitor of apoptosis protein 2 (c-IAP2), an endogenous caspase inhibitor, was increased by ischemia. Nuclear translocation of the apoptosis-inducing factor (AIF), an initiator protein of caspase-independent apoptosis, was also increased during I/R. These results suggest that caspases are inhibited by c-IAP2 induced during ischemia and that AIF may be involved in initiation of apoptosis induced by hepatic I/R without bile duct occlusion.

Aims: Bile acid binding resin (BAR) improves glycaemic control in patients with type 2 diabetes. Although the mechanism is hypothesised to involve the clearance of excess hepatic triglyceride, this hypothesis has not been examined in appropriately designed studies. Therefore, we investigated whether reduced hepatic triglyceride deposition is involved in BAR-mediated improvements in glycaemic control in spontaneous fatty liver diabetic mice without dietary interventions., Methods: Male 6-week-old fatty liver Shionogi (FLS) mice were fed a standard diet without or with 1.5% BAR (colestilan) for 6 weeks. Glucose tolerance, insulin sensitivity, hepatic lipid content, and gene expression were assessed. A liver X receptor (LXR) agonist was also administered to activate the LXR pathway. We also retrospectively analysed the medical records of 21 outpatients with type 2 diabetes who were treated with colestilan for ≥6 months., Results: BAR enhanced glucose tolerance and insulin sensitivity in FLS mice without altering fat mass. BAR improved hepatic insulin sensitivity, increased IRS2 expression, and decreased SREBP expression. BAR reduced hepatic cholesterol levels but not hepatic triglyceride levels. BAR also reduced the expression of LXR target genes, and LXR activation abolished the BAR-mediated improvements in glycaemic control. Colestilan significantly lowered serum cholesterol levels and improved glycaemic control in patients with type 2 diabetes., Conclusions: BAR improved hepatic insulin resistance in FLS mice by reducing hepatic cholesterol without affecting hepatic triglyceride levels or body fat distribution. Our study revealed that BAR improves glycaemic control at least in part by downregulating the hepatic cholesterol-LXR-IRS2 pathway., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Hydrogen sulfide (H2S) ameliorates hepatic ischemia and reperfusion injury (IRI), but the precise mechanism remains elusive. We investigated whether sodium hydrogen sulfide (NaHS), a soluble derivative of H2S, would ameliorate hepatic IRI, and if so, via what mechanism., Methods: Mice were subjected to partial warm ischemia for 75 min followed by reperfusion. Either NaHS or saline was administered intravenously 10 min before reperfusion. The liver and serum were collected 3, 6, and 24 h after reperfusion., Results: In the NaHS(-) group, severe IRI was apparent by the ALT leakage, tissue injury score, apoptosis, lipid peroxidation, and inflammation (higher plasma TNF-α, IL-6, IL-1β, IFN-γ, IL-23, IL-17, and CD40L), whereas IRI was significantly ameliorated in the NaHS(+) group. These effects could be explained by the augmented nuclear translocation of Nrf2, and the resulting up-regulation of HO-1 and thioredoxin-1. Phosphorylation of the PDK-1/Akt/mTOR/p70S6k axis, which is known to mediate pro-survival and anti-apoptotic signals, was significantly augmented in the NaHS(+) group, with a higher rate of PCNA-positive cells thereafter., Conclusion: NaHS ameliorated hepatic IRI by direct and indirect anti-oxidant activities by augmenting pro-survival, anti-apoptotic, and anti-inflammatory signals via mechanisms involving Nrf-2, and by accelerating hepatic regeneration via mechanisms involving Akt-p70S6k.

We have previously reported that phytoceramide and phytosphingosine (PHS) stimulated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in cells. PPARγ is a therapeutic target for type 2 diabetes. We found in this study that an oral administration of PHS improved diet-induced glucose intolerance in mice. Since PHS is highly expressed in yeast, PHS in fermented foods may improve diabetes.

A 50-year-old man with primary biliary cirrhosis underwent living-donor liver transplantation (LDLT) using a graft of a left hemiliver with a left caudate lobe and duct-to-duct hepaticocholedochostomy. Postoperative bile leakage necessitated percutaneous drainage 22 days after LDLT. The patient presented to our hospital 205 days after the LDLT with abdominal distension and fever. Computed tomography showed ascites and a diffusely mottled pattern in the graft. The caudate lobe was swollen, and its bile ducts were dilated. The inferior vena cava was forced to the right by the swollen caudate lobe, and the root of the hepatic vein was stretched. The hepatic vein was not contrasted. Endoscopic retrograde cholangiography showed a biliary anastomotic stricture. Based on these findings, we diagnosed a severe outflow block of the hepatic vein and biliary anastomotic stricture. We performed balloon dilation of the biliary anastomosis and implanted a metallic stent in the hepatic vein. Thereafter, his clinical symptoms improved dramatically.

Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease.

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, β-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.

The liver consists of many cell types with specialized functions. Hepatocytes are one of the main players in the organ and therefore are the most vulnerable cells to damage. Since they are not everlasting cells, they need to be replenished throughout life. Although the capacity of hepatocytes to contribute to their own maintenance has long been recognized, recent studies have indicated the presence of both intrahepatic and extrahepatic stem/progenitor cell populations that serve to maintain the normal organ and to regenerate damaged parenchyma in response to a variety of insults.The intrahepatic compartment most likely derives primarily from the biliary tree, particularly the most proximal branches, i.e. the canals of Hering and smallest ductules. The extrahepatic compartment is at least in part derived from diverse populations of cells from the bone marrow. Embryonic stem cells (ES's) are considered as a part of the extrahepatic compartment. Due to their pluripotent capabilities, ES cell-derived cells form a potential future source of hepatocytes, to replace or restore hepatic tissues that have been damaged by disease or injury. Progressing knowledge about stem cells in the liver would allow a better understanding of the mechanisms of hepatic homeostasis and regeneration. Although a human stem cell-derived cell type equivalent to primary hepatocytes does not yet exist, the promising results obtained with extrahepatic stem cells would open the way to cell-based therapy for liver diseases.

Cell-based technologies to support/restore liver function represent one of the most promising opportunities in the treatment of acute liver failure. However, the understanding of the constituent cell types that interact to achieve liver-specific structure and function has not been achieved in the development of liver assist devices (LADs). Here we show that hepatocytes migrated toward and adhered and formed sinusoids-like structures in conjunction with liver nonparenchymal cells, and that this liver organoid formed sophisticated tissue after 7 days in an implanted LAD in rodents. Hepatocytes only or in combination with human nonparenchymal liver cell lines (endothelial, cholangiocytes, and stellate cells) were cultured in Matrigel. Ultrastructural analysis showed that the hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and lumen. The sinusoid-like structures retained albumin secretion and drug metabolism capabilities. In addition, LADs containing cocultures of human liver nonparenchymal cells were transplanted in animals for a week; the liver tissue formed sophisticated structures resembling the liver. These results demonstrate the importance of nonparenchymal cells in the cellular composition of LADs. The novelty of the culture's sinusoid-like organization and function strongly support the integration of liver nonparenchymal units into hepatocyte coculture-based LADs as a potential destination therapy for liver failure.

Unlabelled: The specific mechanisms of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) pathogenesis remain unknown. In the present study we investigated the differences between NAFL and NASH in terms of liver lipid metabolites and serum lipoprotein. In all, 104 Japanese subjects (50 men and 54 postmenopausal women) with histologically verified NAFL disease (NAFLD) (51 with NAFL, 53 with NASH) were evaluated; all diagnoses were based on liver biopsy findings and the proposed diagnostic criteria. To investigate the differences between NAFL and NASH in humans, we carefully examined (1) lipid inflow in the liver, (2) lipid outflow from the liver, (3) very-low-density lipoprotein (VLDL) synthesis in the liver, (4) triglyceride (TG) metabolites in the liver, and (5) lipid changes and oxidative DNA damage. Most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, VLDL synthesis and lipid outflow from the liver were impaired, and surplus TGs might have been produced as a result of lipid oxidation and oxidative DNA damage in the NASH group., Conclusion: A growing body of literature suggests that a deterioration in fatty acid oxidation and VLDL secretion from the liver, caused by the impediment of VLDL synthesis, might induce serious lipid oxidation and DNA oxidative damage, impacting the degree of liver injury and thereby contributing to the progression of NASH. Therefore, dysfunctional VLDL synthesis and release may be a key factor in progression to NASH.

Hepatocyte transplantation is effective for treating liver failure, but healthy donors as a source of hepatocytes are quite limited. The livers of patients with hepatic fibrosis could be an alternative source; however, few reports have examined the nature of hepatocytes from fibrotic livers (f-hepatocytes). In this study, we compared the growth of f-hepatocytes and hepatocytes from normal livers (n-hepatocytes). Hepatocytes were isolated from normal and CCl(4)-treated wild-type Fischer rats that express dipeptidyl dipeptidase IV (DPPIV) gene (DPPIV(+)). The n- and f-hepatocytes proliferated in culture at similar rates. Both types of hepatocytes were transplanted into DPPIV(-) mutant Fischer rats that had been treated with retrorsine to injure the liver and were partially hepatectomized (PHx) before transplantation. Both n- and f-DPPIV(+)-hepatocytes proliferated and formed colonies. The colony sizes of f-hepatocytes 21 days posttransplantation were approximately three times those of n-hepatocytes. The hepatocytes were analyzed using a fluorescence activated cell sorter (FACS). The FACS profile differed between f- and n-hepatocytes: f-hepatocytes were less granular, less autofluorescent, and smaller than n-hepatocytes. These characteristics of f-hepatocytes resembled those reported for small-sized n-hepatocytes (SHs), which are highly proliferative and preferentially express a unique set of 10 SH genes. However, f-hepatocytes preferentially expressed only five of the SH genes. The expression profile of f-hepatocytes was rather similar to that of proliferating n-hepatocytes in the regenerating liver after PHx. The f-hepatocytes were morphologically normal and did not show any preneoplastic phenotype. These normal and proliferative natures of f-hepatocytes in vivo suggest the fibrotic liver as a source of hepatocytes for transplantation.

Background: Liver fibrosis is the main predictor of the progression of nonalcoholic fatty liver disease. Transient elastography (FibroScan), which measures liver stiffness, is a novel, noninvasive method to assess liver fibrosis., Aim: We investigated the usefulness of liver stiffness measurement in the evaluation of liver fibrosis in nonalcoholic fatty liver disease patients., Study Population: A total of 97 nonalcoholic fatty liver disease patients., Methods: Transient elastography was performed for liver stiffness measurement in 97 nonalcoholic fatty liver disease patients. And the relationship between histological parameters and liver stiffness measurement was studied by multivariate analysis. Moreover, we investigated the relationship between liver stiffness measurement and the serum levels of hyaluronic acid and type IV collagen 7s domain., Results: The liver stiffness was well correlated with the stage of liver fibrosis (Kruskal-Wallis test p < 0.0001). The areas under the receiver-operating characteristic curves were 0.927 for > or = F1, 0.865 for > or = F2, 0.904 for > or = F3, 0.991 for > or = F4. Only fibrosis stage was correlated significantly with liver stiffness measurement by multiple regression analysis. Liver stiffness was also strongly correlated with the serum levels of type IV collagen 7s domain (r = 0.525, p < 0.0001) and hyaluronic acid (r = 0.457, p < 0.0001)., Conclusions: Our results show a significant correlation between liver stiffness measurement and fibrosis stage in nonalcoholic fatty liver disease patients, as confirmed by the results of liver biopsy, which remains the gold standard for evaluation of the severity of liver fibrosis in patients with nonalcoholic steatohepatitis.

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.

Alagille syndrome (AGS, MIM 118450) is an autosomal dominant inherited disease. Paucity of interlobular bile ducts is one of the major abnormalities. To explore the molecular mechanism by which mutation in the human Jagged 1 gene (JAG1, MIM 601920) causes liver defects, we investigated the gene regulation of JAG1 to hepatocyte growth factor gene (HGF). By transfecting wild-type and mutant JAG1 into COS-7 cells in vitro, we found that HGF is a target gene of JAG1 downstream. Wild-type JAG1 is inhibitory for HGF expression and mutant JAG1s relieve the inhibition. Several domain disruptions in mutant JAG1 protein reveal a reduced inhibition to HGF expression at different levels. JAG1 mutations actually result in HGF overexpression. Furthermore, JAG1 controls HGF expression by a dosage-dependent regulation and Notch2 signaling seems to mediate JAG1 function. Given that HGF plays a critical role in differentiation of hepatic stem cells, overexpression of HGF acts on off-balanced cell fate determination in AGS patients. Hepatic stem cells may differentiate towards more hepatocytes but less biliary cells, thus causing the paucity of interlobular bile ducts in liver development of AGS. Our novel findings demonstrated that dosage-dependent regulation by mutations of JAG1 is a fundamental mechanism for liver abnormality in AGS.

Steatosis is one of the most common liver diseases and is associated with the metabolic syndrome. A line of evidence suggests that peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma are involved in its pathogenesis. Hepatic overexpression of PPARgamma1 in mice provokes steatosis, whereas liver-specific PPARgamma disruption ameliorates steatosis in ob/ob mice, suggesting that hepatic PPARgamma functions as an aggravator of steatosis. In contrast, PPARalpha-null mice are susceptible to steatosis because of reduced hepatic fatty acid oxidation. PPARgamma with mutations in its C-terminal ligand-binding domain (L468A/E471A mutant PPARgamma1) have been reported as a constitutive repressor of both PPARalpha and PPARgamma activities in vitro. To elucidate the effect of co-suppression of PPARalpha and PPARgamma on steatosis, we generated mutant PPARgamma transgenic mice (Liver mt PPARgamma Tg) under the control of liver-specific human serum amyloid P component promoter. In the liver of transgenic mice, PPARalpha and PPARgamma agonist-induced augmentation of the expression of downstream target genes of PPARalpha and PPARgamma, respectively, was significantly attenuated, suggesting PPARalpha and PPARgamma co-suppression in vivo. Suppression of PPARalpha and PPARgamma target genes was also observed in the fasted and high-fat-fed conditions. Liver mt PPARgamma Tg were susceptible to fasting-induced steatosis while being protected against high-fat diet-induced steatosis. The opposite hepatic outcomes in Liver mt PPARgamma Tg as a result of fasting and high-fat feeding may indicate distinct roles of PPARalpha and PPARgamma in 2 different types of nutritionally provoked steatosis.

We evaluated the changes in the levels of released cytokines following heat preconditioning of the livers used in rat liver transplantation. The donor rats in the heat preconditioning (HP) group were subjected to heat preconditioning 48 h before graft harvesting. The liver isografts were preserved in Euro-Collins solution for 8 h, and then transplanted orthotopically. The one-week survival rate of the HP group was significantly better than that of the control (C) group. The serum levels of interleukin-6 and interleukin-10 were significantly lower in the HP group than in the C group. Histological staining revealed that the stagnation of red blood cells and infiltration of neutrocytes were reduced in the HP group. The expression of intercellular adhesion molecule-1 was decreased around the central vein in the HP group, as revealed by immunohistochemistry. These results indicate that heat preconditioning downregulates cytokine release and reduces the frequency of microcirculation disorders.

Catechins such as epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), and epigallocatechin (EGC) are polyphenol components of green tea. EGCG is the major component and has been reported to possess a wide range of biological properties including anti-fibrogenic activity. In hepatic fibrosis, activated hepatic stellate cells (HSCs) play a central role. In this study, we investigated the effect of catechins, including EGCG, on collagen production and collagenase activity in rat primary HSCs and activated human HSC-derived TWNT-4 cells. EGCG (50 microM) suppressed type I collagen production in rat HSCs more than ECG (50 microM) did; however, EGC (50 microM) did not show suppressive effects. EGCG also inhibited both collagen production and collagenase activity (active matrix metalloproteinase-1 [MMP-1]) in a dose-dependent manner, but did not affect the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production in TWNT-4 cells. Real-time PCR unexpectedly revealed that EGCG enhanced the transcription of type I collagen and TIMP-1, but did not affect the transcription of alpha-smooth muscle actin (alpha-SMA), and reduced the transcription MMP-1 in TWNT-4 cells. These findings demonstrated that EGCG inhibited collagen production regardless of enhanced collagen transcription and suppressed collagenase activity, and suggested that EGCG might have therapeutic potential for liver fibrosis.

Background: Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF., Methods: We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10-1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal)., Results: When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4alpha, and C/EBPalpha in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal., Conclusions: The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.

For liver-targeted regenerative medicine, embryonic stem (ES) cell-derived hepatocyte-like cells proffer great expectation. In vitro exposure to a combination of various growth factors, such as hepatocyte growth factor and fibroblast growth factor-4, as well as cytokines, leads to differentiation of ES cells into hepatocyte-like cells. We sought to determine the in vivo environment that allowed engraftment of ES cells transplanted to the liver. Thus, we examined the effect of partial hepatectomy (50%) (PHT) and subsequent radiation (RT) of the male Balb/c mouse host liver on ES cell engraftment. ES cells (5 x 10(6)) derived from 129Sv mice were transplanted into the residual liver. The controls were ES cells transplanted into a normal liver. Bromo-deoxy-residine (BrdU)-uptake was performed to evaluate the effect of hepatectomy and RT on hepatocyte regeneration. Mouse ES cells engrafted, forming teratomas in the normal liver without showing any mononuclear infiltration. A liver modified by PHT and RT facilitated engraftment of mouse ES cells compared with a normal liver. Hepatic RT significantly suppressed hepatocytic uptake of BrdU.

Purpose: Uptake and degradation of naked plasmid DNA (pDNA) by liver sinusoidal endothelial cells (LSECs) were investigated., Methods: Tissue distribution and intrahepatic localization were determined after an intravenous injection of 111In- or 32P-labeled pDNA into rats. Cellular uptake and degradation of fluorescein- or 32P-labeled pDNA were evaluated using primary cultures of rat LSECs., Results: Following intravenous injection, pDNA was rapidly eliminated from the circulation and taken up by the liver. Fractionation of liver-constituting cells by centrifugal elutriation revealed a major contribution of LSECs to the overall hepatic uptake of pDNA. Confocal microscopic study confirmed intracellular uptake of pDNA in cultured LSECs. Apparent cellular association of pDNA was similar at 37 degrees C and 4 degrees C. However, trichloroacetic acid (TCA) precipitation experiments showed the TCA-soluble radioactivity in the culture medium increased in an accumulative manner at 37 degrees C. Involvement of a specific mechanism was demonstrated, as the uptake of pDNA was significantly inhibited by excess unlabeled pDNA and some polyanions (polyinosinic acid, dextran sulfate, heparin) but not by others (polycytidylic acid, dextran). These inhibitors also reduced the amount of TCA-soluble radioactivity in the culture medium. CONCLUSION. These results suggest that LSECs efficiently ingested and rapidly degraded naked pDNA in vivo and in vitro and released the degradation products into the extracellular space.

Background and Aims: Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting., Methods: First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T-and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics., Results: The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intra-cellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1., Conclusions: This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

Background: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure., Methods: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure., Results: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression., Conclusions: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability., (Copyright 2004 John Wiley & Sons, Ltd.)

Background: A large-volume intravenous (i.v.) injection of DNA, i.e. a hydrodynamics-based transfection procedure, is known to be an efficient and liver-specific method of in vivo gene delivery. However, little is available on an applicable particle size in the procedure., Methods: We examined the effect of particle size on the hepatic delivery by the hydrodynamics-based procedure, using fluorescein isothiocyanate labeled polystyrene microspheres (MS) of 50, 200 or 500 nm in diameter. MS were injected intravenously to mice by a conventional (normal) or the hydrodynamics-based procedure and their degree of hepatic uptake was determined fluorometrically., Results: For all sizes tested, the two procedures were similar in terms of the apparent degree of hepatic uptake, whereas the intrahepatic localization of MS was apparently different between the procedures as shown by an examination of frozen tissue sections. In mice with gadolinium chloride induced Kupffer cell blockade, the hepatic uptake of MS following the normal procedure was decreased while that of the hydrodynamics-based procedure was less affected. This phenomenon of enhanced hepatic delivery seemed to be more effective for larger particles. Confocal microscopic observation of hepatocyte suspensions indicated that part of the injected MS-50 was delivered intracellularly following the hydrodynamics-based procedure, whereas almost all the observed MS-200 and MS-500 were detected in the extracellular compartment or on the surface of the cells. This was supported by the fact that most of the injected MS existed pericellularly around the transgene-expressing cells., Conclusions: The hydrodynamics-based procedure facilitated extravasation and hepatic delivery of MS. Larger MS were more efficiently extravasated and trapped by the liver, whereas intracellular delivery hardly occurred with them., (Copyright 2004 John Wiley & Sons, Ltd.)

Background: Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT)., Methods: SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS)., Results: One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel., Conclusion: We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-beta receptor (PDGF-betaR), alpha-smooth muscle actin (alpha-SMA), and type I collagen (alpha1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-gamma or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (alpha1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-gamma (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (alpha1), PDGF-betaR, and alpha-SMA expression and suppressed TGF-beta1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

Purpose: Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy-phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132)., Methods: Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured., Results: After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 +/- 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with Km and Vmax of 5.7 microM and 564 pmol/min/10(6) cells, respectively. The uptake was Na+ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydrazone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with Km and Vmax values of 6.1 microM and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats., Conclusions: These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.

Purpose: To investigate the pharmacokinetics and preventive effects of liver-targeted catalase (CAT) derivatives on hepatic injury caused by reactive oxygen species., Methods: The hepatic uptake of 111In-CAT, galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT was investigated in isolated perfused rat livers in a single-pass constant infusion mode. Then, pharmacokinetic parameters were obtained by fitting equations derived from a one-organ pharmacokinetic model to the outflow profile. Their effects in preventing hydrogen peroxide-induced injury were determined by lactate dehydrogenase (LDH) release from the perfused liver., Results: The extraction of CAT derivatives by the liver was dose-dependent, and increased by the chemical modifications described. After being bound to the cell surface, chemically modified CAT derivatives were internalized by the liver faster than CAT. Preperfusion of a CAT derivative significantly reduced LDH release by hydrogen peroxide at least for 30 min, and Man-CAT and Suc-CAT effectively inhibited this release., Conclusions: Internalized CAT derivatives are also effective in degrading hydrogen peroxide and targeted delivery of CAT to liver nonparenchymal cells by mannosylation or succinylation is a useful method for the prevention of hepatic injury caused by reactive oxygen species.

Hepatic uptake and gene expression mechanisms following intravenous administration of naked plasmid DNA (pDNA) by conventional and hydrodynamics-based procedures were studied in mice. After conventional (normal) intravenous injection, (32)P-labeled pDNA was rapidly eliminated from the circulation and predominantly taken up by the liver nonparenchymal cells while no significant gene expression was observed in this organ. The hepatic uptake process was saturable. Involvement of a specific mechanism was demonstrated since the hepatic uptake of [(32)P]pDNA was dramatically inhibited by cold pDNA, calf thymus DNA, and some polyanions [polyinosinic acid (poly I), dextran sulfate], but not by others (polycytidylic acid, chondroitin sulfate). The liver endothelial cells appeared to be a major contributor because gadolinium chloride (GdCl(3))-induced Kupffer cell blockade did not affect the hepatic uptake. After intravenous injection of naked pDNA with a large volume of saline at a high velocity (hydrodynamics-based procedure), the apparent hepatic uptake profile was similar to that after normal injection. The hepatic uptake was not inhibited by prior administration of polyanions, including poly I, dextran sulfate, and heparin. The hydrodynamics-based procedure resulted in marked gene expression in the liver, which was not inhibited by prior administration of polyanions or GdCl(3) treatment. These results indicate that pDNA uptake is a nonspecific process. This hypothesis was supported by the finding that significant hepatic uptake of bovine serum albumin and immunoglobulin G was observed after the hydrodynamics-based procedure.

Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p<0.001). Our finding demonstrated that the DSL domain in JAG1 appears to be essential for normal liver development and function.

We developed a new high-performance liquid chromatographic method using an ODS column and a chiral column for the assay of racemic 4-OH-PL sulfate and enantiomeric 4-OH-PL sulfates, respectively. The method was successfully applied to measure phenolsulfotransferase (PST) activities for 4-OH-PL in cytosolic fractions from livers of Japanese monkeys (Macaca fuscata) and for comparison with its activity of cytosolic fractions from rat, rabbit, dog, and human livers and Hep G2 cells. The activity was ranked as Hep G2 cells > monkeys = humans = dogs = rats > rabbits. To evaluate the Japanese monkey as a nonhuman animal model in drug metabolism studies, we further characterized sulfation of 4-OH-PL as a further metabolic pathway in monkey livers to compare that with human livers. Inhibition studies in which cytosolic fractions were preincubated at 43 degrees C or 2,6-dichloro-4-nitrophenol (DCNP) used as a PST inhibitor indicated that two kinds of PSTs, thermolabile, low-Km and DCNP-resistant PST and thermostable, high-Km and DCNP-sensitive PST were involved in 4-OH-PL sulfation by monkey liver cytosol, which is very similar to the reported profile of 4-OH-PL sulfation by human liver cytosol. Sulfation kinetics in a low concentration range of 4-OH-PL enantiomers demonstrated that apparent Km values were similar between human and monkey liver cytosolic fractions, but the Vmax values were different, so that intrinsic clearance values (Vmax/Km, Clint) were higher in monkeys than in humans. Furthermore, enantiomer selectivity of [R(+)-4-OH-PL > S(-)-4-OH-PL] was observed in the Vmax and CLint values of monkey liver cytosol. These results indicate that the profile of sulfation of 4-OH-PL by liver cytosolic fractions is similar in humans and Japanese monkeys.

Microdialysis combined with tandem mass spectrometry was used to monitor the rapid hydrolysis of CS-866, a new prodrug-type angiotensin II receptor antagonist, in vitro in rat and human plasma as well as in hepatic and intestinal preparations. No chromatographic separation was conducted, and the ion intensity of CS-866 in MS/MS was directly used to perform data acquisition at intervals not longer than several seconds. A methanol-dialyzing medium, flow rate of dialysate, and adoption of sheath liquid were contrived to facilitate this method of measurement. The use of the methanol-dialyzing medium resulted in the effective extraction of the lipophilic analyte through the dialyzing membrane and a substantial reduction of inorganic substances introduced into the ion source. The calibration curve for CS-866 was linear over a concentration range from 0.2 to 20 microM (R(2) = 0.9998), and the intraassay precision was at an acceptable level of not more than 15% in coefficient of variation percentage. CS-866 was hydrolyzed very rapidly to RNH-6270, the pharmacologically active metabolite, in rat and human plasma and rat liver microsomes, and the hydrolysis proceeded extremely rapidly in human plasma with a half-life of less than several seconds., (Copyright 2000 Academic Press.)

1. Using LC-MS and LC-MS/MS, an in vitro study was conducted on the metabolism of human calcitonin (hCT) and salmon calcitonin (sCT) in rat liver and kidney to determine the rates of metabolism and the positions of hydrolytic cleavage in both peptides. 2. In lysosomal fractions of rat liver and kidney, hCT was degraded 9-12 times faster than sCT. Many metabolites of hCT were produced in the lysosomal fractions, whereas the metabolites of sCT were scarcely found. 3. In the case of the cytosolic fractions, three positions of initial endoproteolytic cleavage were found in hCT, leading to the production of many peptide fragments via subsequent exoproteolytic metabolism. The initial cleavage position of sCT could not be identified precisely, but it was postulated that the rate-determining step in the metabolism of sCT is the endoproteolytic hydrolysis. 4. The studies using pure proteases and protease inhibitors indicated that the metabolism of calcitonins proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion, catalysed by an aspartate-protease in lysosomes and by a metalloprotease and cysteine-protease in combination in the cytosol. 5. The result suggested that the higher in vivo pharmacological activity of sCT compared with that of hCT may be due to a slower metabolism of the former.

Hepatocyte transplantation improves the survival of laboratory animals with experimentally induced acute liver failure and the physiological abnormalities associated with liver-based metabolic deficiencies. The role of hepatocyte transplantation in treating decompensated liver cirrhosis, however, has not been studied in depth. To address this issue, cirrhosis was induced using phenobarbital and carbon tetrachloride (CCL(4)) and animals were studied only when evidence of liver failure did not improve when CCL(4) was held for 4 weeks. Animals received intrasplenic transplantation of syngeneic rat hepatocytes (G1); intraperitoneal transplantation of syngeneic rat hepatocytes (G2); intraperitoneal transplantation of a cellular homogenate of syngeneic rat hepatocytes (G3); intraperitoneal transplantation of syngeneic rat bone marrow cells (G4); or intrasplenic injection of Dulbecco's modified Eagle medium (DMEM) (G5). After transplantation, body weight and serum albumin levels deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic hepatocyte transplantation (G1) (P <.01). Prothrombin time (PT), total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation (P <. 01). More importantly, survival was prolonged after a single infusion of hepatocytes and a second infusion prolonged survival from 15 to 128 days (P <.01). Thus, hepatocyte transplantation can improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis and may be useful in the treatment of cirrhosis in humans.

Hepatocyte transplantation (HTX) could be an attractive treatment for patients with liver failure and liver-based metabolic disease. Human primary hepatocytes are ideal in this modality, but the shortage of human livers available for hepatocyte isolation severely limits the use of this form of therapy. A tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to the primary human hepatocytes. To test the feasibility, human hepatocytes were immortalized by a retroviral vector expressing simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. A highly differentiated immortal hepatocyte line NKNT-3 was established. NKNT-3 cells grew in chemically defined serum-free medium, retained highly differentiated liver functions, and were sensitivity to ganciclovir as a prodrug. Essentially unlimited availability of NKNT-3 cells may be clinically useful for HTX and bioartificial liver.

Because of a critical shortage in suitable organs, many patients with terminal liver disease die each year before liver transplantation can be performed. Transplantation of isolated hepatocytes has been proposed for the temporary metabolic support of patients awaiting liver transplantation or spontaneous reversion of their liver disease. A major limitation of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes. A highly differentiated cell line, NKNT-3, was generated by retroviral transfer in normal primary adult human hepatocytes of an immortalizing gene that can be subsequently and completely excised by Cre/Lox site-specific recombination. When transplanted into the spleen of rats under transient immunosuppression, reversibly immortalized NKNT-3 cells provided life-saving metabolic support during acute liver failure induced by 90% hepatectomy.