Role of podoplanin and Kupffer cells in liver injury after ischemia-reperfusion in mice.

AIM: To investigate the relationship between the intrahepatic expression of podoplanin (PDPN) and Kupffer cells (KCs) in ischemia-reperfusion (I/R) liver damage. METHODS: C57Bl/6 mice were injected with 200 µl of clodronate liposomes (macrophage depletion; MDP group) to deplete KCs or control liposomes (control group) via the ophthalmic vein plexus 24 h prior to ischemia. Animals were subjected to 90 min of partial hepatic ischemia (70%), followed by reperfusion, and were then killed at designated time points. Serum and liver tissues were harvested for further analyses. RESULTS: Serum ALT levels, mortality rates, and the percentage of necrotic area in liver sections were significantly higher in the MDP group than in the control group. PDPN was expressed in the lymphatic epithelium, interlobular bile duct epithelium, and in some hepatocytes in each group. Its expression in hepatocytes was down-regulated in the MDP group. The accumulation of platelets in the sinusoid was reduced 6 h after I/R in the MDP group. Tissue HGF and IGF-1 levels decreased in the MDP group. CONCLUSIONS: These results suggest that KCs play a key role in the activation of platelets through direct contact with PDPN-positive hepatocytes in I/R livers.

Changes in Function and Dynamics in Hepatic and Splenic Macrophages in Non-Alcoholic Fatty Liver Disease.

BACKGROUND: The aim of this study was to investigate the populations and functions of hepatic and splenic macrophages (Mfs) in non-alcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: Experiment 1: Wild-type and STAM® mice were given chow or high-fat diets for designated periods. In isolated Mfs, phagocytosis and cytokine production were assessed. Immunohistochemistry for CD68 and F4/80 and expression of CD14 and CD16 were assessed. Experiment 2: Bone marrow cells harvested from enhanced green fluorescent protein (EGFP) mice were transplanted into wild-type mice with or without splenectomy after total body irradiation that was kept on methionine- and choline-deficient diets. RESULTS: Experiment 1: The number of CD68-positive cells and the percentage of F4/80-positive/CD68-positive cells increased with the progression of NAFLD. Production of TNF-α and IL-6 by hepatic Mfs was greater than that by splenic Mfs in mice with NASH. The number of CD14+CD16- Mfs increased in the spleen and decreased in the liver in animals that had progressed to NASH. Furthermore, the number of CD14+CD16+ hepatic Mfs was increased in animals that had progressed to NASH with fibrosis. Experiment 2: EGFP-positive cells were observed in the liver after transplantation. In the splenectomy group, EGFP-positive Mfs were also observed; however, the number was significantly less than that in the sham operation group. CONCLUSION: The populations and functions of hepatic and splenic Mfs are altered during the progression of NAFLD. In addition, increased hepatic Mfs during the progression of NAFLD may migrate from bone marrow to the liver via the spleen.

Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Mortality Caused by Septic Peritonitis in Mice.

The purpose of this study was to investigate whether pituitary adenylate cyclase-activating polypeptide (PACAP) prevents mortality due to sepsis in mice. Mice were given PACAP at designated time points before or after cecal ligation and puncture (CLP), and organ injury and mortality were investigated. Serum inflammatory and anti-inflammatory cytokine levels were assessed after CLP. Plasma corticosterone and adrenocorticotropic hormone levels were also measured. Isolated tissue macrophages (Mfs) were incubated with or without PACAP, and production of cytokines was measured. Activation of NF-κB was investigated in tissue Mfs isolated from CLP animal in the presence and absence PACAP in vitro. PACAP treatment significantly prevented acute lung injury and mortality after CLP. Plasma endotoxin levels and bacterial load were not different between PACAP-treated and nontreated groups. Increased serum TNF-α and HMGB1 levels in animals treated with vehicle were significantly blunted in PACAP-treated animals after CLP. Furthermore, serum IL-10 levels were significantly greater in the PACAP-treated group compared with the vehicle group. Production of HMGB1 and TNF-α by isolated hepatic Mfs was significantly inhibited in the presence of PACAP, whereas production of IL-10 by isolated hepatic Mfs and interstitial lung Mfs was significantly increased. Plasma corticosterone and adrenocorticotropic hormone levels were significantly greater in the animals treated with PACAP compared with vehicle after CLP. Activation of NF-κB was significantly inhibited by PACAP in the hepatic Mfs compared with other tissue Mfs. PACAP prevents mortality due to septic peritonitis by inhibiting inflammation via NF-κB activation and possible effects on the brain.

Effects of Medium-chain Triglycerides Administration in Chemically-induced Carcinogenesis in Mice.

AIM: The aim of this study was to investigate the effects of medium-chain triglycerides (MCTs) on chemically-induced hepatic carcinogenesis (HCC) in mice. MATERIALS AND METHODS: In a first set of experiments, mice were treated with diethylnitrosoamine intraperitoneally at two weeks of age. They were fed chow containing MCT or a normal chow diet and sacrificed after 28 weeks. Incidence of hepatic tumor was compared between the two groups. Expression of oxidative stress, and inflammatory cytokines and chemokines in liver tissues were examined. In a second set of experiments, the histopathological findings of the intraperitoneal adipose tissue were assessed, and expression of adipocytokines in the fat tissue was measured. In a third set of experiments, plasma ß-hydroxybutyrate (HB) concentration was measured in both animals fed chow containing MCT and a normal chow diet. Mouse HCC cells were co-cultured with ß-HB, and the numbers of tumor cells were counted at days 3 and 7. RESULTS: In the first set of experiments, the tumor count observed in the control group was significantly blunted in the MCT group. Maximum tumor diameter also decreased in the MCT group compared to the control group. The expression of inflammatory cytokines and chemokines was significantly decreased by MCT. Furthermore, expression of 4-hydroxynonenal was lower in the MCT group compared to the control group. In the second set of experiments, hypertrophy of the adipocytes was suppressed, and the concentration of adiponectin and leptin in the adipose tissue decreased by MCT. In the third set of experiments, plasma ß-HB concentration increased in the MCT group as expected. ß-HB significantly inhibited the proliferation of HCC cells. CONCLUSION: MCT administration markedly suppresses the incidence of chemically-induced HCC by inhibition of inflammation and increase of ketone bodies.

M-CSF Receptor Antagonists Inhibit the Initiation and Progression of Hepatocellular Carcinoma in Mice.

BACKGROUND/AIM: The aim of this study was to investigate the effects of the macrophage colony-stimulating factor (M-CSF) receptor antagonist on hepatic carcinogenesis in mice. MATERIALS AND METHODS: Mice were injected with diethylnitrosamine (DEN) and treated with M-CSF receptor antagonist GW2580 (GW) or a saline vehicle just after (early treated group) or 2 weeks after (late treated group) DEN injection. Animals were sacrificed after 28 weeks and incidence of tumor was assessed. Isolated Kupffer cells were co-cultured with M-CSF in the presence or absence of GW, and the concentration of VEGF was measured. RESULTS: The incidence of tumors was significantly blunted both in the early- and the late-treated groups. In addition, angiogenesis within the tumor was also suppressed in both groups. The concentration of VEGF increased in Kupffer cells treated with M-CSF compared to those cultured without M-CSF. This increase was blunted by GW. CONCLUSION: M-CSF and its receptor could be novel molecular targets for hepatocellular carcinoma.

Histopathological and Molecular Signatures of a Mouse Model of Acute-on-Chronic Alcoholic Liver Injury Demonstrate Concordance With Human Alcoholic Hepatitis.

Human alcoholic hepatitis (AH) carries a high mortality rate. AH is an acute-on-chronic form of liver injury characterized by hepatic steatosis, ballooned hepatocytes, neutrophil infiltration, and pericellular fibrosis. We aimed to study the pathogenesis of AH in an animal model which combines chronic hepatic fibrosis with intragastric alcohol administration. Adult male C57BL6/J mice were treated with CCl4 (0.2 ml/kg, 2×weekly by intraperitoneal injections for 6 weeks) to induce chronic liver fibrosis. Then, ethyl alcohol (up to 25 g/kg/day for 3 weeks) was administered continuously to mice via a gastric feeding tube, with or without one-half dose of CCl4. Liver and serum markers and liver transcriptome were evaluated to characterize acute-on-chronic-alcoholic liver disease in our model. CCl4 or alcohol treatment alone induced liver fibrosis or steatohepatitis, respectively, findings that were consistent with expected pathology. Combined treatment resulted in a marked exacerbation of liver injury, as evident by the development of inflammation, steatosis, and pericellular fibrosis, pathological features of human AH. E. coli and Candida were also detected in livers of mice cotreated with CCl4 and alcohol, indicating pathogen translocation from gut to liver, similar to human AH. Importantly, liver transcriptomic changes specific to combined treatment group demonstrated close concordance with pathways perturbed in patients with severe AH. Overall, mice treated with CCl4 and alcohol displayed key molecular and pathological characteristics of human AH-pericellular fibrosis, increased hepatic bacterial load, and dysregulation of the same molecular pathways. This model may be useful for developing therapeutics for AH.

Metabolism and Toxicity of Trichloroethylene and Tetrachloroethylene in Cytochrome P450 2E1 Knockout and Humanized Transgenic Mice.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar olefins that can cause liver and kidney toxicity. Adverse effects of these chemicals are associated with metabolism to oxidative and glutathione conjugation moieties. It is thought that CYP2E1 is crucial to the oxidative metabolism of TCE and PCE, and may also play a role in formation of nephrotoxic metabolites; however, inter-species and inter-individual differences in contribution of CYP2E1 to metabolism and toxicity are not well understood. Therefore, the role of CYP2E1 in metabolism and toxic effects of TCE and PCE was investigated using male and female wild-type [129S1/SvlmJ], Cyp2e1(-/-), and humanized Cyp2e1 [hCYP2E1] mice. To fill in existing gaps in our knowledge, we conducted a toxicokinetic study of TCE (600 mg/kg, single dose, i.g.) and a subacute study of PCE (500 mg/kg/day, 5 days, i.g.) in 3 strains. Liver and kidney tissues were subject to profiling of oxidative and glutathione conjugation metabolites of TCE and PCE, as well as toxicity endpoints. The amounts of trichloroacetic acid formed in the liver was hCYP2E1≈ 129S1/SvlmJ > Cyp2e1(-/-) for both TCE and PCE; levels in males were about 2-fold higher than in females. Interestingly, 2- to 3-fold higher levels of conjugation metabolites were observed in TCE-treated Cyp2e1(-/-) mice. PCE induced lipid accumulation only in liver of 129S1/SvlmJ mice. In the kidney, PCE exposure resulted in acute proximal tubule injury in both sexes in all strains (hCYP2E1 ≈ 129S1/SvlmJ > Cyp2e1(-/-)). In conclusion, our results demonstrate that CYP2E1 is an important, but not exclusive actor in the oxidative metabolism and toxicity of TCE and PCE.

Effects of pirfenidone in acute and sub-chronic liver fibrosis, and an initiation-promotion cancer model in the mouse.

Liver fibrosis results from chronic tissue damage and excessive regeneration with accumulation of extracellular matrix proteins; it is a precursor of liver cirrhosis and hepatocellular carcinoma. Liver fibrosis treatments are primarily directed at inflammation, with few options to combat fibrogenesis. Pirfenidone is a drug approved for idiopathic pulmonary fibrosis and this study was focused on anti-fibrotic and anti-cancer potential of pirfenidone in the liver of male B6C3F1/J mice. In a dose-finding study, mice were treated with CCl4 (0.2ml/kg ip, 2×wk for 4weeks) while on a pirfenidone-containing (0-600mg/kg) diet. Pirfenidone at doses of 300 and 600mg/kg had significant anti-fibrotic (collagen) and anti-inflammatory (serum transaminases and "ballooning" hepatocyte) effects. In a sub-chronic study (14weeks), mice received CCl4 while on pirfenidone (300mg/kg) diet. Pirfenidone significantly reduced collagen deposition, but had little effect of inflammation and injury. In an initiation-promotion cancer study with N-nitrosodiethylamine and CCl4, pirfenidone (300mg/kg) did not affect incidence, size, or multiplicity of liver tumors. Overall, we conclude that while pirfenidone exhibits strong anti-fibrotic effects in early stage liver fibrosis, it is less effective in advanced liver fibrosis and was not protective in an initiation-promotion liver cancer.

Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer.

PURPOSE: To investigate the role of podoplanin (PDPN) expression in invasive ductal carcinoma of the pancreas (IDCP) in humans. METHODS: Tumor samples were obtained from 95 patients with IDCP. Immunohistochemical staining was done to evaluate the expression of PDPN in cancer tissues. RESULTS: PDPN was detected predominantly in stromal fibroblasts, stained with α-smooth muscle actin. The cutoff value of PDPN-positive areas was calculated according to a histogram. There was no significant difference in clinicopathologic factors between patients with high vs. those with low PDPN expression. The high PDPN group showed significantly poorer disease-free and disease-specific survival rates than the low PDPN group. Among patients from the high PDPN group, those with lymph node metastases and those with a tumor larger than 20 cm in diameter had significantly poorer prognoses than similar patients from the low PDPN group. Multivariate Cox proportional hazards analysis indicated that a high expression of PDPN was an independent risk factor for disease-specific survival. CONCLUSIONS: PDPN expression in cancer-related fibrotic tissues is associated with a poor prognosis, especially in patients with large tumors or lymph node metastases.

[A case of locally advanced sigmoid colon cancer invading the bladder and seminal vesicle effectively treated with preoperative mFOLFOX6].

A 77-year-old male was admitted to our hospital complaining of dyschezia. Computed tomography (CT) and colonoscopy (CF) revealed a huge sigmoid colon cancer invading the bladder and seminal vesicle. Chemotherapy with mFOLFOX6 was initiated preoperatively, and the tumor shrunk markedly after seven courses of treatment. Pelvic exenteration with negative margins was carried out. The patient is still alive and disease-free 16 months after surgery. It was suggested that mFOLFOX6 may be useful for advanced colon cancer invading other organs when used as neoadjuvant chemotherapy.