Kiyoshi Fukunaga

Platelets contribute to the reduction of liver fibrosis in mice

Background and Aim:  Several recent studies have reported that liver cirrhosis (LC) can be ameliorated, but few adequate strategies are available against liver fibrosis. Although LC clinically shows thrombocytopenia and hypersplenism, the correlation with liver fibrosis and platelets remains unclear. The aim of the present study was to investigate the effect of platelets on liver fibrosis in mouse models.

Hepatic allograft procurement from non-heart-beating donors : Limits of warm ischemia in porcine liver transplantation

To investigate the tolerance to warm ischemia of liver grafts from non-heart-beating donors, porcine orthotopic liver transplantation was performed using grafts obtained at various periods after cardiac arrest. Graft viability was investigated in relation to changes in hepatic adenine nucleotide metabolism. In donors, livers were divided into four groups according to warm ischemic time after cardiac arrest (group 1: 0 min, n=3; group 2: 30 min, n=3; group 3: 60 min, n=5; group 4: 90 min, n=4). Thereafter, the livers were flushed and preserved for 4 hr using 4 degrees C Euro-Collins solution. After surgery, all of the recipients in groups 1, 2, and 3 survived more than 4 days, except for one pig in group 3 that died of bleeding from an arterial catheter on day 2. By contrast, all of the recipients in group 4 died within 12 hr. The serum glutamic oxaloacetic transaminase concentration at 4 hr after reperfusion of the graft was significantly higher in group 4 (mean+/-SE, 2563+/-556 IU/L) than in groups 1, 2, and 3 (298+/-29 IU/L, 1226+/-222 IU/L, and 1181+/-174 IU/L, respectively). The adenylate energy charge of the liver graft recovered at 1 hr after reperfusion of the graft to 0.852+/-0.013, 0.845+/-0.003, and 0.842+/-0.003 in groups 1, 2, and 3, respectively. The recovery was significantly suppressed in group 4 (0.796+/-0.011). The hepatic adenosine triphosphate concentration also was significantly lower in group 4 compared with the other groups. The present study suggests that liver allografts can be used from non-heart-beating donors subjected to warm ischemia for less than 60 min. Postoperative survival is associated with prompt recovery of the adenylate energy charge of the liver graft.

Transforming growth factor-alpha (TGF alpha)-producing gastric carcinoma with acanthosis nigricans: an endocrine effect of TGF alpha in the pathogenesis of cutaneous paraneoplastic syndrome and epithelial hyperplasia of the esophagus.

A case of well-differentiated adenocarcinoma (Borrmann type 3) of the stomach in a 76-year-old man associated with the typical skin manifestations of acanthosis nigricans and with multiple protruding lesions showing epithelial hyperplasia of the esophagus is reported. The advanced tumor was located in the cardiac region of the stomach, and measured approximately 8cm in diameter, with partial invasion to the esophagus. The associated cutaneous lesions were characterized by hyperpigmentation and by protruding verrucous papules on the torso, head, face, neck, upper extremities, perineum, and inguinal region. Histologically, the protruding skin lesions showed keratinocytes proliferation throughout the epidermis, resulting in diffhyperkeratosis, papillomatosis, and acanthosis of the skin. Immunohistological analysis showed coexpression of transforming growth factor alpha (TGF-a) and epidermal growth factor (EGF) receptors in the tumor from the stomach. It is reasonable to conclude from this evidence that gastric carcinoma cells secrete TGF α in an autocrine for auto-stimulation. EGF receptor expression was also noted on the papillomatous hyperplasia of the cutaneous lesion. Serum level of TGF α, determined by an enzyme-linked immunosorbent assay, was high (144pg/ml; normal, 22.0 ±16pg/ml (Mean±SD)). Serum TGF α abruptly decreased to 49pg/ml on day 7 after the total gastrectomy, and then gradually increased to 77pg/ml within 28 days. Amelioration of the cutaneous lesions and the protruding lesions in the esophagus was observed after surgical resection of the gastric carcinoma. This suggests that the TGF α stimulates the proliferation of keratinocytes involved with EGF receptor. Large amounts of circulating TGF α in the blood over a long period released by the primary tumor seem to act as an endocrine-like mechanism causing epidermal and esophageal epithelial cells to proliferate. There is a possible link in the pathogenesis of the acanthosis nigricans as a cutaneous paraneoplastic syndrome, and epithelial hyperplasia of the esophagus.

Activation of human liver sinusoidal endothelial cell by human platelets induces hepatocyte proliferation

BACKGROUND & AIMS We previously reported that platelets promote hepatocyte proliferation. In this study, we focused on the role of platelets in liver sinusoidal endothelial cells (LSECs) in addition to their role in hepatocyte in liver regeneration. METHODS Immortalized human LSECs (TMNK-1) were used. The LSECs were co-cultured with human platelets, and the proliferation of LSECs and the excretion of growth factors and interleukin-6 (IL-6) were subsequently measured. The main factor from platelets which induced the excretion of IL-6 from LSECs was determined using inhibitors of each component contained in the platelets. The need for direct contact between platelets and LSECs was investigated using cell culture inserts. The proliferation of human primary hepatocytes was measured after the addition of the supernatant of LSECs cultured with or without platelets. RESULTS The number of LSECs cocultured with platelets significantly increased. Excretion of IL-6 and vascular endothelial growth factor (VEGF) increased in LSECs with platelets. JTE-013, a specific antagonist for sphingosine 1-phosphate (S1P) 2 receptors, inhibited the excretion of IL-6 from LSECs after the addition of platelets. When the platelets and LSECs were separated by the cell culture insert, the excretion of IL-6 from LSECs was decreased. DNA synthesis was significantly increased in human primary hepatocytes cultured with the supernatant of LSECs with platelets. CONCLUSIONS Platelets promote LSEC proliferation and induce IL-6 and VEGF production. Direct contact between the platelets and LSECs and S1P, that are contained in platelets, were involved in the excretion of IL-6 from LSECs. IL-6 from LSECs induced proliferation of parenchymal hepatocytes.

Platelet dynamics in the early phase of postischemic liver in vivo.

BACKGROUND In liver surgery, ischemia/reperfusion injury occasionally leads to liver failure by activating Kupffer cells (KCs) and leukocytes. However, few reports have demonstrated a relationship between KCs and platelets in vivo. This study investigated the relationship between these cells using intravital microscopy. MATERIALS AND METHODS Male Wistar rats were divided into two groups: (1) KC+ group, receiving 1 mL saline; and (2) KC- group, intravenously injected with liposome-encapsulated dichloromethylene disphosphonate for elimination of KCs. At 48 h after administration, 20 min of total normothermic hepatic ischemia was induced. Rhodamine-6G-labeled platelets and sinusoidal alterations were monitored using intravital microscopy up to 120 min after reperfusion. P-selectin, accumulated leukocytes and morphological damage, and alanine aminotransferase were evaluated. RESULTS In the KC+ group, numbers of adherent platelets increased significantly within 30 min after reperfusion. Endothelial cells of sinusoids in which KCs were mainly located were destroyed and the sinusoids were significantly constricted after reperfusion. Conversely, in the KC- group, adherent platelets in sinusoids were suppressed, and sinusoidal perfusion, endothelial cell damage and serum alanine aminotransferase levels were significantly improved. P-selectin on sinusoidal endothelial cells was not observed up to 120 min after reperfusion in either group. CONCLUSIONS Adherent platelets appear to reflect activation of KCs and lead to leukocyte accumulation, resulting in sinusoidal perfusion disturbance and liver failure. Evaluation of adherent platelets in the microcirculation offers an important marker of hepatic injury.

Increased intracranial pressure in a porcine model of fulminant hepatic failure using amatoxin and endotoxin

BACKGROUND/AIMS The purpose of this study was to develop a clinically relevant porcine model of fulminant hepatic failure (FHF) by means of administration of amatoxin and endotoxin. METHODS Pigs were intraportally administered only saline in group 1 (n = 3), 1 microg/kg of lipopolysaccharide (LPS) in group 2 (n = 4), 0.1 mg/kg of alpha-amanitin in group 3 (n = 5), and amanitin plus LPS in group 4 (n = 9). RESULTS All the pigs in groups 1 and 2 survived with minimal changes in liver function tests. In contrast to the 60% mortality in group 3, all the pigs in group 4 died within 96 h, with a significant increase in aspartate transaminase at 24 h (9,757 +/- 2,167 IU/I). In addition, they demonstrated severe metabolic disorders, such as serum lactate accumulation, hypoglycemia, coagulopathy, plasma amino acid imbalance, and hyperammonemia. The intracranial pressure significantly increased to 17.8 +/- 2.5 mmHg immediately before death. Reversal of FHF in these pigs following orthotopic liver transplantation confirmed that the toxicity is liver-specific and that the graft liver is unaffected. CONCLUSIONS This porcine model of FHF induced by a combination of amanitin and LPS will be of much use in the development of new therapies for human FHF.

Histological and prognostic importance of CD44+/CD24+/EpCAM+ expression in clinical pancreatic cancer

CD44+/CD24+/EpCAM+ cells have been reported to be cancer stem cells in pancreatic cancer; however, the histological and clinical importance of these cells has not yet been investigated. Here we clarified the characteristics of CD44+/CD24+/EpCAM+ cells in clinical specimens of pancreatic cancer using immunohistochemical assay. We used surgical specimens of pancreatic ductal adenocarcinoma from 101 patients. In view of tumor heterogeneity, we randomly selected 10 high‐power fields per case, and triple‐positive CD44+/CD24+/EpCAM+ expression was identified using our scoring system. The distribution, histological characteristics, and prognostic importance of CD44+/CD24+/EpCAM+ cells were then analyzed. As a result, the distribution of CD44+/CD24+/EpCAM+ cells varied widely among the 101 cases examined, and CD44+/CD24+/EpCAM+ expression was correlated with poor glandular differentiation and high proliferation. Survival analysis showed that CD44+/CD24+/EpCAM+ expression was not correlated with patient outcome; however, CD44+/CD24+ expression appeared to be correlated with poor prognosis. In conclusion, CD44+/CD24+/EpCAM+ expression overlapped with poorly differentiated cells and possessed high proliferative potential in clinical pancreatic cancer. In particular, the presence of double‐positive CD44+/CD24+ expression seemed to have clinical relevance, associating with poor prognosis.

Endothelin antagonist treatment for successful liver transplantation from non-heart-beating donors.

BACKGROUND With the shortage of cadaveric donors, non-heart-beating donors (NHBDs) are a potential source of liver allografts. However, warm ischemic injury in NHBDs seriously affects the viability of graft liver. Endothelin (ET)-1 has been reported to be involved in the hepatic microcirculatory disturbances after ischemia-reperfusion. METHODS In a porcine orthotopic liver transplantation model, changes in the serum and liver tissue ET-1 concentration were measured and the effects of an ET receptor antagonist, TAK-044, were evaluated. After cardiac arrest of the donors, liver allografts were subjected to 90 min of warm ischemia, flushed, and preserved for 4 hr at 4 degrees C. The pigs were divided into two groups: a control group (no drug treatment) and a drug-treated group, in which donors and recipients were treated with TAK-044 (10 mg/kg body, drip intravenous injection). Both groups had six donor/recipient pairs. RESULTS -The ET-1 concentration in the hepatic venous blood increased after reperfusion of the graft in the control group recipients. ET-1 in the graft liver significantly increased during the cold preservation period. TAK-044 treatment significantly increased recipient 7-day survival rate. After reperfusion of the graft, the concentrations of serum liver enzymes and arterial lactate in the drug-treated group were significantly lower than in the control group. The postoperative increase in portal venous pressure was significantly reduced in the drug-treated group. Measurements of liver enzymes in the washed-out preservation fluid at the time of graft rinsing indicated that TAK-044 treatment of the donors significantly suppressed liver enzyme release during ischemia. CONCLUSIONS These findings indicate TAK-044 treatment has protective effects on postoperative function of hepatic allografts procured from NHBDs.

Radiofrequency Ablation of the Liver: Determination of Ablative Margin at MR Imaging with Impaired Clearance of Ferucarbotran—Feasibility Study

Institutional review board approval and informed consent were obtained. The feasibility of magnetic resonance (MR) imaging with impaired clearance of ferucarbotran to visualize ablated liver parenchyma surrounding a tumor (ablative margin [AM]) was evaluated after radiofrequency (RF) ablation of the liver. Twenty-one patients with hepatocellular carcinomas underwent RF ablation 2-7 hours after ferucarbotran-enhanced MR imaging. On unenhanced T2*-weighted images acquired after 3-5 days, AMs appeared as hypointense rims. The AM status was related to incidence of residual or recurrent tumors. This technique is feasible for visualization of AM and prediction of residual or recurrent tumors after RF ablation of the liver.

Fatty acid synthase inhibitor cerulenin suppresses liver metastasis of colon cancer in mice

Fatty acid synthase (FAS) is highly expressed in many kinds of human cancers, including colorectal cancer (CRC), and we have investigated the potential use of FAS inhibitors for chemoprevention of liver metastasis of CRC in mice. Expression of FAS was evaluated in murine CRC cell lines Colon 26 and CMT 93. Cerulenin, a natural inhibitor of FAS, induced apoptosis in these cell lines. The ability of cerulenin to prevent development of liver metastatic lesions in Colon 26 was evaluated. The numbers and sizes of liver metastatic CRC tumors were significantly reduced by treating mice with cerulenin. Cerulenin treatment was associated with reduced levels of phosphorylated Akt in Colon 26 cells, suggesting that inhibition of this signal transduction pathway might be involved in the chemopreventive activity of this compound. Based on studies in mouse models, inhibiting FAS would be an effective strategy to prevent and retard growth of liver metastatic tumors of CRC that have high expression of this enzyme. (Cancer Sci 2010; 00: 000–000)