Yasushi Kawazoe

Effects of recombinant human hepatocyte growth factor on the proliferation and function of porcine hepatocytes.

A porcine hepatocyte based bioartificial liver (BAL) is still insufficient to replace liver transplantation. In this experiment, to strengthen the performance of a BAL, the effect of human recombinant hepatocyte growth factor (rhHGF) on the proliferation and function of xenogeneic porcine hepatocytes was studied. Isolated porcine hepatocytes were seeded at various densities (5 x 10(3) to 8 x 10(4) cells/well) on a collagen coated 96 well plate in Dulbeccos modified Eagles medium (DMEM) with 10% FCS. After 4 hours, the medium was changed to DMEM with added insulin and dexamethasone. Subsequently, rhHGF was added at various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 ng/ml) and cultured for an additional 24, 48, and 72 hours, respectively. The proliferation of porcine hepatocytes in response to rhHGF reached a plateau at 2.5 ng/ml at 24 hours and subsequently decreased. The levels of porcine albumin vs protein present in the supernatant increased when cultured at high cell density. In conclusion, rhHGF was found to stimulate proliferation of porcine hepatocytes at low cell density and low concentration. rhHGF can also increase albumin synthesis at higher cell density, thus indicating its potential use in a more satisfactory porcine hepatocyte based BAL.

Protective effect of nafamostat mesilate on injury of porcine hepatocytes by human plasma.

Nafamostat mesilate (NM), a protease inhibitor, possesses a cytoprotective effect and inhibits the activation of complement. The present study investigated whether NM has any protective effect against injury of porcine hepatocytes by human plasma in a bioartificial liver support system. Porcine hepatocytes were harvested and seeded at a density of 2 × 105 cells on a 35-mm collagen-coated plate in Dulbeccos modified Eagles medium (DMEM) with 10% fetal calf serum. Twenty-four hours later, the medium was replaced with human plasma with three concentrations of NM between 3.8 × 10–5 and 3.8 × 10–4 M and then cultured for 6 h. The viability of porcine hepatocytes, lactate dehydrogenase (LDH) levels, lidocaine clearance, porcine albumin production, and changes in complement (C3) levels were measured. The viability of porcine hepatocytes in human plasma decreased significantly to 37.7 ± 11.4% of that in DMEM. NM improved the viability of the hepatocytes, lowered the levels of LDH, and increased lidocaine clearance and albumin production in a concentration-dependent manner. The concentrations of C3, the marker of xenogeneic reactions, did not change significantly, indicating that no hyperacute xenogeneic reaction occurred in our series. Together, our results suggested that NM exerts favorable effects on porcine hepatocytes in human plasma through direct effect such as prevention of protease activity in the plasma membrane of porcine hepatocytes rather than inhibition of complement-dependent immunoreactions.

The efficacy and safety of gene transfer into the porcine liver in vivo by HVJ (sendai virus) liposome

Background. Gene transfer systems using viral vectors are efficient; however, most viral vectors also tend to evoke immunologic reactions, thereby clinically causing serial side effects. HVJ-liposome vector is a hybrid vector consisting of liposome and an inactivated Sendai virus (Hemmagglutinating Virus of Japan [HVJ]), which has been reported to be less immunogenic and can also be repeatedly administered. We examined the usefulness of this vector for hepatic gene therapy in a pig model. Methods. Genes encoding &bgr;-galactosidase and luciferase were used as reporter genes. The pigs were injected with the reporter gene loaded-HVJ-liposome into the portal vein under total vascular exclusion of the liver. The transfection efficiencies were then assessed by &bgr;-galactosidase staining, a luciferase assay, and RT-PCR for LacZ mRNA. Biochemical and histologic analyses were performed to evaluate tissue toxicity after gene transfer. Results. The luciferase gene expression in the liver reached its highest level at 7 days after transfection. It continued to be detected up to 28 days after transfection, while all pigs remained healthy throughout the observation period. The transfection efficiency was 15% in the hepatocytes according to &bgr;-galactosidase staining. Extrahepatic transgene expression was slightly observed in the lung and kidney, but not in the spleen or ovary. Conclusions. These data suggest for the first time that the use of the HVJ-liposome vector is a safe and feasible modality for liver-directed gene transfer in pigs, and it might therefore be suitable for clinical gene therapy trials.

Hepatic expression and serum levels of thrombomodulin reflect the extent of liver injury in rats with fulminant hepatic failure and extensive hepatectomy

In order to evaluate the biological significance of thrombomodulin (TM) in the serum and its expression in the liver, changes in TM were investigated in rats with fulminant hepatic failure (FHF) or after extensive hepatectomy. Forty-two rats were divided into four groups as follows: control (sham, n=6), 70% hepatectomy (70% Hx, n=12), 90% hepatectomy (90% Hx, n=12) and fulminant hepatic failure (FHF, n=12). The soluble-TM levels in the 90% Hx and FHF groups were significantly higher than that in the 70% Hx group. In the FHF group, the soluble-TM level 24 h after induction was significantly higher than that at 12 h (P<0.05). Moreover, the level of soluble TM was significantly higher in the FHF group than that in the 90% Hx group, while hyaluronic acid was not increased at statistical significance. The expression of TM in the liver intensified with time in both FHF and 90% Hx groups, which was more pronounced in zone 3 of the liver in FHF group than in the 90% Hx group. In conclusion, elevation of s-TM in the serum and expression of TM in the sinusoidal endothelial cell are useful markers of hepatic failure and its sinusoidal endothelial injury, especially in the state of FHF, since the presence of necrotic liver tissue is the only difference between FHF and 90% Hx in the model of the rats.

Effects of anticoagulants on porcine hepatocytes in vitro: implications in the porcine hepatocyte-based bioartificial liver.

For the clinical treatment with porcine hepatocyte-based bioartificial liver (BAL), the use of an anticoagulant in the extracorporeal system is essential. In this experiment, we studied the effect of various anticoagulants on cultured porcine hepatocytes. Porcine hepatocytes were isolated and seeded at a density of 2 × 105 cells on a collagen-coated plate in Dulbeccos modified Eagles medium (DMEM) with 10% fetal calf serum (FCS). Twenty-four hours later, the medium was changed to DMEM with various anticoagulants such as nafamostat mesilate (NM), sodium heparin (SH) and sodium citrate (SC) at concentration used clinically. As a control, the hepatocytes were cultured in only DMEM. After culturing for 6 hours, the viability of the porcine hepatocytes, lactate dehydrogenase (LDH) release, lidocaine clearance (cytochrome p450 function) and albumin synthesis were investigated. SC did not affect either the viability or the p450 function of the hepatocytes. In the NM group, the viability of porcine hepatocytes and lidocaine clearance were decreased significantly more than in the other groups. SH did not affect the viability of porcine hepatocytes, however, it seemed to reduce the p450 function. In conclusion, SC may therefore be the optimal anticoagulant available for hepatocyte-based BAL circuit in terms of its cell toxicity.

Efficacy of Nafamostat Mesilate for improving the performance of a bioartificial liver using porcine hepatocytes.

Our bioartificial liver (BAL) consists of porcine hepatocytes attached to beads and plasma perfused through the system. The function of our BAL lasts for approximately 7 hours. The objective of the present study was to investigate the efficacy of Nafamostat Mesilate (NM), a protease inhibitor and potent complement inhibitor, for improving the performance of the BAL. The experimental groups were divided as follows; the NM group (n=7) where the BAL had porcine hepatocytes with 3.8×10−4 M, of NM, and the control group where the BAL had no NM. Plasma obtained from patients suffering from hepatic failure was perfused through the BAL for 10 hours. The viability of the porcine hepatocytes and the levels of alanine aminotransferase (ALT) in the human plasma were measured during perfusion. After the 10-hour perfusion, another human hepatic failure plasma was perfused for an additional 1 hour and then the function of the BAL was evaluated. After the 10-hour perfusion, the viability of the hepatocytes in the NM group was 51± 7 %, whereas that in the control group was rapidly reduced by 35 ± 5 %. Although the levels of ALT in the human plasma in both groups increased with the perfusion time, those in the NM group were significantly lower than those in the control group (p < 0.05). These results suggest that NM prevented damage to the porcine hepatocytes in human hepatic failure plasma as compared to the control group. In the human hepatic failure plasma before perfusion, the partial thrombin time (PT) and the plasma ammonia (NH3) levels were 19.8 ± 12 % and 288 ± 102 μg/dl, respectively. Fischers ratios were 0.98 ± 0.39. Even after the 10- hour perfusion, the BAL in the NM group significantly improved the levels of PT (38 ± 10 %; p < 0.05), NH3 (214 ± 34 μg/dl; p < 0.05) and Fischers ratios (1.4 ± 0.3; p < 0.05). On the other hand, the BAL in the control group did not show any improvement in those parameters. In conclusion, NM was found to help in maintaining the viability of porcine hepatocytes in human hepatic failure plasma, thereby allowing the porcine hepatocyte-based BAL to function much better.