Stimulation of liver regeneration after hepatectomy in mice by injection of bone marrow mesenchymal stem cells via the portal vein.

AIM: To investigate whether mouse bone marrow mesenchymal stem cells (BMC) stimulate liver regeneration after partial hepatectomy. METHODS: Isolated BMCs were purified by density gradient centrifugation. We performed a 70% hepatectomy in male BALB/c mice followed by injection of BMCs into the portal vein (PV-BMC group), or the tail vein (IV-BMC group), or of saline into the portal vein (control group). RESULTS: The wet weight of the liver remnant increased significantly in the PV-BMC group at 3 and 5 days after hepatectomy compared with the IV-BMC and control groups. The Ki-67 labeling index revealed that the increase to result from stimulation of DNA synthesis. The constitutive interleukin-6 and hepatocyte growth factor mRNAs in the remnant liver tended to increase in the PV-BMC group at 3 days after hepatectomy. CONCLUSIONS: These results demonstrated that BMC injection into the portal vein enhanced liver growth after partial hepatectomy in mice.

Change in donor quality of life after living donor liver transplantation surgery: a single-institution experience.

AIM: Changes in quality of life (QOL) of donors after living donor liver transplantation surgery were studied using Short Form 36 (SF-36) to evaluate physical health and the Profile of Mood States (POMS) to evaluate mood. METHODS: Among the 28 donors who had undergone surgery at our hospital, 21 donors replied to our questionnaire (recovery rate: 75%). Changes in the QOL of donors were examined based on the results of pre- and postoperative SF-36 and POMS and a questionnaire survey. RESULTS: The donors included 12 men and nine women with a mean age of 43 years. One donor operation was complicated by biliary stenosis. The SF-36 results showed significant postoperative decline in physical function and increase in pain, but no other significant changes. The POMS results showed no significant differences, with the mean values for anxiety, depression, anger, vitality, and confusion all recovering postoperatively. The questionnaire results indicated that all subjects had recovered physical function, but 24% of subjects felt wound-related physical symptoms and 19% felt anxiety concerning their future health. CONCLUSIONS: Some donors had symptoms that persisted for comparatively long periods postoperatively and considered that long-term outpatient observation was required. No significant psychological changes were found, but donors felt anxiety about their future health and considered that patient meetings would be useful and that adequate outpatient health guidance should be provided.

Active hexose correlated compound inhibits the expression of proinflammatory biomarker iNOS in hepatocytes.

BACKGROUND/AIMS: Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as proinflammatory biomarker in liver injury. The application of active hexose correlated compound (AHCC) as a functional food in complementary and alternative medicine has increased. The possibility that AHCC might inhibit iNOS induction was investigated as a potential liver-protective effect. METHODS: Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured hepatocytes were treated with interleukin-1ß in the presence or absence of AHCC-sugar fraction (AHCC-SF). RESULTS AND CONCLUSION: AHCC-SF inhibited the production of NO and reduced expressions of iNOS mRNA and its protein. AHCC-SF had no effects on either IκB degradation or nuclear factor-κB (NF-κB) activation. In contrast, AHCC-SF inhibited the upregulation of type I interleukin-1 receptor (IL-1RI) through the inhibition of Akt phosphorylation. Transfection experiments with iNOS promoter-luciferase constructs revealed that AHCC-SF reduced the levels of iNOS mRNA at both promoter transactivation and mRNA stabilization steps. AHCC-SF inhibited the expression of iNOS gene antisense transcript, which is involved in iNOS mRNA stabilization. These findings demonstrate that AHCC-SF suppresses iNOS gene expression through a IκB/NF-κB-independent but Akt/IL-1RI-dependent pathway, resulting in the reduction of NO production. AHCC-SF may have therapeutic potential for various liver injuries.

Usefulness of TC-99M GSA liver scintigraphy for the evaluation of liver regeneration in donors after living-donor liver transplantation.

BACKGROUND AND AIMS: We evaluated the impact of steatosis on regeneration and function of the remnant liver by using technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin scintigraphy. METHODS: Twelve living donors were classified into groups with or without mild hepatic steatosis according to the liver-to-spleen attenuation ratio on computed tomography: six donors had a ratio > or = 1.2 (control group) and six had a ratio < 1.20 (fatty liver group). Scintigraphy was performed to determine the hepatic uptake ratio of the tracer (corrected for disappearance from the blood) and the maximum removal rate of the tracer by hepatocytes as parameters of the hepatic functional reserve. RESULTS: The fatty liver group had a significantly lower corrected hepatic uptake ratio and removal rate compared with the control group at 6 and 12 months after partial hepatectomy. The regenerated liver volume estimated by scintigraphy did not differ significantly between the two groups at any time. CONCLUSIONS: Because donors with mild hepatic steatosis showed impaired liver regeneration at 1 year after partial hepatectomy, management of these donors requires more care.

Usefulness of TC-99M GSA liver scintigraphy for the assessment of recurrent hepatitis C after living-donor liver transplantation: a case report.

BACKGROUND AND AIMS: Recurrence of hepatitis C after living-donor liver transplantation was investigated using technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin (Tc-99m-GSA) liver scintigraphy. METHODS: A 55-year-old woman with cirrhosis due to chronic hepatitis C virus (HCV) infection underwent liver transplantation with a graft from her husband. Scintigraphy was used to determine the hepatic uptake ratio of the tracer corrected for disappearance from the blood, as well as the maximal removal rate of the tracer by hepatocytes, as parameters of hepatic functional reserve. RESULTS: Conventional liver function parameters and the graft volume (computed tomography) were almost unchanged up to 18 months after transplantation. Serum HCV RNA was elevated from 3 months after transplantation, and was twofold higher at 12 months compared with 6 months. At 18 months postoperatively, liver biopsy showed an increase of histologic activity, and there was also evidence of recurrent hepatitis C. The corrected hepatic uptake ratio and maximal removal rate were decreased at 3 months postoperatively, and thereafter remained low. CONCLUSIONS: The decrease of scintigraphic parameters at 3 months after transplantation suggested recurrent hepatitis C affecting the graft. Tc-99m-GSA liver scintigraphy is a useful noninvasive method for evaluating graft functional reserve.

Safety of hepatectomy for living donors as evaluated using asialoscintigraphy.

In the living donor operation, accurate estimation of hepatic functional reserve is essential. Technetium-99m-galactosyl-human serum albumin (GSA) is a liver scintigraphy agent that binds to asialoglycoprotein receptors. We evaluated the preoperative assessment of the safety of an elective hepatectomy using GSA liver scintigraphy in 152 patients. GSA scintigraphy was performed after intravenous injection of GSA. The maximal removal rate of GSA (GSA-Rmax) was calculated using a radiopharmacokinetic model. We determined the areas for resection preoperatively depending on the operative procedures and calculated the local GSA-Rmax in the predicted residual liver (GSA-RL). A significant correlation was obtained between the GSA-Rmax and the 15-minute retention rate of indocyanine green. With sub- and monosegmentectomy, 2 patients had postoperative hepatic failure; in those 2 patients, the GSA-RL was 0.127 and 0.133, respectively, but these patients recovered well. Among those having di- and tri-segmentectomy, 5 patients experienced postoperative hepatic failure, in all subjects the GSA-RL was <0.15. Two patients died of postoperative liver failure 1 to 2 months after the operation. We concluded that GSA-RL is useful to select the procedure for hepatectomy in living donors and that GSA-RL should be >0.15 (mg/min/50 kg body weight) to avoid postoperative hepatic failure.

Functional hepatic regeneration following hepatectomy using galactosyl-human serum albumin liver scintigraphy.

In extended hepatectomy and liver transplantation, accurate estimation of functional hepatic regeneration is more important than volumetric regeneration. We investigated the usefulness of measuring the functional hepatic volume by 99m-technetium galactosyl-human serum albumin scintigraphy (GSA). Extended hepatectomy was performed in 32 patients. These patients were divided into subgroups with or without chronic hepatitis or cirrhosis. Functional hepatic volume GSA scintigraphy (GSA-LV) and determination of hepatic volume by CT (CT-LV) measurements were performed preoperatively, at 2 and 4 weeks and at 3 and 6 months after surgery. The preoperative GSA-LV values were significantly correlated with the hepatocyte volume and the 15-minute retention rate of indocyanine green (ICGR15). Similarly, the hepatocyte volume correlated well with the CT-LV and ICGR15. However, the CT-LV correlated only with the ICGR15. Recovery of the GSA-LV was delayed, and about 90% of the volumetric and functional regeneration was observed within 6 months after the hepatectomy. In contrast, the CT-LV in patients with normal liver remnants returned to approximately 90% of the initial volume within 1 month after the hepatectomy, whereas patients with injured livers regeneration showed gradual recovery to approximately 80% of the preoperative value by 6 months after hepatectomy. We conclude that measurement of functional hepatic volume using the GSA-LV is useful to evaluate hepatic function based on hepatocyte volume.

Fibronectin suppresses apoptosis and protects mice from endotoxic shock.

Multiple-organ failure related to septicemia is a common cause of early mortality after liver transplantation. Endotoxemia following living donor hepatectomy may be a cause of postoperative death. Plasma fibronectin (Fn) exerts a broad range of biological effects on cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, reticuloendothelial system function, and ischemic injury. We studied the therapeutic effect of plasma Fn in mice after an intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (GalN). Female Balb/c mice received simultaneous intraperitoneal injection of LPS (50 microg/kg) and GalN (400 mg/kg). Thirty minutes prior to GalN/LPS administration, plasma Fn or bovine serum albumin was given intravenously. A single administration of plasma Fn (500 mg/kg) protected in dose-dependent fashion against lethal shock after GalN/LPS challenge. Plasma Fn significantly reduced the serum tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 levels and significantly increased the serum interleukin-10 levels after GalN/LPS administration. Furthermore, plasma Fn significantly inhibited liver necrosis at 9 hours after GalN/LPS injection. The fraction of apoptotic-positive cells in these plasma Fn-treated mice was significantly lower than in the control group. These results support the protective treatment of endotoxin-induced liver injury by plasma Fn.

Induction of inducible nitric oxide synthase in hepatocytes isolated from rats with ischemia-reperfusion injury.

BACKGROUND: Recent evidence indicates that nitric oxide (NO) has a crucial role in hepatic ischemia-reperfusion (I/R) injury. However, little is known about how I/R influences the gene expression of inducible nitric oxide synthase (iNOS) in hepatocytes. Under inflammatory conditions, we compared the induction of iNOS in hepatocytes isolated from normal and I/R-treated rats. METHODS: Hepatocytes were isolated using the collagenase perfusion method from rats treated with I/R (30-minute ischemia of middle and left lobes, followed by 3-hour reperfusion) or sham operation (control): Primary cultures of rat hepatocytes were incubated with an inflammatory cytokine, interleukin-1beta (IL-1beta), to compare the iNOS induction/NO production between the 2 groups. RESULTS: Both control and I/R groups had no production of nitrite (a stable metabolite of NO) in the absence of IL-1beta. In the control group, IL-1beta stimulated dose- and time-dependent production of NO. The I/R group showed more than 2-fold increased levels of NO production. Western and Northern blot analyses revealed that the I/R group also showed increased levels of iNOS protein and its messenger RNA. CONCLUSION: These results suggest that I/R directly affects the inducibility of the iNOS gene in hepatocytes by IL-1beta. Increased NO may be associated with protective or toxic effects in hepatic I/R injury.

Effects of pirfenidone on endotoxin-induced liver injury after partial hepatectomy in rats.

BACKGROUND: In living donor liver transplantation, restrictions on graft size are a serious obstacle to expand indications for adult recipients. The sequence of gram-negative infection, septicemia, and multiple-organ failure is a common cause of early mortality after liver transplantation. An effective therapy has not been established for endotoxemia following extended hepatectomy in donors or small-for-size grafts in recipients. Pirfenidone (PFD), a new experimental antifibrotic agent, was used to ameliorate on endotoxin-induced liver injury following partial hepatectomy. METHODS: Male Sprague-Dawley rats were intravenously administered lipopolysaccharide (LPS) 48 hours after 70% hepatectomy. Prior to LPS administration, PFD (300 mg/kg) or its vehicle (0.5% carboxymethylcellulose) was given orally twice. RESULTS: The survival rate of the PFD-treated group was markedly improved compared with that of the controls. PFD prevented the increases in the activities of serum enzymes (aspartate transaminase [AST], alanine transaminase [ALT], and lactate dehydrogenase [LDH]) and total bilirubin. The serum and liver tissue levels of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma, and interleukin-6, were significantly lower among the PFD than the control group. Furthermore, the degree of necrosis in the remnant liver was significantly decreased in the PFD-treated rats compared with controls. CONCLUSION: These results indicate that PFD alleviates endotoxin-induced liver injury after partial hepatectomy through the inhibition of production of inflammatory cytokines in the residual liver. PFD may be useful to prevent endotoxin-induced liver injury after hepatectomy.

Effect of hepatocyte growth factor on induction of cytokine-induced neutrophil chemoattractant in rat hepatocytes.

BACKGROUND: Restrictions on graft size are a serious obstacle to the expansion of indications for adult recipients in living donor liver transplantation. Hepatocyte growth factor (HGF) has a crucial role in regeneration following hepatic injury. Rat cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 superfamily in humans, has been implicated in chronic liver diseases or development of liver ischemia-reperfusion injury. Studies were performed to examine whether HGF influences the induction of CINC in hepatocytes. METHODS: Primary cultures of rat hepatocytes were treated with or without recombinant human (rh) HGF. The release of CINC into the culture medium and levels of CINC mRNA were measured using an enzyme-linked immunosorbent assay and Northern blot analysis. Transcription of nuclear factor (NF)-kappa B was detected by electrophoretic mobility shift assays. RESULTS: rhHGF increased the release of CINC in the medium dose- and time-dependently, showing a maximal effect at 100 ng/mL. Genistein (100 mumol/L) blocked the release of CINC stimulated by rhHGF. Levels of CINC mRNA were also increased, reaching a maximum at 8 hours after addition of rhHGF. Electrophoretic mobility shift assays revealed rhHGF activated transcription factor, NF-kappa B. CONCLUSION: These results suggest that HGF stimulates the induction of CINC gene expression through activation of NF-kappa B. CINC may be involved in the function of HGF during liver regeneration.

Pirfenidone protects endotoxin-induced liver injury after hepatic ischemia in rats.

BACKGROUND: Pirfenidone (PFD), an experimental antifibrotic agent, was investigated for its effects on endotoxin-induced liver injury after hepatic ischemia-reperfusion. METHODS: Male Sprague-Dawley rats were subjected to 30 minutes of partial hepatic ischemia, followed by reperfusion for 24 hours. Lipopolysaccharide (LPS) was injected at 30 minutes of reperfusion. PFD (300 mg/kg) or its vehicle (0.5% carboxymethylcellulose) was given orally following LPS administration. RESULTS: PFD prevented the increase in activities of serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase after reperfusion. PFD inhibited the increase of cytokine-induced neutrophil chemoattractant in serum and liver tissue. The number of neutrophils infiltrating the liver was significantly lower in the PFD-treated group than the control group. CONCLUSION: These results indicate that PFD prevents endotoxin-induced liver injury after hepatic ischemia-reperfusion, in part through the decrease of neutrophil infiltration to the liver.

Effect of pirfenidone on induction of chemokines in rat hepatocytes.

BACKGROUND: Hepatic ischemia-reperfusion results in a neutrophil-dependent liver injury. The process of neutrophil recruitment and activation in this injury is at least partially dependent on the induction of chemokines, such as cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) in rats. In the liver, parenchymal cells (hepatocytes), in addition to nonparenchymal cells such as Kupffer cells, have been reported to produce chemokines in the regulation of hepatic inflammation. Pirfenidone (PFD) is a new experimental drug used as an antifibrotic agent. Studies were performed to determine whether PFD influences the production of CINC and MIP-2 stimulated by interleukin (IL)-1beta in a primary culture model of rat hepatocytes. METHODS: Primary cultures of rat hepatocytes were treated with IL-1beta in the presence and absence of PFD. The protein and mRNA of CINC and MIP-2 were analyzed using enzyme-linked immunosorbent assays and Northern blots. RESULTS: IL-1beta increased the release of CINC and MIP-2 into culture media in a dose- and time-dependent manner. PFD inhibited both CINC and MIP-2 release in dose-dependent fashion. However, PFD had no effect on the levels of CINC mRNA induced by IL-1beta. CONCLUSION: These results suggest that PFD inhibits the production of CINC and MIP-2 by IL-1beta at a posttranscriptional step in hepatocytes.

Fibronectin protects endotoxin-induced liver injury after partial hepatectomy in rats.

Endotoxemia following extended hepatectomy may be a cause of postoperative death. Multiple organ failure related to septemia is a common cause of early mortality after liver transplantation. Fibronectins (Fns) are involved in cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, and ischemic injury. Studies were performed to determine whether Fn influences the survival rate of rats subjected to endotoxin-induced liver injury after partial hepatectomy. Male Sprague-Dawley rats were intravenously administered lipopolysaccharide (LPS) 48 hours after 70% hepatectomy. Prior to LPS administration, plasma Fn or bovine serum albumin was given intravenously. The survival rate of the Fn-treated group was higher than that of the controls. Fn prevented increases in serum enzyme activity and total bilirubin related to liver injury. The levels of inflammatory cytokines including tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 interferon-gamma were also significantly lower in the Fn-treated than the control group. Furthermore, the number of apoptotic cells and the degree of necrosis in the remnant liver were significantly decreased in the Fn-treated rats compared with controls. These results indicate that Fn prevents endotoxin-induced liver injury after partial hepatectomy, at least in part through inhibiting the production of inflammatory cytokines, and the necrosis apoptosis in the remaining liver.

Prevention of pleural effusion following hepatectomy using argon beam coagulation.

BACKGROUND: Postoperative pleural effusion occurs frequently after hepatectomy. The value of the argon beam coagulator (ABC) for the prevention of pleural effusion after hepatectomy in patients with hepatocellular carcinoma was studied. METHODS: Sixty patients were divided randomly into two groups: an ABC group (n = 28), in which the cut surface of the hepatic ligaments and bare area of the retroperitoneum were cauterized using an ABC, and a control group (n = 32) in which the ABC was not applied. Patient characteristics, preoperative and postoperative liver function, and postoperative pleural effusion were compared between the two groups. RESULTS: There were no significant differences between the two groups with respect to histological findings, clinical stage, type of resection, operative data, and preoperative and postoperative laboratory data. One of 28 patients in the ABC group and nine of 32 patients in the control group had pleural effusion. The incidence was significantly lower in the ABC group than in the control group (P = 0.01). Pleurocentesis was needed in two of the ten patients and thoracic drainage in four patients. CONCLUSION: Application of an ABC to the cut surface of the hepatic ligaments and bare area of retroperitoneum after liver mobilization may prevent postoperative pleural effusion.

FK506, but not cyclosporin A, prevents mitochondrial dysfunction during hypoxia in rat hepatocytes.

Hepatic ischemia/reperfusion injury occurs in the clinical situations including liver transplantation. FK506 and cyclosporin A (CsA) are reported to be hepatotrophic agents in addition to being a powerful immunosuppressive agent. Studies were performed to determine whether the drugs influence a mitochondrial dysfunction under the hypoxic conditions in primary culture model of rat hepatocytes. The Anaeropack system was used for cell culture to create a hypoxia. Cells were treated with FK506 or CsA under the normoxic and hypoxic conditions. Hypoxia markedly decreased intracellular adenosine 5'-triphosphate (ATP) contents and the ketone body ratio (KBR, acetoacetate/beta-hydroxybutyrate) in culture medium as compared with normoxia. FK506 prevented the decreases of ATP contents and the KBR. In contrast, CsA had no effect on either ATP contents or the KBR. FK506, but not CsA, increased the KBR under the normoxic conditions. Under the hypoxic conditions, heat shock protein 70 (Hsp70) was detected after reoxygenation. FK506 enhanced the induction of Hsp70, but CsA again had no effect on Hsp70 induction. These results indicate that FK506 protects the hypoxia injury in part by preventing the mitochondrial dysfunction in concert with the enhancement of heat shock response in hepatocytes.

High density O-glycosylation of the MUC2 tandem repeat unit by N-acetylgalactosaminyltransferase-3 in colonic adenocarcinoma extracts.

A synthetic peptide corresponding to the human MUC2 tandem repeat unit was glycosylated in vitro using UDP-GalNAc and extracts of colonic adenocarcinoma and paired normal mucosa, followed by fractionation of the products by reverse phase high-performance liquid chromatography. Several peaks of glycopeptides with different numbers of GalNAc residues attached were detected. It is notable that the adenocarcinoma extract was capable of glycosylating peptides to a much greater extent than was normal mucosa. The levels of mRNA for N-acetylgalactosaminyltransferases-1, -2, and -3 were determined by reverse transcription-PCR. Only N-acetylgalactosaminyltransferase-3 mRNA was expressed at a higher level in the adenocarcinoma than in the normal tissue. When the MUC2 tandem repeat peptide was glycosylated with a mixture of the normal mucosa extract and recombinant N-acetylgalactosaminyltransferase-3, larger amounts of glycopeptides with higher contents of GalNAc residues were produced. The MUC2 tandem repeat peptides glycosylated extensively by recombinant N-acetylgalactosaminyltransferase-1, -2, or -3 were prepared and characterized. Substitution at each Thr residue, as revealed by Edman degradation sequencing, in conjunction with evidence obtained on mass spectrometry indicated a heterogeneous pattern of site-specific glycosylation within the MUC2 tandem repeat. It was found that maximum numbers of 6, 8, and 11 GalNAc residues were incorporated by N-acetylgalactosaminyltransferases-1, -2, and -3, respectively, and that only N-acetylgalactosaminyltransferase-3 could completely glycosylate both consecutive sequences composed of three and five Thr residues in the MUC2 tandem repeat unit. These results suggest that O-glycosylation of the clustered Thr residues is a selective process controlled by N-acetylgalactosaminyltransferase-3 in the synthesis of clustered carbohydrate antigens.