Simultaneous transfer of vascular endothelial growth factor and hepatocyte growth factor genes effectively promotes liver regeneration after hepatectomy in cirrhotic rats

BACKGROUND/AIMS: Liver regeneration in a cirrhotic liver is unsatisfactory. In the course of liver regeneration, non-parenchymal cells such as sinusoidal endothelial cells as well as hepatocytes increase in number while the liver structure and physiological functions are maintained. The aim of this study was to examine whether sufficient liver regeneration could be obtained by the simultaneous, preoperative injection of recombinant adenoviral vectors encoding human vascular endothelial growth factor (VEGF), a potent mitogen for sinusoidal endothelial cells, (pAxCAVEGF) and rat hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, (pAxCAHGF) in 70% hepatectomized cirrhotic rats. METHODOLOGY: Forty-eight hours before 70% hepatectomy, dimethylnitrosamine-induced cirrhotic rats were infused intravenously with pAxCAVEGF or with pAxCAVEGF and pAxCAHGF, or with a control virus encoding Escherichia coli beta-galactosidase (pAxCALacZ). RESULTS: Strong VEGF mRNA expressions were shown in the livers of VEGF and VEGF/HGF-treated animals. The plasma HGF concentrations in the VEGF/HGF-treated rats were elevated compared with the other groups. Proliferating cell nuclear antigen immunostaining showed increased labeling indices of hepatocytes in the VEGF/HGF-treated rats at 24 and 48 h after hepatectomy. PCNA labeling indices of SECs were increased in the VEGF and VEGF/HGF-treated rats compared with the control animals at 24 and 48 h after hepatectomy. Moreover, the hepatic regeneration rate after hepatectomy was significantly augmented by the VEGF and VEGF/HGF treatment. CONCLUSIONS: Simultaneous preoperative injection of recombinant adenoviral vectors encoding VEGF and HGF effectively stimulates liver regeneration in cirrhotic rats.     

Ischemic preconditioning improves liver regeneration by sustaining energy metabolism after partial hepatectomy under ischemia in rats.

BACKGROUND: The protective effect of ischemic preconditioning (IPC) has been reported on improvement of survival, reduction of liver necrosis and enhancement of the regenerative capacity of hepatocytes after partial hepatectomy. This study was undertaken to confirm that IPC has a significant impact on regeneration of hepatocytes after partial hepatectomy in ischemically damaged liver. In addition, we sought to examine the role of adenine nucleotides in this process. METHODS: Wistar rats were subjected to 60 min of total hepatic ischemia, followed by 70% hepatectomy. The animals were subdivided into an IPC (10/15 min) group and a non-IPC (control) group. Liver function tests and arginase activity were analyzed. Hepatic adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate were measured using gradient high-performance liquid chromatography. The liver regeneration was identified using relative liver weight and proliferating cell nuclear antigen (PCNA) labeling index. RESULTS: IPC treatment improved serum liver enzymes and tissue arginase activity (P<0.05) when compared with the control group. The preconditioned livers were associated with upregulation of ATP expression and also increased tissue energy charge. Regenerated liver weight in the IPC group was significantly higher than in the control group (P<0.05). The PCNA labeling index in the remnant livers in the IPC group was also significantly increased at 24 and 48 h after partial hepatectomy (P<0.05). CONCLUSION: These results suggest that IPC-augmented liver regeneration after hepatectomy, probably due to the stabilization of energy metabolism in rats.     

Vascular endothelial growth factor does not improve liver regeneration and survival after 90% subtotal liver resection

Aim: Vascular endothelial growth factor (VEGF) has been shown to stimulate liver regeneration after 70% partial hepatectomy (PH). It is unclear, however, whether exogenous administration of VEGF can also be used to improve liver regeneration and survival after 90% subtotal liver resection. The aim of this study was to determine the effect of exogenous and endogenous VEGF after 90% subtotal hepatectomy (SH). Methods: Rats were subjected to 90% SH and treated with VEGF, anti-VEGF or NaCl. Postoperatively (3 h - 5 days) liver body weight ratio (LBR), hepatocyte proliferation and biochemical markers were assessed. ELISA was performed to measure protein levels for VEGF. Gene expression was determined by customized cDNA arrays and quantitative RT-PCR. Results: Administration of VEGF did not enhance LBR or hepatic proliferation, or reduce the serum parameters. VEGF levels were the highest in VEGF-treated animals. The overall survival after 90% SH reached 78% in VEGF-treated animals, but did not differ significantly from that of anti-VEGF or NaCl-treated animals (74% and 75%, respectively). Gene expression analysis showed a modulation of anti-apoptotic and cell cycle control genes that was independent of VEGF. Conclusions: In contrast to PH, liver regeneration and survival after SH cannot be modulated by VEGF. This indicates that the relevant mechanisms that stimulate liver regeneration after hepatectomy at least partially depend upon the extent of liver resection.     

Effects of Bicyclol on Liver Regeneration After Partial Hepatectomy in Rats

Bicyclol is a synthetic antihepatitis drug with antioxidative property. The present study was performed to investigate the effect of bicyclol on liver regeneration after partial hepatectomy in rats. Bicyclol (300 mg/kg) was given to rats subjected to 70% hepatectomy three times before operation. At 6, 24, and 48 h after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBil), hepatic glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, proliferation index, and histopathological examination were evaluated at 48 h after hepatectomy. As a result, bicyclol significantly increased regeneration rate, mitotic index (MI), PCNA labeling index, and proliferation index in PH rats. Additionally, bicyclol remarkably inhibited the elevation of serum ALT and TBil levels, alleviated the formation of liver MDA, restored impaired antioxidant SOD and GSH, increased hepatic glycogen content, and also attenuated hepatic vacuolar degeneration. These results suggested that bicyclol had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative property.     

Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats.

BACKGROUND: The aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats. METHODS: We investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro. RESULTS: The expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h. CONCLUSIONS: These data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.     

Shedding of TNFR1 in regenerative liver can be induced with TNFα and PMA

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated.METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-α, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFα was studied by detecting apoptotic index.RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNF α, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNF α for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNF α and plasma membrane from hepatocytes affected with TNF α for 2 hor from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21% to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too,but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats.CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFα. Membraneanchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.     

Hepatocyte growth factor as well as vascular endothelial growth factor gene induction effectively promotes liver regeneration after hepatectomy in Solt-Farber rats

BACKGROUND/AIMS: Hepatic oval cells play an important role in liver regeneration when proliferation of mature hepatocytes is inhibited. The aim of this study was to examine the effect of hepatocyte growth factor (HGF), or vascular endothelial growth factor (VEGF) on proliferation of oval cells in the Solt-Farber rat model. METHODOLOGY: One hour after 70% partial hepatectomy, 2-acetyl-aminofluorene-induced damaged rats were infected intravenously with recombinant adenoviral vectors, encoding rat HGF or human VEGF, or Escherichia coli beta-galactosidase as a control. RESULTS: The plasma HGF concentrations in the HGF-transferred rats were elevated compared with the other groups at 4 and 7 days after hepatectomy. Oval cells were confirmed by positive staining of both cytokeratin-19 and alpha-fetoprotein. Oval cells around the portal tracts in the HGF or VEGF-transferred rats increased in number compared with the control rats at 7 and 9 days after hepatectomy. The proliferating cell nuclear antigen labeling indices of oval cells and the hepatic regeneration rate after hepatectomy were significantly augmented by the HGF or VEGF treatment. Moreover, cyclin E expression was elevated in the HGF-treated rats. CONCLUSIONS: In the Solt-Farber rat model, HGF or VEGF gene injection effectively promoted liver regeneration after hepatectomy mainly with increased proliferation of hepatic oval cells.     

Interplay among cardiotrophin-1, prostaglandins, and vascular endothelial growth factor in rat liver regeneration

Prostaglandins are hepatoprotective molecules generated in liver regeneration by the rapid induction of cyclooxygenase-2 (COX-2). Cardiotrophin-1 (CT-1) and vascular endothelial growth factor (VEGF) are other hepatoprotective mediators upregulated at 24 hours after partial hepatectomy. The interactions among these molecules during liver regeneration have not yet been defined. Here we show that rats subjected to partial hepatectomy treated with NS-398, a specific COX-2 inhibitor, exhibited cell cycle arrest, increased hepatocyte apoptosis, persistent extracellular signal-regulated kinase (ERK) 1/2 activation, and increased interleukin-6 production. These changes were associated with downregulation of CT-1 and COX-1 and altered pattern of VEGF expression. Administration of an adenovirus encoding CT-1 to NS-398-treated rats restituted normal levels of COX-1, prostaglandins, and VEGF in the liver after partial hepatectomy and restored normal liver regeneration. Furthermore, the stimulation of isolated rat hepatocytes with CT-1 increased COX-1, COX-2, and VEGF messenger RNAs and prostaglandin synthesis. Conversely, the addition of prostaglandin E1 to the culture increased CT-1 and VEGF production. In conclusion, COX-2 activation and production of prostaglandins soon after partial hepatectomy are essential requirements for hepatocyte proliferation and for the correct induction of both CT-1 and VEGF. CT-1 can restore liver regeneration after COX-2 inhibition by increasing VEGF, COX-1 expression, and prostaglandin synthesis.     

Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM:To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft.METHODS:Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF-propagated DCs (group B), GM-CSF+IL-4-propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs(group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation.DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24h, 48h, 72h and 84h,respectively. Liver graft-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with ^51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84h in group C rats and 48h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ production.At the time of the maximal regeneration of liver allograft,the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-y production were detected in group D rats. The apoptotic index of hepatocytes, the activityof liver- resident NK cells, the hepatic TFN-γ mRNA expression level and the serum IFN-γ level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION:The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified Des may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Appearance of an inhibitory cell nuclear antigen in rat and human serum during variable degrees of hepatic regenerative activity

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Effect of serotonin receptor 2 blockage on liver regeneration after partial hepatectomy in the rat liver.

The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. Materials and methods: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. Results: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. Conclusions: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.     

Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM: To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs)on regeneration of partial liver allograft.METHODS: Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF -propagated DCs (group B), GM-CSF+IL-4 -propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E)were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h,respectively. Liver graft-resident NK cell activity, hepatic IFN-y mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with 51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γmRNA expression and recipient serum IFN-γ production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-γ production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-γ mRNA expression level and the serum IFN-γlevel in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION: The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Adenovirus-mediated overexpression of the activin betaC subunit accelerates liver regeneration in partially hepatectomized rats

BACKGROUND/AIMS: The expression level of the activin betaC subunit is high in normal liver and reduces after partial hepatectomy, but its function is controversial. METHODS: To determine the role of the betaC subunit during liver regeneration, we overexpressed the betaC subunit gene in the liver by infusing adenovirus vector encoding the flag-tagged betaC subunit into the portal vein. Adenovirus vector encoding the beta-galactosidase was also infused as a control. Seventy percent hepatectomy was performed 4 days after the infection. RESULTS: Approximately 20% of hepatocytes expressed the flag-tagged betaC subunit at the time of hepatectomy and approximately 50% of hepatocytes expressed the betaC subunit 3 days after hepatectomy. In betaC-infected liver, bromodeoxyuridine labeling was significantly greater at 24 and 48 h after partial hepatectomy compared with the control liver. Consistent with this observation, the liver regeneration rate was significantly greater in betaC-transfected liver at 72 and 96 h after hepatectomy. Many of the bromodeoxyuridine-positive nuclei were observed in or by the betaC-transfected hepatocytes. CONCLUSIONS: These results indicate that liver regeneration is accelerated in betaC-overexpressing liver. The betaC subunit may function to promote replication of hepatocytes during liver regeneration.     

Does steatohepatitis impair liver regeneration? A study in a dietary model of non-alcoholic steatohepatitis in rats

BACKGROUND: Impaired liver regeneration is a feature of alcoholic hepatitis, but the relative importance of alcohol, nutritional imbalance and inflammatory mediators in causing this effect is unclear. Non-alcoholic steatohepatitis (NASH) is a form of liver disease with similar morphology to alcoholic hepatitis, but the effect of this disorder on liver regeneration is unclear. We, therefore, examined the status of liver regeneration in a rat nutritional model of hepatic steatosis with inflammation, which is morphologically identical to NASH in humans. Methods: Male Wistar rats received a methionine-choline-deficient diet (MCDD) for 4 weeks before experiments and both isocaloric pair-fed and ad libitum-fed rats were used as controls. Following partial hepatectomy (68%), the extent of hepatic regeneration was determined 24 h later using [3H]-thymidine incorporation and restitution of liver mass. Results: There was no significant difference of [3H]-thymidine incorporation in MCDD-fed, pair-fed and ad libitum-fed rats (80+/-27, 78+/-11 and 80+/-6.3 d.p.m./microg DNA, respectively). Similarly, restituted liver masses in three groups of rats were not significantly different (17+/-3.8, 18+/-1.8 and 17+/-3.1% initial liver weight, respectively). Conclusions: The similarities in hepatic histology and cytochrome P450 2E1 induction between this nutritional model of hepatic steatohepatitis and alcoholic steatohepatitis imply that these two disorders share pathogenetic mechanisms. However, liver regeneration is not altered by NASH in rats, indicating that the nutritional and inflammatory changes that appear similar to those of alcoholic liver disease do not cause impairment of liver regeneration.