FK506 promotes liver regeneration by suppressing natural killer cell activity

We examined the mechanism of promotion of liver regeneration by tacrolimus hydrate (FK506), a potent immunosuppressant, after partial hepatectomy. The administration of FK506 significantly increased the bromodeoxyuridine (BrdU) labelling index at 36 and 48 h after 70% hepatectomy compared with the placebo group. Using the same model, we examined the effect of FK506 on the expression of hepatocyte growth factor (HGF) and transforming growth factor-beta 1 (TGF-beta 1) and found no changes in HGF and TGF-beta 1 mRNA expression in the liver or in the HGF protein concentration in plasma. We found that pretreatment with FK506 markedly reduced the activity and number of liver-resident natural killer (NK) cells at the time of partial hepatectomy. Our observations suggest that the promotion of liver regeneration by FK506 may be attributable to a reduction in the number of liver-resident NK cells and to inhibition of their activity.     

Connections between the tendons of the musculus flexor digitorum profundus involving the synovial sheaths in the carpal tunnel

We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobes necrosis. After this procedure, lack of regenerative response in the residual viable liver tissue (omental lobes) was associated with elevated plasma hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1) levels and delayed expression of HGF and c-met mRNA in the remnant liver. Here, we investigated whether syngeneic isolated hepatocytes transplanted in the spleen will prolong survival and facilitate liver regeneration in FHF rats. Inbred male Lewis rats were used. Group I rats (n = 46) received intrasplenic injection of 2 x 10(7) hepatocytes and 2 days later FHF was induced. Group II FHF rats (n = 46) received intrasplenic injection of saline. Rats undergoing partial hepatectomy of 68% (PH; n = 30) and a sham operation (SO; n = 30) served as controls. In 20 FHF rats (10 rats/group), survival time was determined. The remaining 72 FHF rats (36 rats/group) were used for physiologic studies (liver function and regeneration and plasma growth factor levels). In Group I rats survival was longer than that of Group II controls (73 +/- 22 hr vs. 33 +/- 9 hr; P < 0. 01). During the first 36 hr, Group I rats had lower blood ammonia, lactate, total bilirubin, PT, and PTT values, lower activity of liver enzymes, and higher monoethylglycinexylidide (MEGX) production than Group II rats. In Group I rats, livers increased in weight at a rate similar to that seen in PH controls and showed distinct mitotic and DNA synthetic activity (incorporation of bromodeoxyuridine and proliferation cell nuclear antigen expression). Plasma HGF and TGF-beta1 levels in these rats decreased and followed the pattern seen in PH rats; additionally, c-met expression in the remnant liver was accelerated. Hepatocyte transplantation prolonged survival in FHF rats and facilitated liver regeneration. Even though the remnant liver increased in weight four times reaching 30% of the original liver mass, the transplant-bearing rats expired due to inability of the regenerating liver to support the rat.     

Immediate onset of DNA synthesis in remnant rat liver after 90% hepatectomy by an administration of follistatin

BACKGROUND/AIM: Follistatin is an antagonist of activins and is effective in promoting liver regeneration after 70% hepatectomy. To examine its efficacy under more critical conditions, we studied the effect of follistatin on liver regeneration in 90% hepatectomized rat. METHODS: Human recombinant follistatin was infused into the portal vein immediately after 90%, hepatectomy in 24-h-starved rats, and changes in the liver regeneration rate and nuclear bromodeoxyuridine labeling were measured. RESULTS: In control rats, nuclear labeling was first detected at 11 h of hepatectomy. In follistatin-treated rats, nuclear labeling was markedly increased at 3 h, and was significantly higher than that in control rats at 24, 72, 96, 120 and 144 h. The liver regeneration rate was significantly higher in follistatin-treated rats at 48, 72, 96, 120, 144 and 168 h. To determine the reason for the accelerated growth in starved rats, we compared the expression of mRNA for c-myc, p53, p21CIP1, p15INK4B, p27KIP1, and subunits of activins in fed and starved rats. mRNA for p21CIP1 and p15INK4B, but not p27KIP1 were decreased in 24 h-starved rats compared to the fed rats. mRNA for betaA subunit of activin was not detected in either fed or 24-h-starved rats, whereas that for betaC subunit was increased in starved rats. CONCLUSION: These results indicate that follistatin induces immediate onset of DNA synthesis in 90% hepatectomized rats and is quite effective in promoting liver regeneration.     

NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotein for superior characteristics in NMR experiments

Despite its obscure and short effect, plasma exchange (PE) remains a mainstay in the treatment of liver disease. However, the question still remains as to whether or not PE suppresses the regeneration of the liver because PE deprives patients of hepatotrophic factors. The effect of PE, which could be a total blood exchange (TBE) in a syngeneic setting, on liver regeneration following a 68% partial hepatectomy (PH) was investigated in rats. In Group 1, 20 ml of blood from normal rats was infused while native blood was removed at 6 and 12 h after PH. In Group 2, 20 ml of blood obtained from PH rats at the same time points was infused. The regeneration rate, labeling index of proliferating cell nuclear antigen (PCNA), and plasma hepatocyte growth factor (HGF) level were determined, and standard liver function tests performed at 24, 48, and 72 h. Although all liver function tests improved in Group 1 at 24 and 48 h, the regeneration rate was significantly impaired. Similarly, the PCNA labeling index was significantly lower in Group 1 than that in Group 2. The plasma HGF level was significantly reduced in Group 1 (6 h blood out versus blood in: 1.1+/-0.5 vs. 0.1+/-0.1 ng/ml, p < 0.05). TBE with normal blood following PH suppressed the early stage of liver regeneration, in part, because of the reduction of HGF even though the blood was purified.     

Effect of RES on liver regeneration and survival after 90% partial hepatectomy

In previous studies, 90% partial hepatectomy in the rat was invariably accompanied by 100% mortality within 40 hr. This paper describes the effect of enhanced reticuloendothelial system (RES) on liver regeneration after 90% partial hepatectomy. RES was activated using 5 K.E. of OK-432 injected intraperitoneally 24 hr before 90% partial hepatectomy. Ninety per cent of the liver mass was resected and rats were provided with tap water or 20% glucose orally and subcutaneously. Survival time was strikingly different. In rats provided with tap water only; 100% of rats died before 42 hr. In rats provided with 20% glucose; 44.2% of rats survived beyond 42 hr. In rats pretreated with OK-432 and provided with 20% glucose; 87.0% of rats survived beyond 42 hr. This regimen results in severe hypoglycemia and dead within 42 hr. When RES was activated before 90% partial hepatectomy, significantly higher blood glucose level was observed. BrdU labeling index was significantly higher in rats pretreated with OK-432 than in control rats. The results indicate that enhancement of RES before 90% partial hepatectomy provides acute metabolic support and enhancement of liver regeneration resulting in improved survival.     

Induction of hepatocyte growth factor in the liver, kidney and lung following total body irradiation in rat

Hepatocyte growth factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6-48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2-fold elevations 1-2 weeks after irradiation (P < 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration.     

The effect of a nucleotide-nucleoside solution on hepatic regeneration after partial hepatectomy in rats

After hepatectomy, purine and pyrimidine metabolism is a key process in the synthesis of DNA and RNA and maintaining cellular energy metabolism. The purpose of this study is to evaluate changes in blood purine and pyrimidine levels after partial hepatectomy and the effect of purine and pyrimidine nucleoside solution injection on hepatic regeneration under the hypothesis that the rat after partial hepatectomy requires substrates for salvage nucleotide synthesis and changes blood nucleoside and nucleobase levels. Blood levels of nucleotides, nucleosides, and nucleobase by high-performance liquid chromatography method and liver ATP level by enzymatic analysis, and the effect of preoperative injection of nucleoside solution (OG-VI) on hepatic regeneration ratio and hepatocytes DNA synthesis, were assessed in rats after 70% partial hepatectomy. Decreased liver adenosine triphosphate and increased plasma xanthine and hypoxanthine after partial hepatectomy indicated an increase in catabolism of purine nucleotides in regenerating liver. Plasma thymidine and cytidine levels increased, then returned to the prevalue, suggesting that the thymidine and cytidine pool was enlarged. OG-VI increased labeling indices of hepatocytes at postoperative d 1 (POD) and hepatic regeneration ratio at POD 14. Blood purine nucleobase and pyrimidine nucleoside levels change after partial hepatectomy and preoperative supply of nucleoside solution is effective for increasing hepatocytes DNA synthesis and hepatic regeneration after partial hepatectomy.     

Role of macrophages in regeneration of liver

In an attempt to clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration among C3H/HeN (LPS-responsive strain) and C3H/HeJ (LPS-resistant strain) mice was investigated. After a 67% partial hepatectomy, an increase in the weight of regenerating liver was significantly delayed in the C3H/HeJ mice, as compared with C3H/HeN mice. The number of hepatocytes labeled with antibody against PCNA reached maximum levels 48 hr after partial hepatectomy, but the PCNA labeling index in C3H/HeJ mice was 20% less than that for C3H/HeN mice. In addition, TNF-alpha activities in serum were enhanced shortly after partial hepatectomy in C3H/HeN strain mice, but were not increased in C3H/HeJ strain mice. Serum IL-6 levels were markedly enhanced in both C3H/HeN and C3H/HeJ mice, but a bimodial peak (14 and 48 hr after partial hepatectomy) was demonstrated in C3H/HeN mice, in contrast to a single peak (at 24 hr) in C3H/HeJ mice. Suppression of Kupffer cells by previous administration of gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF-alpha and IL-6 concentrations, reduced PCNA labeling index of hepatocytes by 20%, and disturbed the regeneration of the liver. Previous administration of antibody against TNF-alpha reduced the PCNA labeling index of hepatocytes by 20% after partial hepatectomy in C3H/HeN strain mice. These results suggest that LPS-responsive macrophages in the liver and their mediators, especially TNF-alpha, could partly play a role in liver regeneration.     

A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration

Limited data exist regarding morphogenesis and differentiation during liver regeneration. We examined the role of epimorphin on liver regeneration. After 70% partial hepatectomy, mouse liver was collected on days 1, 3, 7, and 14 for immunohistochemistry and the detection of epimorphin mRNA and connexin 32. Using primary cultured rat hepatocytes, morphogenesis and differentiation of cells were tested with or without epimorphin. Seven days after cell inoculation, the expression of connexin 32 and the cell-cell communication was tested as a marker of differentiation. Epimorphin was detected exclusively in hepatic stellate cells. Connexin 32 was detected only in hepatocytes. After partial hepatectomy, epimorphin mRNA was detected on day 3 and peaked at day 7, followed by protein expression. Connexin 32 expression showed a similar time course. Cultured hepatocytes formed multicellular spheroids in an active epimorphin-coated culture dish and showed positive dye coupling, whereas the cell-cell communication was lost without active epimorphin. Because epimorphin was expressed late in liver regeneration, it might play a role in morphogenesis and differentiation.     

Proliferating cell nuclear antigen, plasma fibronectin, and liver regeneration rate after seventy percent hepatectomy in normal and cirrhotic rats

BACKGROUND: The difference in liver regeneration rate in relation to proliferating cell nuclear antigen (PCNA) and plasma fibronectin level in the cirrhotic and control liver after 70% hepatectomy were examined in the rat. METHODS: Liver cirrhosis was induced by intraperitoneal injection of thioacetamide for 12 weeks; rats without thioacetamide administration served as controls. On the day before and days 1, 2, 3, and 7 after 70% hepatectomy, PCNA labeling index of hepatocyte, plasma fibronectin level, and percentage of the initial liver weight were determined. RESULTS: Liver regeneration rate as expressed by percent of initial liver weight was impaired in the cirrhotic liver, and significantly lower regeneration rate was observed on days 3 and 7 after hepatectomy in the cirrhotic rats as compared with controls. PCNA labeling index was higher in the cirrhotic liver before hepatectomy. Little change of PNA labeling index was observed in the cirrhotic liver, although the index increased significantly in the control liver; significantly higher values were observed on days 1, 2, and 3, with the maximal value on day 2 after hepatectomy. The plasma fibronectin level was significantly lower in the cirrhotic rat before and after hepatectomy. CONCLUSIONS: The results show that liver regeneration is impaired in the cirrhotic liver associated with little activation of PCNA and with lower plasma fibronectin level.     

Differential expression of transforming growth factor-beta and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl4 administration

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-beta receptor expression contributes to the liver diseases. METHODS: The mRNA expression of TGF-beta1, TGF-beta type I receptor (TGFbetaRI), TGF-beta type II receptor (TGFbetaRII) and TGF-beta type III receptor (TGFbetaRIII) in rat livers injured by CCl4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization. RESULT: The peak of TGF-beta1 mRNA expression was observed 48 h after acute intoxication with CCl4 in nonparenchymal cells. However, the levels of TGFbetaRI and TGFbetaRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFbetaRII mRNA expression was depressed more and for longer than that of TGFbetaRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFbetaRI and TGFbetaRII mRNAs at constant levels during liver regeneration. TGFbetaRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells. CONCLUSIONS: These observations suggest that: (i) whenever TGF-beta1 is increased in CCl4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) the mitoinhibitory effect of TGF-beta1 on hepatocytes is transiently relieved by down-regulation of TGF-beta receptors for 72 h post-damage; and (iii) the resistance to TGF-beta growth inhibition between 24 to 48 h may be predominantly due to down-regulation of the expression of TGFbetaRII.     

Extracellular matrix remodeling at the early stages of liver regeneration in the rat

Previous studies have shown that activity of urokinase-type plasminogen activator (u-PA) increases very rapidly (within 1 minute) after partial hepatectomy. In view of the well-recognized roles of u-PA as one of the major initiators of the matrix proteolysis cascade and as an activator of plasminogen and hepatocyte growth factor (HGF), we studied matrix degradation in liver shortly after partial hepatectomy. The activation of plasminogen to plasmin following partial hepatectomy was examined by Western blot analysis, and a small increase in plasmin at approximately 15 minutes followed by a large elevation at approximately 3 to 6 hours after partial hepatectomy was detected. In addition, we found that fibrinogen, the major substrate for plasmin, begins to be degraded at approximately 15 to 30 minutes following partial hepatectomy. Using immunohistochemical staining, we detected that the distribution of fibrinogen in normal liver is localized to the perisinusoidal space surrounding the periportal region. A decreased distribution of fibrinogen in the periportal region was found by 15 minutes and continued through 24 hours following partial hepatectomy. In addition, the distribution of fibronectin in normal liver was localized to the perisinusoidal space surrounding the periportal and the pericentral regions. A strikingly decreased distribution of fibronectin in the periportal region was found at 5 minutes after partial hepatectomy. Furthermore, we observed that the protein levels of laminin, entactin, and fibronectin in an extracellular matrix (ECM)-enriched preparation decreased shortly after partial hepatectomy, and were restored later. No changes were observed with either vitronectin or the integrin chain alpha(v). In contrast to the protein levels of the ECM components, the messenger RNA (mRNA) levels of fibronectin, integrin chain beta1, and integrin chain alpha(v) gradually increased over 18 hours and then decreased thereafter. Taken together, these results suggest that rapid reorganization of selected ECM components are important for hepatocyte proliferation at the early stages of liver regeneration.     

Human erythrocyte polyamine levels after partial hepatectomy

BACKGROUND/AIM: There are various indices of liver regeneration, but no clinically useful index that reflects the current status of liver regeneration. We assayed human erythrocyte polyamine levels after partial hepatectomy to define the relationship between erythrocyte polyamine levels and liver regeneration. MATERIALS AND METHODS: Levels of human erythrocyte polyamines (putrescine, spermidine, and spermine) were assayed by high-pressure liquid chromatography in 91 patients after partial hepatectomy and in 13 patients after surgery other than partial hepatectomy (controls). Of the patients after partial hepatectomy, 37 underwent hepatectomy of 20% or more of the liver (group A), 27 underwent segmentectomy or subsegmentectomy of the liver amounting to less than 20% of the liver (group B), and 27 underwent an operation smaller in scale than sub-segmentectomy (group C). RESULTS: The greater the proportion of the liver resected, the greater was the percent increase. In groups A, B, and C, erythrocyte levels of spermidine and spermine increased after surgery compared with the base line, and were significantly higher at 7 or 14 days, decreasing later. The differences in spermidine among the three groups were significant. CONCLUSIONS: After partial hepatectomy, the erythrocyte polyamine levels, especially the level of spermidine, were related to the proportion of liver resected. They seemed to reflect the degree of liver regeneration.     

Hepatocyte growth factor stimulates liver regeneration and elevates blood protein level in normal and partially hepatectomized rats

The effects of recombinant human hepatocyte growth factor (HGF) on liver growth and function of normal and partially hepatectomized rats have been examined. HGF was continuously administered into the jugular vein because it was rapidly eliminated from the plasma (t1/2 alpha; approximately 4.5 min) and degraded. In normal rats, the labeling index of hepatocytes was increased about 6 times by the administration of HGF. HGF also decreased the prothrombin time and increased the hepaplastin and serum albumin content. In 70%-hepatectomized rats, HGF stimulated liver regeneration and increased the level of blood proteins such as hepaplastin in a dose-dependent manner. The stimulation of serum protein level seemed to result from not only the increase of hepatic cell number but also the direct effect of HGF on the protein production in hepatocytes, because HGF rapidly enhanced the protein synthesis prior to the increase of cell number and increased the mRNA content of albumin in the liver in vivo. In addition, a combination of heparin with HGF further accelerated the effects of HGF described above, possibly due to the decrease of HGF clearance. These findings suggest that HGF accelerates both the hepatic regeneration and function in vivo, and that rhHGF is clinically expected to be a potent therapeutic agent in hepatectomy and liver injury.     

Hepatocyte growth factor in glycerol-induced acute renal failure

Hepatocyte growth factor (HGF) facilitates the regeneration of injured kidney in acute renal failure (ARF). Here we investigated the HGF production in glycerol-induced ARF rats. HGF mRNA expression levels were elevated in liver, spleen, and lung 6-24 h after glycerol injection. Tissue HGF protein levels determined by an enzyme-linked immunosorbent assay also increased in liver and spleen, whereas they decreased in the injured kidney 24 h after injection. Immunohistochemical studies showed that the number of HGF-producing cells did not increase in the liver. HGF receptor/c-Met mRNA levels were elevated only in the kidney. These results indicate that HGF supplied in an endocrine manner may play an important role in the regenerating process following ARF.     

Rat peripheral mononuclear cell thymidine kinase activity increases during liver regenerative processes after partial hepatectomy

Deoxythymidine kinase (TK; EC 2.7.1.21) activity in the liver has been used as a marker of liver regeneration after partial hepatectomy. In this study we examined TK activity of various organs, plasma and peripheral blood mononuclear cells (PMNC) in 70% partially hepatectomized rats. TK activity of lymph nodes, small intestine, heart, lung, kidney and thymus did not increase significantly during the course of the study, except for spleen at 72 h. On the other hand, PMNC-TK and liver cystosolic TK activity increased in a parallel fashion at all times after partial hepatectomy; they began to increase 12 h after surgery and peaked 48 h post-surgery. Fractionation of PMNC into T cells and B cells revealed that both populations increased and peaked 48 h post-surgery. Plasma TK peaked 12-24 h after surgery, then declined at 36, 48 and 72 h after partial hepatectomy. This change paralleled plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PMNC-TK activity correlated significantly with liver cystosolic TK activity 24 h (r = 0.743; P < 0.05) and 48 h (r = 0.708; P < 0.05) after partial hepatectomy. However, it did not correlate with plasma levels of TK, AST and ALT. The results indicate that in the early stage of liver regeneration PMNC-TK may provide a marker of liver regenerative processes.     

Does left ventricular wall motion of akinetic segment improve after reperfusion therapy in patients with acute myocardial infarction?: assessment by myocardial contrast echocardiography

AIMS/BACKGROUND: The liver clears circulating plasma-kallikrein through a receptor-mediated endocytosis process: an initial fast phase is followed by a slow exponential phase. METHODS: To determine whether the clearance rate of plasma-kallikrein is affected during liver regeneration, we perfused isolated rat livers with rat plasma-kallikrein (rPK) at 0, 1, 2, 3 and 7 days after partial hepatectomy or sham operation. RESULTS: Liver regeneration was followed by the expression of the proliferating-cell nuclear antigen (PCNA) labeling index. The serum concentration of alpha2-macroglobulin, an acute phase protein in rats, was measured. At day 1, the fast phase of rPK clearance rate increased in hepatectomized rats when compared with day 0 (4.9+/-0.4 and 3.7+/-0.4 mU/g liver min, p<0.05). However, at day 2, the rPK fast phase clearance rate dropped significantly (2.6+/-0.2, p<0.05), when compared with day 1. No difference was found among the sham groups at different days of hepatectomy. These changes seem to be independent of the acute phase reaction. The regenerative liver weight increased continuously during the observation period. PCNA expression increased significantly after hepatectomy, with maximal PCNA-labeling indices at days 1 and 2, declining thereafter. CONCLUSION: The rPK fast phase clearance rate changes during liver regeneration, with a zenith occurring when PCNA labeling index is maximal (day 1) and a nadir occurring at the mitotic phase (day 2).