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The purpose of this study was to examine the effect of polyunsaturated phosphatidylcholine (PPC, Essentiale) on the onset of liver regeneration after partial hepatectomy (PH). Hepatectomy was carried out on rats, and immediately after operation doses of 25, 50 or 250 mg PPC/kg b.w. were injected into the vena femoralis. The control group consisted of saline-injected PH rats. The rats were killed 18, 21, 24 and 30 h after PH. The course of liver regeneration was assessed by measuring the incorporation of labelled thymidine into DNA and the hepatocyte mitotic activity. After PPC injection no values of liver DNA specific activity differing from baseline were observed. However, higher values of hepatocyte mitotic activity and lower liver triglyceride levels were found as compared to the saline-treated rats. These observations indicate that the administration of PPC has a favourable effect on liver regeneration after PH.
Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.
BACKGROUND & AIMS: Because impaired regeneration after surgical treatment of the liver is influenced by circulating endotoxin, the underlying molecular mechanism was investigated. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally into mice, followed 24 hours later by 67% partial hepatectomy. We measured serum tumor necrosis factor (TNF) alpha levels as well as proliferating cell nuclear antigen labeling index, transforming growth factor (TGF) beta expression, and plasma kallikrein (PLK) activities in regenerating livers. We also examined the effect of LPS, TNF-alpha, and PLK on latent TGF-beta activation in homotypic and heterotypic cultures of rat or mouse hepatic stellate cells and Kupffer cells. RESULTS: Serum TNF-alpha levels increased after LPS (500 ng/g body wt) injection and after partial hepatectomy, accompanying TGF-beta-mediated suppression of hepatic proliferating cell nuclear antigen labeling index. This suppression was mimicked by a combination of preadministration of 50 ng/g body wt LPS and postoperative administration of 5 ng/g body wt TNF-alpha. In vitro, LPS stimulated Kupffer cells to secrete TNF-alpha, which enhanced PLK activity on the hepatic stellate cell surface through increasing PLK binding, thereby inducing proteolytic activation of latent TGF-beta and its autoinduction. Blockade of TGF-beta, TNF-alpha, or PLK activity prevented LPS-induced impaired regeneration in vivo. CONCLUSIONS: LPS provokes TNF-alpha/PLK-mediated proteolytic activation of latent TGF-beta in hepatic stellate cells, leading to impaired liver regeneration after partial hepatectomy.
BACKGROUND/AIMS: Beside its well-known function as tumour suppressor gene, p53 is supposed to positively regulate cell division and cell differentiation. Because hepatocyte proliferation has been reported to be reduced by blockade of p53 function in vitro, we examined in the present study the impact of p53 inhibition on hepatocyte proliferation in vivo. METHODS: Mice treated with either pifithrin-alpha (PFT), a p53-inactivating agent, or the equivalent volume of vehicle, were subjected to 70% hepatectomy. In addition to assessment of liver mass restitution we examined p53 and p21 protein expression as well as PCNA expression and BrdU incorporation by using Western blot and immunohistochemical techniques. Extent of apoptosis was assessed by TUNEL assay. RESULTS: PFT lowered nuclear but not cytoplasmic p53, and did not inhibit protein expression of regeneration-associated p21. PCNA protein expression as well as PCNA and BrdU immunohistochemistry did not differ between regenerating livers of either PFT- or vehicle-treated animals. Moreover, TUNEL analysis of regenerated liver tissue revealed comparable numbers of apoptotic cells in both groups. CONCLUSIONS: Pharmacological inhibition of p53 did not impair liver regeneration in mice, implying that p53 is functionally redundant in that p53-independent pathways compensate for the blockade of p53 and sufficiently support the process of hepatocyte replication in liver regeneration.
During hepatic regeneration a drop in the liver glycogen content along with a lower blood glucose level have been observed. These data are difficult to correlate with the rise of blood glucagon and the drop of insulin shown at the same times after partial hepatectomy. Therefore, liver glucose-6-phosphatase activity has been studied at 1.5, 4, 15 and 24 h, since that enzyme is involved in the release of glucose from the cell. 4 and 15 h after partial hepatectomy a remarkable decrease in glucose-6-phosphatase activity is observed. These results are discussed in view of the higher metabolic demand of regenerating liver.
Alpha-fetoprotein (AFP) gene expression occurs in the yolk sac, fetal liver and gut, and in the adult liver during regeneration and tumorigenesis. Polymorphism at a single genetic locus, Afr-2 (formerly known as Rif) between inbred mouse strains C3H/He and C57B1/6, results in different levels of AFP expression during liver regeneration. We examined AFP, histone H3, and albumin gene expression during liver regeneration and found that the strain-specific variance in AFP gene expression could not be attributed to a difference in the numbers of dividing cells. Experiments with transgenic mice revealed sequences required for Afr-2 regulation included 172 bp between -1010 and -838 bp and 118 bp immediately upstream of the AFP transcriptional start site-the same regions required for induction during liver regeneration. This suggests that the Afr-2 phenotype may stem from an allelic difference in a gene regulating gene expression during liver regeneration.
The effect of reticuloendothelial system activation on liver regeneration after 90% hepatectomy was investigated. OK-432, a killed streptococcal preparation that increases reticuloendothelial system activity, was administered to rats before 90% partial hepatectomy. Pretreated rats showed marked improvement in long-term survival: 87% (18 of 23) survived beyond 42 hr, compared with only 44.2% (24 of 52) of controls (p < 0.05). Survival was determined by means of life-table analysis and regeneration response by means of bromodeoxyuridine labeling index of hepatic DNA. OK-432 pretreatment had significantly increased bromodeoxyuridine labeling index 18, 24, and 42 hr after partial hepatectomy (p < 0.05). The results indicate that reticuloendothelial system activation by OK-432 before 90% partial hepatectomy enhances liver regeneration and improves survival, but these factors may not be related. The improved survival may be because of less infection in macrophage-stimulated animals or more rapid clearance of hypotension-causing vasoactive compounds.
We studied the effect of aging on rat liver regeneration. We compared the time course of total hepatic mass, DNA and RNA accumulation, and thymidine kinase (TK) messenger RNA (mRNA) content and enzyme activity, after two-thirds partial hepatectomy in 6-week-old (young adult) and 1-year-old rats. Whereas 6-week-old rats had completely regenerated all liver mass, DNA, and RNA by 7 days, the regenerating 1-year-old rat livers at 7 days contained only 60% to 70% of the mass, DNA, and RNA in the normal 1-year-old rat liver. However, rates of tissue regeneration during this time were very similar in both ages. At 28 days the weight and RNA content of the 1-year-old rat livers were 93% of normal, but total DNA was still reduced, at 78% of normal. In the younger rats TK mRNA content and enzyme activity increased substantially after partial hepatectomy and were observed to peak at 24 hours. In the 1-year-old rats TK mRNA was less abundant in normal liver and the peak level observed was lower and delayed until 48 hours. Nonetheless, the incremental increase from baseline to peak was greater in the 1-year-old than in the 6-week-old rats. In contrast to the mRNA, TK activity peaked at 24 hours, but at substantially lower levels than in the 6-week-old rats. Rates of disappearance of TK mRNA and enzyme activity after peak levels were not significantly different by age.(ABSTRACT TRUNCATED AT 250 WORDS)
Hepatocyte growth factor (HGF) is a most potent factor for mature parenchymal hepatocytes in primary culture and may act as a trigger for liver regeneration. We purified HGF from rat platelets to homogeneity and cloned both human and rat HGF cDNA. HGF is a heterodimer molecule composed of the 69 kDa alpha-subunit and the 34 kDa beta-subunit. HGF has no amino acid sequence homology with other known peptide growth factors and possesses the highest potential among known growth factors to stimulate proliferation of hepatocytes in primary culture. HGF is derived from a single chain precursor of 728 amino acid residues and the precursor is proteolytically processed to form a two-chain mature HGF. The alpha-subunit of HGF contains 4 kringle structures and HGF has a homology (38%) with plasmin. Biologically active recombinant human HGF could be expressed from COS-1 cells and CHO cells transfected with cloned cDNA. HGF activity and the HGF mRNA level are markedly increased in the liver following insult such as hepatitis, by the administration of hepatotoxins, ischaemia, physical damage and partial hepatectomy. Moreover, HGF mRNA is induced in the lung and kidney, in the presence of liver injury. In situ hybridization revealed that HGF-producing cells in liver are non-parenchymal liver cells, presumably Kupffer and sinusoidal endothelial cells. Therefore, HGF from neighbouring cells (Kupffer and sinsuoidal endothelial cells) and distal organs (lung and kidney) may function as a trigger for liver regeneration by both a paracrine mechanism and an endocrine mechanism. HGF has mitogenic activity for renal tubular epithelial cells, epidermal melanocytes and keratinocytes as well as mature hepatocytes, and has the potential to promote cell migration for some epithelial cells, including normal human keratinocytes. Since cell growth and cell motility are relevant to tissue repair and embryogenesis, HGF may well have important roles in tissue repair and embryogenesis as well as in liver regeneration.
The change in the hepatic uptake clearance of hepatocyte growth factor (HGF) during liver regeneration in rats was examined. The hepatic uptake clearance per gram liver of 125I-labeled HGF in vivo decreased to a minimum level only 15 min after partial hepatectomy (PH). In the perfused liver, in which the effect of endogenous substances can be ignored, the hepatic uptake clearance of 125I-HGF did not change until at least 3 h after PH, suggesting that the decrease in the clearance in vivo is caused by some endogenous inhibitors. Such a decrease in the clearance cannot be explained only by the elevated endogenous HGF concentration in plasma. In addition, at 48 h after PH the decrease in hepatic uptake clearance was comparable for in vivo and perfused liver (approximately 50 and 60% reduction, respectively), suggesting that the decreased hepatic clearance in vivo at that time was caused mainly by reduction in the binding and/or uptake by the liver itself. In addition, the hepatic uptake clearance of 125I-HGF at that time in the presence of an excess (135 pM) of unlabeled HGF was comparable with that of the control, indicating that only the saturable portion of hepatic uptake clearance is reduced. The internalization rate constant representing the probability of cell surface-bound 125I-HGF being internalized per unit time increased to three times that of the control at 15 min after PH, demonstrating that the internalization of HGF is enhanced immediately after the start of liver regeneration.
TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.
Concern exists that partial liver transplants (either a living donor [LD] or deceased donor [DD] in hepatitis C virus (HCV)-positive recipients may be associated with an increased risk for recurrence. From 1999 to 2003, at our institution, 51 HCV-positive recipients underwent liver transplants: 32 whole-liver (WL) transplants, 12 LD transplants and 7 DD split transplants. Donor characteristics differed in that WL donors were older, and LD livers had lower ischemic times. Recipient characteristics were similar except that mean MELD scores in LD recipients were lower (p < 0.05). With a mean follow-up of 28.3 months, 46 (90%) recipients are alive: three died from HCV recurrent liver disease and two from tumor recurrence. Based on 1-year protocol biopsies, the incidence of histologic recurrence in the three groups is as follows: WL, 81%; LD, 50% and DD split, 86% (p = 0.06 for LD versus WL). The mean grade of inflammation on the biopsy specimens was: WL, 1.31; LD, 0.33 and DD split, 1.2 (p = 0.002 for LD versus WL; p = 0.03 for LD versus DD split). Mean stage of fibrosis was: WL, 0.96; LD, 0.22 and DD split, 0.60 (p = 0.07 for LD versus WL). Liver regeneration does not seem to affect hepatitis C recurrence as much, perhaps, as factors such as DD status, donor age and cold ischemic time.
Although enhanced phosphorylative activity can be a requisite for later DNA synthesis during liver regeneration (LR), mitochondrial generation of reactive oxygen species could lead to altered mitochondrial membrane permeability during the prereplicative phase of LR. Therefore, the role of mitochondrial permeability transition (MPT) was evaluated during rat LR, induced by either partial hepatectomy (PH) or after CCl(4) administration. Parameters indicative of mitochondrial function and membrane potentials, those of oxidative stress, and in vivo changes of the intramitochondrial pool of adenine nucleotides were determined. Twelve hours after PH, mitochondrial oxidative and phosphorylative activities and adenosine diphosphate (ADP) content were increased, reaching a maximal peak at 24 hours after surgery (maximal DNA synthesis). Parameters suggestive of oxidant stress were enhanced, but mitochondrial volume and membrane electrical potential remained unaltered. Interestingly, moderate mitochondrial swelling and depolarization were found at later post-PH times (72 hours). In CCl(4)-treated animals, it was found that an active liver cell necrosis delayed mitotic activity and mitochondrial uncoupled respiration. Starting 12 hours after CCl(4) intoxication, a drastic increase of inorganic phosphate occurred within swollen and strongly depolarized mitochondria, suggesting changes in the MPT. Despite expression of messenger RNA (mRNA) for mitochondrial transcription, factor A showed a similar time course in both experimental models. The so-called augmenter liver regeneration was found significantly elevated only in PH rats. In conclusion, onset of MPT could be associated with cell necrosis and inflammation after CCl(4) treatment, whereas this mitochondrial event could constitute a putative effector mechanism, through which growth or inflammatory factors inhibiting cell proliferation could initiate LR termination.
Proliferation and differentiation of hepatic stem cell progenies (i.e., oval cells) sustain liver regeneration when the replicative and functional capacity of hepatocytes is impaired. The signaling pathways that control stem cell activation remain poorly understood. In this study, we investigated the involvement of nuclear factor-kappa B (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) in oval cell-mediated liver regeneration induced by 2-acetylaminofluorene/partial hepatectomy (AAF/PH) protocol. Using OV1 as a marker for identification and sorting of oval cells, we established that both NF-kappaB and STAT3 were highly activated in the OV1(+) cell population. Three distinct subpopulations of oval cells were defined as OV1(low), OV1(medium), and OV1(high), based on the intensity of OV1 staining. Quantitative polymerase chain reaction analysis revealed that they represent different stages of oval cell differentiation along hepatocyte lineage. OV1(low) cells displayed the least differentiated phenotype as judged by high expression of c-kit and lack of hepatocytic differentiation markers, whereas OV1(high) cells lost c-kit expression, were more proliferative, and acquired more mature hepatocytic phenotype. Notably, NF-kappaB was activated uniformly in all three subpopulations of oval cells. In contrast, phosphorylation of STAT3 was detected only in OV1(high) cells. In conclusion, transcriptional activity supported by NF-kappaB and STAT3 is required for oval cell activation, expansion, and differentiation. The differential induction of NF-kappaB and STAT3 point to a distinct role for these transcription factors at different stages of hepatic stem cell differentiation.
The activity of 2 nonmitochondrial forms of DNA polymerase, designated DNA polymerases alpha and beta, was investigated during liver regeneration in regimented rats. In accord with Barbiroli and Potter, we observed that regimentation of rats with respect to temperature, light and darkness, and availability of food resolves the DNA synthesis response to partial hepatectomy into 2 peaks, one occurring at a fixed time after operation and the other entrained by the environmental conditions. The peaks can be fused or separated depending on the timing of the operation. For this study, operation times were selected to give both patterns of DNA synthesis as measured by the uptake of radioactive thymidine into DNA. For both operation times, DNA polymerase activity in the nuclear extract correlated temporally and qualitatively with radioactive thymidine uptake into DNA. At the times of maximal DNA synthesis and polymerase activity, the DNA polymerase was purified from extracts of isolated nuclei. DNA polymerase alpha represented 70% and DNA polymerase beta represented 30% of the recovered activity from the nuclear extract. This is in agreement with the previous observation in nonregimented rats that DNA polymerase alpha is the major activity in nuclei during liver regeneration. For both operation times, DNA polymerase activity in the postmicrosomal fraction was sedimentable and increased 3 to 4 times above the level observed with this same fraction from normal rat liver. This activity was shown to be due to DNA polymerase alpha only with this subcellular fraction. DNA polymerase alpha activity with this fraction peaked 4 to 6 hr after the time of maximal radioactive thymidine incorporation into DNA. DNA polymerase activity in the microsome fraction did not change significantly after partial hepatectomy. This activity has been shown to represent DNA polymerase beta. Prior administration of cycloheximide and actinomycin abolished the rise in DNA polymerase alpha activity in the nucleus and postmicrosomal fraction. Hydroxyurea did not prevent the rise in DNA polymerase alpha activity with those subcellular fractions but did inhibit over 90% of the uptake of radioactive thymidine into DNA. These data suggest, but do not prove, that DNA polymerase alpha activity is induced in response to the stimulus(i) for liver regeneration.
The effect of omeprazole on liver regeneration was studied in rats following partial (65 per cent) hepatectomy. Omeprazole 0.2 mg/kg increased the relative liver weight (weight of liver as a proportion of body-weight) and mitotic index (P < 0.05). There was no difference in food and water intake. The serum gastrin concentration was significantly higher in animals receiving omeprazole 0.2 mg/kg than in controls (P < 0.05). Omeprazole administration induced an increase in the level of serum alkaline phosphatase (P < 0.05) but had no effect on serum albumin, glutamic-pyruvic transaminase and total bilirubin levels. Omeprazole stimulates liver regeneration after partial hepatectomy and this regeneration may be mediated by gastrin.
OBJECTIVE: To assess the effects of oxygen free radicals on liver regeneration in rats after 80% hepatectomy. DESIGN: Open laboratory study. SETTING: Institute of Biomedical Sciences, University of Milan, Italy. MATERIAL: Female Sprague-Dawley rats. INTERVENTIONS: After laparotomy 122 rats had ischaemia induced by clamping of the portal vein and hepatic artery, and 93 rats had manipulation of the vessels only. They were then allowed to recover before 80% hepatectomy was done. Absolute controls (n = 16) did not have a laparotomy. Mortality was recorded, and surviving rats were killed one, three, five, and seven days after operation. A further 10 rats had 80% hepatectomies and then underwent a 10 minute period of ischaemia 24 hours later. These animals were killed after three days. In yet more experiments 38 rats underwent 80% hepatectomies and were then divided into four groups: 8 were given allopurinol 50 mg/kg/day starting three days before operation; 8 were given superoxide dismutase 4.16 mg/kg intraperitoneally 30 minutes before induction of ischaemia; 12 were given verapamil 0.1 mg/kg 30 minutes before induction of ischaemia; and 10 were given saline (controls). MAIN OUTCOME MEASURES: Incorporation of tritiated thymidine into DNA, differences in liver weights, and lipid peroxide concentrations. RESULTS: 43 rats died after ischaemia/reperfusion and 19 after hepatectomy alone. Ischaemia/reperfusion caused a significant reduction in the incorporation of tritiated thymidine into DNA 24 hours after hepatectomy (p < 0.01), and significant inhibition of recovery of liver weight three (p < 0.01) and five (p < 0.05) days after hepatectomy. These effects were associated with high lipid peroxide concentrations at three days. Allopurinol (p < 0.01, p < 0.05), superoxide dismutase (p < 0.01, p < 0.01) and verapamil (p < 0.01, N.S.) reduced the effects of ischaemia/reperfusion on liver weights or lipid peroxide concentrations three days after hepatectomy. CONCLUSION: A 10 minute period of ischaemia followed by reperfusion temporarily reduces liver regeneration after 80% hepatectomy in rats.