Possible endocrine control by hepatocyte growth factor of liver regeneration after partial hepatectomy.

Hepatocyte Growth Factor (HGF), which is a most potent growth factor for primary cultured hepatocytes, may act as a trigger for liver regeneration. After 70% of the rat liver was removed, HGF activity in the remnant liver began to increase within 24 h. In parallel with the activity, the HGF mRNA level in the remnant liver increased at 12 h after the operation and reached a maximum at 24 h. Increases in HGF activity and in the mRNA level were much lower and later than those in the liver of rats with hepatitis induced with CCl4. However, the first increase in HGF activity in the plasma of hepatectomized rats was noted 3 h after the resection, that is much earlier than the initial DNA synthesis in the remnant liver. Thus, while HGF production was induced in the remnant liver during regeneration after partial hepatectomy, the initial trigger may not be the liver-derived HGF, rather, it may be HGF derived from extrahepatic organs, via blood circulation.     

Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy.

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co-cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen-NPCs), the response of hepatocytes from resting liver to EGF, TGF-alpha, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell-to-cell contact, and conditioned-medium from regen-NPCs, but not normal NPCs, inhibited EGF-induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS-PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin-sensitive but relatively heat-stable. The effects of blocking antibodies established that it was not TGF-beta 1, IL1-beta, or IL6. Investigations of regen-NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy.     

Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.     

Gene expression in regenerating liver in relation to cell proliferation and stress

When hepatocyte proliferation is stimulated in the liver by partial hepatectomy, messenger RNAs coding for fibrinogen, actin, c-myc and topoisomerase I are rapidly accumulated. We distinguish an early phase of accumulation (0-3 h after partial hepatectomy) which is also observed after a sham operation for the four genes, and during inflammation produced by Freund's adjuvant in the case of fibrinogen and c-myc genes. The hepatic response to inflammation appears therefore to mimic events characteristic of the G0/G1 transition, such as the accumulation of the c-myc mRNA. The late phase of mRNA accumulation (beyond 3 h after partial hepatectomy) is typical of liver regeneration. The level of c-myc mRNA is transiently increased (20-fold over normal) 20 h after partial hepatectomy, that is, at the time of DNA synthesis. Topoisomerase-I mRNA level increases between 3 and 24 h after partial hepatectomy (5-10-fold over normal). These results suggest that accumulation of c-myc and topoisomerase-I mRNAs is associated with DNA replication in regenerating liver.     

Conditional Genetic Elimination of Hepatocyte Growth Factor in Mice Compromises Liver Regeneration after Partial Hepatectomy

Hepatocyte growth factor (HGF) has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ER^T transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70–80%) of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH), which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4)-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth.     

Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury

Overlapping cDNA clones for rat hepatocyte growth factor (rHGF) were isolated by cross-hybridization with the cloned cDNA for human hepatocyte growth factor (hHGF) and the nucleotide sequence of the cDNA was determined. The entire primary structure of rHGF was deduced from the sequence. Comparison of the amino acid sequences between rat and human HGFs revealed that the two sequences are highly conserved throughout the protein structures, suggesting that rat and human HGFs may be functionally similar. Responses of the rHGF mRNA during liver regeneration in rats were examined by Northern blot hybridization analysis with the aid of the cDNA probe for rHGF. The mRNA levels increased in the liver and spleen but not in the kidney after administration of carbon tetrachloride. At the maximum level of induction, the rHGF mRNA increased in the liver about 4.5-fold over its normal level. The mRNA levels also increased in the liver and spleen after administration of D-galactosamine. On the other hand, no obvious increase of the mRNA was observed in the liver and spleen after partial hepatectomy. These observations suggest that HGF may function as a regulator of liver regeneration following hepatic injury caused by hepatotoxins.     

Role of cell-surface modulator of DNA synthesis in liver regeneration

A cell-surface modulator of DNA synthesis by cultured rat hepatocytes was studied in relation to the liver regeneration process. When rat hepatocytes isolated 24 h after partial hepatectomy were cultured, the first burst of DNA synthesis peaked at 5-8 h and declined until 24 h, followed by the second burst. Rat liver plasma membranes added 2 h after plating inhibited only the second burst, while in the case of the normal hepatocytes where the DNA synthesis began to increase after 5 h, this inhibition was observed at 16 h but not at 8 h. The inhibition did not differ when the membranes obtained from regenerating livers 12 h after partial hepatectomy were used. Epidermal growth factor binding to the cultured hepatocytes was not hindered by the membranes. These results suggest that the modulator inhibits hepatocyte proliferation at the early G1-phase of the cell cycle and that its action might be controlled by other factors in the process of liver regeneration.     

Kinetics of DNA synthesis in feeding-dependent and independent hepatocyte populations of rats after partial hepatectomy

The effects of food consumption on the kinetics of hepatic DNA synthesis after partial hepatectomy (PH) have been studied in rats. Short-term (4-24 hr) fasting before or after PH resulted in depression and/or delay of DNA synthesis on days 1, 2 and 3 of regeneration. This depression was found in hepatocytes and, to a lesser extent, in littoral cells. Re-feeding resulted in an increase of DNA synthesis within 3-8 hr. The results suggest that two different hepatocyte subpopulations exist in regenerating rat liver: one which proceeds to DNA synthesis without apparent exogenous signals, and another one which needs, in addition to the specific mitogenic action of PH, food intake as a secondary permissive signal in order to initiate DNA synthesis. In the latter population food consumption appears to be required at two different stages: (1) in G0 or the early pre-replicative phase (PRP); (2) in the late PRP 3-8 hr before initiation of DNA synthesis. In the latter stage dietary protein is needed, but no so in the former. The dependence on feeding in the late PRP increases relatively with time after PH. No evidence was found to suggest a different distribution of the two cell populations throughout the liver acinus. The findings support the hypothesis that the known effects of the light-dark rhythm on the timing of DNA synthesis after PH are mediated by the natural feeding rhythm of rats fed ad libitum. In addition they offer a means for improving the synchrony of hepatocyte proliferation in regenerating rat liver.     

Effect of the parenteral administration of amino acid solutions in different phases after partial hepatectomy on the initiation and development of liver regeneration in rats

Amino acid solutions enriched with branched-chain amino acids or pure branched-chain amino acid solutions were administered parenterally to female laboratory rats (pre-operative weight 230 +/- 30 g) which had been subjected to 65-70% partial hepatectomy (PH), and specific liver DNA activity, the hepatocyte mitotic index and other indicators of the initiation of liver regeneration were studied. Both solutions were infused in an hourly dose of 3.3 ml/kg body weight, during the following postoperative intervals: 1-6, 7-12, 1-12, 1-18 and 1-24 hours. The control rats continued to be fed on the standard laboratory diet after the operation. The results show that the infusion of an amino acid solution enriched with branched-chain amino acids had an inhibitory effect on the onset of DNA synthesis in the liver 18 hours after partial hepatectomy whatever the administration interval. The situation in the case of pure branched-chain amino acid solutions was the same. Twenty-four hours after PH, neither type of solution, irrespective of the infusion interval, was followed by an increase in DNA synthesis compared with the controls fed on the standard laboratory diet. Neither the hepatocyte mitotic index, nor the total liver DNA concentration, showed any changes indicative of stimulation of the initiation of liver regeneration. An infusion stress effect, evaluated from the decrease in the weight of the thymus, was found chiefly in the case of infusions lasting 12 h or longer.     

Regulation of hepatocyte growth: alpha-1 adrenergic receptor and ras p21 changes in liver regeneration

Catecholamines, acting via the alpha-1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha-1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]-prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]-myo-inositol was transiently reduced between 8 and 16 hr, when alpha-1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha-1 receptors and ras protein in the early events of liver regeneration.     

Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs.

Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CCl4. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lung-derived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms.     

Long- and medium-chain triacylglycerols in nutritional support of liver regeneration of partially hepatectomized rats

An appropriate choice for a suitable diet during liver regeneration still remains an enigma. To investigate the effect of isocaloric enteral feeding with medium-chain triacylglycerols (MCT) and long-chain triacylglycerols (LCT) supplement (MCT+LCT, 40%:60% w:w) (178 kJ/kg b.w./24 h), rat liver regeneration was studied 24 and 72 h after partial hepatectomy. The liver DNA synthesis 24 h after partial hepatectomy was significantly higher in the MCT+LCT-supplemented rats (30.2+/-8.2 x 10(3) dpm/mg liver DNA) compared to MCT-treated animals (18.1+/-5.7 x 10(3) dpm/mg liver DNA). Liver protein synthesis was non-significantly elevated both 24 and 72 h after surgery in MCT+LCT-supplemented rats (13.7+/-1.1 and 10.9+/-3.1 x 10(3) dpm/mg liver protein). Seventy-two hours after partial hepatectomy, the hepatocyte mitotic activity was significantly increased in MCT+LCT- supplemented group vs. LCT- or MCT-fed rats (3.3+/-0.7 vs. 1.9+/-0.7 or 1.0+/-0.6 mitoses per 1000 hepatocytes), thus exhibiting an increased proliferative potential. The results showed a qualitative difference according to the proportion of MCT to LCT in the enteral supplements. Overfeeding with MCT decreased body weight, increased liver weight by its fatty infiltration, increased rat mortality rate and reduced spontaneous caloric intake. We conclude that the balanced supplement of MCT+LCT (40%:60% w:w) preserves liver regeneration, whereas overfeeding with MCT seems to be deleterious.     

Serum hormone levels following partial hepatectomy in the rat.

Serum levels of insulin, glucagon, growth hormone (somatotrophin) and thyroxine (TT₄) were measured by radioimmunoassay following both sham operation and 70% partial hepatectomy in the rat to evaluate changes in hormone levels during liver regeneration. An eleven fold increase in glucagon was observed (from 112 ± 10 pg/ml to 1500 ± 200 pg/ml) 6 hours following partial hepatectomy but not sham operation. In contrast, insulin levels remained unchanged compared to sham controls for up to 72 hr while growth hormone fell to low levels, 6 to 48 hr after partial hepatectomy. Both total thyroxine and free thyroxine levels also fell 24–72 hours after hepatectomy. These studies suggest that growth hormone, thyroxine and insulin are not primary stimulants of hepatic regeneration although the data suggests that glucagon may modify this growth process.     

Marked induction of hepatocyte growth factor mRNA in intact kidney and spleen in response to injury of distant organs.

Hepatocyte growth factor (HGF) is a potent mitogen for various epithelial cells, including mature hepatocytes and renal tubular cells. Here, HGF mRNA was found to be markedly increased in non-injured kidney and spleen, when the liver or kidney in rats was injured by 70% partial hepatectomy or unilateral nephrectomy. HGF mRNA increased to 3-4 fold higher level than the normal in the kidney and spleen as well as in the remnant liver after partial hepatectomy. Similarly, HGF mRNA markedly increased in the spleen as well as in the remnant kidney after unilateral nephrectomy. These results suggest that the onset of injury to the liver or kidney may be recognized by distal non-injured organs by the signalling of a humoral factor and that HGF derived from these organs may be involved in the regeneration of liver or kidney, through an endocrine mechanism.     

Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.     

Dehydroepiandrosterone inhibits DNA synthesis of rat hepatocytes induced by partial hepatectomy or mitogen (ciprofibrate)

In a previous study we have shown that dehydroepiandrosterone (DHEA) inhibits hepatocyte DNA synthesis after short-term administration and induces hepatocellular carcinomas after long-term administration in the rat. It is not known whether DHEA is also capable of inhibiting replicative and mitogen-induced DNA synthesis. In the present study, we have evaluated the effect of DHEA on DNA synthesis in the rat liver after partial hepatectomy and mitogen administration. After partial hepatectomy, DHEA significantly inhibited DNA synthesis at 20, 26, 32 and 38 h. Similarly, combined administration of ciprofibrate, a peroxisome proliferator and mitogen, and DHEA also resulted in significant hepatocyte DNA synthesis. However, DHEA did not affect liver enlargement caused by ciprofibrate. This experimental system will serve as useful tool to evaluate the role of cell proliferation in carcinogenesis.     

Intravenous injection of an adenovirus encoding hepatocyte growth factor results in liver growth and has a protective effect against apoptosis

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine with mitogenic, motogenic and morphogenic effects for a wide variety of cells. Previous studies have reported that the in vivo infusion in normal, untreated mice of recombinant HGF results in low levels of DNA synthesis and liver proliferation. In this study, we examined whether liver regeneration could be obtained by the in vivo injection of a recombinant adenoviral vector encoding human HGF (Ad.CMV.rhHGF) in normal, intact mice. MATERIALS AND METHODS: C57BL/6 mice were infused intravenously with doses increasing from 1 to 4 x 1011 particles of the recombinant human HGF (rhHGF) adenoviral vector or with a control virus encoding Escherichia coli beta-galactosidase (Ad.CMV.lacZ). At day 5, mice were sacrificed and evaluated for the presence of hepatocyte mitogenesis and liver regeneration (5-bromo-2''-deoxyuridine (BrdU) assays and liver weight determination) and for the presence of liver damage (serum alanine amino-transferase (ALT) measurements and TUNEL assays). RESULTS: In vivo administration of rhHGF stimulated DNA synthesis of hepatocytes and liver weight in a dose-dependent fashion. The maximal effect was seen after the infusion of 3 x 1011 particles which resulted at day 5 in >130% increase in relative liver mass with little cytopathic effect. In contrast, administration of the lacZ adenoviral vector caused little hepatocyte replication, but induced high levels of serum ALT (approximately 3 times higher than the rhHGF vector) and significant apoptotic cell death. CONCLUSIONS: This study shows that a single injection of Ad.CMV.rhHGF alone is able to induce in vivo and in a very short period of time, robust DNA synthesis and liver proliferation in normal mice without liver injury or partial hepatectomy. This recombinant adenoviral vector has a lower toxicity than the control lacZ adenovirus. This suggests that HGF may have a protective effect against adenovirus-induced pathology.     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

Effect of autonomic denervation on DNA synthesis during liver regeneration after partial hepatectomy

The regulatory role of autonomic nerves in liver regeneration after partial hepatectomy was studied in rats by bilateral subdiaphragmatic vagotomy or splanchnicectomy. 1. In control rats the wet weight of the regenerating liver was restored to approximately 80% of the preoperative weight 72 h after partial hepatectomy. Restoration of the liver weight was significantly impaired in vagotomy rats, but not in splanchnicectomy. Increases in the DNA and protein contents of the regenerating liver were also suppressed by vagotomy. 2. Hepatic DNA synthesis, measured as the incorporation of [methyl-3H]thymidine into DNA at various times after partial hepatectomy, was significantly less in vagotomized rats, and slightly more in splanchnicectomized rats than in control rats. The onset of DNA synthesis triggered by partial hepatectomy was also delayed by vagotomy. 3. The increases in activities of hepatic aspartate transcarbamoylase and thymidine kinase, the key enzymes in synthesis of pyrimidine nucleotides via the de novo and salvage pathways respectively, during liver regeneration, were significantly suppressed and retarded in vagotomized rats. Conversely, splanchnicectomy tended to stimulate these enzyme inductions after partial hepatectomy. 4. During starvation the plasma insulin level decreased after partial hapatectomy in control and vagotomized rats, as in sham-operated rats, but showed a transient increase 6 h after partial hepatectomy in splanchnicectomized rats. It is concluded that vagotomy inhibits and delays DNA synthesis and proliferation of liver cells after partial hepatectomy, whereas splanchnicectomy tends to stimulate these processes. The data also suggest that parasympathetic innervation of the liver may play an important regulatory role in liver regeneration.