Expressions of molecules associated with hepatocyte growth factor activation after hepatectomy in liver cirrhosis

BACKGROUND/AIMS: The activation pathway of hepatocyte growth factor (HGF), including HGF activator (HGFA) and HGFA inhibitor-1, 2 (HAI-1, 2), has recently been clarified. The present study examined mRNA expressions of HGF, HGFA and HAI-1 following partial hepatectomy in normal and cirrhotic rats. METHODOLOGY: Liver cirrhosis was induced by intraperitoneal injections of dimethylnitrosamine. Two weeks after, the cirrhotic and normal rats underwent 70% hepatectomy and the liver regeneration rate, DNA synthesis of hepatocytes, plasma HGF level, and mRNA expressions of HGF, HGFA, and HAI-1 in the liver, spleen, and lung were examined at different times. RESULTS: Liver regeneration in the cirrhotic rats was deteriorated with a later peak of hepatocellular DNA synthesis. Hepatic HGF mRNA and splenic HAI-1 mRNA were upregulated and liver HGFA mRNA was downregulated in the cirrhotic rats. CONCLUSIONS: Insufficient HGF activation both by a reduced expression of hepatic HGFA and an increased expression of splenic HAI-1 may be one of the reasons for the impaired liver regeneration in cirrhosis.     

Effects of cycloheximide on hepatic and uterine insulin-like growth factor-I mRNA

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.     

Hepatic regeneration in insulin-like growth factor binding protein-1 transgenic mice

BACKGROUND/AIMS: Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1. METHODS: Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice. RESULTS: Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice. CONCLUSIONS: Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.     

Testosterone effect on growth and growth mediators of the GH-IGF-I axis in the liver and epiphyseal growth plate of juvenile rats.

Several studies have suggested that testosterone may have a direct, GH-independent effect on growth. In order to assess possible mechanism(s) whereby testosterone exerts its growth-promoting effect, we evaluated its effect on growth mediators of the GH-IGF-I axis, in both the liver and the epiphyseal growth plate (EGP). Testosterone was administered to peripubertal rats and the responses of mRNA of GH receptor, IGF-I, IGF-I receptor and IGF-binding proteins-1 and -3 (IGFBP-1 and IGFBP-3) as well as circulating IGF-I were evaluated in two time-related models: over 12 h after a single injection (short-term study) and 10 days after continuous administration (long-term study). Rats in the short-term study were castrated and were killed 1, 4, 6 and 12 h post injection. Rats in the long-term study were divided into two groups: castrated vs castrated and hypophysectomized, in order to assess the effect of testosterone in the presence and absence of GH. mRNA levels were determined by RNase protection assay, and serum IGF-I by RIA. Testosterone enhanced weight gain in the rats treated for 10 days, a change that was similar in the presence or absence of GH. This effect was relatively small, however, by comparison with the total weight gained without testosterone. Testosterone had no effect on hepatic IGF-I mRNA abundance but induced a reduction in circulating IGF-I levels, in both the short- and long-term study. Testosterone had no effect on hepatic GH receptor and IGFBP-3 mRNA levels but resulted in a transient, short-term elevation in IGFBP-1 mRNA levels that was maximal 4 h post injection.In the EGP, neither testosterone administration nor hypophysectomy had any effect on IGF-I and IGF-I receptor mRNA levels. However, testosterone increased GH receptor mRNA abundance after 10 days of continuous administration in hypophysectomized rats only.These data suggest that the effect of testosterone on growth (as assessed by weight gain) is small and is not mediated by changes in hepatic gene expression of IGF-I, IGF-I receptor, IGFBP-1, IGFBP-3 or circulating IGF-I. At the EGP, the testosterone effect on linear growth is not mediated through changes in mRNA abundance of IGF-I and IGF-I receptor. The small but significant elevation of GH receptor mRNA levels in hypophysectomized rats may suggest a testosterone-mediated augmentation of a GH effect at the target organ.     

Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat

Thyroid hormones influence growth in part by altering the secretion and effects of GH. GH, in turn, mediates its effects by regulating the synthesis and secretion of insulin-like growth factor-I (IGF-I). IGF-I is a pleiotropic growth factor that is synthesized by many tissues and acts on many tissues to regulate both cellular replication and differentiated function. We have studied the direct effects of thyroid hormones and the combined effects of thyroid hormones and GH on the regulation of IGF-I synthesis and secretion in hypophysectomized (hypox) rats in vivo. All rats, except normal littermates and a hypox control group, received 100 micrograms hydrocortisone/100 g BW for 10 days. Circulating IGF-I was measured by specific RIA (normal rats, 1 U/ml), and hepatic IGF-I mRNA was measured by Northern blot hybridization with an antisense cRNA probe. 1) Hypox rats treated with hGH (75 micrograms, ip, twice daily) for 10 days gained 17 g BW vs. 70 g for normal littermates. GH markedly increased hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 U/ml 12 h after the last GH injection vs. 0.03 +/- 0.02 for hypox controls). 2) T4 (1 micrograms/100 g BW, ip) for 10 days increased neither weight, hepatic IGF-I mRNA, nor circulating IGF-I. 3) Rats treated with T4 for 10 days followed by a single injection of 1 mg GH, ip, increased hepatic IGF-I mRNA and circulating IGF-I levels comparably as in rats receiving acute GH alone (IGF-I, 12 h, 0.31 +/- 0.09 vs. 0.36 +/- 0.06 U/ml). 4) Hypox rats treated with a single injection of T3 (1.5 micrograms/100 g BW, ip) had slightly increased hepatic IGF-I mRNA, but showed no significant change in circulating IGF-I levels. 5) A single injection of T3 plus GH to hypox rats increased IGF-I mRNA levels above those in rats injected with GH alone and increased serum IGF-I levels to 0.48 +/- 0.12 U/ml compared to 0.36 +/- 0.06 U/ml for GH alone. 6) After 10 days of GH treatment, a single injection of T3 lowered both hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 to 0.16 +/- 0.06 U/ml, 6 h after T3). These studies demonstrate that thyroid hormones have relatively little direct effect on IGF-I synthesis but can have major effects on GH-stimulated IGF-I synthesis and secretion. The pattern of these effects depends on the integrity of the pituitary gland, prior exposure of the liver to GH and/or thyroid hormones, and the temporal relationship between GH and thyroid hormone administration.     

Serum response of hepatocyte growth factor, insulin-like growth factor-I, interleukin-6, and acute phase proteins in patients with colorectal liver metastases treated with partial hepatectomy or cryosurgery

BACKGROUND/AIMS: The aim of the study was to compare the serum response of regeneration factors and acute phase proteins in patients treated with partial hepatectomy or cryosurgery. METHODS: The responses of serum hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I) (free and total), interleukin-6 (IL-6) and the acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) were examined in patients with colorectal liver metastases treated with partial hepatectomy (n = 14) or cryosurgery (n = 10). RESULTS: In both groups, IL-6 peak levels at the end of the operation were followed by peak levels at day 1 for HGF and CRP. SAA peak levels occurred on day 1 (hepatectomy group) and on day 4 (cryo group). The total HGF, IGF-I, and IL-6 responses were comparable in both groups. CRP and SAA responses were higher in the patients treated with cryosurgery than in patients after hepatectomy. Free IGF-I trough levels were lower in partial hepatectomy patients than in cryosurgery patients. CONCLUSIONS: In patients with colorectal liver metastases the responses of the regenerating factors HGF, IGF-I, and IL-6 are comparable to those in patients treated with partial hepatectomy. Upregulation of acute phase protein production is higher in patients after cryosurgery than in patients after partial hepatectomy.     

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.     

Insulin-like growth factor-II/mannose-6-phosphate receptors are increased in hepatocytes from regenerating rat liver

Insulin-like growth factor-II (IGF-II) receptor levels were determined in hepatocytes from sham-operated and two thirds hepatectomized rats. [125I]IGF-II binding to confluent cultures increased 2-fold in cells isolated 24 and 48 h after hepatectomy compared to that in cells from sham-operated rats. Receptor levels increased from 1.74 +/- 0.14 x 10(4)/cell (sham-operated) to 3.60 +/- 0.31 x 10(4)/cell (hepatectomized; P = 0.002), with no change in affinity of IGF-II binding (Ka = 6.4 x 10(9) M-1). As previously reported, receptor levels increased in cells plated at low density, but this effect was decreased in cells from hepatectomized rats (80% increase) compared to that in cells from control rats (300% increase). Serum IGF-I levels decreased 50% 24 h after partial hepatectomy (P less than 0.001), but returned to normal levels by 48 h. However, IGF-I synthesis was not decreased in hepatocytes isolated 24 h after partial hepatectomy, suggesting that decreased serum levels are due to decreased liver mass. Circulating IGF-II levels were not altered by partial hepatectomy, and IGF-II production was not detected in hepatocytes from sham-operated or hepatectomized rats. Transforming growth factor-beta is thought to terminate hepatocyte proliferation upon complete liver regeneration. In hepatocytes from sham-operated or hepatectomized rats transforming growth factor-beta totally blocked DNA synthesis, but had no effect on elevated IGF-II receptor levels after partial hepatectomy. Concomitant with increased IGF-II receptor levels, hepatocytes from hepatectomized rats were more sensitive to IGF-II stimulation of DNA synthesis. [3H]Thymidine incorporation in the presence of epidermal growth factor (50 ng/ml) was stimulated 66% by IGF-II (300 ng/ml) in control cells compared with 220% in cells from hepatectomized animals. These results suggest that IGF-II and the IGF-II receptor may play a role in liver regeneration.     

Role of growth hormone (GH) in liver regeneration

Liver regeneration is a fundamental mechanism by which the liver responds to injury. This process is regulated by endogenous growth factors and cytokines, and it involves proliferation of all mature cells that exist within the intact organ. To understand the role of the GH/IGF-I axis in liver regeneration, we performed partial hepatectomies in three groups of mice: GH antagonist (GHa) transgenic mice, in which the action of GH is blocked; liver IGF-I-deficient mice that lack IGF-I specifically in the liver and also lack the acid-labile subunit (ALS; LID+ALSKO mice), in which IGF-I levels are very low and GH secretion is increased; and control mice. Interestingly, the survival rate of GHa transgenic mice was dramatically reduced after partial hepatectomy (57%) compared with the survival rate of controls (100%) or LID+ALSKO mice (88%). In control mice, the liver was completely regenerated after 4 d, whereas liver regeneration required 7 d in LID+ALSKO mice. In contrast, in GHa mice, liver regeneration reached only 70% of the original liver mass after 4 d and did not improve thereafter. Strikingly, 36 and 48 h after hepatectomy, the livers of control and LID+ALSKO mice, respectively, exhibited intense 5-bromo-2'-deoxyuridine (BrdU) staining, whereas BrdU staining was dramatically decreased in the livers of GHa-treated mice. These results suggest that GH plays a critical role in liver regeneration, although whether it acts directly or indirectly remains to be determined.     

Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats.

AIMS/BACKGROUND: Liver regeneration is a physiological mechanism which leads to restoration of the hepatic parenchyma following hepatectomy or toxic injury. This process is mediated by a wide variety of cytokines and growth factors. The aim of the present study was to evaluate the influence of hepatectomy extent on the levels of intrahepatic mRNAs for cell-cycle markers and growth factors in rats submitted to a 30%, two-third or 80% hepatectomy. METHODS: Cyclins, thymidine kinase and growth factors mRNA levels were quantitatively assessed by RT-PCR at different time points post-hepatectomy (2h, 6h, 12h, days 1, 2, 6). RESULTS: As compared with a two-third hepatectomy, cyclins and thymidine kinase mRNA levels were increased but with a delayed peak at day 2 in the 80% hepatectomy group and showed a progressive increase until day 6 in the 30% hepatectomy group; mRNA levels for HGF or TGFalpha were increased with a delayed peak at 12 h or day 2 in the 80% hepatectomy group, respectively and this delay was more pronounced in the 30% hepatectomy group with a peak at day 1 or day 6. CONCLUSION: A regenerative response occurs whatever the extent of hepatectomy but the course of regeneration and expression of growth factors differs according to the volume of resected liver. A better knowledge of these events could improve the clinical results of hepatic resection for primary or metastatic liver disease.     

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.     

Identification and characterization of "injurin," an inducer of expression of the gene for hepatocyte growth factor.

The marked and rapid increase of hepatocyte growth factor (HGF) mRNA in the intact lung of rats after partial hepatectomy or unilateral nephrectomy suggests the existence of a humoral factor mediating a signal of injury to distal organs and may induce the expression of HGF gene in these organs. We have now identified a proteinous factor in the sera of rats with injury of liver or kidney that increases HGF mRNA in the intact lung. When the serum of rats with liver insult caused by partial hepatectomy or ischemic treatment was injected i.p. into normal noninjured rats, it induced a marked HGF mRNA expression in the lung of the recipient rats. The addition of serum from rats with various hepatic or renal injuries to MRC-5 human embryonic lung fibroblasts in culture also led to the induction of HGF mRNA expression, so that the production of HGF by MRC-5 cells after treatment with the sera was remarkably increased in the culture medium. However, serum from the normal intact rat induced no HGF production and no HGF mRNA in the lung in vivo and lung fibroblasts in vitro. This factor, which increases HGF production, was purified greater than 200-fold from sera of CCl4-treated rats. The factor proved to be an acid- and heat-stable protein with an apparent molecular mass of 10-20 kDa in SDS/PAGE. Its activity markedly increased within 3-6 hr in the plasma of rats after various treatments that injured the liver or kidney. These results suggest that the factor specifically appears in the blood of rats with organ injury and may be involved in organ regeneration through the potential to increase the synthesis of HGF. Since the factor seems to mediate various organ injuries, we named it "injurin."     

Heparin accelerates liver regeneration following portal branch ligation in normal and cirrhotic rats with increased plasma hepatocyte growth factor levels

BACKGROUND/AIMS: Heparin is widely used as a general anticoagulant, and has been recently reported to elevate plasma hepatocyte growth factor (HGF) levels by releasing HGF sequestrated in the extracellular matrix. Therefore, we investigated the effects of heparin administration on liver regeneration following portal branch ligation (PBL) in normal and cirrhotic rats. METHODS: Dimethylnitrosamine-induced cirrhotic rats and control rats underwent portal ligation of the left lateral and median branches, followed by intraperitoneal heparin injections, every 12 h. To examine the feasibility of an extensive hepatectomy in the cirrhotic livers, cirrhotic rats with or without heparin treatment underwent resection of occluded lobes at 72 h after the PBL. RESULTS: Heparin injections significantly augmented liver regeneration after PBL in both normal and cirrhotic rats, following an increase in hepatocellular DNA synthesis at 24 h after the PBL. The plasma HGF concentrations were elevated by heparin treatment in both groups. In addition, heparin administration dramatically improved the survival rate after an extensive hepatectomy in the cirrhotic rats. CONCLUSIONS: Heparin treatment significantly accelerated liver regeneration following the PBL, with an increase in the plasma HGF levels in both normal and cirrhotic rats. Heparin administration may make an extensive hepatectomy clinically feasible even for cirrhotic livers.     

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.