Liver Growth Factor Research Articles

Decrease in fibrosis, and increase in number of hepatocytes, in Cl 4 C-cirrhotic rats after injection of the liver growth factor. LGF : [formula omitted]. Clínica Puerta de Hierro and Universidad Complutense, Madrid, Spain

IGF actions are modulated by a family of six proteins, the IGFBPs. This review discussed structural aspects of the proteins and their genes, emphasizing in particular the posttranslational modifications—glycosylation, phosphorylation and proteolysis—which are believed to be important modifiers of IGFBP activity. The IGFBP genes all contain four coding exons, with only the IGFBP-3 gene having a fifth, non-coding exon. Consistent with a relatively conserved gene structure, the proteins themselves, all approximately 20–30 kDa in size, are highly conserved in primary structure, each consisting of three domains: cysteine-rich amino-terminal and carboxy-terminal domains with marked sequence similarity among all of the proteins, and central domains showing no conservation. Putative sites of N-linked glycosylation (three in IGFBP-3, and one in IGFBP-4 and -6) occur in this region, and appear occupied by carbohydrate in IGFBP-3 and -4, whereas IGFBP-1, -5, and -6 have varying amounts of O-linked carbohydrate. All of the IGFBPs bind both IGF-I and IGF-II, with IGFBP-6 having an outstanding preferential affinity for IGF-II. IGFBP-3, alone among the IGFBPs, binds to another ligand, the 85 kDa, leucine-rich acid-labile subunit, with which it forms a ternary complex, together with IGF-I or IGF-II, in the circulation.Certain of the IGFBPs can associate with cell surfaces or matrix, in the case of IGFBP-1 and -2, via an Arg-Gly-Asp motif which interacts with receptors of the integrin class, and in the case of IGFBP-3 and -5 via a highly basic motif in their carboxy-terminal domain. IGFBP-1, -3, and -5 can be serine-phosphorylated, resulting in changes in their cell-binding and/or IGF affinity. Finally, limited proteolysis by enzymes of a variety of classes can convert IGFBPs to stable forms of lower molecular weight with reduced IGF-binding affinity. These proteases are postulated to be of major importance in modulating the ways in which IGFBPs affect IGF activity in the cellular environment, and may also be important in determining the bioavailability of circulating IGFs.

Intracellular pH regulation in hep G2 cells: Effects of epidermal growth factor, transforming growth factor-α, and insulinlike growth factor-II on Na+/H+ exchange activity

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Intracellular pH regulation in hep G2 cells: Effects of epidermal growth factor, transforming growth factor-α, and insulinlike growth factor-II on [formula omitted] exchange activity

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na + H + exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na + H + exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-α (TGFα), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGFα, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na + removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH 4C1) by an amiloride inhibitable Na + H + exchange. In KRB, an additional, DIDS-inhibitable, Na + and HCO 3-dependent, but Cl --independent acid extruder (Na:HCO 3 cotransport) was activated. No evidence was found for a Cl/HCO 3 exchange acting as acid loader. Administration of EGF and TGFα, but not of IGF-II, induced a dosedependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na + H + exchange activity, shifting to the right the JH/pHi curve. Finally, 3Hthymidine incorporation in Hep G2 cells, in the presence of FCS or TGFα, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na + H + exchange, which activity can be stimulated by EGF and TGFα, but not by IGF-II. Administration of TGFα stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na + H + exchanger. These data suggest that Na + H + exchange activation may play a critical role in the growth of some hepatic tumors.

Ultrastructural location of human hepatocyte growth factor in human liver

Human hepatocyte growth factor has been purified from the plasma of patients with fulminant liver failure, but where this factor is produced in organs or cells of subjects with liver diseases is unknown. Therefore, we used a monoclonal antibody to human hepatocyte growth factor to stain cells in three normal and 29 diseased liver tissues by immunohistochemical techniques. By light microscopy, the immunostained cells seemed to be polymorphonuclear leukocytes because of their segmented nuclei. Some biliary epithelial cells also were stained. Electron microscopy confirmed that the immunostained cells with segmented nuclei were polymorphonuclear leukocytes and that the stained grains were on the membranes of rough endoplasmic reticulum, around specific or azurophilic granules and in the cell sap. Stained grains in the biliary epithelial cells were found sporadically on the inside and outside of the membranes of rough endoplasmic reticulum near the nuclei. Human hepatocyte growth factor is now known to be the same protein as scatter factor and tumor cytotoxic factor, both of which are produced by human fibroblasts in culture, but our results suggest that polymorphonuclear leukocytes in diseased livers are one cellular source of circulating human hepatocyte growth factor. The immunostaining properties of biliary epithelial cells in diseased livers also suggest that the cells produce and secrete human hepatocyte growth factor.

Ultrastructural location of human hepatocyte growth factor in human liver

Human hepatocyte growth factor has been purified from the plasma of patients with fulminant liver failure, but where this factor is produced in organs or cells of subjects with liver diseases is unknown. Therefore, we used a monoclonal antibody to human hepatocyte growth factor to stain cells in three normal and 29 diseased liver tissues by immunohistochemical techniques. By light microscopy, the immunostained cells seemed to be polymorphonuclear leukocytes because of their segmented nuclei. Some biliary epithelial cells also were stained. Electron microscopy confirmed that the immunostained cells with segmented nuclei were polymorphonuclear leukocytes and that the stained grains were on the membranes of rough endoplasmic reticulum, around specific or azurophilic granules and in the cell sap. Stained grains in the biliary epithelial cells were found sporadically on the inside and outside of the membranes of rough endoplasmic reticulum near the nuclei. Human hepatocyte growth factor is now known to be the same protein as scatter factor and tumor cytotoxic factor, both of which are produced by human fibroblasts in culture, but our results suggest that polymorphonuclear leukocytes in diseased livers are one cellular source of circulating human hepatocyte growth factor. The immunostaining properties of biliary epithelial cells in diseased livers also suggest that the cells produce and secrete human hepatocyte growth factor. (HEPATOLOGY 1994;19:1157–1163.)

In vivo response of hepatocytes to growth factors requires an initial priming stimulus

Although growth factor effects have been studied in cultured hepatocytes, little information exists as to whether these factors can trigger hepatocyte replication in vivo. In this study we infused epidermal growth factor, transforming growth factor-alpha and hepatocyte growth factor directly into the portal vein of rats for 24 hr to see whether they could induce DNA synthesis in normal livers or in livers subjected to one-third hepatectomy. Infusion of transforming growth factor-alpha or epidermal growth factor at doses up to 80 micrograms/24 hr had little effect on hepatic DNA synthesis in normal liver, whereas the monomeric and heterodimeric forms of hepatocyte growth factor generally produced increases of less than threefold in hepatic DNA synthesis. In contrast, after one-third hepatectomy infusion of epidermal growth factor, transforming growth factor-alpha or hepatocyte growth factor produced dose-dependent increases in hepatic DNA synthesis. At a dose of 40 micrograms/24 hr, epidermal growth factor increased DNA synthesis threefold, whereas transforming growth factor-alpha or hepatocyte growth factor increased DNA synthesis to greater than six times that in rats that had undergone hepatectomy alone. Furthermore, infusion of these growth factors, with or without one third-hepatectomy, induced the expression of transforming growth factor-alpha mRNA in the liver. The pattern of protooncogene expression induced by one-third hepatectomy was studied to determine the effect of this procedure in sensitizing the liver to the growth factors. Compared with the well-characterized two-thirds hepatectomy system, there was a similar but smaller increase in c-myc expression but no induction of c-jun expression.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo response of hepatocytes to growth factors requires an initial priming stimulus

Although growth factor effects have been studied in cultured hepatocytes, little information exists as to whether these factors can trigger hepatocyte replication in vivo. In this study we infused epidermal growth factor, transforming growth factor-α and hepatocyte growth factor directly into the portal vein of rats for 24 hr to see whether they could induce DNA synthesis in normal livers or in livers subjected to one-third hepatectomy. Infusion of transforming growth factor-α or epidermal growth factor at doses up to 80 μg/24 hr had little effect on hepatic DNA synthesis in normal liver, whereas the monomeric and heterodimeric forms of hepatocyte growth factor generally produced increases of less than threefold in hepatic DNA synthesis. In contrast, after one-third hepatectomy infusion of epidermal growth factor, transforming growth factor-α or hepatocyte growth factor produced dose-dependent increases in hepatic DNA synthesis. At a dose of 40 μg/24 hr, epidermal growth factor increased DNA synthesis threefold, whereas transforming growth factor-α or hepatocyte growth factor increased DNA synthesis to greater than six times that in rats that had undergone hepatectomy alone. Furthermore, infusion of these growth factors, with or without one third-hepatectomy, induced the expression of transforming growth factor-α mRNA in the liver. The pattern of protooncogene expression induced by one-third hepatectomy was studied to determine the effect of this procedure in sensitizing the liver to the growth factors. Compared with the well-characterized two-thirds hepatectomy system, there was a similar but smaller increase in c-myc expression but no induction of c-jun expression. The results suggest that a small functional deficit in the liver that by itself causes little DNA synthesis “primes” hepatocytes for replication and that the loss of liver mass and growth factor infusions complement each other by providing essential stimuli needed for DNA synthesis. (Hepatology 1994;14:489–497).

Epidermal growth factor and transferrin receptors expression in human fetal liver

Epidermal growth factor and transferrin receptors expression in human fetal liver

Detection of hepatic rejection by monitoring of the liver growth factor, h-LGF, in liver transplant recipients

Detection of hepatic rejection by monitoring of the liver growth factor, h-LGF, in liver transplant recipients

Improvement of DMN-cirrhotic rats after injection of the Liver Growth Factor

Improvement of DMN-cirrhotic rats after injection of the Liver Growth Factor

In the liver growth factor the earliest sign of hepatic regeneration?

In the liver growth factor the earliest sign of hepatic regeneration?

Cell-specific expression of hepatocyte growth factor in liver. Upregulation in sinusoidal endothelial cells after carbon tetrachloride.

The cellular origin of hepatocyte growth factor (HGF), a polypeptide implicated in liver regeneration, was examined in normal liver and in hepatic regeneration induced by carbon tetrachloride. In normal liver, HGF and its mRNA were abundant in lipocytes, with smaller amounts present also in sinusoidal endothelial and Kupffer cells. In regenerating liver, HGF gene expression increased exclusively in endothelial cells. HGF mRNA levels rose sixfold in these cells, peaking at 6 h after toxin administration and returning to near normal by 24 h. The rise in HGF mRNA was accompanied by a 5.4-fold increase in HGF secretion. CCl4 did not alter HGF expression by either Kupffer cells or lipocytes; nor did it induce HGF expression by hepatocytes. Nonparenchymal liver cells contained two HGF transcripts: one predicting a full-length molecule of 728 amino acids; and the other encoding a functional five-amino acid deletion variant of HGF. The variant was less abundant than the full-length transcript, but increased in parallel with native HGF mRNA in response to CCl4. The response of nonparenchymal cells to HGF was examined by plating endothelial cells and lipocytes in the presence of recombinant human HGF. Under the conditions examined, the growth factor exerted neither mitogenic nor scatter factor activity on these cells.

Administration of hepatic stimulatory substance alone or with other liver growth factors does not ameliorate acetaminophen-induced liver failure

Sixty-two beagle dogs were given three doses of acetaminophen over a period of 24 hr in a fulminant liver failure model that is 70% lethal in 72 hr. Treatment of the animals with hepatic stimulatory substance alone or in a mixture with insulin, transforming growth factor-α and insulin-like growth factor II had no effect on mortality. Evidence of maximum regeneration with a mitotic index 20 to 25 times resting was the same in treated and untreated animals. Similarly, the biochemical and hematological indexes of liver injury were unaffected by therapy. These studies illustrate the futility of treating fulminant liver failure with exogenous growth factors that apparently are already present in large amounts in the natural response to liver injury. The results suggest that on-going liver injury by mechanisms other than lack of growth factors is the central problem of fulminant liver failure. If so, provision of regeneration-stimulating substance is an inappropriate therapeutic strategy. (HEPATOLOGY 1993;17:429–433.)

Administration of hepatic stimulatory substance alone or with other liver growth factors does not ameliorate acetaminophen-induced liver failure

Sixty-two beagle dogs were given three doses of acetaminophen over a period of 24 hr in a fulminant liver failure model that is 70% lethal in 72 hr. Treatment of the animals with hepatic stimulatory substance alone or in a mixture with insulin, transforming growth factor-alpha and insulin-like growth factor II had no effect on mortality. Evidence of maximum regeneration with a mitotic index 20 to 25 times resting was the same in treated and untreated animals. Similarly, the biochemical and hematological indexes of liver injury were unaffected by therapy. These studies illustrate the futility of treating fulminant liver failure with exogenous growth factors that apparently are already present in large amounts in the natural response to liver injury. The results suggest that on-going liver injury by mechanisms other than lack of growth factors is the central problem of fulminant liver failure. If so, provision of regeneration-stimulating substance is an inappropriate therapeutic strategy.

Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers

Immunolocalization of desmin in fetal rat livers shows that on day 12 of gestation a high number of liver cells express desmin. This number decreases from day 14 onward. On day 20 about the same density of desmin-containing cells is found in fetal rat livers as is found in adult rat livers. Desmin-containing cells show two types of labeling patterns, especially on days 12 and 13 of gestation: (a) a basketlike network of intermediate filaments throughout the whole cell, similar to the labeling pattern of cytokeratin in hepatocytes; and (b) more strongly labeled intermediate filaments developing long and slender processes between adjacent cells, close to the labeling pattern of Ito cells in adult rats. From day 14 of gestation, the first type becomes rare, and from day 18 only the second type remains. Double-labeling experiments show that coexpression of desmin and cytokeratin is found in cells of the first type on days 12, 13 and 14 of gestation. Cells containing desmin with the labeling pattern of the second type never express cytokeratin. Coexpression of vimentin and cytokeratin is never found in fetal hepatocytes, even on day 12 of gestation. Numerous nonhepatocyte cells coexpress desmin and vimentin, but some cells contain vimentin or desmin alone. In desmincontaining cells the labeling pattern is of the first type (basketlike network). These results suggest that in early stages of fetal liver development, desmin is found in two different types of liver cells. Cells of the first type contain cytokeratin and desmin, and those of the second type contain desmin and vimentin, as Ito cells in adult liver tissue. Cells of the first type might represent a particular type of fetal hepatocyte. The existence of these two types of desmin immunolocalization is in convergence with electron microscopic data showing numerous lipid droplets both in fetal hepatocytes and in perisinusoidal cells. It is generally admitted that fetal liver differentiation is the result of the close association between endodermal and mesenchymal cells. Among the latter, desmin-containing cells (fetal Ito cells) might play a crucial role, especially because they are known to contain vitamin A and to express a hepatocyte growth factor in the normal adult liver. (HEPATOLOGY 1993;17:293–300.)

Appearance of exogenous epidermal growth factor in liver, bile, and intestinal lumen of suckling rats

This study was performed to investigate the distribution and the degradation of IV administered [125I]rat epidermal growth factor (rEGF) in the liver and gastrointestinal tract of suckling rats. The bile duct of anesthetized rats was cannulated, and [125I]rEGF was injected (with or without 2500-fold excess unlabeled rEGF) into the femoral vein. After 5, 30, 60, and 120 minutes, the radioactivity in the liver, stomach, small intestine, blood, kidney, bile, and luminal contents of the stomach and small intestine was measured. The extracted radioactivity was then analyzed by immunoaffinity chromatography and binding to EGF-specific receptors. High levels of radioactivity were found in the liver (57% of total administered) and small intestine (10%) at 5 minutes, which gradually decreased. On the contrary, radioactivity secreted in the bile and luminal contents of the small intestine increased with time. The radioactivity in the bile represented 2.4% and 4.5% of the total administered at 60 and 120 minutes, respectively. During the first 60 minutes, more than 90% of the radioactivity in the liver, small intestine, bile, and intestinal contents was immunoreactive. Thirty-four to seventy percent of the radioactivity in the bile and liver and 20%–41% of radioactivity in the small intestinal wall and contents were capable of binding to EGF-specific receptors. Radioactivity detected in the liver, bile, small intestine, and intestinal contents was profoundly reduced by the coinjection excess of unlabeled EGF. These studies show that IV administered [125I]rEGF is rapidly taken up by the liver and the gastrointestinal tract and secreted into the bile and intestinal luminal contents of suckling rats in form(s) capable of binding to anti-EGF antibody and EGF-specific receptors. The uptake and secretion by the liver and the small intestine appear to be receptor mediated.

Stimulation of putrescine production by epidermal growth factor in rat liver after partial hepatectomy

When epidermal growth factor was given to rats after partial hepatectomy, hepatic putrescine content was significantly increased at 4, 6 and 10 hr compared with control rats. Ornithine decarboxylase activity was also increased. Hepatic ornithine decarboxylase messenger RNA content was significantly greater than control levels at 2 hr after epidermal growth factor treatment, but not at 10 hr, when the amount of ornithine decarboxylase messenger RNA in control animals was four times that at 2 hr. When actinomycin D was administered 6 hr after partial hepatectomy, hepatic ornithine decarboxylase activity at 10 hr was reduced to half the control levels. This reduction was attenuated by epidermal growth factor treatment at 6 and 8 hr. Hepatic immunoreactive ornithine decarboxylase protein content showed a highly positive correlation with hepatic ornithine decarboxylase activity at 4, 6 and 10 hr, irrespective of epidermal growth factor treatment. Hepatic spermidine N1-acetyltransferase activity was significantly increased at 6 hr compared with control rats. These results suggest that, after partial hepatectomy in rats, exogenous epidermal growth factor may stimulate hepatic putrescine production by increasing ornithine decarboxylase messenger RNA content and altering posttranscriptional ornithine decarboxylase regulation, as well as enhancing spermidine N1-acetyltransferase activity.

Human hepatocyte growth factor in blood of patients with fulminant hepatic failure

Human hepatocyte growth factor (hHGF) was purified from the plasma of six patients with fulminant hepatic failure due to hepatitis B in two and non-A, non-B hepatitis in four. The purified hHGF from each patient contained two major protein bands having molecular weights of 79,000 and 86,000 and several minor bands having molecular weights between 76,000 and 92,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreduced conditions. After reduction with 2-mercaptoethanol, three major bands having molecular weights of 58,000, 34,500, and 31,500 were evident. In addition, a band having a molecular weight of 21,000 was detected. hHGF activity was destroyed by its reduction. The hHGF purified from patients demonstrated a dose response in terms of an increase in DNA synthesis using cultured hepatocytes. The hHGF concentration in the plasma of the patients with grade III-IV hepatic coma was calculated to be in the range of 1.8-3.0 nM. Finally the heavy chain of hHGF was not recognized by an anti-human albumin antibody, indicating that hHGF is not biliprotein, an albumin-bilirubin complex, that has been reported to be a putative liver growth factor.

Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis

Specific binding of 125I-labeled human recombinant HGF to the primary cultured rat hepatocytes or liver plasma membranes was observed to be temperature- and time-dependent. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 24-32 pM, a value in good accord with half maximum dose for HGF activity and a receptor density of about 500-600 sites/cell. Affinity cross-linking of the receptor with 125I-HGF revealed the HGF receptor in rat liver membranes to be a polypeptide of Mr approximately 220,000. After partial hepatectomy, specific binding of 125I-HGF to the membranes of residual livers decreased by 60-70% between 3 and 6 h, and was scanty at 12 h after hepatectomy. After one week, the binding was recovered to the 1.7 fold level in the untreated rat liver. This rapid down-regulation of HGF receptors was also observed in plasma membranes of rat livers in the presence of hepatitis induced by CCl4. We propose that HGF which can be immediately supplied to the liver after hepatic injury will function as a trigger for regeneration of this organ.

Liver contains heparin-binding growth factors as the major growth factor for cultured fibroblasts

The presence of heparin-binding growth factors in liver was investigated by measuring the DNA synthesis stimulatory activity of liver extracts using quiescent fibroblasts as target cells. It was found that cytosolic fractions of mouse, rat and human liver, as well as isolated rat hepatocytes, contain a large amount of growth stimulatory activity. Most liver cytosolic activity is due to heparin-binding growth factors, because > 90% of the activity bound to a heparin affinity column in the presence of 0.8 mol/L NaCl, and was quantitatively eluted with 2 mol/L NaCl. Purification of these factors from both mouse and rat liver indicated the presence of both heparin-binding growth factor-1 and 2 in liver extracts. The level of the heparin-binding growth factors, as estimated from the biological activity, is approximately 1 μg/gm mouse liver and 0.1 μg/gm rat and human liver. Heparin-binding growth factor-1–like factors were 10 times as abundant as heparin-binding growth factor-2–like factors. These data indicate that the cytosolic fractions of mouse, rat and human liver contain heparin-binding growth factors as the primary growth factor for fibroblasts, and heparin binding growth factor-1–like molecules account for most of the cytosolic activity in both mouse and rat liver. (HEPATOLOGY 1991;13:6–14).