Sparse distribution of hepatocyte growth factor-producing cells inside hepatocellular foci in rats treated with hepatocarcinogens

The distribution of hepatocyte growth factor (HGF)-synthesizing cells in rat liver during development of glutathione S-transferase P form (GST-P)-positive nodules after diethylnitrosamine initiation followed by promotion with 2-acetylaminofluorene plus partial hepatectomy (PH) was investigated using in situ hybridization. HGF-producing cells were non-parenchymal in nature, and were suspected to be mainly of Kupffer type. They were mostly located outside GST-P-positive lesions, in the surrounding parenchyma. In the oval cell proliferation phase 1 week after PH, they increased and they were mainly localized around the portal triads. It is concluded that HGF is directly involved in an endogenous paracrine growth pathway controlling proliferation in oval cells and in normal, but not GST-P-positive, hepatocytes.     

Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.     

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

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

FK506 promotes liver regeneration by suppressing natural killer cell activity

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

Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis

BACKGROUND: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro. METHODS: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgen-independent), and LNCaP (androgen-dependent). We studied the expression of the HGF receptor c-met proto-oncogene (c-met) by Western blot analysis, and also determined the effects of HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay. RESULTS: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells. CONCLUSION: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Regeneration of transplanted human liver: gene expression at the time of graft implantation

The liver tissue mRNA expression of protooncogenes c-fos, c-myc, c-Ha-ras, c-met and c-erb B1, and TGF alpha and TGF beta genes is sequentially and temporarily increased in the early stages after partial hepatectomy, ischaemia or other mitogenic stimuli. These gene expressions were studied in 38 samples of liver tissue from 24 patients who underwent orthotopic liver transplantation, at different evolution stages. Eleven samples were obtained from surgical liver biopsies before graft implantation at day 0 (Group A), 14 samples from percutaneous liver biopsies during the post-operative period from day 9 to day 48 (Group B) and 13 samples in the long-term follow-up period from day 102 to day 1,382 (Group C). Gene expression was studied using 32P-labeled cDNA and oligonucleotidic probe hybridization in slot blots. A GAPD gene was used as a control gene. All expression values of protooncogenes were related to those of the GAPD gene. After cold ischaema, the relative gene expression (quantity of specific mRNA/quantity of GAPD mRNA ratio) tended to diminish in most cases. The relative expressions of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha gene were correlated at day 0. During and after the liver transplantation, an overexpression of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha was observed in different pathological conditions such as cold ischaemia and conservation injury, acute rejection, and cytomegalovirus infection. In three cases the relative expression values of c-fos, c-myc and c-Ha-ras increased over long-term follow-up without any associated acute pathology. These results suggest the necessity of an intercellular mediation--by means of graft reperfusion--in induction of hepatocyte proliferation and regeneration of the transplanted liver.     

Internal AU-rich elements modulate activity of two competing 3'' splice sites in plant nuclei

Histopathological and immunohistochemical studies were carried out on D-galactosamine (GalNAc)-induced acute hepatitis in rats of the JCI: Wistar TgN (ARGHGEN) 1 Nts strain (Mini rats), in which expression of the growth hormone gene is suppressed by an antisense transgene. Hepatitis characterized by hepatocellular acidophilic necrosis with inflammatory cell infiltration was most prominent at 2 days after GalNAc (1000 mg/kg)-injection, when proliferation of Ito cells and deposition of fibronectin and laminin were found along the sinusoidal linings. At 72 hours after GalNAc-injection, Ito cell proliferation with deposition of laminin and fibronectin became more prominent, and marked proliferation of small epithelial cells was observed in the periportal area. At 7 days after GalNAc-injection, quite a number of alpha-smooth muscle actin-positive Ito cells, surrounded by abundant fibronectin, laminin and type IV collagen, were still observed in close juxtaposition to rapidly proliferating small epithelial cells. The small epithelial cells were found to be positive for both alpha-fetoprotein and cytokeratin 7 and were therefore considered to be so-called oval cells. The results suggest that there may be some relation between oval cell proliferation, Ito cell activation and extracellular matrix accumulation in GalNAc-induced acute hepatitis in Mini rats.     

Modulation of motility and proliferation of glioma cells by hepatocyte growth factor

Invasive proliferation is a critical biological characteristic of gliomas. We evaluated the activities of hepatocyte growth factor (HGF) on proliferation and motility of glioma cells, comparing them with the effects of other growth factors (EGF, bFGF, PDGF-BB, TGF-beta 1). Seven primary culture lines all expressed c-met and HGF mRNA, and secreted HGF. HGF stimulated 3H-thymidine uptake of every glioma cell line (30 to 70% upregulation). Boyden chamber assay and scattering assay revealed that HGF promoted cell motility with chemokinetic and strong chemotactic activities. Concentric circle assay showed that HGF promoted two-dimensional expansion (proliferation and motility) most strongly among the growth factors studied. Further, we analyzed 23 paraffin-embedded sections of surgically resected gliomas (7 grade II, 8 grade III, and 8 grade IV) by immunohistochemistry. Expression of HGF and Met increased with malignant progression of gliomas, suggesting that gliomas stimulated their invasive proliferation by autocrine HGF production. Neurons and vasculature were HGF-positive, and Met-positive glioma cells gathered around them. The data indicate that neurons and vasculature, which are the main tracks of glioma invasion, augment chemotactic invasion and proliferation of gliomas by paracrine HGF secretion. Clearly HGF plays a critical role in invasive proliferation of glioma cells and it is therefore a candidate target of therapeutic intervention.     

Effects of benfluorex metabolites on membrane fluidity and insulin-related processes

Hepatocyte growth factor (HGF) is a potent mitogen for the maturation of hepatocytes in vitro which plays a role in liver regeneration in vivo. In addition, transforming growth factor-beta 1 (TGF-beta 1) is also a potent regulator of liver regeneration. In attempting to clarify the mechanisms related to liver regeneration after partial hepatectomy, we investigated the expression of HGF and TGF-beta 1 in rats with liver cirrhosis (LC). A rat model of LC was prepared using carbon tetrachloride (CCl4). The expression of HGF mRNA in both the LC and control groups showed a similar time-course with the highest expression seen at 18h after a 70% hepatectomy. The expression of TGF-beta 1 mRNA peaked at 18h after partial hepatectomy in the LC group and at 48h in the control group. The 5-bromo-2'-deoxyuridine (BrdU) labeling index for the LC group at 24, 48, and 72 h after partial hepatectomy was 9.2%, 5.9%, and 1.8%, while for the control group it was 7.0%, 11.7%, and 6.8%, respectively. The BrdU labeling index in the LC group was thus suppressed earlier than that in the control group. We therefore postulate that regeneration of the remnant liver in the presence of LC accelerates immediately after partial hepatectomy, but the extent of regeneration is insufficient because of an early cessation due to an early expression of TGF-beta 1.     

Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates

Hepatocyte growth factor (HGF) decreases transforming growth factor beta1 (TGFbeta1) levels in the liver and attenuates hepatic fibrosis caused by dimethylnitrosamine in rats. In the liver, HGF is presumed to act predominantly on parenchymal cells, and TGFbeta1 is produced mainly by mesenchymal cells. In hepatic fibrosis, stellate cells play a central role with undergoing activation, which also occurs when the cells are cultured on plastic. Thus, we wondered if HGF could act directly on stellate cells. c-Met was detected in rat stellate cells activated by culture for 10 days, but not in the cells cultured for 3 days. Specific binding of HGF to the activated cells was determined, and Scatchard analysis indicated an apparent Kd of 1.5 nM. c-Met mRNA was detected in freshly isolated stellate cells from rats treated with carbon tetrachloride for 8 weeks, but not in those cells from normal rats. These results indicate that stellate cells express c-met when activated in vitro and in vivo. HGF enhanced TGFbeta1 production and DNA synthesis in the activated cells.     

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

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

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

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

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

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

In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer

We have studied the proliferation of cells in two models of chemical hepatocarcinogenesis. The cells were genetically labeled in vivo using retrovirally mediated transfer of the Escherichia coli beta-galactosidase marker gene coupled to a nuclear localization signal (nls-lacZ gene). In the first carcinogenic model, rats were fed a choline-deficient diet containing 2-acetylaminofluorene, and their livers were perfused with recombinant retrovirus at the onset of oval cell proliferation. The second model was based on the administration of diethylnitrosamine coupled with a partial hepatectomy and is thought to induce cancer with no involvement of oval cells. Analysis of beta-galactosidase expression in the liver at various times after gene transfer revealed the presence of large clusters of positive cells in both models. Moreover, the beta-galactosidase-positive cells displayed morphologic, antigenic, and enzymatic profiles consistent with a hepatocyte phenotype. Our results, therefore, provide evidence for a strikingly similar clonal proliferation of apparently normal hepatocytes during the course of 2-acetylaminofluorene- as well as diethylnitrosamine-induced liver carcinogenesis.     

Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes

Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.