Variant Of Hepatocyte Growth Factor Research Articles

Pathogenic variants in HGF give rise to childhood-to-late onset primary lymphoedema by loss of function.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

Dimer Interface in Natural Variant NK1 Is Dispensable for HGF-Dependent Met Receptor Activation.

NK1, a splicing variant of hepatocyte growth factor (HGF), binds to and activates Met receptor by forming an NK1 dimer and 2:2 complex with Met. Although the structural mechanism underlying Met activation by HGF remains incompletely resolved, it has been proposed that the NK1 dimer structure participates in this activation. We investigated the NK1 dimer interface’s role in Met activation by HGF. Because N127, V140, and K144 are closely involved in the head-to-tail NK1 dimer formation, mutant NK1 proteins with replacement of these residues by alanine were prepared. In Met tyrosine phosphorylation assays, N127-NK1, V140-NK1, and K144-NK1 showed 8.3%, 23.8%, and 52.2% activity, respectively, compared with wild-type NK1. Although wild-type NK1 promoted cell migration and scattering, N127-NK1, V140-NK1, and K144-NK1 hardly or marginally promoted them, indicating loss of activity of these mutant NK1 proteins to activate Met. In contrast, mutant HGFs (N127-HGF, V140-HGF, and K144-HGF) with the same amino acid replacements as in NK1 induced Met tyrosine phosphorylation and biological responses at levels comparable to those of wild-type HGF. These results indicate that the structural basis responsible for NK1-dependent Met dimer formation and activation differs from, or is at least distinguishable from, the structural basis responsible for HGF-dependent Met activation.

Antiproliferation activities of NK4 on multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy. The hepatocyte growth factor (HGF) has been demonstrated to promote MM cell growth. NK4, a splice variant of HGF in which the heavy chain consists of the N-terminal domain and the four kringle domains, is a specific antagonist of HGF that competes with HGF for tyrosine-protein kinase receptor binding. The current study aimed to examine the antiproliferative activity of NK4 on human MM cells and to investigate the underlying mechanism. The results indicated that NK4 suppressed proliferation and induced apoptosis in RPMI 8226 cells. In addition, NK4 altered the expression of cell cycle and apoptosis-associated proteins in RPMI 8226, including cyclin-dependent kinase 4, cyclin D1, cyclin-dependent kinase inhibitor 1B, apoptosis regulator Bcl-2, apoptosis regulator BAX, cleaved caspase-9 and caspase-3. Furthermore, NK4 inhibited the activation of the RAC-α serine/threonine-protein kinase (Akt)/serine/threonine-protein kinase mTOR (mTOR) signaling pathway and reduced the levels of phosphorylated (p)-Akt, p-mTOR, ribosomal protein S6 kinase beta-1 and eukaryotic initiation factor 4E binding protein 1 in RPMI 8226 cells. In conclusion, NK4 inhibited the proliferation of human MM RPMI 8226 cells, which may be attributed to the induction of apoptosis and the inhibition of the Akt/mTOR signaling pathway.

High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli.

Hepatocyte growth factor (HGF) is an effective anti-fibrotic factor because of its bioactivity in inhibiting fibrosis-related proteins in the development of hepatic fibrosis. However, high-level production of bioactive mature form HGF is difficult because of its complex structure. Here, we report a non-fusion protein expression system to obtain truncated variant of N-terminal hairpin and first kringle domains of HGF (tvNK1) in Escherichia coli to determine its anti-fibrotic effects on hepatic stellate cells (HSCs). Under the selected conditions of cultivation and isopropyl-β-D-1-thiogalactopyranoside induction, the expression level of tvNK1 accounted for approximately 65 % of the total cellular protein and 50 % of fusion protein in the supernatant of whole cell lysates. The recombinant protein could be purified in one step with Ni(2+)-affinity chromatograph. Finally, about 65 mg recombinant tvNK1 was obtained from 1 l fermentation culture with no <95 % purity. In vitro, the final purified tvNK1 was shown to inhibit the proliferation of HSCs and decrease the mRNA and protein expression levels of fibrosis-related COL1A1 and α-smooth muscle actin genes.

Regulatory expression of MMP-8/MMP-9 and inhibition of proliferation, migration and invasion in human lung cancer A549 cells in the presence of HGF variants

Hepatocyte growth factor (HGF), a potent cytokine of mesenchymal origin, exhibits polytrophic physiological responses, including proliferation, migration, and invasion, in a wide variety of cells. Although it is known that inhibition of the responses by HGF variants was via signal transducers and activators of the transcription pathway, the mechanisms of action of the variants involved in the production of matrix metalloproteinases (MMPs) were not clearly understood. Thus, recombinant HGF variants, NK1, NK2, NK3, and NK4 were topically applied to assays for proliferation, migration, invasion, and expression of MMPs in the human lung cancer cell line A549 and compared to that of control medium and a glutathione-s-transferase control. Results showed that these recombinant HGF variants significantly inhibited proliferation, migration, and invasion of A549 at >4 nM, downregulated expression of MMP-9, and upregulated expression of MMP-8. The study clearly suggests that binding of the HGF variants to the cell surface c-Met resulted in the downregulation of MMP-9, and upregulation of MMP-8 protein expressions might be key molecular signals against proliferation, migration, and invasion of A549 cells.

Targeted Disruption of Heparan Sulfate Interaction with Hepatocyte and Vascular Endothelial Growth Factors Blocks Normal and Oncogenic Signaling

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal–epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell–cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.

NK3 and NK4 of HGF enhance filamin production via STAT pathway, but not NK1 and NK2 in human breast cancer cells

The purpose of this study was to reveal the effects of hepatocyte growth factor (HGF) variants on human breast cancer cells and the differential signaling pathways of the variants in controlling cell proliferation and invasion. Four HGF variants (NK1, NK2, NK3, and NK4) were created by gene engineering, and the variant DNA fragments were cloned into pGEM-T for DNA sequencing and then transferred to a pTrcHis-A plasmid for expression. Recombinant proteins were purified from Escherichia coli, and a series of assays, including cell proliferation and invasion were carried out. Phosphorylated components in the HGF-c-Met and STAT (signal transducers and activators of transcription) pathways were detected by immunoprecipitation-Western blots. All the HGF variants inhibited the vigorous growth of the cancer cells differently and dose-dependently, but the effect of NK3 or NK4 was 7.5-fold higher than NK1 or NK2. In addition, the assays for the phosphorylation of the components in the HGF-c-Met pathway showed that NK3 and NK4 inhibited invasion via the STAT pathway, whereas NK1 and NK2 were via the HGF-c-Met pathway. The engineered HGF variants inhibited the proliferation of human breast cancer cells via different signaling pathways, NK1 and NK2 via the HGF-c-Met pathways, and NK3 and NK4 via the STAT pathway, the latter being a possible key route for the inhibition of cell invasion. All of the HGF variants have the potential to become pharmaceutical drugs in the treatment of human cancer.

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl4) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl4-induced liver fibrosis. NK2 transgenic mice and wild-type (WT) mice were injected intraperitoneally with CCl4 twice a week. The extent of hepatic fibrosis was evaluated by Azan-Mallory staining. Expression levels of mRNAs of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase-13 (MMP-13) were examined by real-time polymerase chain reaction. The protein levels of alpha-smooth muscle actin (alpha-SMA), c-Met and its phosphorylation were determined by Western blot analysis. Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl4 administration increased the expression levels of TGF-beta1 mRNA and alpha-SMA protein, and decreased the expression of MMP-13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c-Met protein expression in the liver was compatible with the degree of fibrosis. As for c-Met activation, no difference was found between NK2 and WT livers. Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl4-induced chronic liver injury.

A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist

Hepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.

Mesenchymal Stem Cells Over-expressing Hepatocyte Growth Factor Improve Small-for-size Liver Grafts Regeneration

Ischemia-reperfusion (I/R) associated with small-for-size liver transplantation (SFSLT) impairs liver graft regeneration. Mesenchymal stem cells (MSCs) have the capability, under specific conditions, of differentiating into hepatocytes. Hepatocyte growth factor (HGF) has potent anti-apoptotic and mitogenic effects on hepatocytes during liver injury, and has been utilized in many experimental and clinical applications. In this study, we implanted HGF-expressing MSCs into liver grafts via the portal vein, using a 30% small-for-size rat liver transplantation model. HGF, c-met expression, hepatic injury and liver regeneration were assessed after liver transplantation. Our study demonstrated that MSCs over-expressing HGF prevented liver failure and reduced mortality in rats after SFSLT. These animals also exhibited improved liver function and liver weight recovery during the early post-transplantation period. Using green fluorescent protein (GFP) gene as a marker, we demonstrated that the engrafted cells and their progeny incorporated into remnant livers and produced albumin. These findings suggest that MSCs genetically modified to over-express HGF and implanted in the liver graft, may offer a novel approach to promoting liver regeneration after small-for-size transplantations.

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

Instant Hepatic Differentiation of Human Embryonic Stem Cells Using Activin a and a Deleted Variant of HGF

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.

Differentiation of Human Embryonic Stem Cells to Hepatocytes Using Deleted Variant of HGF and Poly-amino-urethane-Coated Nonwoven Polytetrafluoroethylene Fabric

Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10(-7) M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 microg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.

Transplantation of Human Hepatocytes Cultured with Deleted Variant of Hepatocyte Growth Factor Prolongs the Survival of Mice with Acute Liver Failure

Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF. We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10-1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal). When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4alpha, and C/EBPalpha in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal. The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.

Hepatocyte growth factor prevents chronic allograft dysfunction in liver-transplanted rats.

Hepatocyte growth factor (HGF) is a growth factor with multiple biologic properties, including mitogenic, morphogenic, anti-apoptotic, and antifibrogenic activities. Long-term administration of the deletion variant of HGF (dHGF) might contribute to the prevention of chronic liver allograft dysfunction, which is attributed to immunologic and nonimmunologic reactions. Low-dose tacrolimus was administered to rat-liver recipients after transplantation. Effects of dHGF on transplanted livers treated with low-dose tacrolimus were investigated. Rats receiving liver transplants treated with only low-dose tacrolimus administration showed chronic allograft dysfunction. Treatment with dHGF prolonged the survival time of rats that received liver allografts and suppressed fibrosis of liver allograft. Treatment with dHGF also suppressed the expression levels of interleukin (IL)-1beta, caspase-1, and transforming growth factor (TGF)-beta mRNAs in liver allografts. The findings indicate that dHGF may prevent chronic liver-allograft dysfunction and thus may become a novel treatment for chronic liver-allograft dysfunction.

Reduction of monocrotaline-induced hepatic injury by deleted variant of hepatocyte growth factor (dHGF) in rats.

Monocrotaline is a hepatotoxic agent which exerts predominant toxicity to central veins and centrilobular sinusoids. In this study, we investigated the effects of deleted variant of hepatocyte growth factor (dHGF) on monocrotaline-induced hepatic injury in rats. 100 mg/kg monocrotaline was gavaged to male rats twice with a 4-days' interval. Treatment of dHGF was started 4 days before the initial administration of monocrotaline and 500 microg/kg was intravenously injected twice daily for 11 days. Monocrotaline induced severe damage of central veins and destruction of central zone of hepatic lobules concurrent with derangement of blood levels of total protein, albumin, alanine-aminotransferase, total bilirubin, direct bilirubin, and hepaplastin time. dHGF reduced the structural and blood-chemical abnormalities induced by monocrotaline. These results suggest that dHGF prevented and repaired the monocrotaline-induced hepatic injury, and could have therapeutic potency in hepatic failure with sever centrilobular destruction.

Biological Significance of C-met Over Expression in Papillary Renal Cell Carcinoma

Hereditary papillary renal cell carcinoma is associated with mutations of the c-met proto-oncogene. Similar aberrations have been described at a molecular level in up to 13% of sporadic papillary renal cell carcinomas. We assessed c-met expression in papillary renal cell carcinomas and evaluated the prognostic significance of c-met expression in patients with this tumor. We performed immunohistochemical testing to identify c-met expression in archival specimens of 55 papillary renal cell carcinomas in 51 patients. Only 1 patient reported a family history of renal malignancy. We identified c-met protein expression in the cytoplasm and cell membrane of 80% and 56% of these tumors, respectively. c-met expression significantly correlated with higher stage tumors (p = 0.004) but it was not associated with Fuhrman nuclear grade (p = 0.157). A trend toward a higher overall survival rate was noted in patients in whom tumors did not express c-met but this association failed to achieve statistical significance (p = 0.07). Our study indicates that c-met over expression may be associated with an aggressive phenotype in these tumors.

Beneficial effect of deletion variant of hepatocyte growth factor for impaired hepatic regeneration in the ischemically damaged liver.

This study was conducted to determine the influence of hepatic ischemia and reperfusion (HIR) injury on liver regeneration and the effect of the deletion variant of hepatocyte growth factor (dHGF) under these conditions. Male Sprague-Dawley rats were subjected to 60 minutes of total hepatic ischemia, and two-thirds hepatectomy was performed just before reperfusion. Animals received intravenous administration of either vehicle buffer (vehicle control group) or dHGF (1 mg/kg) (HGF group) at the end of the period of hepatic ischemia and again 6 hours after reperfusion. At 8 hours after hepatectomy, plasma HGF levels in the vehicle control group were significantly lower than those in the nonischemic controls. Plasma aspartate transaminase levels in the vehicle control group reached 3,462 +/- 1,039 IU/L, but levels in the HGF group were significantly inhibited to 1,849 +/- 605 IU/L. The relative liver weight in the vehicle control group was significantly greater than in the HGF group, a finding that was implicated in focal liver necrosis with sinusoidal congestion. Less histological damage was observed in the HGF group. Twenty-four hours after hepatectomy, an increase in the relative liver weight in nonischemic controls and in the HGF group was higher than that in vehicle control group, and the 5-bromo-2?deoxyuridine (BrdU) labeling index in the HGF group was 23% versus 18% in the nonischemic controls. Administration of dHGF significantly improved the 7-day survival to 82% versus 40% in the vehicle control group. dHGF has potential benefit as a pharmacological agent to ameliorate impairment of the hepatic microcirculation and to potentiate a regeneration response in the ischemically damaged liver after hepatectomy and/or liver transplantation.

Experimental study of the effects of deletion variant of hepatocyte growth factor on hepatic ischaemia-reperfusion injury.

Hepatic ischaemia-reperfusion (IR) injury is still a serious complication following liver surgery. The effect of the deletion variant of hepatocyte growth factor (dHGF) on hepatic IR injury was examined in rats. Male Wistar rats were divided into two groups after 90 min of partial liver ischaemia: the dHGF group which was given dHGF 0.5 mg/kg intravenously immediately after reperfusion, followed by 0.5 mg/kg every 12 h, and the control group, which received vehicle buffer only. Serum chemistry, histopathological findings and liver weights were compared between the groups. In the dHGF group, the increase in serum alanine transaminase and hyaluronic acid levels was significantly reduced, and the serum albumin level increased after reperfusion. The extent of hepatic necrosis 24 h after reperfusion was decreased in the dHGF group. Moreover, the proportion of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labelling-positive hepatocytes 6 h after reperfusion was reduced in the dHGF group. The non-ischaemic-, ischaemic- and whole-liver weight : body-weight ratio significantly increased in the dHGF group after reperfusion. The proportion of proliferating cell nuclear antigen-positive hepatocytes in the dHGF group markedly increased after 6 h after reperfusion in the non-ischaemic lobes, while in the ischaemic lobes it increased 24 h after reperfusion. These data suggest that dHGF not only improves recovery from IR injury, but also accelerates recovery from these injuries. dHGF may be an effective pharmacological agent for prevention and treatment of hepatic IR injury.