[Hepatocyte growth factor paracrined from human placental mesenchymal stem cells of fetal origin alleviates the injury of human pulmonary microvascular endothelial cells induced by lipopolysaccharide].

Objective To investigate the protective function of paracrine hepatocyte growth factor (HGF) derived from human placental mesenchymal stem cells of fetal origin (hfPMSCs) cultured in serum-free medium against endothelial cell injury induced by lipopolysaccharide (LPS). Methods The hfPMSCs were cultured in serum-free medium and surface antigen CD73, CD90, CD105, CD14, CD34, CD45 and HLA-DR were analyzed by flow cytometry. Using TranswellTM co-culture system [human pulmonary microvascular endothelial cell (HPMECs) were added into the upper chambers of TranswellTM inserts, and hfPMSCs were added into the lower chambers of TranswellTM inserts], the influence of hfPMSCs paracrine HGF on the permeability of HPMECs in LPS condition was identified. Then four different co-culture conditions were used as follows: 100 ng/mL LPS treatment group; hfPMSCs co-culture group; HGF neutralization group; HPMECs normal control. After 100 µg FITC-dextran was added into the upper chambers of TranswellTM inserts, the effect of stain permeability was detected by fluorescence microplate reader. The expression of VE-cadherin, caveolin-1 and cleaved caspase-3, cleaved PARP-1 in HPMECs were measured by Western blot analysis. Results The hfPMSCs showed the classic morphology of mesenchymal stem cells and expressed the surface markers CD73, CD90 and CD105, but did not express CD14, CD34, CD45 and HLA-DR. Compared with LPS treatment group, the co-culture with hfPMSCs dramatically inhibited the permeability of HPMECs, significantly up-regulated the expression of VE-cadherin, and reduced the expression of caveolin-1, cleaved caspase-3, cleaved PARP1. In contrast, neutralizing HGF with anti-HGF antibody reversed the above effects of hfPMSCs-HPMECs co-culture. Conclusion Owing to paracrine HGF, hfPMSCs possess the capability to availably inhibit the permeability of HPMECs induced by LPS.

Amniotic fluid stem cell exosomes: Therapeutic perspective.

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158-167, 2018.

Isolation and molecular cloning of hepatocyte growth factor from guinea pig (gHGF), and expression of truncated variant of gHGF with improved anti-fibrotic activity in Escherichia coli.

Hepatocyte growth factor (HGF) is an attractive target for anti-fibrotic therapy because it attenuates excessive transforming growth factor-ß1 (TGF-ß1) which plays an important role in hepatic fibrosis. In the study, we reported on the isolation and molecular cloning of the open reading frame (ORF) of guinea pig HGF (gHGF), encoding a protein of 729 amino acids, with an apple-like (hairpin) domain, four kringle domains and a trypsin-like serine protease domain. Moreover, the truncated variant of gHGF (a double mutant of N-terminal hairpin and first kringle domains of gHGF, K132E and G134E, gmNK1) protein fused with His6 tag, the molecular weight of which was about 20.0kDa, which was expressed in Escherichia coli BL21 (DE3) and purified with Ni2+-affinity chromatography. Furthermore, gmNK1 inhibited protein expression levels of fibrosis-related type I collagen (Col I) and α-smooth muscle actin (α-SMA) genes in TGF-ß1-activated HSC-T6 cells and CCl4-induced liver fibrosis in rat. In addition, gmNK1 ameliorated liver morphology and fibrotic responses in fibrosis animal. Taken together, we first reported on the sequence of HGF from guinea pig and determined the anti-fibrotic activity of gmNK1 in hepatic fibrosis, which will be helpful for investigations into the biological roles of gHGF in this important animal model.

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and ß chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-ß1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells.

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12 mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.

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-ß-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.

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta.

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His(6)-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni-nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P.angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.

Isolation and functional identification of a novel human hepatic growth factor: hepatopoietin Cn.

UNLABELLED: Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. CONCLUSION: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.

Urokinase-type plasminogen activator supports liver repair independent of its cellular receptor.

BACKGROUND: The urokinase-type (uPA) and tissue-type (tPA) plasminogen activators regulate liver matrix remodelling through the conversion of plasminogen (Plg) to the active protease plasmin. Based on the efficient activation of plasminogen when uPA is bound to its receptor (uPAR) and on the role of uPA in plasmin-mediated liver repair, we hypothesized that uPA requires uPAR for efficient liver repair. METHODS: To test this hypothesis, we administered one dose of carbon tetrachloride (CCl4) to mice with single or combined deficiencies of uPA, uPAR and tPA, and examined hepatic morphology, cellular proliferation, fibrin clearance, and hepatic proteolysis 2-14 days later. RESULTS: Absence of uPAR alone or the combined absence of uPAR and tPA had no impact on the resolution of centrilobular injury, but the loss of receptor-free uPA significantly impaired the clearance of necrotic hepatocytes up to 14 days after CCl4. In response to the injury, hepatocyte proliferation was normal in mice of all genotypes, except for uPAR-deficient (uPAR degrees) mice, which had a reproducible but mild decrease by 33% at day 2, with an appropriate restoration of liver mass by 7 days similar to experimental controls. Immunostaining and zymographic analysis demonstrated that uPA alone promoted fibrin clearance from centrilobular regions and efficiently activated plasminogen. CONCLUSION: uPA activates plasminogen and promotes liver matrix proteolysis during repair via a process that neither requires its receptor uPAR nor requires a contribution from its functional counterpart tPA.

Active hepatocyte growth factor is present in skeletal muscle extracellular matrix.

When skeletal muscle is stretched or injured, satellite cells are activated to proliferate, and this process can be mediated by release of the active form of hepatocyte growth factor (HGF) from the extracellular matrix. The objective of these experiments was to determine whether the mechanism of release includes proteolytic activation of pro-HGF. Extracellular HGF in uninjured adult rat extensor digitorum longus muscle was extracted by treatment with 1 M NaCl or heparinases I and III in the presence of a cocktail of serine protease inhibitors. Active HGF heterodimer was the predominant form present, but both pro-HGF and active HGF were extracted when muscle was incubated with Triton X-100 or crush-injured. Incubation of exogenous pro-HGF with uninjured or crush-injured skeletal muscle resulted in cleavage to the active form, indicating that endogenous extracellular proteases are present and capable of rapidly converting pro-HGF to active HGF. Finally, treatment with sodium nitroprusside, a nitric oxide (NO) donor, resulted in liberation of active HGF. These experiments indicate that the active form of HGF is present in the extracellular compartment of uninjured skeletal muscle; therefore, the mechanism of HGF release in response to stretch and NO does not require proteolytic activation of pro-HGF.

Identification and characterization of a novel isoform of hepatopoietin.

AIM: To isolate a novel isoform of human HPO (HPO-205) from human fetal liver Marathon-ready cDNA and characterize its primary biological function. METHODS: 5'-RACE (rapid amplification of cDNA 5' ends) was used to isolate a novel isoform of hHPO in this paper. The constructed pcDNA(HPO-205), pcDNA(HPO) and pcDNA eukaryotic expression vectors were respectively transfected by lipofectamine method and the stimulation of DNA synthesis was observed by (3)H-TdR incorporation assay. Proteins extracted from different cells were analyzed by Western blot. RESULTS: A novel isoform of hHPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated production of 23 kDa was isolated and distinguished from the previous HPO that lacked the N-terminal 80 amino acids. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (Mitogen-activated protein kinase) phosphorylation by Western blot analysis revealed that HPO-205 might have the stronger activity of stimulating hepatic cell proliferation than that of HPO. CONCLUSION: A novel isoform of hHPO (HPO-205) was isolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure and function of hHPO, and provide the new way of thinking to deeply elucidate the biological roles of HPO/ALR.

Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study.

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.

High levels of soluble syndecan-1 in myeloma-derived bone marrow: modulation of hepatocyte growth factor activity.

Syndecan-1 is a heparan sulfate proteoglycan expressed on the surface of, and actively shed by, myeloma cells. Hepatocyte growth factor (HGF) is a cytokine produced by myeloma cells. Previous studies have demonstrated elevated levels of syndecan-1 and HGF in the serum of patients with myeloma, both of negative prognostic value for the disease. Here we show that the median concentrations of syndecan-1 (900 ng/mL) and HGF (6 ng/mL) in the marrow compartment of patients with myeloma are highly elevated compared with healthy controls and controls with other diseases. We show that syndecan-1 isolated from the marrow of patients with myeloma seems to exist in an intact form, with glucosaminoglycan chains. Because HGF is a heparan-sulfate binding cytokine, we examined whether it interacted with soluble syndecan-1. In supernatants from myeloma cells in culture as well as in pleural effusions from patients with myeloma, HGF existed in a complex with soluble syndecan-1. Washing myeloma cells with purified soluble syndecan-1 could effectively displace HGF from the cell surface, suggesting that soluble syndecan-1 can act as a carrier for HGF in vivo. Finally, using a sensitive HGF bioassay (interleukin-11 production from the osteosarcoma cell line Saos-2) and intact syndecan-1 isolated from the U-266 myeloma cell line, we found that the presence of high concentrations of syndecan-1 (more than 3 microg/mL) inhibited the HGF effect, whereas lower concentrations potentiated it. HGF is only one of several heparin-binding cytokines associated with myeloma. These data indicate that soluble syndecan-1 may participate in the pathology of myeloma by modulating cytokine activity within the bone marrow.

Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients.

OBJECTIVES: To determine bronchoalveolar lavage (BAL) fluid concentrations of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), two potent growth factors for alveolar type II epithelial cells, in patients with acute respiratory distress syndrome (ARDS). DESIGN: Prospective study. SETTING: An adult trauma/surgical intensive care unit in an urban teaching hospital. PATIENTS: A total of 32 ventilated patients with pulmonary infiltrates prospectively identified with ARDS (n = 17) or without ARDS (n = 15), including eight patients with hydrostatic edema (HE), and ten nonventilated patients serving as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: BAL was performed 2.88 days +/- 2.4, 3.5 days +/- 2.4, and 2.3 days +/- 2.2 after the lung insult in ARDS, HE, and other non-ARDS patients respectively (p = .32). KGF was detected in BAL fluid in 13 of the 17 ARDS patients (median, 31.6 pg/mL), in one patient with HE, and in none of other non-ARDS patients. In ARDS patients, detection of KGF in BAL was associated in BAL fluid with the detection of type III procollagen peptide (PIIIP), a biological marker of fibroproliferation. In ARDS patients, detection of KGF in BAL was associated with death (p = .02). HGF was detected in 15 ARDS patients (median, 855 pg/mL), in seven patients with HE (median, 294 pg/mL; p = .05 for the comparison with ARDS group), in six of other non-ARDS patients (median, 849 pg/mL; p = .32 with ARDS group). HGF concentrations were higher in nonsurvivors than in survivors (p = .01). None of the ten BAL of controls contained either KGF or HGF. CONCLUSION: KGF was detected almost exclusively in BAL fluid from ARDS patients and correlated with a poor prognosis in this group. In contrast, HGF was detected in the BAL fluid from a majority of patients with or without ARDS. Elevated HGF concentrations were associated with a poor prognosis in the overall group.

Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium.

Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF-containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum-free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro-HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells.

Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity.

The liver regenerates by replication of differentiated hepatocytes after damage or removal of part of the liver. Although several growth factors and signaling pathways are activated during regeneration, it is unclear as to which of these are essential for hepatocyte replication. We show here that low- (1 mg/kg) and high- (10 mg/kg) dose hepatocyte growth factor (HGF) induced replication of 2.1% and 11.1% of hepatocytes in rats, respectively. Lipopolysaccharide (LPS), an inducer of the acute phase response, augmented hepatocyte replication in response to low- and high-dose HGF by 4- and 2-fold, respectively. HGF alone induced moderate levels of c-Jun-N-terminal kinase (JNK) and p44/p42 mitogen-activated protein kinase (MAPK), resulting in moderate levels of AP-1-DNA binding activity. The combination of LPS + HGF increased JNK and AP-1-DNA binding activity more than levels seen with LPS or HGF alone. The activation of Stat3 that was observed after administration of LPS + HGF, but not HGF alone, could contribute to increased transcription of AP-1 components. Because phosphorylation of the c-Jun component of AP-1 by JNK increases its ability to activate transcription, the AP-1 in hepatocytes from animals treated with LPS + HGF may be more active than in rats treated with LPS or HGF alone. LPS may contribute to hepatocyte replication by potentiating the effect of HGF on the activation of both AP-1-DNA binding and transcriptional activity.

Natural hepatocyte growth factor (HGF) from human serum and a bound form of recombinant HGF with heparan sulfate are indistinguishable in their physicochemical properties.

Natural hepatocyte growth factor (nHGF) purified from human serum showed a difference in molecular mass (Mr) between SDS PAGE (76-90 kDa) and gel filtration chromatography on a Sephadex G-200 column (> 200 kDa), whereas nHGF or recombinant HGF (rHGF) from cell culture medium did almost the same Mr (74-100 kDa). A bound form of rHGF with heparan sulfate (or heparin), and an aggregate form of rHGF itself showed a homogeneous band with a Mr of 76-90 kDa on SDS PAGE, but showed a Mr larger than 200 kDa on a Sephadex G-200 column. Both nHGFs, rHGF and the bound form were basic, but the aggregate form was acidic in ionic nature. No significant difference was found in affinity for heparin among these HGF preparations. The bound form treated by the procedures for purification of nHGF from human serum still showed a larger molecular form. The bound form mimicked physicochemical properties of nHGF purified from human serum. These results suggest that a possible form of nHGF in human serum may be a bound form with heparin-like molecules such as heparan sulfate, which are found in the circulation and on cell surface, and purified as the bound form.

Co-expression of hepatocyte growth factor and its receptor in human prostate cancer.

Hepatocyte growth factor acts differently depending on the organs or tumours involved. It may be produced simultaneously with its receptor, c-Met, in several types of malignant tumour cells and may exercise an autocrine regulation. To analyse the effect of hepatocyte growth factor in human prostate cancer, we conducted immunohistochemistry, in situ hybridization and the reverse transcriptase polymerase chain reaction. The first two techniques revealed the growth factor in prostate cancer cells, and the polymerase chain reaction confirmed this expression. c-Met is expressed in prostate cancer cells, but not in interstitial cells. Hepatocyte growth factor is expressed in interstitial cells, especially in hormone-treated cancer tissue, indicating that the growth factor pathway changes with the hormonal status. Low-grade tumours expressed c-Met at the plasma membrane. Higher grade tumours tended to express it in the cytoplasm, suggesting that the role of c-Met as the hepatocyte growth factor receptor was blocked in higher grade tumours. The relationship between the growth factor and its receptor is thus influenced by hormonal status and differentiation in prostate cancer and is not explained simply in terms of autocrine or paracrine action.

Purification and characterization of hepatocyte growth factor (HGF) from human seminal plasma.

Naturally occurring forms of hepatocyte growth factor/scatter factor (HGF/SF) have been purified by heparin-Sepharose chromatography, followed by cation exchange chromatography from a pool of human seminal plasma. Using an enzyme-linked immunosorbant assay, MDCK scatter assay, and Western blot analysis, it was found that, after heparin-Sepharose chromatography, human HGF/SF present in seminal plasma eluted in two different fractions, between 0.72 and 0.85 M NaCl (fraction I) and between 0.95 and 1.10 M NaCl (fraction II). Further purification of fraction I by cation exchange chromatography resulted again in two fractions which eluted at 0.2-0.4 and at 0.6-0.8 M NaCl. The fraction which eluted at 0.2-0.4 M NaCl consisted of two biologically less active heavy chains of the heterodimeric form of HGF/SF (107.1 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. The second fraction, which eluted at 0.6-0.8 M NaCl, revealed three bands with molecular weights of 87, 65 and 62 kDa, respectively. The 87 kDa form is thought to be a single chain precursor of HGF/SF devoid of biological activity. After subjecting fraction II to cation exchange chromatography, only one major peak eluted between 0.9 and 1.0 M NaCl, and consisted of two biologically active heavy chains of the heterodimeric form of HGF/SF (708.3 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. Nonreducing conditions for both fraction I and fraction II revealed only one band with a molecular weight of 68 kDa. The ratio ofpro-HGF/SF and less biologically active HGF/ SF (fraction I) over mature heterodimeric HGF/SF (fraction II) was approximately 1:3, in seminal plasma from sperm donors. In seminal plasma, pro-HGF/SF represents an 87 kDa glycoprotein which, apparently, is converted by limited proteolysis into several bands with molecular weights of 65 and 62 kDa. This is the first report showing the presence of pro-HGF/SF and heterodimeric mature HGF/SF, as well as less biologically active forms of HGF/SF in human seminal plasma.