Recombinant Human Hepatocyte Growth Factor Research Articles

Hepatocyte Growth Factor Suppresses Transforming Growth Factor-Beta-1 and Type III Collagen in Human Primary Renal Fibroblasts

Tubulointerstitial changes in the diabetic kidney correlate closely with renal fibrosis, and transforming growth factor-beta-1 (TGF-beta1) is thought to play a key role in this process. In contrast, hepatocyte growth factor (HGF) has shown therapeutic effects on injured renal tubules in animal models. This study was undertaken to test the hypothesis that the preventive effects of HGF may result from interventions in TGF-beta1-mediated signaling and collagen III secretion. We examined the expression of HGF/HGF receptor (c-Met) and TGF-beta1 in renal fibroblasts at multiple time points. The effects of recombinant human HGF on TGF-beta1 expression were studied by RT-PCR and Western blotting, and the levels of collagen III were measured by ELISA. In the high-glucose condition, the expression of HGF and c-Met in renal fibroblasts was detected as early as 6 hours following cell culture while the level of TGF-beta1 peaked at 96 hours. The addition of recombinant human HGF to the culture media dose-dependently inhibited TGF-beta1 mRNA expression and reduced collagen III secretion by 34%. These results indicate that, during hyperglycemia, HGF inhibits TGF-beta1 signaling and type III collagen activation in interstitial fibroblasts. Furthermore, we should recognize that changes in the balance between HGF and TGF-beta1 might be decisive in the pathogenesis of chronic renal fibrosis. Therefore, administration of HGF to restore this balance may offer a novel therapeutic intervention in managing renal fibrogenesis in diabetic nephropathy.

Overproduction of recombinant human hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multi-functional protein that affects morphogenesis, cell migration, organ regeneration, and tumor invasion in various tissues, and has thus been considered to have potential as a therapeutic target in various diseases. In our current study, we established Chinese hamster ovary (CHO) cells overexpressing recombinant human HGF (rhHGF) protein and in a 5 day batch culture process using a 7.5l bioreactor (5l working volume) and serum-free medium these cells could produce over 13 mg/l of rhHGF protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in about a 35% recovery rate. This purified rhHGF was found to be a mixture of inactive pro-HGF and an active heterodimeric form of this protein with a higher molecular weight than its counterpart expressed from insect cells. This finding suggests that the glycosylation of rhHGF protein in CHO cells differs from that in insect cells. Inactive pro-HGF was found to rapidly convert to the active heterodimeric form of HGF in the presence of FBS (Fetal Bovine Serum), suggesting that this process would occur also when injected into human body. We further demonstrate in cell proliferation and scattering activity assays that our purified rhHGF protein preparation is functionally active with a half-maximal effective concentration of 36.3 ng/ml.

Translational research to identify clinical applications of hepatocyte growth factor

Hepatocyte growth factor (HGF), originally purified from the plasma of patients with fulminant hepatic failure, has been shown to carry out various physiological functions. HGF not only stimulates liver regeneration, but also acts as an antiapoptotic factor in in vivo experimental models. Therefore, HGF is a promising therapeutic agent for the treatment of fatal liver diseases, including fulminant hepatic failure. After performing a number of preclinical tests, our group began an investigator-initiated registered phase I/II clinical trial of patients with fulminant hepatic failure to examine the safety and clinical efficacy of recombinant human HGF. In this article, we will discuss the basic research results as well as the translational research that underpins current attempts to use HGF in various clinical settings.

Intratracheal Delivery of Hepatocyte Growth Factor Directly Attenuates Allergic Airway Inflammation in Mice

Hepatocyte growth factor (HGF) has an important role in many biological events such as angiogenesis and cell proliferation, as well as anti-fibrotic and anti-apoptotic effects. In addition, we found that HGF suppresses antigen-induced immune responses in the airway by suppressing dendritic cell functions, using a HGF-producing plasmid vector. In the present study, we examined whether delivery of the HGF protein in the lung attenuates allergic airway inflammation in a mouse model. Generally, HGF is rapidly cleared from organs. So, to achieve the efficient delivery of HGF, we prepared a slow-releasing form by mounting recombinant human (rh) HGF protein in biodegradable gelatin hydrogels. BALB/c mice were immunized and then challenged with ovalbumin (OVA) to induce eosinophilic airway inflammation. Intratracheal delivery of a very small amount of gelatin-coupled rhHGF (0.3 μg) just once before the inhalation of OVA significantly suppressed eosinophilic airway inflammation. In addition, cytokine production in thoracic lymph nodes and the antigen-presenting capacity of lung CD11c+ cells were reduced. In contrast, delivery of 1.0 μg of rhHGF did not exhibit any significantly suppressive effect. These results suggest that the controlled release of rhHGF protein can suppress antigen-induced allergic immune responses in the lung. Therefore, HGF could be a novel therapeutic option for asthma.

Hepatocyte Growth Factor Treatment Improves Alveolarization in a Newborn Murine Model of Bronchopulmonary Dysplasia

Background: Bronchopulmonary dysplasia (BPD) is thought to be one form of developmental arrest of the lung. Hepatocyte growth factor (HGF) participates in normal lung growth and in lung regeneration. Objectives: The purpose of this study is to investigate whether HGF can improve alveolarization and attenuates functional abnormalities of a murine model of BPD induced by hyperoxia. Methods: Three-day-old CD-1 mice were exposed to 90% of oxygen or room air (control group) for 7 days. These animals were then kept in room air for the next 7 days. Recombinant human (rh) HGF (100 μg/g b.w., divided 3 times, rhHGF group) or vehicle (vehicle group) was administered intraperitoneally during hyperoxia. On day 17, the pulmonary function test and bronchial hyperresponsiveness (BHR) were examined. Mean linear intercepts (MLI) were measured as parameters of alveolarization. Cell renewal (on day 10) and vascularization of the lung were also evaluated. Results: Exposure to hyperoxia induced increased airway resistance and BHR. These animals showed a severely simplified alveolar structure, increased MLI, decreased cell renewal (16.1 ± 2.4 vs. 29.6 ± 2.4%, p < 0.05), and decreased vascularization (15.1 ± 0.3 vs. 18.4 ± 1.5 vessels/hpf, p < 0.05, vehicle vs. control group, respectively). rhHGF treatment during exposure to hyperoxia significantly reduced all of these changes (27.9 ± 1.7%, 18.2 ± 0.5 vessels/hpf for cell renewal and vascularization, respectively; all values are p < 0.05 against vehicle animals). Conclusion: HGF partially protects against the inhibition of alveolarization and improves functional abnormality in the hyperoxia-induced neonatal mice model of BPD.

Translational research on HGF: A phase I/II study of recombinant human HGF for the treatment of fulminant hepatic failure

Hepatocyte growth factor (HGF) is a potential therapeutic agent for fatal liver diseases, including fulminant hepatic failure (FHF). After performing a number of preclinical tests with recombinant human HGF (rh-HGF), we started a phase I/II study in September 2005 of patients with FHF or late-onset hepatic failure (LOHF), to examine the safety and clinical efficacy of rh-HGF. We first administered rh-HGF (0.6 mg/m(2)/day) for 13 days to a 67-year-old Japanese man with FHF. All data from this patient were reviewed by the independent data monitoring committee, and the safety of rh-HGF was recognized. Finally, a clinical trial of rh-HGF was approved to be continued. As of August 2007, we have administered rh-HGF to four patients with FHF or LOHF. All patients showed a moderate decrease in systolic blood pressure during rh-HGF administration, while the urinary excretion of albumin did not increase in all cases. In the first and third patients, hepatic failure gradually progressed, and they died 66 and 29 days, respectively, after encephalopathy occurred. The second and fourth patients are presently still alive. In conclusion, we started a clinical trial that examined the effects of rh-HGF in patients with FHF or LOHF, and in the four patients with FHF or LOHF enrolled in this study, repeated doses of rh-HGF did not produce any severe side effects.

Hepatocyte Growth Factor/c-MET Axis-mediated Tropism of Cord Blood-derived Unrestricted Somatic Stem Cells for Neuronal Injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.

Noncompetitive Inhibition of Hepatocyte Growth Factor-dependent Met Signaling by a Phage-derived Peptide

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC 50 of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC 50 of ∼ 20 μM. The 2D 1H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.

Hepatocyte growth factor suppresses ischemic cerebral edema in rats with microsphere embolism

The present study was aimed at determining whether human recombinant hepatocyte growth factor (HGF) ameliorates cerebral edema induced by microsphere embolism (ME). Rats were injected with 700 microspheres (48μm in diameter). Continuous administration of HGF at 13μg/3 days/animal into the right ventricle was started from 10min after embolism to the end of the experiment by using an osmotic pump. On day 3 after the ME, the rats were anesthetized, and their brains were perfused with an isotonic mannitol solution to eliminate constituents in the vascular and extracellular spaces. Thereafter, tissue water and cation contents were determined. A significant increase in tissue water content of the right hemisphere by ME was seen. This ME-induced increase in water content was associated with increases in tissue sodium and calcium ion contents and decreases in tissue potassium and magnesium ion contents of the right hemisphere. The treatment of the animal with HGF suppressed the increases in water and sodium and calcium ion contents, but not the decreases in potassium and magnesium ion contents. These results suggest that HGF suppresses the formation of ischemic cerebral edema provoked intracellularly in rats with ME.

Recombinant human hepatocyte growth factor for liver failure

The purpose of this paper is to evaluate the efficacy and safety of recombinant human hepatocyte growth factor (rh-HGF) for liver failure (LF) using meta-analysis of data from the literature involving available randomized controlled trials of rh-HGF plus comprehensive therapy (CT) compared with that of CT alone (Therapy I versus II) in treating LF. We searched the Cochrane Library, MEDLINE, EMBASE, CBMdisc, and CNKI as well as employing manual searches.Based on our search criteria, we found 21 trials, involving 5902 patients. Our results showed that Therapy I, compared with therapy II, significantly reduced the overall mortality (RR=0.62; 95% CI, 0.59–0.66; p=0.0001). Compared to two clinical types of LF (acute and acute-on-chronic), therapy I perhaps had significant effect on mortality due to sub-acute LF, RR and 95% CI were 0.76 [0.65, 0.89], 0.66 [0.60, 0.74], and 0.58 [0.53, 0.64], respectively. Additionally, there was a reduction in mortality of patients that had evidence for an early stage of LF compared to the two other clinical stages of LF (Middle and Advanced); RR and 95% CI were 0.34 [0.24, 0.49], 0.49 [0.44, 0.55], and 0.87 [0.82, 0.93], respectively. No serious adverse events were reported. We conclude that Therapy I may reduce mortality in LF, especially in sub-acute LF and the early stage of LF. However, considering the strength of the evidence, additional randomized controlled trials are needed before Therapy I can be recommended routinely.

Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-kappaB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-kappaBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH precursor, significantly reduced Jo2-induced cell death. Conversely, the GSH-depleting agent buthionine sulfoximine completely abolished the protective effects of N-acetylcysteine in Met-KO hepatocytes. In conclusion, genetic inactivation of c-Met in mouse hepatocytes caused defects in redox regulation, which may account for the increased sensitivity to Fas-induced apoptosis and adaptive up-regulation of NF-kappaB survival signaling. These data provide evidence that intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous reactive oxygen species.

Hepatocyte Growth Factor Suppresses Proinflammatory NFκB Activation through GSK3β Inactivation in Renal Tubular Epithelial Cells

Activation of NFkappaB is a fundamental cellular event central to all inflammatory diseases. Hepatocyte growth factor (HGF) ameliorates both acute and chronic inflammation in a multitude of organ systems through modulating NFkappaB activity; nevertheless, the exact molecular mechanism remains uncertain. Here we report that HGF through inactivation of GSK3beta suppresses NFkappaB p65 phosphorylation specifically at position Ser-468. The Ser-468 of RelA/p65 situates in a GSK3beta consensus motif and could be directly phosphorylated by GSK3beta both in vivo and in vitro, signifying Ser-468 of RelA/p65 as a putative substrate for GSK3beta. In addition, the C terminus of RelA/p65 harbors a highly conserved domain homologue of the consensus docking sequence for GSK3beta. Moreover, this domain was required for efficient phosphorylation of Ser-468 and was indispensable for the physical interaction between RelA/p65 and GSK3beta. HGF substantially intercepted this interaction by inactivating GSK3beta. Functionally, phosphorylation of Ser-468 of RelA/p65 was required for the induced expression of a particular subset of proinflammatory NFkappaB-dependent genes. Diminished phosphorylation at Ser-468 by HGF resulted in a gene-specific inhibition of these genes' expression. The action of HGF on proinflammatory NFkappaB activation was consistently mimicked by a selective GSK3beta inhibitor or GSK3beta knockdown by RNA interference but largely abrogated in cells expressing the mutant uninhibitable GSK3beta. Collectively, our findings suggest that HGF has a potent suppressive effect on NFkappaB activation, which is mediated by GSK3beta, an important signaling transducer controlling RelA/p65 phosphorylation specificity and directing the transcription of selective proinflammatory cytokines implicated in inflammatory kidney disease.

Intrathecal Delivery of Hepatocyte Growth Factor From Amyotrophic Lateral Sclerosis Onset Suppresses Disease Progression in Rat Amyotrophic Lateral Sclerosis Model

Hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. We showed that introduction of the HGF gene into neurons of G93A transgenic mice attenuates motor neuron degeneration and increases the lifespan of these mice. Currently, treatment regimens using recombinant protein are closer to clinical application than gene therapy. To examine its protective effect on motor neurons and therapeutic potential we administered human recombinant HGF (hrHGF) by continuous intrathecal delivery to G93A transgenic rats at doses of 40 or 200 microg and 200 microg at 100 days of age (the age at which pathologic changes of the spinal cord appear, but animals show no clinical weakness) and at 115 days (onset of paralysis), respectively, for 4 weeks each. Intrathecal administration of hrHGF attenuates motor neuron degeneration and prolonged the duration of the disease by 63%, even with administration from the onset of paralysis. Our results indicated the therapeutic efficacy of continuous intrathecal administration of hrHGF in transgenic rats and should lead to the consideration for further clinical trials in amyotrophic lateral sclerosis using continuous intrathecal administration of hrHGF.

Hepatocyte Growth Factor Prevents Pulmonary Ischemia–Reperfusion Injury in Mice

Ischemia-reperfusion (IR) injury after lung transplantation leads to significant morbidity and mortality in recipients, which remains the major obstacle in clinical lung transplantation. To reduce pulmonary graft dysfunction and improve prognosis after lung transplantation, prevention of IR-induced lung injury in the peri-operative period is required. In the present study, we investigated the effects of recombinant hepatocyte growth factor (HGF) on pulmonary IR injury using a murine model system. To assess the protective effect of HGF against lung injury, mice with pulmonary IR were divided into two groups and injected with 500 microg/kg of human recombinant HGF or the same dose of saline alone as a control. After pulmonary IR injury, the lung injury score increased in a time-dependent manner up to 24 hours. A significant reduction of lung injury score was observed with the administration of exogenous HGF. Moreover, the ratio of apoptotic cells was significantly reduced in mice treated with HGF. Significantly increased expression of Bcl-xL was observed after IR in mice administered HGF as compared with saline-treated controls. In contrast, expression of Bax was reduced significantly in HGF-treated mice. Serum levels of endogenous murine HGF were increased significantly in HGF-treated mice. Our findings indicate that administration of exogenous HGF ameliorates the pulmonary tissue injury induced by IR, which may provide an alternative for prevention of IR-induced lung injury in the peri-operative period in lung transplantation.

HGF ameliorates a high-fat diet-induced fatty liver

Hepatocyte growth factor (HGF) has various effects especially on epithelial cells. However, the precise role of HGF on lipogenesis is still not fully understood. A high-fat diet was administered to HGF transgenic mice and wild-type control mice in vivo. Furthermore, recombinant human HGF (rhHGF) was administered to HepG2 cell line in vitro. We performed an analysis regarding the factors relating to lipid metabolism. An overexpression of HGF dramatically ameliorates a high-fat diet-induced fatty liver. HGF transgenic mice showed an apparently reduced lipid accumulation in the liver. The activation of microsomal triglyceride transfer protein (MTP) and apolipoprotein B (ApoB) accompanying higher triglyceride levels in the serum were found in HGF transgenic mice on a normal diet. Interestingly, this upregulation of the MTP activation became more apparent in the high-fat diet. In addition, the administration of rhHGF stimulated MTP and ApoB expression while reducing reduced the intracellular lipid content in HepG2 cell line. However, this induction of MTP and ApoB by HGF was clearly inhibited by PD98059 (MAPK inhibitor). In conclusion, the data presented in this study indicated that HGF ameliorates a high-fat diet-induced fatty liver via the activation of MTP and ApoB.

Hepatocyte growth factor (HGF) promotes oligodendrocyte progenitor cell proliferation and inhibits its differentiation during postnatal development in the rat

Hepatocyte growth factor (HGF) was initially cloned as a mitogen for hepatocytes and has been identified as a neurotrophic factor for a variety of neurons. However, few attempts have assessed the role of HGF in cells of oligodendrocyte lineage. The purpose of this study was to elucidate the role of HGF in such cells during development. Double immunostaining for either c-Met/HGF receptor or phospho-c-Met with either NG2 or RIP in rat striatum at postnatal day 3 (P3), P7, and P14 revealed that c-Met was phosphorylated on tyrosine residues and thereby activated in NG2 + oligodendrocyte progenitor cells (OPCs) at P3–P14 and in RIP + oligodendrocytes at P14. Intrastriatal injections of recombinant human HGF at both P7 and P10 revealed that the relative ratio of BrdU +/NG2 + cells per total number of NG2 + cells increased, while BrdU +/MBP + oligodendrocyte numbers decreased. Western blot analysis showed a down-regulation of myelin basic protein (MBP) after HGF injection. Electron microscopy revealed that the numbers of myelinated nerve fibers decreased after HGF treatment. Furthermore, administration of anti-HGF IgG into the striatum increased the number of BrdU +/MBP + oligodendrocytes. These findings demonstrated that HGF increases proliferation of OPCs and attenuates their differentiation into myelinating oligodendrocytes, presumably by favoring neurite outgrowth that may be inhibited by the myelin inhibitory molecules on oligodendrocytes. Down-regulation of HGF mRNA in the striatum from P7 to P14, as revealed by quantitative real-time RT-PCR, may be favorable for OPC differentiation into myelinating oligodendrocytes. Our findings suggest that c-Met signaling, together with HGF regulation, plays an important role in developmental oligodendrogenesis.

Transfection of human hepatocyte growth factor gene ameliorates secondary lymphedema via promotion of lymphangiogenesis

Conclusion: In this model, expression of human hepatocyte growth factor (HGF) via plasma transfer promotes lymphangiogenesis and improves lymphedema. Summary: It is possible to stimulate lymphangiogenesis (Develop Biol 1997;188:96-109). It is also suggested hepatocyte growth factor (HGF) promotes formation of lymphatics (EMBO J 2005;24:2885-95). In this study, the authors investigated the potential therapeutic efficacy of HGF in an animal model of lymphedema. Immunofluorescence techniques can be used to identify lymphatic specific markers on canine primary lymphatic endothelial cells (cLECs). These markers include vascular endothelial growth factor receptor-3, LYBE-1, podoplanin and prox-1. The HGF receptor c-Met can also be identified. A rat-tail lymphedema model of secondary lymphedema was used (Surgery 1999;229:421-5). The superficial lymphatic networks of the rat’s tail are severed with a circumferential incision about the base of the tail. There were 60 rats randomly assigned to one of six groups: human HGF plasma, human BEGF-165 plasma, green fluorescent protein plasma, saline, operation only, and no operation. Measures of tail thickness before and after treatment were used as measurements of phenotypic expression of lymphangiogenesis. When cLECs were treated with human recombinant HGF, there was a dosed-dependent increase in cell migration and cell growth. There was also increased activity of extracellular single-regulated kinase and Akt and c-Met. HGF treatment increased cell migration. Growth was dose-dependent. C-FOS motor activity was also increased via transfection of human HGF plasma DNA into canine LECs. Expression of cLEC markers (LYBE-1 and prox-1) also increased with HGF injection. Comment: These data suggest the phenotypic changes associated with secondary lymphedema can be attenuated with HGF gene transfer and that this occurs through local lymphangiogenesis induced by HGF. HGF is proving to be an extraordinarily interesting molecule. It appears to be a substance that allows targeted angiogenesis of both arteries and lymphatics. The huge current interest in HGF certainly seems justified.

A Hepatocyte Growth Factor (HGF)/Receptor Autocrine Loop Regulates Constitutive Self‐Renewal of Human Periodontal Ligament Cells but Reduces Sensitivity to Exogenous HGF

In addition to its prominent role in liver regeneration, hepatocyte growth factor (HGF) is now generally thought to be produced by mesenchymal cells to promote the regeneration of epithelial tissue by a paracrine mechanism. However, it is not known how or if HGF could be involved in the regeneration of periodontal tissues. The purpose of this study was to characterize the ability of normal human periodontal ligament (PDL) cells to produce or respond to HGF. PDL cells derived from healthy young volunteers were used from passages four through 10. HGF receptors were detected both by immunocytochemical staining and Western-blotting analysis. Both DNA synthesis (by bromo-deoxyuridine [BrdU]-incorporation) and secreted HGF were quantified by enzyme-linked immunosorbent assays. Mitogen-activated protein kinase (MAPK) phosphorylation was also analyzed by Western blot. Despite the immunocytochemical demonstration of HGF receptor protein in the cytoplasm and on the plasma membrane of PDL cells, exogenous recombinant human HGF did not exert the mitogenic effects expected. As reported for other mesenchymal cells, PDL cells were found to secrete HGF. Treatments with neutralizing anti-HGF antibody significantly suppressed constitutive PDL cell proliferation and sustained the receptor protein at higher levels than in non-treated cells. Under these conditions, exogenous HGF rapidly phosphorylated extracellular signal-regulated kinase (ERK), an action linked to the cell proliferation and downregulation of cell-surface receptors. Unlike other known mesenchymal or epithelial cells, these findings suggest that normal PDL cells from young donors possess a constitutive HGF/receptor autocrine loop that normally regulates their replacement self-proliferation but reduces sensitivity to exogenously applied HGF by acute receptor downregulation.

Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood–brain barrier and decreases in expression of tight junctional proteins in cerebral vessels

Hepatocyte growth factor (HGF) exerts its physiological activities as that of an organotropic factor for regeneration and can prevent ischemia-induced injuries; however, its effect and mechanism of action under in vivo pathophysiological conditions remains to be determined. Recently, we demonstrated that treatment with human recombinant HGF (hrHGF) attenuated the disruption of the blood–brain barrier (BBB) observed after microsphere embolism-induced sustained cerebral ischemia. To see if tight junctional proteins were involved in this attenuation, in the present study, we investigated the effects of HGF on the levels of occludin and zonula occludens (ZO)-1 in cerebrovascular endothelial cells after microsphere embolism. Sustained cerebral ischemia was induced by the injection of 700 microspheres (48 μm diameter) into the right internal carotid artery of rats. hrHGF was injected into the right ventricle of the brain by using an osmotic pump at a dose of 30 μg/7 days per animal. The levels of tight junctional proteins in the endothelial cells were examined by immunohistochemical analysis. Treatment with hrHGF attenuated the decrease in the expression of occludin and ZO-1 proteins in the endothelial cells that occurred after sustained cerebral ischemia. Furthermore, treatment with hrHGF resulted in retention of these tight junctional proteins in fluorescein isothiocyanate (FITC)-albumin-perfused cerebral vessels, which did not leak FITC–albumin in the ipsilateral cortex. These results suggest that HGF-mediated maintenance of the tight junctional proteins in the endothelial cells may be a possible mechanism for the protective effect of HGF against the disruption of the BBB after cerebral ischemia.

Transfection of Human Hepatocyte Growth Factor Gene Ameliorates Secondary Lymphedema via Promotion of Lymphangiogenesis

Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs) were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c-fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF- and vascular endothelial growth factor 165-injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.