Form Of Hepatocyte Growth Factor Research Articles

Novel rAAV vector mediated intrathecal HGF delivery has an impact on neuroimmune modulation in the ALS motor cortex with TDP-43 pathology.

Recombinant adeno-associated virus (rAAV)-based gene therapies offeran immense opportunity for rare diseases, such as amyotrophic lateral sclerosis (ALS), which is defined by the loss of the upper and the lower motor neurons. Here, we describe generation, characterization, and utilization of a novel vector system, which enables expression of the active form of hepatocyte growth factor (HGF) under EF-1α promoter with bovine growth hormone (bGH) poly(A) sequence and is effective with intrathecal injections. HGF's role in promoting motor neuron survival had been vastly reported. Therefore, we investigated whether intrathecal delivery of HGF would have an impact on one of the most common pathologies of ALS: the TDP-43 pathology. Increased astrogliosis, microgliosis and progressive upper motor neuron loss are important consequences of ALS in the motor cortex with TDP-43 pathology. We find that cortex can be modulated via intrathecal injection, and that expression of HGF reduces astrogliosis, microgliosis in the motor cortex, and help restore ongoing UMN degeneration. Our findings not only introduce a novel viral vector for the treatment of ALS, but also demonstrate modulation of motor cortex by intrathecal viral delivery, and that HGF treatment is effective in reducing astrogliosis and microgliosis in the motor cortex of ALS with TDP-43 pathology.

Abstract 3922: Novel hepatocyte growth factor and macrophage stimulating protein antagonists for the treatment of castration-resistant prostate cancer

Abstract Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer that is refractory to androgen deprivation therapy and characterized by aggressive growth, extensive metastasis formation, and poor prognosis. CRPC is temporarily manageable by the first-line chemotherapeutic docetaxel, but will eventually progress as a chemoresistant cancer, prompting a need for additional therapeutics to either replace docetaxel or to be used in combination therapies that increase the effectiveness of chemotherapy. The aberrant activation of the tyrosine kinase receptor c-Met by the active, dimeric form of hepatocyte growth factor (HGF) and the tyrosine kinase receptor RON by macrophage stimulating protein (MSP) results in the overactivation of signaling pathways that encourage cell proliferation, migration, and survival, contributing to the malignant behavior and chemoresistance of many cancers. The onset of metastatic prostate cancer is associated with both increased serum levels of HGF and expression of c-Met and RON in metastatic growths. Our laboratory has developed small molecules that inhibit HGF dimerization, c-Met activation, and malignant cell behaviors in several cancer cell types. Considering the high degree of homology between HGF and MSP, we predict that our strategy for inhibiting the activation of HGF can be extended to develop compounds that dually inhibit HGF and MSP. We hypothesize that inhibiting HGF and MSP with our antagonists will correspondingly reduce activation of c-Met and RON and suppress tumor growth and metastasis in CRPC. We further anticipate a therapy that combines our antagonists and docetaxel will increase the efficacy of chemotherapy by blunting the pro-survival properties of the HGF/c-Met and MSP/RON systems. Preliminary results indicate that our HGF dimerization antagonist Norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] significantly inhibits the migration of CRPC cells and increases their susceptibility to docetaxel treatment in vitro. The primary objective of this study is to investigate the feasibility of HGF, MSP, and HGF/MSP dual antagonists as a therapy for CRPC either alone or in combination with docetaxel. Citation Format: Brett Vanderwerff, Kevin Church, Leen Kawas, Joseph Harding. Novel hepatocyte growth factor and macrophage stimulating protein antagonists for the treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3922. doi:10.1158/1538-7445.AM2015-3922

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

A novel and effective human hepatocyte growth factor kringle 1 domain inhibits ocular neovascularization

Neovascularization is the critical pathological process and the leading cause of blindness in a variety of clinical conditions. This angiogenesis process is still uncertain. Human hepatocyte growth factor 1 (HGFK1) is derived from the mature form of hepatocyte growth factor, which contains four kringle domains in its α-chain. This study aimed to investigate the antiangiogenic activity of HGFK1 using in vitro and in vivo assays. HGFK1 was added into the DMEM to test the proliferation and migration of human umbilical vascular endothelial cells (HUVEC), and it was intravitreously injected in laser photocoagulation-induced choroidal neovascularization (NV) model, oxygen-induced retinopathy model and rho/VEGF transgenic mice to test its antiangiogenic effect. The results showed that HGFK1 effectively inhibited VEGF-stimulated HUVEC proliferation and migration, and also had anti-NV activity in choroidal NV and retinal NV. It is suggested that HGFK1 has antiangiogenic activity in vitro and in vivo. It may lead to new potential drug discoveries and the development in addition to anti-VEGF therapy in the future clinical anti-angiogenesis treatment.

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.

Generation of a Fully Human High Affinity Neutralizing Antibody Against MT-SP1/Matriptase and Its Potential Role for the Treatment of B Cell Lymphoma.

Abstract Membrane-type serine protease 1 (MT-SP1)/Matriptase is a type II transmembrane serine protease that is primarily expressed in epithelial cells and is over-expressed in several cancers. MT-SP1 has been implicated in the processes of tumor cell invasion and metastasis, primarily due to its ability to degrade extracellular matrix proteins and to activate the latent forms of hepatocyte growth factor (HGF), urokinase type plasminogen activator and protease-activated receptor 2. To further explore the role of this protein in human cancer, we developed a fully human IgG1, high affinity, neutralizing antibody against MT-SP1 (95/96) using XenoMax® technology. Using this antibody, we confirmed the expression of MT-SP1 on epithelial cells. In addition, we observed MT-SP1 expression on human peripheral blood B lymphocytes and monocytes. Furthermore, expression was also observed on several B cell lymphomas including Ramos, Raji and Daudi cell lines where, in contrast to epithelial cells, MT-SP1 was found in the absence of its inhibitor and cognate receptor, HGF activator inhibitor -1 (HAI-1). As 95/96 is a potent inhibitor of MT-SP1 enzymatic activity, we used the antibody to examine the functional role of MT-SP1 in B cell lymphoma. Using a tripeptide fluorogenic substrate of MT-SP1, we detected cell surface enzymatic activity on Ramos cells that was completely inhibited by 95/96, implying that the MT-SP1 expressed on Ramos cells is catalytically active. The ability of 95/96 to modulate the invasive properties of Ramos lymphoma cells was evaluated using a rodent hind-limb paralysis model. The median survival of SCID mice systemically inoculated with Ramos lymphoma cells, and receiving 95/96, was significantly extended compared to the animals receiving an isotype-matched control antibody (35 days vs. 26 days, p < 0.01). 95/96 had no effect on the in vitro proliferation or invasion of Ramos cells and was unable to induce cell death in a whole blood assay or by complement-dependent cytolysis. Collectively, these data suggest a role for MT-SP1 catalytic activity in the invasion or metastasis of Ramos B cell lymphoma.

Serum Hepatocyte Growth Factor Activator (HGFA) in Benign Prostatic Hyperplasia and Prostate Cancer

Objectives: Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor (HGF). We attempted to clarify whether serum levels of HGFA could be used as a marker for prostate cancer. Material and methods: Serum levels of total HGF and HGFA were measured by enzyme-linked immunosorbent assay in 99 healthy controls, 27 patients with benign prostatic hyperplasia (BPH) and 119 patients with prostate cancer. Results: The mean ± S.D. serum levels of HGFA in untreated prostate cancer and BPH cases were 0.42 ± 0.24 and 0.50 ± 0.26 ng/ml, respectively (no significant difference). Serum HGFA was significantly elevated in hormone-refractory prostate cancer (stage D3) compared to other stages, while HGF did not significantly differ with regard to clinical stage. Conclusions: Serum HGFA tends was elevated in patients with advanced stage prostate cancer. Further studies in large groups of patients are needed to clarify the clinical value of HGFA.

Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice

Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.

Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells

Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs. Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on. In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs. Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture. Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by 3H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media. The optimum timing for gene transfection was also evaluated. To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice. The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% ± 12.3%) was achieved with culture media, which also enabled the highest 3H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation. In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation. In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system. (H EPATOLOGY 2002;36:1488-1497.)

Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells

Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs. Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on. In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs. Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture. Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by 3H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media. The optimum timing for gene transfection was also evaluated. To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice. The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% ± 12.3%) was achieved with culture media, which also enabled the highest 3H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation. In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation. In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system. (Hepatology2002;36:1488-1497).

Pretreatment with a deleted form of hepatocyte growth factor (dHGF) prevents the mortality of plasma-loss-induced hypovolemic shock in rats.

Severe trauma, infection, burn, pancreatitis and major surgery often induce circulatory collapse leading to multiple organ failure and death. It is hypothesized that therapy for the attenuation of circulatory collapse may improve the prognosis in these diseases. Previous work has documented that pretreatment with a deleted form of hepatocyte growth factor (dHGF) in normal rats increases the circulating plasma volume that reflects its accelerating action of hepatic protein synthesis. Therefore, the effects of pretreatment with dHGF on hypovolemic shock models were studied in rats. Rats were intravenously administered dHGF (1 mg/kg, twice daily for 5-6 days) or vehicle, and subjected to a 25% total body surface area full-thickness burn or a trypsin-induced acute pancreatitis. In rats that were receiving vehicle, survival rates on day 7 after injury induction were 12% in the burn model and 5% in the pancreatitis model, respectively. In both models, hematocrit values were apparently increased and circulating plasma volumes were decreased compared to sham-operated rats at 6 h after injury induction. The pretreatment of animals with dHGF increased the survival rates on day 7 to 40% in the burn model and 29% in the pancreatitis model. dHGF-treatment in normal rats decreased the hematocrit values and increased the circulating plasma volumes, and these changes of hematocrit value and circulating plasma volume were also maintained after injury induction. These findings suggest that dHGF pretreatment prevents the mortality in the severe burn and acute pancreatitis, and that its effect may contribute to ameliorating the progressing of plasma-loss-induced hypovolemia.

Continuous intravenous infusion of deleted form of hepatocyte growth factor attenuates hepatic ischemia–reperfusion injury in rats

Background/Aims : Although beneficial roles of hepatocyte growth factor (HGF) and its variants on several hepatic disorders have been reported, their effects on hepatic ischemia–reperfusion (IR) injury remain undetermined. We investigated the action of a deleted form of HGF (dHGF) on hepatic IR injury in rats. Methods : dHGF or phosphate-buffered saline was continuously infused intravenously for 20 h prior to a 20-min occlusion of hepatic vessels. Samples were taken before and after IR, for measurement of serum dHGF and released enzymes, liver γ-glutamylcysteinyl glycine (GSH) level, as well as histological and immunohistochemical examinations. Results : After reperfusion, histological injury, as well as increase in the serum activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase-BB were significantly attenuated in the dHGF-treated rats. dHGF maintained a high GSH level and suppressed oxidative stress and intercellular adhesion molecule-1 (ICAM-1) expression on sinusoidal endothelial cells (SECs), on which c-Met was not detected. IR caused activation of c-Met expression, which was milder in the dHGF-treated group, in hepatocytes at the pericentral region. Conclusions : dHGF attenuated liver injury after IR. It also maintained a higher GSH level, depressed oxidative stress and inhibited ICAM-1 expression on c-Met negative SECs, suggesting a paracrine effect of dHGF.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Specific Cell Surface Binding Protein of Hepatocyte Growth Factor Activator (HGFA) and Regulates HGFA Activity in the Pericellular Microenvironment

Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA.HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1beta accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA.HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity.

A deleted form of human hepatocyte growth factor stimulates hepatic lipogenesis and lipoprotein synthesis in rats

Here we report the effect of the recombinant human deleted form of hepatocyte growth factor (dHGF) on lipid metabolism in rats. In primary cultured rat hepatocytes, dHGF accelerated incorporation of [14C]acetate into cellular lipids in a concentration-dependent manner. dHGF also increased the gene expression and enzyme activity of glucose-6-phosphate dehydrogenase, a rate-limiting enzyme of the pentose phosphate pathway, in hepatocytes. These results suggest that dHGF stimulates hepatocyte lipogenesis through upregulation of the pentose pathway and NADPH formation. Injection of dHGF into normal rats induced elevation of the serum triglyceride, phospholipid and cholesterol levels dose-dependently and in the same time course as the liver growth. dHGF injections stimulated the [14C]acetate incorporation into the liver lipids, but not into the adipose tissue nor the small intestine. Serum very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) levels were elevated by dHGF injections. [14C]Leucine incorporation into VLDL and LDL was also increased by dHGF injections. In rats with alcohol-induced fatty livers, dHGF treatment markedly diminished the accumulated liver triglyceride, while elevating serum lipid concentrations. The present results indicate that dHGF stimulates exclusively hepatic lipogenesis and increases serum lipoprotein levels in rats.

Tri-iodothyronine and a deleted form of hepatocyte growth factor act synergistically to enhance liver proliferation and enable in vivo retroviral gene transfer via the peripheral venous system.

Retroviral vectors integrate into the target cell genome in a stable manner and therefore offer the potential for permanent correction of the genetic diseases that affect the liver. These vectors, however, usually require cell division to occur in order to allow provirus entry into the nucleus. We have explored clinically acceptable methods to improve the efficiency of retroviral gene transfer to the liver, which avoid the need for liver damage. Tri-iodothyronine (T3) and recombinant hepatocyte growth factor have previously been used to induce hepatocyte proliferation in rat livers and allow in vivo retroviral gene transfer. We investigated the combined effects of these growth factors, with their differing mechanisms of action, on hepatocyte proliferation in vivo and assessed their effectiveness in priming cells for retroviral gene transfer. During the phase of hepatocyte proliferation retrovirus was administered via either the portal or tail vein. Acting synergistically, T3 and a truncated form of recombinant hepatocyte growth factor (dHGF) induced 30% of hepatocytes in normal rat liver to enter DNA synthesis at 24 h. This increased proliferation enabled the liver to be transduced in vivo by retroviral vectors via either the portal or peripheral venous system, achieving transduction efficiencies of 6.9 +/- 1.6% and 4.3 +/- 0.4% respectively. Thus, the liver can be simply and conveniently transduced in vivo with integrating vectors, introduced via the peripheral venous system during a wave of growth factor-induced proliferation, pointing the way to clinically applicable gene transfer techniques.

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.

Continuous intravenous administration of delete form of hepatocyte growth factor prevents ischemia-reperfusion injury in the rat liver

Continuous intravenous administration of delete form of hepatocyte growth factor prevents ischemia-reperfusion injury in the rat liver

Differential expression of hepatocyte growth factor and its receptor (c-Met) in a rat artificial anus model.

Hepatocyte growth factor is a multifunctional polypeptide that has been implicated in cancer growth, tissue development and wound repair. It is mainly synthesized in mesenchymal cells and acts on epithelial cells, where its actions are dependent on binding to a specific cell-surface hepatocyte growth factor receptor (c-Met). In an artificial anus, two different types of epithelial cells (ductal cells of the colon and squamous cells of the skin) intermingle with each other. In the present study, we examined the localization of hepatocyte growth factor and c-Met during the process of repair in a rat artificial anus model, and attempted to clarify the cell types that express hepatocyte growth factor or c-Met messenger RNA by in situ hybridization. Western blot analysis revealed abundant localization of the mature form of hepatocyte growth factor in artificial anal tissues. Moderate hepatocyte growth factor immunoreactivity was noted in regenerated squamous cells in the skin and colonic ductal cells, and strong expression in macrophages and fibroblasts. Moderate c-Met immunoreactivity was present in regenerated epithelial cells in the skin and colon. Throughout the repair process, hepatocyte growth factor and c-Met immunoreactivities were more prominently localized in the squamous cells of the skin than in colonic ductal cells. Competitive reverse transcription-polymerase chain reaction analysis revealed that hepatocyte growth factor mRNA was maximal on day 14 after the operation; however, c-Met mRNA expression had two peaks, on day 1 and day 7. Hepatocyte growth factor mRNA was expressed more in the stromal fibroblasts, macrophages and endothelial cells, and c-Met mRNA was predominant in regenerated squamous cells of the skin. These findings suggest the possibility that hepatocyte growth factor may act in a paracrine manner to mainly enhance the growth of squamous cells of the skin and to a lesser extent the ductal cells of the colon in the artificial anus.

Deleted form of hepatocyte growth factor ameliorates the mortality rate of severe thermal injury in rats

The modulating effects of the deleted form of hepatocyte growth factor (dHGF) on burn-induced mortality rates and hepatic protein synthesis were studied in rats. Rats were anesthetized, subjected to a 40% full-thickness scald burn, and divided into 2 groups receiving dHGF and vehicle. In normal rats, dHGF-treatment (1 mg/kg intravenously, twice daily) for 5 days increased the circulating plasma volume. In burned rats that were receiving vehicle, the survival rate on day 23 after the burn was 27%. The serum albumin levels were decreased and did not reverse to the normal levels until day 23 after the burn. Serum alpha 2-concentration in the injured rats was increased, whereas serum levels of transferrin, total protein, and high-density lipoprotein-cholesterol were decreased. The treatment of animals with dHGF (1 mg/kg intravenously, 3 times daily) for 3 days increased the survival rate on day 23 by 64%. In the animals treated with dHGF for 3 or 6 days, serum alpha 1-, alpha 2-, and beta-globulin concentrations were increased by the dHGF treatment. The serum levels of albumin, transferrin, total protein, and high-density lipoprotein-cholesterol reversed to normal levels or higher. Our data show that dHGF treatment may attenuate the decrease of the circulating plasma volume after burn and reduce a high risk of burn shock. It is also indicated that dHGF accelerates synthesis of not only acute-phase reactants but also other hepatic proteins such as albumin and transferrin on severe burn injury. These findings suggest that the appropriate upregulation of hepatic protein synthesis induced by dHGF may accelerate the physiologic recovery process after thermal injury and contribute to ameliorating the burn-induced death.

Deleted form of hepatocyte growth factor ameliorates the mortality rate of severe thermal injury in rats

Deleted form of hepatocyte growth factor ameliorates the mortality rate of severe thermal injury in rats