OBJECTIVETo study the influence of hepatocyte growth factor (HGF) antibody on the lung expression level of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1).

METHODSThirty male Wistar rats were randomly divided into 3 groups: control group, model group, and intervention group. Endotoxin was intratracheally infused in the model and intervention groups. HGF antibody was injected in the rats of the intervention group from day 1 to day 14, while the same volume of saline was injected in the control group. The rats were sacrificed on day 28 after endotoxin treatment. The amounts of MMP-9 mRNA and TIMP-1 mRNA were measured by reverse transcription-polymerase chain reaction, and protein expression levels of MMP-9 and TIMP-1 were measured by immunohistochemistry.

RESULTSIn the model group, both mRNA and protein expression levels of TIMP-1 were significantly increased, the same as MMP-9. In the intervention group, the increase of TIMP-1 was remarkably reduced compared with the model group, while the mRNA and protein expression levels of MMP-9 were still increased.

CONCLUSIONHGF activity may accelerate the repair of lung injury through contrary regulating the expression levels of TIMP-1 and MMP-9.

Acute Lung Injury ; metabolism ; pathology ; Animals ; Antibodies ; immunology ; Hepatocyte Growth Factor ; physiology ; Male ; Matrix Metalloproteinase 9 ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; genetics

OBJECTIVETo explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transfected with adenoviral vector carrying hepatocyte growth factor (HGF, Ad-HGF) on burn wound healing.

METHODSBMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum (FBS). Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection (MOI). The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) respectively. Thirty-two female rats were subjected to 90 degree centigrade water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells (MSCs) treatment group (Group A), Ad-HGF treatment group (Group B), Ad-HGF-modified MSCs treatment group (Group C) and saline control group (Group D). On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected. Transplanted cells and the expression of (sex-determining region Y) SRY gene were detected by in situ hybridization and polymerase chain reaction (PCR), while the expression of HGF in wound tissues was detected by ELISA.

RESULTSThe result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h, 72 h and 96 h (P less than 0.01). Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into the derma of burn wounds. Sirius red stain showed that the content of collagen I in Group C was much less compared with that in other groups 21 days postburn. In situ hybridization revealed an expression of SRY gene in burned female rats, consistent with the finding of PCR. Immunohistochemistry demonstrated the largest increase of HGF expression in Group C, whose contents of hydroxyproline, however, decreased on day 7 postburn. Compared with other groups, the content of HGF in the wounds of Group C increased obviously on day 14 after transfection (P less than 0.05) and there was no significant difference among Groups A, B and D.

CONCLUSIONOur study suggests that transplantation of MSCs modified with Ad-HGF has positive effects on the healing of burn wounds probably through differentiation and release of relevant cytokines.

Adenoviridae ; genetics ; Animals ; Burns ; metabolism ; therapy ; Cells, Cultured ; Female ; Genetic Therapy ; Hepatocyte Growth Factor ; analysis ; genetics ; Humans ; Hydroxyproline ; analysis ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Rats, Wistar ; Transfection ; Wound Healing

BACKGROUNDSo far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms.

METHODSTwenty male Wistar rats were randomly divided into four groups: control group (group C), model group (group M), early intervention group (group I) and late intervention group (group II). Groups M, I and II were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group I on day 7, 14 and 21. Group II received the same treatment like Group I on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-beta1 (TGF-beta1), cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR. Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-beta1 and COX-2 in lung parenchyma.

RESULTSThe plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups I and II. Pulmonary fibrosis levels of Groups I and II were obviously lower than that of group M (P < 0.05). Expression of TGF-beta1 protein and mRNA in lung tissue was markedly decreased in Groups I and II compared with Group M (P < 0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups I and II than in Group M (P < 0.05).

CONCLUSIONSInjection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced lung fibrosis. Exogenous HGF may inhibit the expression of TGF-beta1 and regulate the crosstalk between AECs and mesenchymal fibroblasts.

Animals ; Bleomycin ; toxicity ; Cyclooxygenase 2 ; genetics ; Electroporation ; Genetic Therapy ; Hepatocyte Growth Factor ; genetics ; Hydroxyproline ; analysis ; Injections, Intramuscular ; Lung ; chemistry ; Male ; Muscle, Skeletal ; metabolism ; Plasmids ; Pulmonary Fibrosis ; chemically induced ; therapy ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; analysis ; genetics

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-beta1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.
Animals ; Cell Engineering ; Cells, Cultured ; *Genetic Engineering ; Hepatocyte Growth Factor/analysis/*genetics ; Humans ; Liver/metabolism/pathology ; Liver Cirrhosis/pathology/*therapy ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*metabolism ; Rats ; Rats, Sprague-Dawley ; *Up-Regulation

OBJECTIVETo characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro.

METHODSRat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology.

RESULTSTen weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation.

CONCLUSIONCombined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.

Albumins ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Epidermal Growth Factor ; pharmacology ; Glutathione Transferase ; biosynthesis ; genetics ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Keratin-18 ; metabolism ; Protein Array Analysis ; Pyruvate Kinase ; biosynthesis ; genetics ; RNA, Messenger ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.
Amnion/cytology/*immunology ; Cell Differentiation/immunology ; Coculture Techniques ; Dinoprostone/genetics/immunology ; Female ; Hepatocyte Growth Factor/genetics/immunology ; Humans ; Immunologic Factors/*immunology ; Immunophenotyping ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/immunology ; Interferon-gamma/immunology ; Interleukin-10/analysis/immunology ; Interleukin-17/analysis/immunology ; Leukocytes, Mononuclear/cytology/immunology ; Mesenchymal Stem Cells/cytology/*immunology ; Pregnancy ; RNA, Messenger/chemistry/genetics ; Regenerative Medicine/methods ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta/genetics/immunology

Hepatocyte growth factor (HGF) is a potent mitogen and promoter of proliferation of insulin producing beta cells of pancreatic islets. To study the role of HGF, an adenoviral vector carrying the human HGF (Ad.hHGF) gene was transfected into the streptozotocin-induced diabetic mice and evaluated the effect on the blood glucose metabolism and the insulin-secreting beta cells of pancreatic islets. Ad.hHGF gene transfection resulted in amelioration of hyperglycemia and prolongation of survival period in the diabetic mice. Concomitantly adenoviral- mediated hHGF gene therapy slightly increased serum insulin concentration and the expression of insulin in the pancreatic islet. Although the proliferation of beta-cell mass was not noticeable, the beneficial effect of HGF is significant to an almost deteriorated pancreatic islets. Taken together, these data suggest that the Ad.hHGF gene therapy into diabetic mice may prevent the further destruction and present as a beneficial remedy for type 1 diabetic patients.
Adenoviridae/*genetics/physiology ; Animals ; Blood Glucose/analysis ; Body Weight ; Diabetes Mellitus, Experimental/blood/genetics/*metabolism/*therapy ; *Gene Therapy ; Hepatocyte Growth Factor/genetics/*metabolism ; Humans ; Hyperglycemia/blood/complications/genetics/*therapy ; Insulin/blood ; Islets of Langerhans/metabolism/*pathology ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/genetics/metabolism ; Survival Rate

BACKGROUNDVascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.

METHODSHepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA. The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.

RESULTSSerum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.

CONCLUSIONThis present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

Butadienes ; pharmacology ; Cell Line, Tumor ; Chromones ; pharmacology ; Colorectal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Hepatocyte Growth Factor ; blood ; pharmacology ; Humans ; MAP Kinase Signaling System ; physiology ; Morpholines ; pharmacology ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; physiology ; Phosphorylation ; Proto-Oncogene Proteins c-met ; metabolism ; RNA, Messenger ; analysis ; Signal Transduction ; physiology ; Vascular Endothelial Growth Factor A ; genetics

Met tyrosine kinase receptor, the receptor of hepatocyte growth factor/scatter factor (HGF/SF), is present in mouse tissues as two major isoforms differing by a 47-aminoacid segment in the juxtamembrane domain via alternative splicing of exon 14. We found that the smaller isoform of Met (Sm-Met) was highly transformable in both in vitro and in vivo tumorigenesis assays. In this report, close examination of the transforming activity of the Sm-Met showed that the expression of Sm-Met conferred the cells serum independence and anti- apoptotic property when treated with doxorubicin. These properties of Sm-Met seemed to be originated from its far longer maintenance of tyrosine kinase activity after the binding of HGF/SF. Interestingly, the longer maintenance of activated status was accompanied with more increase of tyrosine phosphorylation of Stat3 protein. Moreover, we have tried to find (an) animal tumorigenesis model(s) showing the increase in the expression of this transforming variant of Met. In gamma-ray-induced mouse thymic lymphoma model, the expression of the mRNAs for Sm-Met was significantly increased as well as those of wild type Met and HGF/SF, suggesting a possible role of the Sm-Met in tumorigenesis in vivo.
Animals ; *Apoptosis ; Cell Proliferation ; Cell Survival ; *Cell Transformation, Neoplastic ; DNA-Binding Proteins/metabolism ; Doxorubicin/pharmacology ; Hepatocyte Growth Factor/pharmacology ; Lymphoma/*etiology/genetics/metabolism ; Mice ; NIH 3T3 Cells ; Phosphorylation ; Protein Isoforms/genetics/metabolism ; Proto-Oncogene Protein c-met/genetics/*metabolism ; RNA, Messenger/analysis/metabolism ; Research Support, Non-U.S. Gov't ; Serum/metabolism ; Thymus Gland ; Trans-Activators/metabolism