This study was purposed to investigate the angiogenesis-promoting activities of human mesenchymal stem cells (hMSCs) modified by hepatocyte growth factor (HGF) and the underlying mechanisms. The hMSCs were transfected by recombinant adenoviral vector carrying human HGF gene and seeded onto the chicken chorioallantoic membrane. Three days later, the number of blood vessels was counted and their angiogenic response was compared with those of hMSCs of same generation, recombinant basic fibroblast growth factor (bFGF) and alpha-MEM as control. The expression levels of bFGF, VEGF, angiopoietin-1 and angiopoietin-2 were evaluated by RT-PCR assay. The results showed that gene-modified hMSCs exhibited greatest activity to promote angiogenesis while the angiogenic response was nearly same between groups treated by hMSCs and bFGF, all of which were significantly higher than that observed in control (p < 0.01). RT-PCR analysis revealed that hMSCs constitutively expressed multiple angiogenesis-associated growth factors and their levels seemed up-regulated by HGF gene transfer. It is concluded that HGF gene-modified hMSCs show a potent angiogenesis-promoting function and may be useful in the treatment of ischemic disorders.
Animals ; Cells, Cultured ; Chick Embryo ; Chickens ; Hepatocyte Growth Factor ; genetics ; Humans ; Mesenchymal Stromal Cells ; Neovascularization, Physiologic ; genetics ; Transfection

OBJECTIVETo obtain high expression of human hepatocyte growth factor (HGF) in passaged rabbit bone marrow-derived mesenchymal stem cells (BMSCs) via recombinant adenovirus vector mediated HGF gene transfection, and explore the feasibility of this strategy for local treatment of avascular necrosis of the femoral head (ANFH).

METHODSHGF gene was subcloned into the adenovirus shuttle plasmid pDC316, and the products were co-transfected into HEK293 cells with the helper plasmid pBHGlox deltaE1,3Cre. The recombinant adenovirus Ad-HGF was generated by homologous recombination of the 2 plasmids in HEK293 cells. After PCR identification, Ad-HGF was amplified and purified and its titer measured by TCID50 assay before transfected into the second passage of rabbit BMSCs. The transcription and expression of HGF gene in the transfected BMSCs was detected by RT-PCR, in situ hybridization, and immunological histochemistry.

RESULTSAd-HGF was successfully constructed with a titer of 2.6x10(10) TCID50/ml. The expressions of HGF mRNA and protein were confirmed in the transfected BMSCs.

CONCLUSIONAd-HGF transfection allows efficient expression of HGF protein in rabbit BMSCs, and this success may facilitate further study of local treatment of ANFH using HGF-expressing BMSCs.

Adenoviridae ; genetics ; Animals ; Cell Line ; Genetic Vectors ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Rabbits ; Transfection

AIMTo construct a plasmid carrying hepatocyte growth factor gene and investigate its effects in vitro.

METHODSA complementary DNA (cDNA) clone for human HGF was isolated from human placental cDNA, then subcloned into pUDK vector, which was constructed by ourselves, to form the pUDKH plasmid. The transfection efficiency and the expression level of HGF and VEGF were evaluated by transfecting pUDK or pUDKH into primary rat skeletal muscle cells. The biological effects of HGF-expressing product at different doses on endothelial cells were investigated in vitro, and assessed by MTT.

RESULTSThe primary rat skeletal muscle cells could be transfected efficiently with pUDKH (0.057%), and secreted HGF(16 -18 ng/4 x 10(5) cells) and VEGF proteins. The expressing product could significantly stimulate proliferation of human umbilical vein endothelial cells, in a dose-dependent manner (P < 0.05).

CONCLUSIONpUDKH has the potential application in vivo to treat ischemic diseases.

Animals ; Cells, Cultured ; DNA, Complementary ; genetics ; Genetic Vectors ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Plasmids ; Rats ; Rats, Wistar ; Transfection

OBJECTIVETo investigate the effect of adenoviral-mediated exogenous HGF (Ad-HGF) gene transfer on lung angiogenesis in the rabbit lung in rabbits with hyperkinetic pulmonary artery hypertension.

METHODSA thoracotomy was performed through a midsternal incision in 1-month-old immature rabbit and an anastomosis between the left innominate artery and the pulmonary trunk was made to establish a chronic patent left to right shunt. Three months later, animals were randomly assigned to receive either Ad-HGF (2 x 10(9) Pfu in 0.2 ml PBS, H1 group), repeated administration of Ad-HGF after one week (H 2 group), Ad-GFP (2 x 10(9) Pfu in 0.2 ml PBS, G group), or PBS (0.2 ml, C group) by the intratracheal method of gene transfection. After two weeks, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical examination was performed to identify HGF mRNA and HGF protein expression. The capillary density and small pulmonary artery density were determined by immunostained with antibodies against factor VIII and alpha-SMA. After 1 month, the collateral vessels were evaluated by angiogram under digital subtraction angiography (DSA).

RESULTSTwo weeks after Ad-HGF transfection, 484 bp bands could be found by RT-PCR in H1 and H2 groups, but not in other groups. The expression of HGF protein could be detected on alveolar epithelium and pulmonary vessel endothelium by immunohistochemistry examination. The number of factor VIII-positive pulmonary capillaries was also significantly increased in the H1 and H2 groups compared with the C and G groups (P < 0.05). The capillary density reached (17.0 +/- 3.3) mm(2) and (19.7 +/- 2.8) mm(2) in the H1 and H2 group, respectively, whereas it remained (13.2 +/- 3.2) mm(2) in the C group and (13.5 +/- 2.4) mm(2) in the G group (P < 0.05). One month after Ad-HGF transfection, the number of small pulmonary arteries was significantly increased in H1 and H2 group compared with control groups (P < 0.05). The collateral vessels were more abundant in HGF transfection groups than that in the two control groups reviewed by angiogram under digital subtraction angiography (DSA).

CONCLUSIONIn vivo gene transfection with HGF by means of the intra-tracheal injection could induce pulmonary angiogenesis in the early stage and small pulmonary arterial angiogenesis in later stage.

Animals ; Disease Models, Animal ; Female ; Hepatocyte Growth Factor ; genetics ; Hypertension, Pulmonary ; physiopathology ; Lung ; blood supply ; Male ; Neovascularization, Physiologic ; Rabbits ; Transfection

BACKGROUND: Hemoglobin concentration reportedly is positively associated with muscle strength, for example, handgrip strength. However, hemoglobin cannot repair muscle directly, but is beneficial only in a supportive role. Since hepatocyte growth factor (HGF) regulates muscle satellite cell production and differentiation, which is stimulated by organ injury, the supportive effect of hemoglobin should thus be stronger for participants with high HGF than for those with low HGF. However, the association between hemoglobin concentration and handgrip strength in relation to HGF levels remains unknown. METHODS: We conducted a cross-sectional study of 255 Japanese elderly men aged 60-69 years who participated in annual health check-ups in 2014-2015. The study population was categorized on the basis of a median value of HGF of 300.6 pg/mL. RESULTS: Among present study population, 128 participants showed low HGF. For participants with low HGF, hemoglobin concentration showed no significant association with handgrip strength (standardized parameter estimate (β) = 0.03, p = 0.767), but for those with high HGF, hemoglobin concentration was significantly positively associated with handgrip strength (β = 0.23, p = 0.014). CONCLUSIONS A significant positive association between hemoglobin level and handgrip strength was established for elderly Japanese men aged 60-69 years with high HGF but not for participants with low HGF. Our finding indicates that HGF levels could determine the relationship of hemoglobin concentration with handgrip strength in elderly Japanese men aged 60-69 years. This result can be expected to serve as an effective tool for the clarification of the roles played by HGF and hemoglobin concentration in maintenance of muscle strength.
Aged ; Cross-Sectional Studies ; Hand Strength ; physiology ; Hemoglobins ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Japan ; Male ; Middle Aged

Animals ; Connective Tissue Growth Factor ; Genetic Therapy ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Liver Cirrhosis ; therapy ; Transforming Growth Factor beta ; biosynthesis ; genetics

In order to trace the expression of exogenous hepatocyte growth factor (HGF) in eukaryotic cells exactly, a recombinant plasmid that expresses the fusion protein of HGF and red fluorescent protein (RFP) was constructed. The gene encoding HGF without stop code was amplified from pMD19-T-HGF by PCR technique and then cloned in pDsRed-express-N1. The recombinant plasmid (pDsRed-HGF) was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. pDsRed-HGF was transfected into 293T cells. The expression of HGF mRNA, HGF and red fluorescent protein was detected. The results showed that the target gene sequence in pDsRed-HGF was completely in conformity with HGF cDNA in GenBank, and it was expressed highly in 293T cells. In this study, pDsRed-HGF was successfully constructed and expressed in eukaryotic cells.
Cell Line ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Hepatocyte Growth Factor ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism

Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

The present investigation was to construct lentiviral vector carrying the human gene NK4 and transfect the human bone mesenchymal stem cells (hBMSCs) and to determine the expression of NK4 gene in hBMSCs after transfection. The NK4 gene was obtained from HGF cDNA by polymerase chain reaction(PCR), and the pGC-FU-NK4 plasmid was constructed by double restriction enzyme digestion and gene recombinant. The titer of virus was tested by real-time quantitative PCR. After transfected by lentivirus, the green fluorescent protein (GFP) in hBMSCs was observed using fluorescence microscope, and the expression of NK4 in culture supernatant was detected by enzymelinked immunosorbent assay (ELISA). The sequence of the PCR product was consistent with the data of GeneBank by DNA sequencing. The virus titer was 2 X 10(8)TU/ml. Strong green fluorescence was observed in the cell membrane and cytoplasm of hBMSCs with fluorescent microscopy. The expression of NK4 in culture supernatant was increased with time extension. The hBMSCs can be transfected by NK4 gene expressing lentiviral vector safely and effectively, and the expressin and secretion of NK4 was persistent and stable.
Bone Marrow Cells ; metabolism ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection