Effect Of HGF Transfected Into Skeletal Muscle Cells On Apoptosis Of Vascular Endothelial Cells Under Hypoxic Condition In Vitro

Angiogenic growth factors play important roles in angiogenic responses, such as vasculogenesis and angiogenesis in response to hypoxia. Hepatocyte growth factor(HGF) with the C-met proto-oncogene product as its receptor, is specific to vascular endothelial cells(Ecs), and has the most potent mitogenetic role, besides antiapoptotic effect, endothelial barrier function enhancement, endothelium-dependent relaxtion, and so on. Because hypoxia is generally considered to represent a fundamental stimulus for angiogenesis, the aim of the present study was to investigate the effects of HGF on apoptosis induced by hypoxia in rat vascular endothelial cells.ObjectiveThe plasmid pIRES2-EGFP-HGF were transfected into primary cultured skeletal muscle cells of rats in vitro. Then the HGF protein secreted in culture medium were measured and the function of secreted HGF was evaluated by studying its antiapoptotic action that prevented primary cultured vascular endothelial cell apoptosis induced by hypoxia.Method1. The more purified skeletal muscle cells(satellite cells) were obtained from Wistar neonate rats and isolated by trypsin digestion method, and purified by velocity sedimentation. The cells were observed under inverted microscope every day and identified by immunochemical stain for Desmin and electron microscope. Vascular Ecs were isolated from thoracic explants of the Wistar rat and identified by immunofluorescence for von Willebrand factor.2. Extraction and amplification the plasmid of pIRES2-EGFP-HGF, The plasmid was comfirmed by BamH I digestion. Transfected plasmid into primary cultured skeletal muscle cells with cationic liposome (Lipofectamine^TM 2000). Transfection efficiency was examed by GFP. The amount of human HGF in the conditioned medium of skeletal muscle cells was measured using ELISA kit.3. Vascular Ecs were treated with different concentrations of HGF (0.5ng/ml,1.0 ng/ml,2.0ng/ml,4.0ng/ml) in the medium for 12 hours in the hypoxic condition(2ï¼…O₂). The morphological change was analyzed using Hoechst staing by microscopy. The levels of apoptosis were quantitated using Annexin V-FITC and PI staing by FCM.Results1. The purification of the skeletal muscle cells was about 95ï¼…by their immunochemical stain for Desmin. The skeletal muscle cells are myoblast and may confluent, differentiate to myobute. The rat vascular ECs exhibited a typical "cobble-stone" pattern when cultured vascular ECs were seeded at high density to achieve confluence. The vascular ECs were identified by immunofluorescence for yon Willebrand factor.2. The skeletal muscle cells were transfected with pIRES2-EGFP-HGF using Lipofectamine^TM 2000 at the ratio of plasmid 1μg to liposome 2μl. Green fluorescent protein(GFP) were observed through fluorescence microscope. Transfection efficiency was 10.8ï¼…. The level of HGF protein in supernatant was 743.5±170.5 pg/ml(1d),1592.5±135.8 pg/ml(2d),3424.5±160.3 pg/ml(3d),5402.0±227.9 pg/ml(4d),6883.8±191.3 pg/ml(5d),3563.0±253.9 pg/ml(7d),775.1±121.6 pg/ml(9d) after gene transfer.3. Compared with control group, HGF could inhibit the apoptosis of vascular Ecs induced by hypoxia (P＜0.05). The apoptosis of the vascular Ecs cells was increased with the decreasing of the HGF concentration. The antiapoptotic effect of HGF in response to hypoxia on the rat vascular ECs was significantly.Conclusions1. Rat skeletal muscle cells and vascular endothelium cells were isolated and cultured successfully in vitro.2. pIRES2-EGFP-HGF were transfected into primary cultured rat skeletal muscle cells successfully.3. The secreted HGF protein can prevents primary cultured vascular endothelium cells apoptosis induced by hypoxia through its antiapoptotic action, in a dose-dependent manner.