Human Mesenchymal Stem Cells Modified By Hepatocyte Growth Factor Gene Promote Angiogenesis

Partâ… Isolation, Culture-expansion, modification by Ad-HGF and identification of human marrow MSC (hMSC)Objective To obtain human bone marrow mesenchymal stem cells modified by an adenoviral vector carrying human hepatocyte growth factor cDNA (Ad-HGF) for the experiments below. Methods Human bone marrow mononuclear cells were isolated by Percoll gradient centrifugation and cultured for 72 hours. The un-attached cells were discarded and the culture was maintained till the confluence density reached up to 80%. The adherent cells were passaged for further investigations or stored in liquid nitrogen. hMSC were infected by Ad-HGF and maintained in a medium without serum supplement for 2 hours. Afterwards, the cells were incubated in alpha-MEM containing fetal bovine serum for 48 hours. The morphological and phenotypical features of Ad-HGF-modified human MSC (hMSC/Ad-HGF) were observed. The secretion of HGF by hMSC/Ad-HGF was analyzed with enzyme-linked immunoabsorption assay (ELISA). Results Primary hMSC were successfully isolated and culture-expanded. hMSC infected by Ad-HGF maintained the fibroblast-like morphology. Similarly to hMSC, hMSC/Ad-HGF were positive for CD44 and CD29, and negative for CD31, CD34, CD45 and CD 14. hMSC/Ad-HGF secreted significantly higher amount of HGF than hMSC (P<0.001). Conclusion HGF gene-modified hMSC, whilst overexpressing HGF, maintain the original morphology and phenotype characteristics.Partâ…¡hMSC/Ad-HGF promote chicken embryonic angiogenesis Objective To observe the angiogenesis-promoting activities of hMSC modified by HGF gene and investigate the underlying mechanisms. Methods hMSC were infected by Ad-HGF 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 hMSC, recombinant basic fibroblast growth factor, and alpha-MEM as a control. The expression levels of bFGF, VEGF, angiopoietin-1 and angiopoietin-2 were evaluated by RT-PCR assay. Results Gene-modified hMSC exhibited greatest activity to promote angiogenesis while the angiogenic response was comparable between hMSC and bFGF treatment, all of which were significantly obvious than that observed in control (P<0.01). RT-PCR analysis revealed that hMSC constitutively expressed multiple angiogenesis-associated growth factors and their levels seemed up-regulated by HGF gene transfer. Conclusion HGF gene-modified hMSC adopt potent angiogenesis-promoting function and might be useful in the management of ischemic disorders.Partâ…¢hMSC/Ad-HGF promote angiogenesis of local limb ischemia in ratsObjective To evaluate the angiogenesis-promoting activities of human mesenchymal stem cells (hMSCs) modified by hepatocvte growth factor (HGF) in vivo and further investigate the underlying mechanisms. Methods hMSC/Ad-HGF were injected intramuscularly into the ischemic area of a rat limb ischemia model and 21 days later, laser Doppler perfusion imaging was performed to detect the blood flow of the limbs. Microvessel density (MVD) was evaluated by H&E staining and immunohistochemistry on skeletal muscle sections. Evaluation of proliferation, migration and forming tube-like structures on ECV304 cells impacted by hMSC/Ad-HGF was determined by MTT, Transwell migration and tubercular structure formation assays. Results Injection of hMSC/Ad-HGF and hMSC, compared with that of controls, significantly improved the blood flow perfusion of the ischemic limbs (P<0.01). The relative means of blood perfusion in hMSC/Ad-HGF were comparable to those of sham-controls and greater than those of hMSC-transplanted rats (P<0.05). The amounts of microvessels per high-fold field were highest in the hMSC/Ad-HGF-treated muscles, followed by those of sham-controls and hMSC, and all of them were greatly higher than those of controls (P<0.01). The supernatants of hMSC/Ad-HGF were shown to promote the proliferation of ECV304 cells and the stimulation activity was greater than that from recombinant epithelial growth factor at a concentration of 20ng/ml (P<0.001). In agreement, the conditioned medium of hMSC/Ad-HGF exhibited a greater activity than epithelial growth factor to enhance the trans-membrane migration and tubercular structure formation of ECV304 cells. Conclusion HGF gene-modified human mesenchymal stem cells promote the proliferation, migration and vascular formation of endothelial cells, thus activating the angiogenesis process.