Effect Of Hypoxia-inducible Factor 1α Gene Transfected Into Human Mesenchymal Stem Cells On Angiogenesis In Vivo

Objective To evaluate the effect of hypoxia-inducible factor la(HIF-1α)gene transfected into human mesenchymal stem cells (hMSCs)on angiogenesis in vivo. Thereby,provide a novel method and strategy for facilitating angiogenesis of tissue engineered bone.Methods1. Transfection of hypoxia-inducible factor la gene into human mesenchymal stem cells: Recombinant retrovirus vector of human HIF-1α gene was transfected into packaging cell PT67. After G418 screening and amplification,cell clones producing high level recombinant virus were obtained and expanded in vitro.The virus supernatant from infected PT67 cell culture was collected and used to infect hMSCs. After selection by culturing with 200 μg/ml G418, hMSCs with stable expression of HIF-1α gene were obtained. Transgene expression in infected hMSCs was detected by RT-PCR analysis, and HIF-1α protein was detected under hypoxic conditions and normoxic conditions respectively by immunohistochemistry.2. Evaluation of angiogenesis using the chick chorioallantioic membrane (CAM): On day 13 of incubation, transgenic hMSCs and nontransgenic controls were implanted onto the surface of the CAM respectively. After 3 days, CAMs were photographed in ovo under a stereomicroscope and then fixed in Bouin's fluid, dehydrated in ethanol, embedded in resin, mounted on glass slides.Specimens were photographed and capillary areas were measured and analysed using Image-Pro Plus 4.5, data were analyzed for statistical significance using t-test.Results1. After selection by culturing with G418, clones of transgenic hMSCs were obtained and showed bright EGFP expression.2. HIF-1α mRNA expression in transgenic hMSCs increased remarkably comparedwith nontransgenic controls in hypoxia.3. Cell nucleus that stained positively for HIF-la were observed in transgenic hMSCs culturing under hypoxic conditions but no in those culturing under normoxic conditions.4. Macroscopically, in the CAMs implanted with transgenic hMSCs,numerous allantoic vessels developed in the areas surrounding the implants and converged radially toward it. In nontransgenic controls, no vascular response was detectable in the area surrounding the implants.5. In agreement with the macroscopic observations, in the CAMs implanted with transgenic hMSCs numerous newly formed blood vessels were recognizable arranged radially arround the implants with a spoke-wheel pattern. Moreover,dense networks of newly formed capillaries were visible within the transgenic hMSCs nodules.Cell aggregates from nontransgenic controls did not induce any angiogenic response.A higher microvessel density was detectable within the CAMs implanted with transgenic hMSCs than in those treated with nontransgenic controls.6. Image analysis revealed a statistically significant increase in capillary areas in transgenic hMSCs compared with nontransgenic controls (p<0.0\ ).Conclusions1. HIF-la genes have been transfected into hMSCs and expressed effectively.2. Within nucleus of transgenic hMSCs HIF-la protein was stable in hypoxia,whereas unstable in normoxia.3. Transgenic hMSCs appeared to significant induction of angiogenesis compared with nontransgenic controls.4. HIF-la overexpression may represent a promising therapeutic strategy for promoting neovascularization in tissue engineered bone.