Publications (2)0 Total impact
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ABSTRACT: To investigate the effect of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1alpha (Ad-HIF-1alpha-564/402/803) in modulating angiogenesis in vitro. The recombinant adenoviruses Ad-lacZ, Ad-Null, Ad-HIF-1alpha-nature, and Ad-HIF-1alpha-564/402/803 were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions, and the adenoviral titer was determined by end-point dilution assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis, and the infection efficiency was observed by X-gal staining. Human microvascular endothelial cells (HMVECs) were infected with Ad- HIF-1alpha-564/402/803, Ad- HIF-1alpha-nature, or Ad-Null to compare the number of capillary-like tube structures in vitro. The effect of Ad- HIF-1alpha-564/402/803, Ad-HIF-1alpha-nature, and Ad-Null on angiogenesis was evaluated using a chick embryo chorioallantoic membrane (CAM) model. PCR and gene sequencing suggested the correct construction of the recombinant adenovirus HIF-1alpha, and the adenoviral titer reached 1011-1012 PFU/ml. Infection of the hMVECs with Ad-HIF-1alpha-564/402/803 at the optimal multiplicity of infection of 100 pfu/cell resulted in a significantly greater number of capillary-like tube structures than infection by Ad-HIF-1alpha-nature and Ad-Null (P=0.000). Ad-HIF-1alpha-564/402/803 group showed significantly higher microvessel density than Ad-HIF-1alpha-nature, Ad-Null, and PBS groups, with also higher angiogenesis area to CAM area ratio (P=0.01, 0.000, and 0.000, respectively). The triple mutant Ad-HIF-1alpha-564/402/803 can obviously promote the formation of capillary-like tube structures in vitro and modulate angiogenesis in the CAM model, suggesting the capacity of Ad-HIF-1alpha-564/402/803 in promoting angiogenesis under normoxic condition.Nan fang yi ke da xue xue bao = Journal of Southern Medical University 04/2010; 30(4):686-9.
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ABSTRACT: To investigate the effect of recombinant adenovirus-mediated hypoxia-inducible factor-1alpha (Ad-HIF-1alpha) at different doses on angiogenesis in a rabbit model of hind limb ischemia. Left hind limb ischemia was induced in 45 Zealand white rabbits by ligation of the left femoral artery. The rabbits were randomly divided into 5 groups (n=9) to receive intramuscular injections of 0.5 ml saline, 2x10(10) PFU empty vector (Ad-null), or different doses of Ad-HIF-1alpha (2x10(9), 2x10(10) or 2x10(11) PFU) immediately after the operation. On the 7th day after the operation, real-time PCR was used to detect the expression of HIF-1alpha mRNA in the skeletal muscles. Immediately and on the 14th and 28th days after the operation, contrast enhanced ultrasound (CEU) was used to observe the blood perfusion of the hind limb. On the 28th day postoperatively, immunohistochemistry for CD31 was performed to evaluate the microvascular density (MVD). Real-time PCR showed that Ad-HIF-1alpha significantly increased the expression of HIF-1alpha mRNA (P<0.01) in a dose-dependent manner as compared with that in the saline and Ad-null groups (P<0.01). CEU revealed greater blood perfusion in the hind limb of rabbits in association with increased dose of Ad-HIF-1alpha (P<0.05 or P<0.01); similar changes in the MVD was observed following Ad-HIF-1alpha injections as shown by immunohistochemistry (P<0.05 or P<0.01). No significant differences were found either in the blood perfusion or MVD between saline and Ad-null groups (P>0.05). Ad-HIF-1alpha can dose-dependently promote the angiogenesis in the ischemic limb of rabbits.Nan fang yi ke da xue xue bao = Journal of Southern Medical University 02/2010; 30(2):210-3.