[Effects of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1alpha in modulating angiogenesis].

Abstract

OBJECTIVE:

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.

METHODS:

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.

RESULTS:

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).

CONCLUSION:

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.

• Authors: Xuan Wei, Yu Tao, Jing-xian Pei, Ming-yan Li, Jian-wei Chen, Cheng Liu, Ping-sheng Wu
• Journal: Nan fang yi ke da xue xue bao = Journal of Southern Medical University (Nan Fang Yi Ke Da Xue Xue Bao) Vol. 30 Issue 4 Pg. 686-9 (Apr 2010) ISSN: 1673-4254 [Print] China
• PMID: 20423826 (Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• HIF1A protein, human
• Hypoxia-Inducible Factor 1, alpha Subunit
• Recombinant Proteins

Topics

• Adenoviridae (genetics, metabolism)
• Animals
• Capillaries
• Chick Embryo
• Chorioallantoic Membrane (blood supply)
• Endothelial Cells (cytology, metabolism)
• HEK293 Cells
• Humans
• Hypoxia-Inducible Factor 1, alpha Subunit (biosynthesis, genetics)
• Mutation
• Neovascularization, Physiologic (genetics)
• Recombinant Proteins (biosynthesis, genetics)