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


CONCLUSIONS
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.
Wei
Xuan
X
Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. weixuan_xuan@126.com
Tao
Yu
Y
Pei
Jing-xian
JX
Li
Ming-yan
MY
Chen
Jian-wei
JW
Liu
Cheng
C
Wu
Ping-sheng
PS
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002196:Capillaries; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011994:Recombinant Proteins