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Hypoxic response elements control expression of human vascular endothelial growth factor 165 genes transferred to ischemia myocardium in vivo and in vitro
Author(s) -
Jiang Bo,
Dong Hongyan,
Zhang Zhongming,
Wang Wei,
Zhang Yiqian,
Xu Xiahong
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1070
Subject(s) - vascular endothelial growth factor , in vivo , western blot , ischemia , gene expression , angiogenesis , andrology , immunohistochemistry , genetic enhancement , messenger rna , microbiology and biotechnology , northern blot , hypoxia (environmental) , biology , gene , medicine , immunology , chemistry , vegf receptors , organic chemistry , oxygen , biochemistry
Background Vascular endothelial growth factor (VEGF) gene transfer with recombinant adeno‐associated viral (rAAV) vector for ischemia heart disease therapy is being increasingly studied. However, uncontrolled long‐term expression of VEGF may cause some side effects. Therefore, an attempt to develop an effective gene control system for safeguarding against such side effects should be made. Pathphysiologically, an ideal control system for VEGF gene expression is letting it respond to hypoxia. We used nine copies of hypoxic response element (HRE) to regulate expression of h VEGF 165 in the myocardium, and tried to elucidate the feasibility and safety of the application of the HIF‐1‐HRE system. Methods Cardiomyocytes of neonatal Sprague Dawley rats were cultured and incubated with rAAV‐9HRE‐ h VEGF 165 , and pig ischemic heart models were established and rAAV‐9HRE‐ h VEGF 165 was injected into ischemia myocardium. RT‐PCR, Western blot, ELISA, and immunohistochemistry were used to determine h VEGF 165 expressions of cultured cardiomyocytes and myocardium under hypoxic and reoxygenation conditions. Results The results of RT‐PCR and ELISA determinations revealed that, in cultured cardiomyocytes, expressions of h VEGF 165 mRNA and protein were up‐regulated under hypoxic conditions. After 4 h of reoxygenation, h VEGF 165 mNRA expression was decreased, and disappeared following 8 to 12 h of reoxygenation ( P < 0.01). RT‐PCR and Western blot also showed that, under myocardial ischemia, h VEGF 165 expression was increased significantly ( P < 0.01). Following myocardial reperfusion, both h VEGF 165 mRNA and protein expressions were inhibited ( P < 0.01). The new vessels in the reperfusion condition were decreased. Conclusions This study suggested that 9HRE can effectively control h VEGF 165 gene expression in vivo and in vitro . It has feasibility for using the HIF‐1‐HRE system for regulation of angiogenic factor expression in ischemia heart. Copyright © 2007 John Wiley & Sons, Ltd.