Hypoxic response elements control expression of human vascular endothelial growth factor(165) genes transferred to ischemia myocardium in vivo and in vitro.
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 hVEGF(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-hVEGF(165), and pig ischemic heart models were established and rAAV-9HRE-hVEGF(165) was injected into ischemia myocardium. RT-PCR, Western blot, ELISA, and immunohistochemistry were used to determine hVEGF(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 hVEGF(165)mRNA and protein were up-regulated under hypoxic conditions. After 4 h of reoxygenation, hVEGF(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, hVEGF(165) expression was increased significantly (P < 0.01). Following myocardial reperfusion, both hVEGF(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 hVEGF(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.
Jiang
Bo
B
Department of Cardiothoracic Surgery, Affiliated Hospital of Xuzhou Medical College; Xuzhou, Jiangsu, PR China.
Dong
Hongyan
H
Zhang
Zhongming
Z
Wang
Wei
W
Zhang
Yiqian
Y
Xu
Xiahong
X
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D000229:Dependovirus; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D008856:Microscopy, Fluorescence; D017202:Myocardial Ischemia / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020218:Response Elements / Q000235:genetics*; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
D016895:Culture Media, Serum-Free; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A