Hypoxic response elements control expression of human vascular endothelial growth factor165 genes transferred to ischemia myocardium in vivo and in vitro

✍ Scribed by Bo Jiang; Hongyan Dong; Zhongming Zhang; Wei Wang; Yiqian Zhang; Xiahong Xu

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 406 KB
• Volume: 9
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.1070

✦ Synopsis

Abstract

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.