Hypoxia-inducible vascular endothelial growth factor gene therapy for ischemic myocardium using the oxygen dependent degradation domain

Abstract

Gene therapy with angiogenic factors is a promising strategy for the treatment of cardiovascular diseases. A number of genes are currently under investigation for the therapeutic purpose of cardiovascular diseases, vascular endothelial growth factor (VEGF) is one of the most effective therapeutic genes for neovascularization. However, uncontrolled and sustained expression of exogenous VEGF could induce adverse effects on normal tissues, such as tumor growth, hemangioma, and retinopathy. Thus, the controllable and diseases-specific gene expression systems are required for safe and successful gene therapy. Most hypoxia specific expression systems have been developed to induce gene expression under hypoxia. However, leaky expression by the basal promoter activity under normoxia should also be reduced to avoid pathological angiogenesis. In this study, a hypoxia-inducible luciferase expression vector with the oxygen dependent degradation (ODD) domain and the erythropoietin (Epo) enhancer, pEpo-SV-Luc-ODD, was constructed. The ODD domain is located in the central region of HIF-1 and stabilizes HIF-1 under hypoxia. Therefore, the ODD domain was used for post-translational induction of VEGF expression under hypoxia. The combination of the Epo enhancer and the ODD domain effectively increased gene expression in HEK 293 cells or primary cardiomyocytes under hypoxia. The hypoxia-inducible VEGF expression vector, pEpo-SV-VEGF-ODD, was constructed and injected into rat ischemic myocardium. pEpo-SV-VEGF or normal saline was injected as a control. Five days after gene transfer, VEGF expression increased in all groups, and the highest expression was obtained in the pEpo-SV-VEGF-ODD group. Masson`s trichrome staining and -smooth muscle actin ( -SMA) staining showed significantly less fibrotic areas in the pEpo-SV-VEGF-ODD group as compared with other groups. In addition, apoptosis of cardiomyocytes was reduced in the pEpo-SV-VEGF-ODD group. These results demonstrate in vivo efficacy of an ODD mediated hypoxia-inducible system in ischemic myocardium. Therefore, the VEGF gene under the control of hypoxia-specific expression system may be useful for treatment of ischemic heart disease.