Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Korea.
Pharm Res. 2010 Oct;27(10):2075-84. doi: 10.1007/s11095-010-0206-7. Epub 2010 Jul 7.
A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease.
Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively.
The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments).
An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.
构建并测试了一种具有缺氧诱导型 VEGF 表达系统和氧依赖性降解（ODD）结构域，旨在用于缺血性心肌疾病的基因治疗。
构建了带有或不带有 ODD 结构域的荧光素酶和 VEGF 表达载体系统：pEpo-SV-Luc（或 pEpo-SV-VEGF）和 pEpo-SV-Luc-ODD（或 pEpo-SV-VEGF-ODD）。在体外，在常氧和缺氧条件下，用 HEK 293 细胞评估每种载体类型的基因表达效率。通过 ELISA 估计 VEGF 蛋白的量。将带有或不带有 ODD 结构域的 VEGF 表达载体注射到缺血性大鼠心肌中。通过 Masson 三色染色、α-平滑肌肌动蛋白（α-SMA）免疫染色和 TUNEL 检测分别评估纤维化、新生血管形成和心肌细胞凋亡。
含有 ODD 的质粒载体显著提高了缺氧条件下 VEGF 蛋白的表达水平。VEGF 蛋白产量的增加归因于由于缺氧导致的蛋白稳定性增加。在大鼠心肌缺血模型中，与对照组（生理盐水和 pEpo-SV-VEGF 处理组）相比，pEpo-SV-VEGF-ODD 组的心肌纤维化程度较低，微血管密度较高，心肌细胞凋亡较少。
一种 ODD 介导的 VEGF 表达系统，可在缺氧条件下促进 VEGF 的产生，可能对缺血性心脏病的治疗有用。
