Chen Feng, Tan Zui, Dong Chang-yuan, Chen Xiao, Guo Shu-fang
Department of Vascular Surgery, Zhongnan Hospital, Wuhan University, Wuhan 430071, China.
Acta Pharmacol Sin. 2007 Apr;28(4):493-502. doi: 10.1111/j.1745-7254.2007.00527.x.
Angiopoietin-1 (Ang1) and vascular endothelial growth factor A (VEGF) play important roles in vascular formation and maturation, suggesting a combination of these 2 would be a promising therapy for ischemic diseases. So we constructed an adeno-associated virus (AAV) vector, simultaneously encoding human VEGF(165) and Ang1 (AAV-VEGF/Ang1), and investigated its therapeutic effect in a rabbit ischemic hind-limb model.
Four experimental groups were used to prepare the rabbit ischemic hind-limb model following AAV vectors intramuscular administration as follows: PBS (phosphate buffered solution), AAV-VEGF, AAV-Ang1, AAV-VEGF/Ang1.
Eight weeks after administration, human VEGF(165) and Ang1 were detected by RT-PCR, Western blotting and histochemical staining methods in AAV-VEGF/Ang1 transduced muscles. Group AAV-VEGF/Ang1 showed a significantly increased blood-flow recovery in ischemic hind-limbs compared with the other groups. Histological staining for alkaline phosphatase showed that capillary density of group AAV-VEGF/Ang1 or AAV-VEGF was significantly higher than that of group PBS or AAV-Ang1. Histological immunostaining for smooth muscle alpha-actin (alpha-SMA) revealed that group AAV-VEGF/Ang1 had the highest density of alpha-SMA-positive vessels compared with the other groups. Vascular leakage, one of the major adverse effects induced by VEGF, was very severe in group AAV-VEGF, but the permeability was obviously reduced when VEGF was co-expressed with Ang1 in group AAV-VEGF/Ang1.
AAV vectors can simultaneously encode 2 proteins which can be efficiently and stably co-expressed in transduced tissues. AAV-mediated VEGF and Ang1 gene transfer enhances neovascularization, prevents capillary leakage, and improves blood flow in a rabbit hind-limb ischemic model. These findings suggest that intramuscular administration of AAV-VEGF/Ang1 may be useful in the treatment of ischemic diseases.
血管生成素-1（Ang1）和血管内皮生长因子A（VEGF）在血管形成和成熟过程中发挥重要作用，这表明二者联合应用可能是治疗缺血性疾病的一种有前景的疗法。因此，我们构建了一种同时编码人VEGF（165）和Ang1的腺相关病毒（AAV）载体（AAV-VEGF/Ang1），并在兔缺血后肢模型中研究其治疗效果。
采用四个实验组，在肌肉注射AAV载体后制备兔缺血后肢模型，具体如下：磷酸盐缓冲液（PBS）组、AAV-VEGF组、AAV-Ang1组、AAV-VEGF/Ang1组。
给药8周后，通过RT-PCR、蛋白质印迹法和组织化学染色法在AAV-VEGF/Ang1转导的肌肉中检测到人VEGF（165）和Ang1。与其他组相比，AAV-VEGF/Ang1组缺血后肢的血流恢复显著增加。碱性磷酸酶组织学染色显示，AAV-VEGF/Ang1组或AAV-VEGF组的毛细血管密度显著高于PBS组或AAV-Ang1组。平滑肌α-肌动蛋白（α-SMA）组织学免疫染色显示，与其他组相比，AAV-VEGF/Ang1组α-SMA阳性血管密度最高。VEGF诱导的主要不良反应之一血管渗漏在AAV-VEGF组非常严重，但在AAV-VEGF/Ang1组中当VEGF与Ang1共表达时通透性明显降低。
AAV载体可同时编码2种蛋白质，它们能在转导组织中高效稳定地共表达。AAV介导的VEGF和Ang1基因转移可增强新生血管形成，防止毛细血管渗漏，并改善兔后肢缺血模型中的血流。这些发现表明肌肉注射AAV-VEGF/Ang1可能对缺血性疾病的治疗有用。
