Background Therapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion. Vascular endothelial growth factor （VEGF） plays an important role in angiogenesis. However, it has side effects that limit its therapeutic utility in vivo, especially at high concentrations. This study aimed to investigate whether an intramuscular injection of a genetically engineered zinc finger VEGF-activating transcription factor modulates the endothelial progenitor cells （EPC） and promotes therapeutic angiogenesis in a hindlimb ischemia model with type 1 diabetes. Methods AIIoxan （intravenous injection） was used to induce type I diabetes in C57BL/6 mice （n=58）. The ischemic limb received ZFP-VEGF （125 pg ZFP-VEGF plasmid in 1% poloxamer） or placebo （1% poloxamer） intramuscularly. Mice were sacrificed 3, 5, 10, or 20 days post-injection. Limb blood flow was monitored using laser Doppler perfusion imaging. VEGF mRNA and protein expression were examined using real-time PCR and ELISA, respectively. Capillary density, proliferation, and apoptosis were examined using immunohistochemistry techniques. Flow cytometry was used to detect the EPC population in bone marrow. Two-tailed Student＇s paired t test and repeated-measures analysis of variance were used for statistical analysis. Results ZFP-VEGF increased VEGF mRNA and protein expression at 3 and 10 days post-injection, and increased EPC in bone marrow at day 5 and 20 post-injection compared with controls （P〈0.05）. ZFP-VEGF treatment resulted in better perfusion recovery, a higher capillary density and proliferation, and less apoptosis compared with controls （P〈0.05）. Conclusions Intramuscular ZFP-VEGF injection promotes therapeutic angiogenesis in an ischemic hindlimb model with type 1 diabetes. This might be due to the effects of VEGF on cell survival and EPC recruitment.
Objective To construct the zinc finger protein-activating transcription factor (ZFP-ATF) plasmid and evaluate its efficacy in inducing vascular endothelial growth factor (VEGF) expression in EY.HY926 endothelial cells. Methods Firstly, we constructed the ZFP-ATF plasmid, then testified the quantity of VEGF protein in EY.HY926 endothelial cells after transfected with ZFP-ATP plasmid by Western blot, finally, we used the RT-PCR to testify whether the ZFP-ATF can stimulate expression of VEGF splice variants. Results The ZFP-ATF DNA sequences were located the multiclone sites of PVAX1 vector between the site of BamH Ⅰ and Xhol. Western blot result showed VEGF expression in EY.HY926 endothelial cells transfected with ZFP-ATF plasmid was significantly higher than that in cells transfected with VEGF165 (19.95±3.95 vs. 12.15±1.55 μg/μL, P<0.01). RT-PCR result showed VEGF-A mRNA expression level induced by ZFP-ATF was high than that induced by VEGF165. Conclusion ZFP-ATF can up-regulate the VEGF-A expression in comparison with VEGF165, which might have beneficial effects in angiogenesis process.
Li-shan LianYao-guo YangWei LiuLi-long GuoHeng GuanChang-wei LiuYong-jun Li
目的观察体外增殖的人脐带血CD34+细胞对缺血肢体血管生成的影响,探讨CD34+细胞与血管生成的关系。方法采集人脐血细胞并进行CD34+细胞的提取、培养。建立小鼠左后肢缺血模型并随机分为CD34+细胞增殖组(n=5)、CD34+细胞组(n=5)和空白对照组(n=5)3组。采用Di I标记CD34+细胞示踪显像和抗人核抗原抗体(HNA)免疫组织化学方法评估人CD34+细胞向缺血区域的迁移能力,肢体温度、CD31免疫组织化学方法和转化生长因子β1(TGF-β1)m RNA表达评估缺血肢体血流改善情况。结果 Di I标记示踪及HNA显色可见扩增的CD34+细胞出现在小鼠缺血下肢的肌肉组织中并分布在血管周围。注射液体后第14天(t=5.421,P=0.001;t=0.616,P=0.000)和第28天(t=10.780,P=0.000;t=12.123,P=0.000),CD34+细胞增殖组和CD34+细胞组的温度改变百分比均明显高于对照组。注射液体后第28天,CD34+细胞增殖组和CD34+细胞组毛细血管密度分别为592.3±24.6(t=26.386,P=0.000)和530.7±25.5(t=21.502,P=0.000),均明显高于对照组的219.7±19.9;CD34+细胞增殖组和CD34+细胞组的TGF-β1mRNA表达量分别为(0.578±0.050)(t=12.376,P=0.000)和(0.504±0.080)copies(t=7.098,P=0.000),均明显高于对照组的(0.224±0.040)copies。结论体外培养的人脐带血CD34+细胞能向缺血区富集并诱导血管生成,促进血流复通,有望成为治疗下肢缺血的有效途径。
