Zinc Finger Protein-activating Transcription Factor Up-regulates Vascular Endothelial Growth Factor-A Expression in Vitro

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.

Nerve Growth Factor Promotes Angiogenesis and Skeletal Muscle Fiber Remodeling in a Murine Model of Hindlimb Ischemia

Background： Therapeutic angiogenesis has been shown to promote blood vessel growth and improve tissue perfusion.Nerve growth factor （NGF） has been reported to play an important role in both physiological and pathological angiogenesis.This study aimed to investigate the effects of NGF on angiogenesis and skeletal muscle fiber remodeling in a murine model of hindlimb ischemia and study the relationship between NGF and vascular endothelial growth factor （VEGF） in angiogenesis.Methods： Twenty-four mice were randomly allocated to normal control group （n =6）, blank control group （n =6）, VEGF gene transfection group （n =6）, and NGF gene transfection group （n =6）.The model of left hindlimb ischemia model was established by ligating the femoral artery.VEGF165 plasmid （125 μg） and NGF plasmid （125 μg） was injected into the ischemic gastrocnemius of mice from VEGF group and NGF group, respectively.Left hindlimb function and ischemic damage were assessed with terminal points at 21th day postischemia induction.The gastrocnemius of four groups was tested by hematoxylin-eosin staining, proliferating cell nuclear antigen and CD34 immunohistochemistry staining, and myosin ATPase staining.NGF and VEGF protein expression was detected by enzyme-linked immunosorbent assay.Results： On the 21th day after surgery, the functional assessment score and skeletal muscle atrophy degree of VEGF group and NGF group were significantly lower than those of normal control group and blank control group.The endothelial cell proliferation index and the capillary density of VEGF group and NGF group were significantly increased compared with normal control group and blank control group （P 〈 0.05）.The NGF and VEGF protein expression of NGF group showed a significant rise when compared with blank control group （P 〈 0.05）.Similarly, the VEGF protein expression of VEGF group was significantly higher than that of blank control group （P 〈 0.05）, but there was no significant difference of the NGF protein expression between VEGF group and blank control group （P 〉 0.05）.The type Ⅰ skeletal muscle fiber proportion in gastrocnemius of NGF group and VEGF group was significantly higher than that of blank control group （P 〈 0.05）.Conclusions： NGF transfection can promote NGF and VEGF protein expression which not only can induce angiogenesis but also induce type Ⅰ muscle fiber expression in ischemic limbs.