[Delayed disappearance of h-VEGF₁₆₅ mRNA and protein under regulation of hypoxic response element]

Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach for angiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects, such as hemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxic response element (HRE) to control the expression of human VEGF(165) (h-VEGF(165)) under anoxic condition at cell level and also observed the synchron of h-VEGF(165) mRNA and protein expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by using the three-plasmid system and cotransfected to human embryo kidney 293T cells by the calcium phosphate precipitates method. The rAAV vector was purified by chloroform-PEG8000/NaCl-chloroform and added to cultured myocardiocytes. Myocardiocytes of Sprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured under normoxic conditions (21% O2) for 8 h as control; Group II: cultured under anoxic conditions (1% O2) for 8 h; Group III: cultured under normoxic conditions (21% O2) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O2) for 8 h with gene transfer; Group V, VI, VII: cultured under anoxic conditions (1% O2) for 8 h with gene transfer and then tured to normoxic conditions (21% O2) for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O2) for 20 h with gene transfer. After completion of cell culture, the amount of h-VEGF(165) protein in culture supernatant was quantified by using enzyme linked immunosorbent assay (ELISA). Expression of h-VEGF(165) protein in cultured cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR was employed to detect the expression of h-VEGF(165) mRNA. The results revealed that there were no expressions of h-VEGF(165) mRNA and protein in groups I, II, III, VI and VII. After gene transfer, the expressions of h-VEGF(165) protein and mRNA were significantly higher in groups IV and VIII than those in other groups (P<0.01); Immunofluorescence positive cells were observed in groups IV, V and VIII. RT-PCR revealed that a 484-bp strip can be found in groups IV and VIII, but unavailable in other groups. We conclude that HRE is a promising regulator for h-VEGF(165) gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF(165) gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF(165) was inhibited. Although expression of h-VEGF(165) mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF(165) protein was hysteretic to h-VEGF(165) mRNA expression.