Secretory Expression Of PR39 Following Adeno-associated Viral-encoding Fusion Gene Transfer Induces Angiogenesis In Hypoxia Chick Embryo

Objective: Exogenous delivery of PR39 may provide a useful approach to the treatment of myocardial ischemia. The purpose of this study is to assess the value of AAV-mediated NT4-TAT-His-PR39 fusion gene expression on anti-apoptosis in hypoxia vascular endothelial cell and on angiogenesis in chicken embryos, so as to discuss the protection of PR39 for myocardial ischemia.Methods: PR39 cDNA was connected with NT4, TAT, 6×His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively transducted stably with AAV-NT4-TAT-His-PR39 and empty vector (EV), the fusion protein expression were confirmed by immunocytochemistry using mouse 6×His antibody. Hypoxia (1%O2) resulted in an increase of apoptosis in ECV304, as determined by flow cytometry (FCM) detection analysis and HIF-1αactivity by immunocytochemistry. Thirty chicken embryos were infected randomly with AAV expressing NT4-TAT-His-PR39 fusion protein or equal volume EV and phosphate-buffered saline (PBS) respectively in hypoxia (5% O2) and normoxia environment. The vessel density of the chicken embryos chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software.Results: The expression of the 6×His fusion protein and HIF-1αprotein were significantly higher in ECV304 cells transduced with PR39 fusion gene than in the control group (Paired Samples Statistics: t=51.68, P<0.001 and Statistics: t=11.23,P<0.001, respectively ). Hypoxia-induced cellular apoptosis existed markedly difference between the AAV-NT4-TAT-His-PR39 group and the PBS group by FCM analysis (AAV-PR39, 7.35%±0.43%; PBS, 32.58%±0.39%; P<0.005). There was no difference in the vessel density of CAM among the three groups in normoxia environment (PBS, 11.9%±0.5%; EV, 11.0%±0.4%; AAV-PR39, 9.8%±0.5%; P>0.05). However, in hypoxia environment, the difference in the vessel density of CAM between the AAV-NT4-TAT-His-PR39 group and the control EV group or the PBS group was significant (5.65%±0.6% versus 2.20%±0.5%, P<0.001 and 5.65%±0.6% versus 2.23%±0.4%, P<0.001).Conclusion: When cultured ECV304 cells were exposed to hypoxia, the recombinant AAV vector-delivered fusion gene NT4-TAT-His-PR39 could increase its expression of HIF-1αand could inhibit hypoxia-induced apoptosis in cultured endothelial cells. Chicken embryos cultured under hypoxic conditions showed a considerable increase in angiogenesis of CAM that were exposed to AAV vectors expressing NT4-TAT-His-PR39.