Cloning and Construction of Adenovirus Expressing Human Angiopoietin-1 or Vascular Endothelial Growth Factor

Document Type

Article

Publication Date

2-1-2003

Description

Aim: We aimed to clone angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) full-length DNAs of human origin and construct replication-deficient adenovirus encoding for either of these two genes which can be potentially served for clinical applications. Methods: VEGF165 and Ang1 full-length cDNAs of human origin were amplified by RT-PCR, verified by sequencing, cloned into a pShuttle-CMV vector, recombined with a E1 and E3 regions double-deleted adenovirus, packaged in 293A cells, and purified by ultracentrifugation. The titers of Ad-Ang1 and Ad-VEGF165 were determined by a tissue culture infectious dose50 method. Expression of Ang1 and VEGF165 proteins in H9C2 cardiac myoblasts was examined by Western blot. To examine the protective properties of Ad-Ang1 and Ad-VEGF165, DNA fragmentation induced by H2O2 was analyzed in H9C2 cells 24 hours after transfection. Ad-GFP served as a vehicle control. Results: Sequencing analysis indicated that there is one base difference at site 1206 (t) in Ang1 compared with that of GeneBank (c, U83508) although the coded amino acids are the same (Ileucine). VEGF165 cDNA sequence was same as that of GeneBank (AB021221). Western blot showed that protein levels of Ang1 and VEGF165 were increased 3.53 and 11.53 fold respectively 24 h after transfection as compared to control. Examination of DNA fragmentation suggested that Ang1 and/or VEGF165 significantly protected H9C2 cells from H2O2 induced apoptosis. Conclusions: The two constructed adenoviral vectors, Ad-Ang1 and Ad-VEGF165, functionally expressed target proteins. We demonstrated, for the first time, that the combined utilization of Ang1 and VEGF165 inhibited apoptosis, in addition to their angiogenesis properties.

This document is currently not available here.

Share

COinS