A new strategy for therapeutic angiogenesis using dual-transgene selection plasmid vector in a mouse model of hindlimb ischemia

Abstract

Critical limb ischemia (CLI), as a severe manifestation of peripheral artery disease, is emerging as a major concern for aging society worldwide. Cell based gene therapy has been involved in a treatment for CLI to induce angiogenesis in ischemic area. Despite the many studies demonstrated its efficacy as promising results, claudication and amputation along with cardiovascular events are critical complications that need advanced therapy. In the present study, we have established a simplified method to improve the transduced gene effect based on cell-based gene therapy using the selection plasmid vector with sorting system. We constructed the selection plasmid vector by incorporating vascular endothelial growth factor (VEGF) coding sequences as a target gene into pEF1/HisC vector, and subsequently cloned enhanced green fluorescence protein (EGFP) as a selection marker gene with its CMV promoter. By using the selection plasmid vector (pEF1/HisC:VEGF:EGFP), both VEGF and EGFP genes were expressed evenly in cardiomyocytes (H9C2 cells) and mesenchymal stem cells (MSCs) in vitro. Dual-genes expressing MSCs were collected solely via flow cytometry sorting system and the sorted cells were injected into the ischemic hindlimb of mouse. A significant improvement of blood perfusion was displayed at day 21 after hindlimb ischemia induction compared to that in a typical cell-based gene delivery, which means without sorting system, or intact MSCs control. These results suggest that the selection plasmid vector together with sorting system efficiently improved therapeutic angiogenesis in ischemic hindlimb by augmenting the presence of transduced VEGF genes in the based MSCs. Furthermore, this selection plasmid vector could be a practical use in a wide range of disease, which use cell-based gene therapy as a treatment tool, by simply replacing the target gene or based cell source.