A potential therapeutic angiogenesis using a naked DNA containing angiogenin cDNA
Authors: Keerang Park, Soo-Ik Chang, Young-Ill Lee, Heesoon Chang, Young-Hwa Cho, Bong-Su Kang, Jeong-Eun Lee, Ji-Eun Yun, Hyun Park
Journal: Molecular Therapy (2004)

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A promising therapeutic angiogenesis using gene transfer of angiogenic regulators has been extensively investigated and developed for the treatment of various diseases including cardiovascular diseases such as coronary and peripheral artery disease, ischemic disease, restenosis, vein-graft failure, as well as ulcers. Many clinical trials and animal studies have shown that administration of angiogenic factors can increase permeability and nutrient perfusion to induce the formation of supplemental collateral blood vessel. In those studies, the best angiogenic factors are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). However, other angiogenic factors showing better angiogenic activity need to be screened. Although angiogenin has been known that it interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses including cell migration, invasion, proliferation, and neovascularization, a possibility of therapeutic approach using angiogenin remains unclear. Here we investigated a potential therapeutic angiogenesis using a naked DNA containing angiogenin cDNA. Angiogenin cDNA that contains either of whole open reading frame including signal peptide (SPANG1) or only mature peptide region (ANG1) was inserted into the linearized pEGFP-N1 vector (Clontech) to generate two different subclones, so called pSPANG1-EGFP and pANG1-EGFP. HEK293 or TSA-201 cells (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) were used for transfection to express those of two different constructs. The angiogenin-GFP protein expression was monitored by a confocal microscopy and the expression pattern of angiogenin-GFP was examined using western blot analyses. Both of angiogenin-GFP proteins containing the signal peptide or not migrated to the same position of 43 kD. The angiogenin-GFP protein containing the signal peptide in the cell lysates began to be detected at 48 hr post-transfection, whereas the angiogenin-GFP protein of the mature peptide in the cell lysates was first detected at 24 hr after transfection. On the other hand, the secreted angiogenin-GFP from the cells expressing pSPANG1-EGFP was first detected at 72 hr post-transfection, in contrast, the secreted angiogenin-GFP from the cells expressing pANG1-EGFP was first measurable at 24 hr after transfection. The angiogenin-GFP expression from the cells transfected with both type of constructs was maintained to 96 hr post-transfection. The functional activity of the expressed angiogenin-GFP proteins was tested by a wound-healing assay. More studies using pCIneo-SPANG1 and pCIneo-ANG1 are currently under investigation to verify that GFP tagging does not interfere angiogenic activity of angiogenin. Animal studies using four different types of naked DNA containing angiogenin cDNA remain to be done.

Taken together, these data suggest that the naked DNA containing angiogenin cDNA may be a potential therapeutic angiogenesis for the treatment of cardiovascular diseases.