1119. Isolation of a Novel Therapeutic Angiogenic Peptide, AG-30, by High Throughput Screening Using the HVJ-E Vector

Abstract

Top of pageAbstract In a large proportion of critical limb ischemia patients, the anatomic extent and distribution of arterial occlusive disease make the patients unsuitable for operative or percutaneous revascularization. Thus, novel strategies, such as therapeutic angiogenesis, are expected to play an increasing role in the treatment of these patients. The goal of the present study was to screen a human cDNA library to isolate potent and powerful angiogenic factors that may produce better patient outcomes. Our study utilizes Hemagglutinating Virus of Japan (HVJ; Sendai virus) envelope vector (HVJ-E) for high throughput screening, based on the advantage of this vector demonstrated in previous studies. The advantages of high throughput functional screening system based on HVJ-E included: 1) rapid preparation of the vector containing the DNA library; 2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity; and 3) easy cloning of candidate genes by transformation of E. coli. These advantages result in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. In fact, the present experiment was able to isolate candidate genes within four weeks. A human heart cDNA library (approximately 1 |[times]| 107 independent clones) was amplified, and approximately 4 |[times]| 106 clones were randomly infused into HVJ-E vector, which were transferred to human aortic endothelial cells (HAEC). This assay identified three of the genes that were superior to VEGF in terms of c-fos promoter activity. One gene in particular showed marked HAEC proliferative activity and c-fos promoter activity in comparison to VEGF and was used for subsequent experiments. Further study on this clone revealed that 96 bp segment is the active site of the clone. Thus, 30-amino acid peptide (AG-30) that derived from the segment was synthesized and used for further analysis. AG-30 showed a dose-dependent increase in HAEC viability and cell migration. Further, to confirm the angiogenic ability, an in vitro tube formation assay was performed. Tube formation was greater in response to AG-30 treatment than to recombinant VEGF. The in vivo angiogenic activity of AG-30 has been evaluated by an established mouse Matrigel plug assay. Neovessels containing intact red blood cells were observed in the Matrigel plug containing AG-30. The increased presence of neovessels in the AG-30-treated group relative to the other groups was confirmed by immunostaining with anti-CD31 antibody. Real-time RT PCR revealed that AG-30 strongly and specifically upregulate the mRNA of IGF-1 of HAEC in a time-dependent manner for 72 hours. The relatively durability of the effect of AG-30 makes it potentially suitable for clinical angiogenic therapy. Secondary effects of AG-30 may include: 1) IGF-1-mediated upregulation of VEGF expression, and 2) IGF-1-mediated Akt activation, thereby further promoting angiogenesis. Since small peptides are typically not stable in vivo, development of a delivery system to establish slow release of this peptide would be required for clinical utility.