Abstract 17139: In Vivo selection of Target Peptides to Dysfunctional Endothelial Cells Using M13 Phage Peptide Display Library in Mice
Background: Atherosclerotic lesions develop predominantly at branches, bends, and bifurcations in the arterial tree because these sites are exposed to low or disturbed blood flow, which exerts low-oscillatory shear stress (OSS) on the vessel wall. In vivo biopanning of phage display is a powerful strategy for directly identifying peptides that target the vasculature of normal or disturbed flow lesion in living animals. In this study, we performed phage display to identify specific peptide sequences that bind selectively to dysfunctional endothelial cells (ECs) induced by disturbed flow.
Method and results: As a model of acutely induced atherosclerosis by disturbed flow, we used partial carotid artery ligation model in C57BL/6 mouse. The endothelium of partially ligated left carotid artery (LCA) was exposed to OSS and showed increased atheroma formation within 2 weeks. 7-amino acid peptide display library (Ph.D. C7C library, NEB) was injected into mice at 3 days after LCA ligation. Phages from ligated LCA was recovered, amplified and then subjected to three rounds of in vivo Biopanning. After the third round, phages DNA of all colonies from carotid arteries were sequenced. Six dysfunctional ECs specific peptides (DESP 1~6) were selected and bioinformatic analysis was performed. To confirm specificity, selected peptide-expressing phages were prepared and phage binding assay was performed in cultured mouse aortic endothelial cells (iMAECs) and HUVECs under laminar (LSS) and OSS. In-Vivo phage binding assay was also compared by en face staining of carotid arteries. In three of six peptides (DESP3,4,6), we confirmed that binding of these peptide-expressing phages to ECs significantly increased in OSS condition compared to LSS condition. In addition, In-Vivo phage binding assay showed that DESP3 and DESP6-expressing phages specifically bind to ligated LCA in en face staining.
Conclusions: These results show that in vivo biopanning using a peptide phage display library make it possible to identify peptides bind to dysfunctional ECs induced by disturbed flow, efficiently and specifically. DESP could be used to deliver therapeutic agents including drugs and nucleic acid specifically to the atherosclerotic dysfunctional ECs. (Funded by NRF-2011-0019695)
- © 2013 by American Heart Association, Inc.