Figure 1. Selection and in vitro validation of specific phages for pro-atherogenic endothelium under disturbed flow.

At 3 days post-partial ligation surgery on the left carotid ligation (LCA), the C57BL/6 mice (n = 3) were injected with a CX₇C peptide library (1 × 10¹¹ pfu) via the tail-vein. Ten minutes after the injection, the mice were perfused with saline and phages were recovered from various organs, including the carotid arteries. (a) Enrichment of phages in each screening round. Phages from the ligated LCA were recovered, amplified and re-injected in two consecutive rounds. Bars represent the titers of phages bound to the ligated LCA or non-ligated RCA in each selection round. Shown are means ± SEM of three independent experiments. *Significant difference (LCA vs. RCA, p < 0.05). (b) In vivo distribution of selected phages in mice. Organs were removed, homogenized and the number of phages in the tissues was determined at third rounds in vivo panning. At 3 days after surgery, the C57BL/6 mice (n = 3) were injected with eluted phages (1 × 10¹¹ pfu) in second round. Ten minutes after the injection, the mice were perfused with saline and phages bound to organs including carotid arteries were collected. Bound phages were counted in various organs. Bars represent the titers of phages from the third round of amplification in various tissues. *Significant difference compared with other organs, with the exception of the liver and spleen (p < 0.05). (c,d) In vitro validation of individually selected phage clones in ECs. iMAECs (c) or HUVECs (d) were exposed to flow or static conditions for 48 h, and cells were incubated with each selected peptides (SPs)-displaying phage individually for 1 h. Cells were washed and bound phages were stained using an anti-M13 bacteriophage antibody. Bars represent the percent of phage positive ECs and are presented as means ± SEM of three independent experiments. *Significant difference compared with LSS conditions (p < 0.05). ^#Significant difference compared with static conditions (p < 0.05).