Identification of circulating cells interacted with integrin α4β1 ligand peptides REDV or HGGVRLY

Recently, artificial blood vessels modified by integrin α4β1 ligand, such as REDV, showed endothelialization improvement and antithrombotic properties have been reported. Early endothelialization was affected by the type of circulating cells captured by the peptide in the initial transplantation state, however, it is still not clarified. In this study, we identified in vitro circulating cells bound with the peptides arginine-glutamic acid-aspartic acid-valine (REDV) or histidine-glycine-glycine-valine-arginine-leucine-tyrosine (HGGVRLY). The effect of free C- or N-terminal of HGGVRLY on the type of peptide-binding cells was also studied. The rat circulating cells were isolated from blood and incubated with 5(6)-carboxyfluorescein (5/6-FAM, F) labeled F-REDV (C-terminal free), F-HGGVRLY (C-terminal free), or HGGVRLY-F (N-terminal free). Furthermore, peptide-binding cells were identified by co-staining with various antibodies labeled with PE, PerCP/Cy5.5, or APC. N-terminal free HGGVRLY-F was found to bind to more circulating cells than C-terminal free F-REDV and F-HGGVRLY. The ratio of integrin α4β1 positive cell bound with F-REDV, F-HGGVRLY, or HGGVRLY-F reached over 90 %, demonstrating that HGGVRLY is also a ligand of integrin α4β1. Among identified cell types, we found that F-REDV mainly bounds with EPC and BMSC, while F-HGGVRLY with BMSC. HGGVRLY-F bounds with EPC and BMSC, exhibiting a higher EPC binding ratio than F-REDV and F-HGGVRLY.