P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists.

P-selectin is a leukocyte adhesion receptor expressed on activated vascular endothelium and platelets that mediates leukocyte rolling and attachment. Because P-selectin is critically involved in inflammation, we used phage display libraries to identify P-selectin-specific peptides that might interfere with its proinflammatory function. Isolated phage contained a highly conserved amino acid motif. Synthetic peptides showed calcium-dependent binding to P-selectin, with high selectivity over E-selectin and L-selectin. The peptides completely antagonized adhesion of monocyte-derived HL60 cells to P-selectin and increased their rolling velocities in flow chamber experiments. Peptide truncation and alanine-scanning studies indicated that an EWVDV (single-letter amino acid codes) consensus motif sufficed for effective inhibition. Intriguingly, the apparent avidity of the peptides was increased 200-fold when presented in a tetrameric form (2 microM versus 10 nM), which is consistent with the proposed divalent interaction of P-selectin glycoprotein ligand 1 (PSGL-1) with P-selectin. As the EWVDV peptides inhibit the binding of an established glycoside ligand for P-selectin (sulfated Lewis A), it is conceivable that EWVDV interacts with or in close proximity to the actual carbohydrate recognition domain of P-selectin, without being a direct structural mimic of sialyl Lewis(x). These ligands are among the most potent antagonists of P-selectin yet designed. Their high affinity, selectivity, and accessible synthesis provide a promising entry to the development of new anti-inflammatory therapeutics and might be a powerful tool to provide important information on the binding site of P-selectin.

Uptake and processing of modified bacteriophage M13 in mice: implications for phage display.

Internalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries. Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice. (35)S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively. Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t(1/2) = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage. Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min. In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle.