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ISSN 1672-9145 Acta Biochim Biophys Sin 2005, 37(4): 227–233 CN 31-1940/Q

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Identification of Peptides Inhibiting Adhesion of Monocytes to the Injured Vascular Endothelial Cells through Phage-displaying Screening

Yu GUO1, Jia ZHANG2, Ji-Cheng WANG2, Feng-Xiang YAN1, Bing-Yang ZHU1, Hong-Lin HUANG1, and Duan-Fang LIAO1,2*

1Institute of Pharmacy and Pharmacology, and 2Institute of Single Nucleotide Polymorphism, Nanhua University, Hengyang 421001, China

Abstract Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the “adsorption-elution-amplification” procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

Key words random peptide phage library; vascular endothelial cell; whole-cell screening; cell adhesion; caveolin-1; eNOS

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Received: November 26, 2004 Accepted: March 1, 2005

This work was supported by the grants from the National Natural Science Foundation of China (No. 30171084 and No. 30470719) and the National Basic Research Program of China (No. G2000056905)

*Corresponding author: Tel/Fax, 86-734-8281308; E-mail, [email protected]