Biochemical characterization of the binding of cyclic RGDyK to hepatic stellate cells.

Activated hepatic stellate cells (HSCs) play a crucial role in the development of liver fibrosis. Noninvasive monitoring of the activation of HSCs has been challenging due to the lack of specific receptors or motifs on the cells. The present study provides the evidence that integrin alpha v beta 3 expressed on HSCs is a biomarker reflecting the activation of HSCs. Solid-phase synthesis of cRGDyK (Arg-Gly-Asp-(D)Tyr-Lys) peptide and FAM-conjugated peptide were employed for binding to integrin alpha v beta 3. The increased expression of integrin alpha v and beta 3 at mRNA and protein levels was detected during HSC activation. The affinity of cRGDyK to integrin alpha v beta 3 was examined by both radioligand binding assay and FAM-conjugated peptide binding measurements. Quantitative RT-PCR and Western blotting showed a less dramatic, but significant increase in alpha v and beta 3 integrin mRNA and protein expression following activation of rat HSCs. Radioiodinated cRGDyK binds to both purified and membrane-bound integrin alpha v beta 3 with high affinity in a dissociable manner. FAM-conjugated cRGDyK was coupled to activated HSCs in a time- and dose-dependent, receptor-mediated manner. Activated HSCs express sufficient number of integrin alpha v beta 3 receptor. cRGDyK peptide binds to both purified and membrane-bound integrin alpha v beta 3 with high affinity in a reversible fashion. Thus, the cRGDyK peptide represented a new agent potentially useful for the diagnosis of liver fibrosis.