Highly stable, fluorescence-labeled heptapeptides substituted with a D-amino acid for the specific detection of oxidized low-density lipoprotein in plasma.

Probes that can detect oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques can be useful for the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε-amino group of N-terminus Lys in the absence/presence of 6-amino-n-caproic acid (AC) linker to FITC-(FITC)KP6 and (FITC-AC)KP6-can be useful as fluorescent probes for the specific detection of ox-LDL. In this study, to develop the fluorescent peptides with high plasma stability for the specific detection of ox-LDL, we investigated the interaction of (FITC)KP6 and (FITC-AC)KP6 substituted with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6-with ox-LDL, and the in vitro stability of these peptides in mouse plasma. (FITC)dKP6 and (FITC-AC)dKP6 bound with high specificity to ox-LDL in a dose-dependent manner, and also to ox-LDL in the mouse plasma. Furthermore, (FITC)dKP6 was more stable than (FITC)KP6 in mouse plasma (102.1% versus 69.0% remained after 1 h). These findings strongly suggest that (FITC)dKP6 and (FITC-AC)dKP6 may be effective fluorescent probes with higher plasma stability than (FITC)KP6 and (FITC-AC)KP6 for the specific detection of ox-LDL.

C-reactive protein specifically enhances platelet-activating factor-induced inflammatory activity in vivo.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. C-reactive protein (CRP) is an acute-phase plasma protein that increases rapidly and dramatically in response to inflammation. In this study, we investigated the effect of the interaction between CRP and PAF on inflammatory responses in vivo. From binding analysis using a time-resolved fluorometric assay, CRP bound to PAF and its precursor/metabolite lyso-PAF in a concentration-dependent manner. In addition, CRP bound to several phospholipids containing lysophosphatidylcholine, which bears structural resemblance to PAF and lyso-PAF, sphingosylphosphorylcholine, and lysophosphatidylethanolamine more readily than to lysophosphatidic acid and lysophosphatidylserine. In in vivo experiments using a rat model of hind paw oedema, CRP increased PAF-induced rat paw oedema in a dose-dependent manner, without causing the oedema itself, but it did not increase histamine and serotonin-induced paw oedema. Furthermore, the receptor for CRP, lectin-like oxidized low-density lipoprotein receptor 1 was not involved in the increase in PAF-induced inflammatory responses caused by CRP. These results indicate that CRP can specifically enhance PAF-induced inflammatory activity through binding to PAF and lyso-PAF. Therefore, CRP may accelerate the pathogenesis of numerous inflammatory diseases caused by PAF.

Novel fluorescently labeled peptide compounds for detection of oxidized low-density lipoprotein at high specificity.

The probes for specific detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the ε-amino group of K at the N-terminus to FITC in the presence/absence of 6-amino-n-caproic acid (AC) linker to FITC--(FITC-AC)KP6 and (FITC)KP6--both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the ε-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC)KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL.