Protein sulfhydryl group oxidation and mixed-disulfide modifications in stable and unstable human carotid plaques

Abstract

Objectives: Oxidative stress has been implicated in the outcome of atherosclerotic plaques. However, at present, no data are available neither on the degree of plaque protein sulfhydryl groups oxidation nor on its relationship with plaque vulnerability. We investigated the entity of protein-SH oxidative modifications, focusing on low molecular weight thiols adduction, in human carotid plaque extracts in relation to plaque stability/instability.

Methods: Plaque stability/instability was histologically assessed. The extent of protein-SH oxidative modifications was established by a differential proteomic approach on fluorescein-5-maleimide-labeled plaque extracts and corresponding plasma samples from 48 endarterectomized patients. The analysis on protein thiolation was performed by capillary zone electrophoresis.

Results: We observed a higher protein-SH oxidation of both plasma-derived and topically expressed proteins in unstable plaques, partly due to higher levels of S-thiolation. Conversely, in plasma, none of the investigated parameters discriminated among patients with stable and unstable plaques.

Conclusions: Our results suggest the presence of a more pronounced oxidative environment in unstable plaques. Identifying specific oxidative modifications and understanding their effects on protein function could provide further insight into the relevance of oxidative stress in atherosclerosis.

Figure 1

Representative SDS-PAGE patterns of F5M-labeled proteins from stable and unstable plaque extracts (a) and the corresponding CBB staining (b). In (a), inverted fluorescent image is reported.

Figure 2

Graphic representation of results obtained by SDS-PAGE of F5M-labelled proteins extracted from stable (black bars) and unstable (empty bars) plaques. The fluorescence intensity signals (arbitrary units) of single bands (a, b, and c) and single lanes (d) were normalized for the corresponding signals obtained after Blue Coomassie G-250 staining.

Figure 3

Representative 2D electrophoretic patterns of F5M-labelled proteins and the corresponding CBB staining from stable ((a) and (c)) and unstable ((b) and (d)) plaque extracts. In (a) and (b), inverted fluorescent images are reported. The molecular weight scale was constructed from protein standards (Invitrogen BenchMark Protein Ladder) run alongside the focused strip in the second dimension. The pI scale is based on the linear immobilized pH gradient over 7 cm strips. The numbers indicated on the gels correspond to the spot numbers given in Table 1.

Figure 4

Typical electropherograms of total (a) and protein-bound (b) LMW-thiols from carotid plaque extracts. Cys-Gly: cysteinylglycine; Hcy: homocysteine; Cys: cysteine; GSH: glutathione; Glu-Cys: glutamylcysteine; I.S.: internal standard.