Abstract 237: The Apolipoprotein A-I Mimetic Peptide D-4F Acts As A Scavenger For Myeloperoxidase-Derived Hypochlorous Acid
Background: Myeloperoxidase (MPO) and its product hypochlorous acid (HOCl) are proatherogenic molecules that functionally modify lipoproteins and impair endothelial cell function. MPO- and HOCl-induced changes in HDL function are thought to occur via modifications at the level of apolipoprotein (apo) A-I.
Objective: We tested the hypothesis that the apo A-I mimetic peptide D-4F, which possesses HDL-like properties, prevents endothelial dysfunction by acting as a reactive substrate for HOCl.
Methods and Results: Rat aortic ring segments were isolated and suspended in an isolated tissue bath and exposed to HOCl (10μM, 30min, n=7). Acetylcholine (Ach)-induced relaxation was inhibited by 51% compared to saline-treated controls (n=8). Concurrent exposure to HOCl and D-4F (10μM, n=9) significantly improved the response to Ach (15% inhibition vs control). Incubation of tissues with D-4F alone (n=9) did not impair endothelial function. Since increased superoxide formation has been implicated in HOCl-induced endothelial dysfunction, we tested whether D-4F influences formation of this oxidant. Electroparamagnetic resonance spectroscopy, in conjunction with BMPO (25mM) spin trapping, was used to determine whether D-4F reduces superoxide generated by xanthine (1mM)/xanthine oxidase (15mU). There was no difference in BMPO resonance spectra in the presence and absence of D-4F at concentrations up to 300 μM. In subsequent studies, effects of HOCl on structural properties of D-4F were assessed. Fluorescence spectroscopy revealed a significant reduction in tryptophan fluorescence of D-4F (20μM) in the presence of HOCl (10μM). Time of flight mass spectrometry was used to characterize reaction products formed by HOCl and D-4F. MS analysis of D-4F (20μM) alone yielded a product with the expected m/z ratio of 2,310. Addition of 10μM HOCl to D-4F yielded a new product with a net gain of 16 mass units. MS/MS analysis confirmed the addition of this mass to the tryptophan residue, reflecting oxidation of this residue.
Conclusion: While D-4F was previously shown to inhibit lesion formation and inflammation in experimental models of atherosclerosis, results of the current studies suggest that D-4F may also serve as a scavenger of MPO-derived HOCl.