The myeloperoxidase product hypochlorous acid oxidizes HDL in the human artery wall and impairs ABCA1-dependent cholesterol transport

Abstract

Although oxidatively damaged lipoproteins are implicated in vascular injury, there is little information regarding the role of high-density lipoprotein (HDL) oxidation in atherogenesis. One potential pathway involves hypochlorous acid (HOCl) produced by myeloperoxidase (MPO), a heme protein secreted by phagocytes. We previously showed that 3-chlorotyrosine is a specific product of HOCl. Therefore, to explore the role of oxidized HDL in the pathogenesis of vascular disease, we used MS to quantify 3-chlorotyrosine in HDL isolated from plasma and atherosclerotic tissue. HDL from human aortic atherosclerotic intima had an 8-fold higher level of 3-chlorotyrosine than plasma HDL. Tandem MS analysis identified MPO as a component of lesion HDL, suggesting that the two interact in the artery wall. Moreover, immunohistochemical studies found that specific epitopes derived from HOCl colocalized with apolipoprotein A-I, the major protein of HDL. These observations strongly support the hypothesis that MPO promotes HDL oxidation in the human artery wall. Levels of 3-chlorotyrosine were elevated in HDL isolated from the blood of humans with established coronary artery disease, suggesting that circulating levels of oxidized HDL represent a unique marker for clinically significant atherosclerosis. HDL or lipid-free apolipoprotein A-I exposed to HOCl was less able to remove cholesterol from cultured cells by a pathway requiring the cell membrane transporter ATP-binding cassette transporter A1. The detection of 3-chlorotyrosine in HDL isolated from vascular lesions raises the possibility that MPO, by virtue of its ability to form HOCl, may promote atherogenesis by counteracting the established antiatherogenic effects of HDL and the ATP-binding cassette transporter A1 pathway.

Fig. 1.

Immunohistochemical analysis of apoA-I, MPO, and proteins modified by HOCl in human atherosclerotic intima. Photomicrographs of adjacent sections of an atherosclerotic coronary artery demonstrating immunostaining for apoA-I (A), proteins modified by HOCl (B), macrophages (C), and MPO (D) are shown. Positive immunohistochemical staining is indicated by a red reaction product. HOCl-modified epitopes colocalize with extracellular apoA-I (A and B, arrows), whereas MPO staining is primarily associated with macrophages (C and D, arrowheads). Original magnification, ×100; hematoxylin counterstain.

Fig. 2.

MS detection of 3-chlorotyrosine in HDL isolated from atherosclerotic human tissue harvested at surgery. Atherosclerotic tissue was obtained from subjects undergoing carotid endarterectomy. HDL was isolated from the supernatant of tissue powder by sequential ultracentrifugation. ¹³C-Labeled internal standards were added, and the protein was hydrolyzed with acid. (A) Western blot analysis of circulating HDL (1) and lesion HDL (2) with an Ab monospecific for apoA-I. Arrow, monomeric apoA-I. (B) Analysis of derivatized amino acids derived from HDL by isotope dilution negative-ion electron capture GC/MS with selected ion monitoring.

Fig. 3.

MS quantification of 3-chlorotyrosine in HDL isolated from plasma and human atherosclerotic lesions. Plasma was obtained from healthy humans and humans with established coronary artery disease (CAD). Human atherosclerotic tissue was obtained at surgery from subjects undergoing carotid endarterectomy. HDL was isolated from plasma and tissue by sequential ultracentrifugation. Oxidized amino acids isolated from hydrolyzed HDL proteins were quantified by isotope dilution GC/MS with selected ion monitoring.

Fig. 4.

Detection of MPO in lesion HDL by 2D liquid chromatography tandem MS analysis. (A) Sequence of MPO. (B) HDL isolated from human lesions was digested with trypsin and subjected to liquid chromatography-electrospray ionization-MS/MS analysis. Four peptides (underlined in A) unique to MPO were identified. The MS/MS spectrum of one peptide (WDGERLYQEARK) from the heavy chain of MPO is shown.

Fig. 5.

Cholesterol efflux activities of native and HOCl-oxidized HDL, apoA-I, and peptide 18A. [³H]Cholesterol-labeled mock- (A and C) or ABCA1-transfected BHK cells were incubated for 4 h with native (Ctrl), H₂O₂-oxidized, HOCl-oxidized HDL (20 μg/ml), or apoA-I (5 μg/ml) (A), for 2 h with 5 μg/ml apoA-I oxidized with the indicated mol ratio of HOCl (B) or for 2 h with control (-) or HOCl-oxidized (+) peptide Ac-18A-NH₂ (20 μg/ml) (C). At the end of the incubation, [³H]cholesterol efflux to the acceptor particle was measured. ^*, P < 0.01 compared with controls.