Immunohistochemical evidence for the myeloperoxidase/H₂O₂/halide system in human atherosclerotic lesions

Abstract

The`oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol-engorged foam cells. To identify HOCl-modified-epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl-modified LDL) that do not cross-react with other LDL modifications, i.e. peroxynitrite-LDL, hemin-LDL, Cu 21 -oxidized LDL, 4-hydroxynonenal-LDL, malondialdehyde-LDL, glycated-LDL, and acetylated-LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl 2 added as reagent or generated by the MPO/H 2 O 2 /halide system. Immunohistochemical studies revealed pronounced staining for HOCl-modified-epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion-prone location (type I). HOCl-oxidationspecific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H 2 O 2 /halide system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.

Class B Scavenger Receptors CD36 and SR-BI Are Receptors for Hypochlorite-modified Low Density Lipoprotein

“…HOCl-LDL enhances platelet reactivity and release reaction (21,22), and most importantly, HOCl converts LDL into a high uptake form for mouse peritoneal macrophages (23), leading to the formation of cholesteryl ester (CE)-laden foam cells, which are the hallmark of fatty streaks and the earliest recognizable lesion of atherosclerosis. The presence of 3-chlorotyrosine in human atherosclerotic lesions (12), the presence of HOCl-modified epitopes inside and outside monocytes/macrophages, endothelial cells, and smooth muscle cells in human and rabbit lesion material (7,24,25), and the presence of HOCl-modified apolipoprotein (apo) B-100 extracted from advanced human atherosclerotic lesions (26) supported the view that the MPO/hydrogen peroxide/chloride system converts LDL into an atherogenic form under in vivo conditions. However, the cellular mechanisms leading to HOCl-LDL uptake/processing by macrophages has not yet been addressed.…”

“…HOCl-modified apoB-100 is present in human atherosclerotic lesions, and HOCl-modified (lipo)proteins are predominantly cell-associated and present in monocytes and macrophages (7,26,36). Despite intense interest in the role of HOCl in inflammatory injury and vascular disease, including recognition of HOCl-LDL by macrophages (23,47) at physiologically relevant HOCl concentrations, information on specific binding properties and corresponding receptors mediating HOCl-LDL uptake is lacking.…”

G-CSF Induces Membrane Expression of a Myeloperoxidase Glycovariant that Operates as an E-selectin Ligand on Human Myeloid Cells

“…Our in vitro studies of myeloid cells coincubated with HUVECs indicate that G-CSFtreated myeloid cells are angiotoxic to E-selectin-expressing endothelial cells; this effect is ameliorated by interruption of E-selectin receptor/ligand interactions and by inhibition of MPO activity. Although further studies are necessary to directly assess the role(s) of MPO-EL in vasculopathy, these findings offer novel perspectives on the association between MPO expression and vascular injury: in vivo, MPO-EL could serve as a key effector of sickle cell crises (28), myocardial infarction and coronary artery disease (29,30), atherosclerosis (31,32), and stroke (33), conditions which have all been linked to MPO. Indeed, G-CSF administration can trigger angina and myocardial infarction (34,35).…”

Probucol Protects against Hypochlorite-induced Endothelial Dysfunction

“…Vascular endothelial cells overlying atherosclerotic lesions contain myeloperoxidase and proteins modified by its principle product HOCl (13,41), and blood vessels exposed to HOCl exhibit a defect in endothelium-derived ⅐ NO bioactivity manifested as impaired endothelium-dependent arterial relaxation (16). Here we showed that dietary or exogenously added probucol attenuated this vascular dysfunction induced by HOCl and that HOCl oxidized probucol to DPQ with intermittent formation of DTBP.…”