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Objective- Inflammation-driven endothelial dysfunction initiates and contributes to the progression of atherosclerosis, and MPO (myeloperoxidase) has been implicated as a potential culprit. On release by circulating phagocytes, MPO is thought to contribute to endothelial dysfunction by limiting NO bioavailability via formation of reactive oxidants including hypochlorous acid. However, it remains largely untested whether specific pharmacological inhibition of MPO attenuates endothelial dysfunction. We, therefore, tested the ability of a mechanism-based MPO inhibitor, AZM198, to inhibit endothelial dysfunction in models of vascular inflammation. Approach and Results- Three models of inflammation were used: femoral cuff, the tandem stenosis model of plaque rupture in Apoe -/- mice, and C57BL/6J mice fed a high-fat, high-carbohydrate diet as a model of insulin resistance. Endothelial dysfunction was observed in all 3 models, and oral administration of AZM198 significantly improved endothelial function in the femoral cuff and tandem stenosis models only. Improvement in endothelial function was associated with decreased arterial MPO activity, determined by the in vivo conversion of hydroethidine to 2-chloroethidium, without affecting circulating inflammatory cytokines or arterial MPO content. Mechanistic studies in Mpo -/- mice confirmed the contribution of MPO to endothelial dysfunction and revealed oxidation of sGC (soluble guanylyl cyclase) as the underlying cause of the observed limited NO bioavailability. Conclusions- Pharmacological inhibition of MPO is a potential strategy to limit endothelial dysfunction in vascular inflammation. Visual Overview- An online visual overview is available for this article.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque., Methods and Results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene., Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

Atherosclerosis is the main pathophysiological process underlying coronary artery disease (CAD). Acute complications of atherosclerosis, such as myocardial infarction, are caused by the rupture of vulnerable atherosclerotic plaques, which are characterized by thin, highly inflamed, and collagen-poor fibrous caps. Several lines of evidence mechanistically link the heme peroxidase myeloperoxidase (MPO), inflammation as well as acute and chronic manifestations of atherosclerosis. MPO and MPO-derived oxidants have been shown to contribute to the formation of foam cells, endothelial dysfunction and apoptosis, the activation of latent matrix metalloproteinases, and the expression of tissue factor that can promote the development of vulnerable plaque. As such, detection, quantification and imaging of MPO mass and activity have become useful in cardiac risk stratification, both for disease assessment and in the identification of patients at risk of plaque rupture. This review summarizes the current knowledge about the role of MPO in CAD with a focus on its possible roles in plaque rupture and recent advances to quantify and image MPO in plasma and atherosclerotic plaques. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO) activity contributes to arterial inflammation, vascular dysfunction and disease, including atherosclerosis. Current assessment of MPO activity in biological systems in vivo utilizes 3-chlorotyrosine (3-Cl-Tyr) as a biomarker of hypochlorous acid (HOCl) and other chlorinating species. However, 3-Cl-Tyr is formed in low yield and is subject to further metabolism. Recently, we reported a method to selectively assess MPO-activity in vivo by measuring the conversion of hydroethidine to 2-chloroethidium (2-Cl-E + ) by liquid chromatography with tandem mass spectrometry (LC–MS/MS) (J. Biol. Chem., 289, 2014, pp. 5580–5595). The hydroethidine-based method has greater sensitivity for MPO activity than measurement of 3-Cl-Tyr. The current methods paper provides a detailed protocol to determine in vivo and ex vivo MPO activity in arteries from mouse models of vascular inflammation and disease by utilizing the conversion of hydroethidine to 2-Cl-E + . Procedures for the synthesis of standards, preparation of tissue homogenates and the generation of 2-Cl-E + are also provided in detail, as are the conditions for LC–MS/MS detection of 2-Cl-E + . [ABSTRACT FROM AUTHOR]

Abstract: Smokers have an elevated risk of atherosclerosis but the origin of this elevated risk is incompletely defined, though increasing evidence supports a role for the oxidant-generating enzyme myeloperoxidase (MPO). In previous studies we have demonstrated that smokers have elevated levels of thiocyanate ions (SCN−), relative to nonsmokers, and increased thiol oxidation, as SCN− is a favored substrate for MPO, and the resulting hypothiocyanous acid (HOSCN) targets thiol groups rapidly and selectively. In this study we show that increased HOSCN formation by MPO diminishes damage to nonthiol targets on both model proteins and human plasma proteins. Thus high SCN− levels protect against HOCl- and MPO-mediated damage to methionine, tryptophan, lysine, histidine, and tyrosine residues on proteins. Furthermore, levels of the HOCl-mediated marker compound 3-chlorotyrosine and the cross-linked product dityrosine are decreased. Plasma protein 3-chlorotyrosine levels induced by HOCl exposure in nonsmokers are elevated over the levels detected in smokers when exposed to identical oxidative insult (P<0.05), and a strong inverse correlation exists between plasma SCN− levels and 3-chlorotyrosine concentrations (r=0.6182; P<0.0001). These correlations were also significant for smokers (r=0.2724; P<0.05) and nonsmokers (r=0.4141; P<0.01) when analyzed as individual groups. These data indicate that plasma SCN− levels are a key determinant of the extent and type of protein oxidation induced by MPO on isolated and plasma proteins and that smoking status and resulting high SCN− levels can markedly modulate the levels of the widely used biomarker compound 3-chlorotyrosine. [Copyright &y& Elsevier]

The involvement of myeloperoxidase (MPO) in human atherosclerotic plaque progression is well documented, including its potential role in plaque rupture that can cause acute myocardial infarction. Despite this, establishing a causal relationship between MPO and atherosclerosis-associated complications has proven difficult in mouse models. In the present study we employed a mouse model of atherosclerotic plaque instability (Circ Res 113:252) to examine the role of MPO in endothelial dysfunction and atherosclerotic plaque vulnerability. Unstable plaques were introduced in apolipoprotein E gene knockout (Apoe –/– ) mice fed a Western diet for 13 weeks by placing a tandem stenosis around the right carotid artery after 6 weeks of diet, and MPO activity was partially blocked by oral administration of a 2-thioxanthine (AZM198). Unstable carotid plaques, but not stable plaques in the aortic root and arch, contained measurable MPO activity as assessed by the in vivo conversion of hydroethidine to 2-chloroethidium and non-invasive magnetic resonance imaging using MPO-gadolinium (bis-5-hydroxytryptamine-DTPA-Gd). Administration of AZM198 did not change circulating white blood cell counts, plasma concentrations of pro-inflammatory cytokines and lipids, nor did it affect atherosclerotic lesion size in the aortic root. In contrast, AZM198 significantly attenuated arterial MPO activity in unstable plaques and endothelial dysfunction in the abdominal aorta. Inhibition of MPO activity was associated with increased plaque stability in the carotid artery, as indicated by the increase in cap thickness and the cap-to-lesion ratio, without affecting necrotic core size and arterial monocytes. Together, these data indicate that vulnerable but not stable atherosclerotic lesions in Apoe –/– mice exhibit MPO activity, and that pharmacological inhibition of MPO stabilizes vulnerable plaques. [ABSTRACT FROM AUTHOR]