Elsevier

Atherosclerosis

Volume 235, Issue 2, August 2014, Pages 554-561
Atherosclerosis

Niacin decreases leukocyte myeloperoxidase: Mechanistic role of redox agents and Src/p38MAP kinase

https://doi.org/10.1016/j.atherosclerosis.2014.05.948Get rights and content

Highlights

  • Niacin, by inhibiting reactive oxygen species production, decreases leukocyte MPO release and its activity.

  • Niacin, by decreasing MPO activity, attenuates HDL/apo-AI degradation and decreases formation of dysfunctional HDL.

  • Niacin acts as an inhibitor of MPO and explain niacin's role on HDL function and its impact on atherosclerosis.

Abstract

Objectives

Leukocyte myeloperoxidase (MPO) is a major player in the pathogenesis of various chronic diseases including atherosclerosis. This study proposes the novel concept that niacin, through reactive oxygen species (ROS)-mediated signaling, decreases neutrophil MPO release and its activity, protects apolipoprotein-AI (apo-AI) modification and improves HDL function.

Methods

Human blood leukocytes and leukocytic cell line HL-60 cells were treated with niacin, and stimulated with phorbol myristate acetate (PMA). Cellular and released MPO activity in the medium was measured by assessing chlorination of MPO-specific substrate. MPO protein release in the medium and apo-AI degradation was measured by Western blot analysis. Monocyte adhesion to human aortic primary endothelial cells was measured to assess biological function of HDL/apo-AI.

Results

PMA significantly increased leukocyte MPO activity in both intracellular extract and medium. Niacin (0.25–0.5 mM) decreased PMA-induced MPO activity (cellular and released in the media). Niacin also decreased MPO protein mass in the medium without affecting its mRNA expression. Increased NADPH oxidase and ROS production by PMA were also significantly inhibited by niacin. Studies with specific inhibitors suggest that ROS-dependent Src and p38MAP kinase mediate decreased MPO activity by niacin. Niacin blocked apo-AI degradation, and apo-AI from niacin treated cells decreased monocyte adhesion to aortic endothelial cells.

Conclusions

These findings identify niacin as a potent inhibitor of leukocyte MPO release and MPO-mediated formation of dysfunctional HDL. Niacin and niacin-related chemical entities may form important therapeutic agents for MPO-mediated inflammatory diseases.

Introduction

MPO is one of the major neutrophil bactericidal proteins involved in the host defense mechanism against infection through the formation of reactive oxidants and diffusible radical species. MPO utilizes hydrogen peroxide (H2O2) and chloride to generate the powerful oxidant hypochlorous acid (HOCl) with potent host microbicidal activity for invading pathogens [1]. However, MPO-derived oxidants have been proposed to play critical roles in tissue injury and the pathogenesis of various chronic diseases including cancer, renal disease, lung injury, atherosclerosis, multiple sclerosis, Alzheimer's and Parkinson's disease [2]. Specifically, emerging evidence suggests that MPO may be one of the major players in the initiation and progression of atherosclerotic CVD.

Several clinical observations indicated that MPO levels in blood neutrophils were associated with the risk of coronary artery disease (CAD), and patients with the highest quartile of neutrophil-MPO levels had increased risk of CAD compared with those in the lowest quartile [3]. Elevated levels of blood MPO are associated with CAD, predict incident risks for myocardial infarction and cardiac death in subjects with acute coronary syndrome, and predict future risk of CAD in healthy individuals [3], [4], [5], [6], [7]. Furthermore, MPO deficiency in subjects is shown to be cardio-protective [8], [9], [10], [11]. During the past 10–15 years, major research efforts have been focused on understanding mechanisms of action of MPO on atherogenesis. The pathophysiological significance of MPO in atherosclerosis is highlighted by the presence of MPO and MPO-generated specific oxidation products in human atherosclerotic lesions [12], [13]. A growing body of evidence indicates that MPO-generated oxidants modify HDL–apo-AI resulting in increased generation of HDL protein-bound 3-chlorotyrosine and 3-nitrotyrosine [14], [15]. MPO-modified HDL–apo-AI exhibited significantly decreased cholesterol effluxing and anti-inflammatory properties [14], [16], [17], suggesting that MPO participates in HDL–apoAI oxidative modification and the formation of “dysfunctional HDL” with impaired anti-atherosclerotic properties [18], [19], [20]. Additionally, MPO has also been shown to limit the bioavailability of nitric oxide and causes endothelial dysfunction [21], [22], [23].

Despite its critical role in atherosclerosis and other inflammatory diseases, development and/or identification of pharmacological agents that inhibit MPO release and activity have not been extensively studied. MPO is mainly synthesized, packaged and stored in cytoplasmic membrane-bound primary granules of neutrophils. Upon stimulation, neutrophils secrete the granule contents into the membrane-bound phagosomes and extracellular space through a process referred as degranulation or exocytosis [24], [25]. Although mechanisms of neutrophil degranulation and MPO release are not clearly understood, evidence indicates that oxidative stress with increased production of ROS and ROS-mediated cellular activation of Src and p38MAP kinase signaling play major regulatory roles in neutrophil MPO release [26], [27]. Based on our previous work on niacin's ability to inhibit cellular ROS via increased redox state [28], in this study we investigated the novel concept that niacin, through ROS-mediated signaling mechanisms, inhibits neutrophil MPO release and activity, protects apoAI modification and improves HDL's anti-inflammatory and other functions.

Section snippets

Cells and materials

Normal human blood samples for isolation of leukocytes were commercially obtained from Innovative Research Inc. Blood leukocytes were isolated using Ficoll-Paque plus reagent (StemCell Technologies) according to the reagent manufacturer's instructions. Human leukocytic cell line HL-60 cells were obtained from American Type Culture Collection (ATCC). HL-60 cells were grown in Iscove's Modified Dulbecco's medium containing 20% FBS. Normal human aortic endothelial cells (HAEC) were purchased from

Experimental results

Neutrophils are the major source of blood MPO, and accounts for more than 95% of MPO in the circulation. Neutrophils and neutrophil-derived MPO have been extensively used for investigating the pathophysiological role of MPO including its impact on plasma apo-AI oxidation and HDL function [2], [15], [16], [29], [31], [32]. In addition to the freshly isolated human blood neutrophils, human neutrophilic cell line HL-60 was also commonly used and shown to have similar neutrophilic characteristics

Discussion

Neutrophil activation and degranulation are of considerable significance in host defense mechanism as well as in various inflammatory disorders. Among four different types of neutrophil granules, primary (azurophilic) granules are the main production and storage site of MPO. MPO is mainly produced in promyelocytes of primary granules during myelopoiesis and the production of MPO ceases at the promyelocyte/myelocyte transition. Primary granules undergo partial degranulation/exocytosis in

Sources of funding

This study was supported by a Merit Review Grant from the Department of Veterans Affairs.

Disclosures

None.

Conflict of interest

Authors do not have any conflict of interest.

Acknowledgments

We thank Ximing Xiong for technical assistance in Western blot analysis.

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