Inhibition of myeloperoxidase-mediated endothelial dysfunction by glycocalyx-targeted nitroxides

Abstract

The leukocyte-derived heme enzyme myeloperoxidase (MPO) represents a key therapeutic target in vascular inflammatory diseases, where it mediates endothelial dysfunction, which manifests as the impaired bioactivity of endothelial-derived nitric oxide (NO•). During inflammation, MPO is released by activated leukocytes and accumulates within the sub-endothelium of the vessel wall. Here, it mediates endothelial dysfunction by promoting NO• consumption via its NO• oxidase activity and producing reactive oxidants (e.g. hypochlorous acid (HOCl) and NO2•) that oxidatively damage the extracellular matrix, stimulate vascular superoxide anion radical production and impair vascular NO• bioactivity. Tempol and related nitroxides have been shown to exert protection in a variety of inflammatory conditions, with this usually attributed to their broad actions as radical scavengers and superoxide dismutase mimetics. Recent reports also show that nitroxides inhibit MPO-catalysed HOCl production and scavenge NO2• in simple model systems. The capacity of nitroxides to protect the endothelium against MPO-mediated damage, as well as structural features that may optimise this activity, have not yet been examined. Here, we synthesised a range of novel nitroxides conjugated to polyamines in order to optimise nitroxide delivery into the subendothelium where MPO and its oxidants localise. We show for the first time that both simple and novel endothelial-targeted nitroxides efficiently inhibit MPO-catalysed chlorination and nitration reactions in the vascular endothelium, protect against MPO-mediated endothelial dysfunction in isolated rat aorta, and inhibit MPO-catalysed NO• consumption in human plasma. Furthermore, our data identified a novel nitroxide (putrescine TEMPO) that is maximally internalised into the endothelium and optimally protects against MPO chlorination, nitration and NO• oxidase activities. Overall, nitroxides are ideal for development as MPO-targeted therapeutics. This work also examined a diverse range of pharmacological agents, including nitroxides, with established redox activities as MPO inhibitors or substrates and/or radical scavengers, for their effects on MPO NO• oxidase activity in human plasma and physiological model systems containing endogenous MPO substrates and antioxidants. Overall, our data reveal novel insights into how widely-used pharmacological agents strongly influence MPO NO• oxidase activity. Kinetic analyses revealed the mechanistic basis of drug activities and identified criteria for developing drugs with improved activity against MPO NO• oxidase activity.