The role of myeloperoxidase in endothelial dysfunction

Abstract

Endothelial dysfunction (ED) is a clinically relevant feature of cardiovascular disease (CVD), which manifests as impaired bioactivity of endothelial-derived nitric oxide (NO), produced by endothelial-NO synthase (eNOS). Increasing studies implicate the pro-inflammatory enzyme myeloperoxidase (MPO) as a mediator of ED in CVD. MPO employs hydrogen peroxide (H2O2) and chloride ions to form the powerful oxidant hypochlorous acid (HOCl), and can also utilize nitrite (NO2-) or thiocyanate (SCN-) to yield nitrogen dioxide (*NO2) or hypothiocyanous acid (HOSCN), respectively. A pre-requisite for MPO-mediated ED is its sequestration in the endothelium, where it catalyses localized oxidative reactions that impact on endothelial function. This thesis investigated the implications of these oxidative reactions on eNOS activity and NO bioactivity. Incubation of endothelial cells (EC) with MPO resulted in its time-dependent accumulation into the sub-endothelium, accompanied by an intracellular re-distribution of eNOS from the plasma membrane to the cytosol. MPO-containing EC showed enhanced H2O2 consumption that coincided with the production of HOCl. In MPO-containing EC, acute exposure (0-10 min) to H2O2 induced a rapid increase in intracellular calcium (Ca2+) levels due to capacitative Ca2+ entry and this coincided with increased eNOS activity and coordinated changes to its phosphorylation at Ser-1177 and de-phosphorylation at Thr-495. Inhibition of Ca2+ and calmodulin-signalling, but not protein kinases known to phosphorylate eNOS at Ser-1177, inhibited MPO-induced eNOS phosphorylation at this site, indicating the involvement of a novel, Ca2+/calmodulin-dependent signalling pathway. Prolonged exposure (>30 min) of MPO-containing EC with H2O2 resulted in reduced cellular uptake of the eNOS substrate L-arginine, eNOS activity and protein stability, and increased production of superoxide anion radical (O2*-), events attenuated by supplementation with NO2- or SCN-. Isolated vessel studies indicated that endothelial-localized MPO impaired NO bioactivity and endothelial-dependent vasorelaxation by increasing O2*- production, which was prevented with the O2*− scavenger, polyethylene glycol O2*- dismutase, or supplementation with NO2- or SCN-. Together, these studies show that despite acutely activating eNOS, MPO-derived HOCl impairs endothelial function in a superoxide-dependent manner, which is attenuated by NO2- or SCN- supplementation. These studies provide important insights into the cell signalling and oxidative pathways by which endothelial-localized MPO promotes ED.