Objective: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. This study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. Approach: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) were used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. Results: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. Conclusion: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, this study highlights an important role for NOX5 in atherosclerosis.
Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis
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Josiane Fernandes da Silva, Juliano V Alves, Julio A Silva-Neto, Rafael Menezes Costa, Karla Bianca Neves, Rheure Alves-Lopes, Livia De Lucca Camargo, Francisco Rios, Augusto C Montezano, Rhian M Touyz, Rita C Tostes; Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis. Clin Sci (Lond) 2021; CS20210468. doi: https://doi.org/10.1042/CS20210468
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