Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis

Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14+ Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14+ Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca2+-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca2+ ionophore. Exposure of Mac to increasing concentrations of IFNγ (5–100 ng/ml) or oxidized LDL (5–100 μg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca2+ concentration. Immunohistochemical staining revealed that Nox5 is present in CD68+ Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis. •Nox5 variants are expressed in human THP-1 Mon and primary CD14+ Mon.•Nox5 contributes to ROS formation in human Mac.•Nox5 protein expression levels are up-regulated by IFNγ and oxLDL.•Nox5 is localized in the CD68+ Mac-rich area within human atherosclerotic plaques.