Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress*

The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS