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

Abstract

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.

Apocynin reduces blood pressure and restores the proper function of vascular endothelium in SHR

“…apocynin, but NOX1, NOXO1, and NOX4 expression remains unaltered NOX1 is regulated [49] and colocalized [50], and it requires NOXO1 for its activation [51] and ROS production [52]. The study groups did not differ with respect to NOX1 NOX2 is activated and colocalized with p47phox to assemble the active enzyme, and this interaction is essential for ROS production [49][50][51][52].…”

“…The study groups did not differ with respect to NOX1 NOX2 is activated and colocalized with p47phox to assemble the active enzyme, and this interaction is essential for ROS production [49][50][51][52]. NOX2 (Fig.…”

NOX isoforms in the development of abdominal aortic aneurysm

“…Different isoforms separately or together display different functions. NOX1 has been shown to be involved in diabetic endothelial dysfunction and endothelial cell response to atherogenic oscillatory shear stress [15], [16]. On the contrary, NOX4 plays a critical role in cardiac I/R injury [9].…”

“…For example, NOX1, but not NOX2 or NOX4, underlies endothelial dysfunction in streptozotocin-induced type 1 diabetes mellitus [15]. While protective laminar shear stress assembles NOX2-p47phox at basal levels to increase eNOS phosphorylation and nitric oxide (NO) production, atherogenic oscillatory shear stress activates NOX1-NOXO1 complex to uncouple eNOS and sustain oxidative stress [16]. On the other hand, NOX4 but not NOX1/2, is activated to mediate eNOS uncoupling in ischemia/reperfusion (I/R)-injured heart, and involved in cardiac damage and remodeling [9], [17], [18].…”

Mitochondrial Ca 2+ transport in the endothelium: regulation by ions, redox signalling and mechanical forces

“…/ROS and a concomitant decrease in NO production/bioavailable levels ( figure 3c,d) [196]. Based on the higher levels of mROS/cytosolic ROS in OS-exposed ECs compared with PS (or SS)- figure 3c,d), which may be the reason behind the OS-induced EC inflammation and decreased cell resistance to apoptosis.…”