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.

Keywords: NADPH oxidase; nitric oxide; nitric oxide synthase; oxidative stress; shear stress; signal transduction.

FIGURE 1.

A, schematic of the shear stress protocol. B, LSS and OSS regulation of superoxide production. Superoxide production was measured using electron spin resonance at different time points after exposure of bovine aortic endothelial cells to LSS or OSS (15 dynes/cm²). Data shown are mean ± S.E. from four to seven individual experiments. *, p < 0.05 compared with the static condition; #, p < 0.05 compared with corresponding LSS.

FIGURE 2.

Colocalization of NOX catalytic and regulatory subunits in endothelial cells at baseline. Bovine aortic endothelial cells were double-labeled with NOXs and p47phox (A–F) or NOXs and NOXO1 (G–L) to study colocalization of different membrane catalytic subunits with p47phox-NOXO1. Colocalization of NOX1 with p47phox (C) or NOXO1 (I) is demonstrated by overlap of immunofluorescent images of NOX1 (A or G, green) with p47phox (B, red) or NOXO1 (H, red). Colocalization of NOX2 with p47phox (F) or NOXO1 (L) is demonstrated by overlap of immunofluorescent images of NOX2 (D or J, green) with p47phox (E, red) or NOXO1 (K, red).

FIGURE 3.

Shear stress regulation of NOX isoforms and eNOS expression. Expression of the NOX isoforms and eNOS was determined by Western blotting analyses from bovine aortic endothelial cells harvested 24 h after exposure to different shear stresses. The representative Western blotting analyses are shown at the top of each panel, and the grouped densitometric data are shown at the bottom (n = 5). The blots are of NOX1 (A), NOX2 (B), and eNOS (C); *, p ≤ 0.05.

FIGURE 4.

The role of p47phox in LSS/OSS regulation of NO and superoxide production. siRNA was used to inhibit the NOX regulatory subunit p47phox to examine its role in the endothelial responses to LSS and OSS. A, expression of p47phox in response to LSS/OSS with and without siRNA treatment (n = 4). B, superoxide measured from control or p47phox siRNA-treated cells under static, LSS, or OSS conditions (n = 3). C, NO measured from control or p47phox siRNA-treated cells under static, LSS, or OSS conditions (n = 3); *, p ≤ 0.05.

FIGURE 5.

The role of NOXO1 in LSS/OSS regulation of NO and superoxide production. siRNA was used to inhibit the NOX regulatory subunit NOXO1 to examine its role in the endothelial responses to LSS and OSS. A, expression of NOXO1 in response to LSS/OSS with and without siRNA treatment (n = 4). B, superoxide measured from control or NOXO1-siRNA treated cells under static, LSS, or OSS conditions (n = 3). C, NO measured from control or NOXO1 siRNA-treated cells under static, LSS, or OSS conditions (n = 3); *, p ≤ 0.05.

FIGURE 6.

LSS activates eNOS via p47phox-dependent H₂O₂ production. siRNA against p47phox was used to examine its role in LSS-stimulated NO production. A, Western blotting data of total and phosphorylated eNOS in bovine aortic endothelial cells treated with p47phox siRNA and LSS (n = 4; *, p < 0.01). B, H₂O₂ levels in LSS- and OSS-stimulated cells indicating a modest increase with LSS and a marked increase with OSS (n = 6–7; *, p < 0.05; #, p < 0.01). C, Western blotting data of total and phosphorylated eNOS in LSS and PEG-catalase (PEG-CAT)-treated endothelial cells (n = 3; *, p < 0.01; #, p < 0.01 versus all others); *, p < = 0.05.

FIGURE 7.

NOX2 regulates LSS production of NO via eNOS phosphorylation. siRNA against NOX2 was used to examine whether NOX2 plays a role in LSS activation of eNOS. A, Western blotting data of total and phosphorylated eNOS in bovine aortic endothelial cells treated with NOX2 siRNA and LSS (n = 4; *, p < 0.001; #, p < 0.001 versus all others). Of note, NOX2 siRNA inhibited LSS activation of eNOS. B, superoxide production in cells treated with NOX2 siRNA and LSS (n = 4; *, p < 0.001 versus static; #, p < 0.05 versus static control siRNA; &, p < 0.05 versus LSS control siRNA). C, NO production in cells treated with NOX2 siRNA and LSS (n = 4; #, p < 0.01 versus all others). Consistent with eNOS activation data, NOX2 siRNA attenuated NO production in response to LSS.

FIGURE 8.

Inhibition of NOXO1 restores eNOS activity uncoupled by OSS. siRNA against NOXO1 was used to examine whether OSS-induced changes in superoxide and NO production are related to the uncoupling of eNOS. Bovine aortic endothelial cells were treated with control or NOXO1 siRNA prior to being subjected to static or OSS conditions. Superoxide was measured from these cells in the presence or absence of L-NAME, a NOS inhibitor, to assess the coupling state of eNOS. Obviously, L-NAME inhibited superoxide production under OSS conditions, implicating uncoupling of eNOS. This response was fully reversed by the treatment with NOXO1 siRNA (n = 3); *, p ≤ 0.05.

FIGURE 9.

Schematic of the pathways elucidated in this work. Under LSS, p47phox and NOX2 complex, causing a modest increase in H₂O₂, which results in activation of eNOS, a consequent increase in NO production, and a decline in superoxide levels. Under OSS, NOX1 and NOXO1 complex, resulting in uncoupling of eNOS. This leads to increased production of superoxide and a decrease in NO bioavailability.