Abstract 21188: Differential Roles of NOX1/NOXO1 and NOX2/p47phox Complexes in Endothelial Responses to Laminar and Oscillatory Shear Stress
Introduction: Whereas laminar shear stress (LSS) is vasoprotective, oscillatory shear stress (OSS) is atherogenic. Their potentially differential effects on vascular NAD(P)H oxidase (NOX) complexes however, remain unclear.
Hypothesis: Different NOX machineries differentially account for endothelial responses to different shear stress.
Methods and Results: A software-controlled valve-operated device-OsciFlow was used to apply LSS or OSS to endothelial cells for 2 hr, and cells were harvested at 1, 6 and 24 hr later. Some cells were transfected with siRNAs specific for different NOX isoforms before application of shear. Production of superoxide and nitric oxide was determined by electron spin resonance. Co-localization of NOX isoforms with their regulatory subunits was assessed by immunocytochemical staining, which revealed that p47phox is co-localized with NOX1, NOX2 and NOX4 in endothelial cells, and that NOXO1 (NADPH oxidase organizer 1) is co-localized with NOX1. Under static condition, RNAi inhibition of NOX2 decreased superoxide production whereas RNAi inhibition of p47phox increased nitric oxide production, implicating a “housekeeping” role of NOX2-p47phox complex in basal superoxide production. LSS induced a transient increase in superoxide production at 1 hr, followed by attenuation of superoxide production after 6 hr. OSS on the other hand induced a time-dependent increase in superoxide production. NOX2 expression was downregulated by either LSS or OSS. Despite that NOXO1 and p47phox were both robustly upregulated by OSS, only NOXO1 inhibition by RNAi decreased superoxide production and boosted nitric oxide bioavailability. Inhibition of p47phox however had no effect. Of note, LSS and OSS both increased nitric oxide production although LSS is much more potent.
Conclusions: OSS seems to induce a sustained increase in superoxide production through a NOXO1/NOX1 machinery. Downregulation of the “house-keeping” NOX2/p47phox complex by LSS may contribute to its stimulatory effects on NO production. The simultaneous production of superoxide and nitric oxide in response to OSS however, may facilitate peroxynitrite-dependent vascular injury.
- © 2010 by American Heart Association, Inc.