Abstract 355: NOX Deficiency Alters Endothelial Response to Blood Flow--Induced Shear Stress
Increased levels of Reactive oxygen species (ROS) lead to vascular complications like atherosclerosis. Important producers of ROS are the family of NADPH oxidases (NOXes). NOX subtypes have recently been shown to be responsive to blood flow-induced shear stress. Shear stress is also responsible for the focal nature of atherosclerosis by regulating expression of the athero-protective transcription factors KLF2 and Nrf2. These transcription factors inhibit pro-inflammatory and promote antioxidant gene expression. High, pulsatile blood flow in straight vessel segments induces KLF2 and Nrf2. In contrast, disturbed flow at vessel bends and bifurcations inhibits them, thus rendering the vessel wall susceptible to systemic risk factors for vascular disease. Several studies indicate that ROS might influence KLF2/Nrf2 expression and/or activation. In this study we set out to determine the interrelationship between shear stress-induced NOX subtypes and KLF-2/Nrf2 activation.
HUVEC were exposed to 1.5 Pascal pulsatile shear stress (athero-protective) for 6 days in IBIDI flow chambers and compared to static conditions (athero-prone). NOX 1 and NOX 5 were expressed at undetectable levels for PCR analyses in static HUVEC. NOX 2 and NOX 4 were expressed in static HUVEC. Upon exposure to shear stress, NOX 2 expression decreased to undetectable levels while NOX 4 was reduced by 50%. This shift in NOX 2/NOX 4 balance resulted in a ROS shift from O2- to H2O2, as determined by DHE and CMDCF staining. Subsequently, we silenced NOX 2 and 4 by transducing HUVEC with lenti-viral shRNAs, using non-targeting shRNA and non-transduced cells as controls. Silencing did not affect static or shear stress-induced KLF2 and Nrf2 expression, activation, or target gene expression. However, we did observe an Nrf2-independent effect of NOX silencing on antioxidant expression, which is currently under further investigation.
Our preliminary results show a shift in NOX/ROS balance under shear stress, without affecting KLF2 or Nrf2. Together with the anti-oxidant activities of KLF2 and Nrf2, this shift in oxidant balance might be causative in the quiescent, anti-atherogenic effect of high, pulsatile shear stress on ECs.
Author Disclosures: L.C.J. Winkel: None. R.D. Fontijn: None. R.J.P. Musters: None. J.J. Wentzel: None. A.J.G. Horrevoets: None. K. van der Heiden: Research Grant; Significant; K.v.d.H. is funded by an NWO-Veni grant 916.11.015.
- © 2014 by American Heart Association, Inc.