Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kappaB in endothelial cells.

NF-kappaB signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-kappaB activity is transient, our earlier studies have demonstrated a persistent activation of NF-kappaB in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-kappaB signaling kinases (IKKalpha and beta) that may be responsible for the sustained activation of NF-kappaB in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKKbeta, but it is also persistently induced for a prolonged time frame. The IKK activity (both alpha and beta) is blocked by apocynin (400 microM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 microM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-kappaB activation selectively in low shear-induced endothelial cells.

IkappaBalpha-dependent regulation of low-shear flow-induced NF-kappa B activity: role of nitric oxide.

We have investigated the role of inhibitor kappaBalpha (IkappaBalpha) in the activation of nuclear factor kappaB (NF-kappaB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm(2). Low shear for 6 h resulted in a reduction of IkappaBalpha levels, an activation of NF-kappaB, and an increase in kappaB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IkappaBalpha in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IkappaBalpha is the major factor in the low shear-induced activation of NF-kappaB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IkappaBalpha/NF-kappaB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-kappaB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 microM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IkappaBalpha and concomitantly reduced NF-kappaB binding activity and kappaB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IkappaBalpha levels in HAEC and that the application of low shear flow increases NF-kappaB activity by attenuating NO generation and thus IkappaBalpha levels.