Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

Abstract

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.

Figure 1

Hemodynamic forces acting on the blood vessel wall and the potential sensors initiating mechanotransduction. (A) Hemodynamic forces experienced by the blood vessel wall including: 1) shear stress, which is the tangential frictional force acting on the vessel wall due to blood flow, defined as force/wall area (e.g., dyn/cm²); 2) normal stress, which is the force acting perpendicularly on the vessel wall due to hydrostatic pressure; and 3) tensile stress, which is the force acting on the vessel wall in the circumferential direction due to stretch of the vessel wall. (B) Potential mechano-sensors likely to initiate mechanotransduction in endothelial cells, including G protein-coupled receptor (GPCR), mechano-activated ion channels, growth factor receptor, glycocalyx, caveolae, membrane lipids (fluidity), junction proteins, cytoskeleton network, integrins, focal adhesion kinase (FAK), etc. [5]. In mechanotransduction process the mechanical signals trigger the perturbation of these mechano-sensors, thus generating biochemical signals and initiating mechano-sensitive signaling cascades that lead to downstream gene expression.

Figure 2

Roles of ROS/NO in mechano-sensor mediated redox signaling in ECs exposed to shear stress. Hemodynamic shear stress is detected by various mechano-sensors present on the membrane of ECs, triggering a network of signaling pathways that alter gene and protein expression, eventually leading to anti-atherogenic or pro-atherogenic effects on ECs. In this process, ROS triggers oxidative modification and NO triggers S-nitrosation of many target molecules, together with activation of antioxidant and pro-oxidant enzymes to regulate the redox status of ECs. Shear stress with a regular flow pattern (steady or pulsatile) produces lower levels of ROS (hence to be anti-atherogenic) than shear stress with an irregular flow pattern (disturbed or oscillatory) that is pro-atherogenic.

Figure 3

Devices used to carry out in vitro studies to examine the influence of flow (shear stress) on ECs. (A) Parallel-plate flow chamber. In a parallel-plate flow chamber system ECs monolayers are exposed to well-defined flow and thus shear stress (denoted by τ) in a small channel with fixed height. (B) Cone-and-plate flow chamber. In a cone-and-plate flow chamber system ECs monolayers are exposed to shear stress (τ) generated by a rotating cone. The magnitude of shear stress can be calculated using the respective formula shown in A and B.

Figure 4

Classification and description of flow patterns. (A) Illustration of regular flow and irregular flow. The flow pattern in a parallel-plate flow chamber is laminar with a parabolic-like velocity profile and the flow condition is termed regular flow, which can be steady or pulsatile. In contrast, the flow pattern in a vertical step-flow chamber is disturbed with the formation of eddies and separation of streamlines and thus the flow condition is termed irregular flow, which can be disturbed or oscillatory. (B) Demonstration of various types of flow. According to the magnitude of shear stress and variation of shear stress with time, they can be categorized as static control, steady flow, pulsatile flow, and reciprocating (oscillatory) flow. For static control, no shear stress is produced because there is no flow. For steady flow, a physiological level of shear stress (τ) is produced by the flow. For pulsatile flow and reciprocating (oscillatory) flow, cyclic change (e.g. 1 Hz) in the level of shear stress is maintained, but the average level of shear stress (τ) of pulsatile flow is relatively higher in comparison with reciprocating (oscillatory) flow, for which the average level of shear stress is zero or very low.

Figure 5

Relative levels of ROS in ECs exposed to various flow patterns. (A) Steady flow (step shear stress increase from 0 to 13.5 dyn/cm² and then maintained for 10 or 30 min), (B) Pulsatile flow (periodic variation in shear stress from 3 to 25 dyn/cm², 1 Hz), (C) Impulse flow (step increase in shear stress from 0 to 13.5 dyn/cm² for 3 seconds). ROS levels in ECs exposed to various flow patterns were determined by measuring the 6-carboxy-DCF (an ROS probe) fluorescence and normalized to the static control. Data represent the means ± S.E. of three experiments. # P <0.05 vs. static control. (Yu-Chih Tsai, Master’s Thesis, Department of Chemical Engineering, National Taiwan University, 2002).

Figure 6

Pro- or anti- atherogenic effect of flow patterns through different redox signalings and genes expression. A regular flow pattern (steady or pulsatile) produces lower levels of ROS and pro-oxidant activity, yet higher NO bioavailability and anti-oxidant activity, that result in an anti-oxidative state, favoring the activation/regulation of key transcription factors such as Nrf2, KLF2 to promote anti-atherogenic environment by enhancing the expression of SOD, HO-1, etc. On the other hand, an irregular flow pattern (disturbed or oscillatory) produces higher levels of ROS and pro-oxidant activity, yet lower NO bioavailability and anti-oxidant activity, that result in an oxidative state, favoring the activation/regulation of key transcription factors such as AP-1, NF-κB for pro-atherogenic environment by enhancing the expression of MCP-1, ICAM-1, etc. ++: relatively higher; +: relatively lower.

Figure 7

Model of the effect of shear stress on S-nitrosation of redox-sensitive Cys-containing proteins in ECs. Shear stress activates endothelial nitric oxide synthase (eNOS), leading to an increased level of NO and increased S-nitrosation (S-NO) of proteins via NO carrier proteins or peptides. Shear stress-induced protein’s S-nitrosation may prevent the irreversible oxidative modification of proteins (S-OH and S-O₂/O₃H) that otherwise would occur during severe inflammation [73].