Stop the flow: a paradigm for cell signaling mediated by reactive oxygen species in the pulmonary endothelium

Abstract

The lung endothelium is exposed to mechanical stimuli through shear stress arising from blood flow and responds to altered shear by activation of NADPH (NOX2) to generate reactive oxygen species (ROS). This review describes the pathway for NOX2 activation and the downstream ROS-mediated signaling events on the basis of studies of isolated lungs and flow-adapted endothelial cells in vitro that are subjected to acute flow cessation (ischemia). Altered mechanical stress is detected by a cell-associated complex involving caveolae and other membrane proteins that results in endothelial cell membrane depolarization and then the activation of specific kinases that lead to the assembly of NOX2 components. ROS generated by this enzyme amplify the mechanosignal within the endothelial cell to regulate activation and/or synthesis of proteins that participate in cell growth, proliferation, differentiation, apoptosis, and vascular remodeling. These responses indicate an important role for NOX2-derived ROS associated with mechanotransduction in promoting vascular homeostasis.

Figure 1

Reactive oxygen species (ROS) generation upon chemotransduction or altered mechanotransduction. Chemical stimuli or stop of shear activate NADPH oxidase, type 2 (NOX2) and endothelial nitric oxide synthase (eNOS). The resultant generation of oxygen ( ${\mathrm{O}}_2^{·-}$)- and nitrogen (^·NO)-centered radicals results in signaling or injury, depending on the level of production and antioxidant defenses of the cell.

Figure 2

The assembled NADPH oxidase, type 2 (NOX2) complex. gp91^phox (NOX2) and p22^phox are integral plasma membrane components and together constitute cytochrome b₅₅₈. The remaining components are normally cytosolic proteins that translocate to the plasma membrane following their phosphorylation. ${\mathrm{O}}_2^{·-}$ that is generated by NOX2 is converted to H₂O₂, which can diffuse across the plasma membrane, possibly through aquaporin (AQP) channels, to initiate cell signaling.

Figure 3

Apparatus for flow-adapting endothelial cells in vitro to shear stress. Cells are seeded, are allowed to attach for 24 h, and are then subjected to flow (generally at shear stresses of 5–10 dyn cm⁻²) for 24–72 h. (a) Procedure for flow-adapting cells for fluorescence imaging. The upper-panel chamber (Warner Instruments, Hamden, CT) shows the parallel plate. The middle panel shows a schematic of the perfusion circuit. Ceils are seeded on cover slips inserted into the chamber. Flow-adapted wild-type, caveolin-1-null, and K_IR6.2 (K_ATP channel)-null cells were evaluated under continuous-flow (control) or stopped-flow (ischemia) conditions in the presence of the membrane potential–sensitive fluorophore bisoxonol; images were acquired by confocal microscopy (lowerpanel). Depolarization indicated by increased fluorescence was observed in wild-type but not in caveolin-1-null and K_ATP channel-null cells (91). (b) Procedure for cellular and biochemical analysis of flow-adapted cells. Cells are seeded on fibronectin-coated polycarbonate capillaries encased in a housing for perfusion via luminal or abluminal ports (upper panel). For the initial cell attachment and the subsequent ischemia phases of the experiment, medium is perfused through the abluminal ports, which removes the shear stress but allows adequate oxygenation and provision of nutrients (black arrows). For flow adaptation, medium is perfused through the luminal ports (red arrows). The lower panel shows images of cells that have been trypsinized from the capillaries and labeled with PKH for analysis by fluorescence-activated cell sorting; cell generations are indicated by the differently colored peaks. Continuous laminar flow results in an antiproliferative state. Increased proliferation is shown by wild-type cells that were flow adapted and then subjected to flow cessation; increased proliferation is not seen in cells that do not generate ROS, either NOX2-null cells or cells treated with a K_ATP channel agonist (cromakalim) to prevent NOX2 activation (91).

Figure 4

Possible mechanism for ${\mathrm{O}}_2^{·-}$ -induced ${\mathrm{O}}_2^{·-}$ release by mitochondria. ${\mathrm{O}}_2^{·-}$ generated extracellularly by NOX2 can penetrate the pulmonary endothelial cell membrane through Cl⁻3 channels (Cl3 C) and can interact with the inositol 1,4,5-triphosphate (InsP3) receptor, resulting in Ca²⁺ release, depolarization of the mitochondrial inner membrane, and mitochondrial ${\mathrm{O}}_2^{·-}$ generation by auto-oxidation of components of the electron transport chain (62).

Figure 5

Key elements in endothelial cell sensing and signal transduction following an abrupt decrease in flow in the pulmonary circulation. The signaling cascade leads to NOX2 activation and ROS generation. Abbreviations: MAP kinase, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3 kinase; PLA₂, phospholipase A₂; Prdx6, peroxiredoxin 6.

Figure 6

Downstream signaling by NOX2-generated ROS. ROS can activate several transcription factors that result in a diverse array of physiological effects. AP-1, activator protein-1; IκB, inhibitor of NFκB activation.

Figure 7

Mechanism for increased intracellular Ca²⁺ following an abrupt cessation of flow. The acute decrease in shear results in K_ATP channel closure, leading to endothelial cell membrane depolarization. Such depolarization in turn results in the opening of T-type voltage-gated Ca²⁺ channels (VGCC) in the plasma membrane, permitting Ca²+ entry (103). The increased intracellular Ca²⁺ can associate with calmodulin (CaM) to activate endothelial nitric oxide synthase (eNOS), resulting in ^·NO generation (89, 93).