Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

Alveolar epithelial cells (AEC) maintain integrity of the blood-gas barrier with gasket-like intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We hypothesize that stretch rapidly reorganizes actin (<10 min) into a perijunctional actin ring (PJAR) in a manner that is dependent on magnitude and frequency of the stretch, accompanied by spontaneous movement of actin-anchored receptors at the plasma membrane. Primary AEC monolayers were stretched biaxially to create a change in surface area (DeltaSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min, or held tonic at 25% DeltaSA for up to 60 min, or left unstretched. By 10 min of stretch PJARs were evident in 25% and 37% DeltaSA at 0.25 Hz, but not for 12% DeltaSA at 0.25 Hz, or at tonic 25% DeltaSA, or with no stretch. Treatment with 1 muM jasplakinolide abolished stretch-induced PJAR formation, however. As a rough index of remodeling rate, we measured spontaneous motions of 5-mum microbeads bound to actin focal adhesion complexes on the apical membrane surfaces; within 1 min of exposure to DeltaSA of 25% and 37%, these motions increased substantially, increased with increasing stretch frequency, and were consistent with our mechanistic hypothesis. With a tonic stretch, however, the spontaneous motion of microbeads attenuated back to unstretched levels, whereas PJAR remained unchanged. Stretch did not increase spontaneous microbead motion in human alveolar epithelial adenocarcinoma A549 monolayers, confirming that this actin remodeling response to stretch was a cell-type specific response. In summary, stretch of primary rat AEC monolayers forms PJARs and rapidly reorganized actin binding sites at the plasma membrane in a manner dependent on stretch magnitude and frequency.

Alveolar epithelial cells (AECs) maintain the pulmonary blood-gas barrier integrity with gasketlike intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We have previously shown that AEC monolayers stretched cyclically and equibiaxially undergo rapid magnitude- and frequency-dependent actin cytoskeletal remodeling to form perijunctional actin rings (PJARs). In this work, we show that even 10 min of stretch induced increases in the phosphorylation of Akt and LIM kinase (LIMK) and decreases in cofilin phosphorylation, suggesting that the Rac1/Akt pathway is involved in these stretch-mediated changes. We confirmed that Rac1 inhibitors wortmannin or EHT-1864 decrease stretch-stimulated Akt and LIMK phosphorylation and that Rac1 agonists PIP3 or PDGF increase phosphorylation of these proteins in unstretched cells. We also confirmed that Rac1 pathway inhibition during stretch modulated stretch-induced changes in occludin content and monolayer permeability, actin remodeling and PJAR formation, and cell death. As further validation, overexpression of Rac GTPase-activating protein β2-chimerin also preserved monolayer barrier properties in stretched monolayers. In summary, our data suggest that constitutive activity of Rac1, which is necessary for stretch-induced activation of the Rac1 downstream proteins, mediates stretch-induced increases in permeability and PJAR formation.

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

Ventilation of septic patients often leads to the development of edema and impaired gas exchange. We hypothesized that septic alveolar epithelial monolayers would experience stretch-induced barrier dysfunction at a lower magnitude of stretch than healthy alveolar epithelial monolayers. Alveolar epithelial cells were isolated from rats 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Following a 5-day culture period, monolayers were cyclically stretched for 0, 10, or 60 minutes to a magnitude of 12% or 25% change in surface area (ΔSA). Barrier function, MAPk and myosin light chain (MLC) phosphorylation, tight junction (TJ) protein expression and actin cytoskeletal organization were examined after stretch. Significant increases in epithelial permeability were observed only in 2CLP monolayers at the 12% ΔSA stretch level, and in both 2CLP and sham monolayers at the 25% ΔSA stretch level. Increased permeability in 2CLP monolayers was not associated with MAPk signaling or alterations in expression of TJ proteins. 2CLP monolayers had fewer actin stress fibers before stretch, a more robust stretch-induced actin redistribution, and reduced phosphorylated MLCK than sham monolayers. Jasplakinolide stabilization of the actin cytoskeleton in 2CLP monolayers prevented significant increases in permeability following 60 minutes of stretch to 12% ΔSA. We concluded that septic alveolar epithelial monolayers are more susceptible to stretch-induced barrier dysfunction than healthy monolayers due to actin reorganization.

No abstract

Alveolar epithelial cells (AECs) maintain integrity of the blood‐gas barrier via gasket‐like transcellular tight junction proteins bound to the actin cytoskeleton. AEC monolayers stretched biaxially undergo rapid actin cytoskeletal remodeling. We hypothesize that Rac1 and downstream effectors LIMK½, cofilin, Akt, and SSH1L are activated in stretched AECs. Furthermore we hypothesize that Rac1 pathway inhibition will attenuate actin remodeling and preserve paracellular permeability during AEC stretch. Primary AEC monolayers were stretched biaxially to create a change in surface area of 37% at ¼ Hz for 10 or 60 min. Additional monolayers were pretreated with Rac1 pathway inhibitors wortmannin, EHT‐1864, or IPA‐3 and stretched. Unstretched treated and untreated AECs served as two control groups. Without treatments, biaxial stretch rapidly increased Rac1, Akt, LIMK½, and cofilin activity, and resulted in rapid actin remodeling and increased monolayer permeability. Compared to stretched vehicle control monolayers, Rac1 pathway inhibitors reduced Rac1, Akt, LIMK½, and cofilin activity, attenuated actin remodeling, and preserved monolayer permeability in stretched monolayers. This study provides a foundation for developing treatment strategies aimed at reducing cellular injury during high magnitude biaxial deformation. NIH R01 HL057204