Sepsis Enhances Epithelial Permeability with Stretch in an Actin Dependent Manner

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

BODIPY staining increases with stretch magnitude.

<p>The area (μ ± SE) stained by BODIPY-conjugated ouabain was determined in sham and 2CLP monolayers following 1 hour of cyclic stretch (0.25 Hz) to 0% (NS), 12%, or 25% ΔSA (N≥3 isolations). Area stained in sham monolayers did not increase above unstretched levels, while stained areas in 2CLP monolayers significantly increased above unstretched control levels following stretch to both magnitudes. Confocal micrographs of Na+/K+-ATPase pumps on the basal membrane of unstretched and stretched (1 hour, 25% ΔSA) 4-day old sham and 2CLP monolayers demonstrated that increases in BODIPY staining was not due to differences in receptor expression (Scale bar in Sham – NS = 100 µm). Significant differences between stretch groups were determined by Tukey tests. Significance defined as p<0.01, bar indicates significant difference between sham and 2CLP at same stretch magnitude, *significantly different than 2CLP unstretched. (mean ± SE).</p

Phosphorylation of MAPk signaling pathways (ERK and JNK) following cyclic stretch for 1 hour to 12% ΔSA in sham and 2CLP cell monolayers.

<p>No increases in MAPk phosphorylation were observed in either cell population when stretched to 12% ΔSA for 10 or 60 minutes. (mean ± SE, N≥6 isolations).</p

Solubility of cytoskeleton-associated tight junction proteins in unstretched monolayers.

<p>The ratio of the amount of soluble to insoluble junctional protein that precipitated with the cytoskeleton of sham (▪) and 2CLP (□) monolayers was higher in the 2CLP population, but did not reach significance. (mean ± SE, N = 4 isolations).</p

Phosphorylation of MLCK following low-magnitude stretch (12% ΔSA) of sham and 2CLP monolayers.

<p>Phosphorylation of MLCK was consistently reduced in 2CLP but not sham monolayers following 60 minutes of cyclic stretch (p<0.05, bar). Total levels of protein (tMLCK) remained unchanged in the cell monolayers. Actin is shown as a loading control. (N = 3 isolations, representative western blots shown).</p

Expression of tight junction proteins following 60 minutes of stretch.

<p>Tight junction protein expression in sham (▪) and 2CLP (□) with and without stretch. *significantly different from sham, bar indicates significantly different between sham and 2CLP, p<0.05 (mean ± SE, N≥3 isolations).</p

Phalloidin staining of actin in sham and 2CLP monolayers in unstretched, stretched cyclically for 60 minutes to 12% ΔSA, and stretched cyclically for 60 minutes to 25% ΔSA wells.

<p>Similar staining patterns were observed in sham unstretched monolayers and sham monolayers stretched to 12% ΔSA. Intensity of the actin stain in unstretched 2CLP monolayers was reduced compared to unstretched sham, and a thin cortical actin ring is apparent (arrow). In 2CLP monolayers stretched to 12% ΔSA, stress fibers form a circular band throughout the cytoplasm of the cell, and become more prominent compared to unstretched monolayers. In cells stretched to 25% ΔSA, cortical rings form (arrow), which are also more prominent in 2CLP monolayers compared to sham. Scale bar 100 µm. Representative of images from 2 isolations with 2 wells per isolation.</p

Sham and 2CLP monolayers were cyclically stretched to a magnitude of 12% ΔSA for 60 minutes following a 1-hr incubation with either Jasplakinolide (1 µM, 2CLP) or DMSO (Sham and 2CLP), and permeability to BODIPY-ouabain was analyzed.

<p>Stretch resulted in significant permeability increases in DMSO-treated 2CLP monolayers only, compared to unstretched sham and 2CLP monolayers. Treatment with Jasplakinolide prevented all permeability increases. Significance defined as p<0.05 with multiple comparison correction (β = 4), *significantly different than stretched sham (mean ± SE, N≥3 isolations).</p

Surface characterization of nanoparticles using near-field light scattering

The effect of nanoparticle surface coating characteristics on colloidal stability in solution is a critical parameter in understanding the potential applications of nanoparticles, especially in biomedicine. Here we explored the modification of the surface of poly(ethylene glycol)-coated superparamagnetic iron oxide nanoparticles (PEG-SPIOs) with the synthetic pseudotannin polygallol via interpolymer complexation (IPC). Changes in particle size and zeta potential were indirectly assessed via differences between PEG-SPIOs and IPC-SPIOs in particle velocity and scattering intensity using near-field light scattering. The local scattering intensity is correlated with the distance between the particle and waveguide, which is affected by the size of the particle (coating thickness) as well as the interactions between the particle and waveguide (related to the zeta potential of the coating). Therefore, we report here the use of near-field light scattering using nanophotonic force microscopy (using a NanoTweezerTM instrument, Halo Labs) to determine the changes that occurred in hydrated particle characteristics, which is accompanied by an analytical model. Furthermore, we found that altering the salt concentration of the suspension solution affected the velocity of particles due to the change of dielectric constant and viscosity of the solution. These findings suggest that this technique is suitable for studying particle surface changes and perhaps can be used to dynamically study reaction kinetics at the particle surface