(TLC)) [16]. Therefore, we postulated that sepsis and mechanical stretch would act synergistically to increase epithelial permeability via a MAPk signaling pathway. In the current study, we tested the hypothesis that the cyclic stretch magnitude required to induce barrier dysfunction would be lower for monolayers formed by cells from septic lungs compared to monolayers formed by healthy epithelial cells. We utilized a previously described culture model of septic epithelia to test the barrier function of septic epithelial monolayers following low (12% DSA or 70% TLC) and moderate (25% DSA or 90% TLC) magnitudes of cyclic stretch [17,18]. Furthermore, we hypothe- sized that MAPk regulation of TJ protein expression and disruption of the actin cytoskeletal organization are mechanisms of barrier dysfunction, and evaluated these mechanisms with inhibitors to MAPk activation and actin depolymerization. Methods Ethics Statement All animal use was done in accordance with, and with the approval of, the IACUC in the Office of Regulatory Affairs of the University of Pennsylvania. Cecal Ligation and Double Puncture Male Sprague-Dawley rats (Charles River, Boston, MA) weighing 240–260 grams underwent cecal ligation and double puncture (2CLP) or control (sham) surgery as previously described [13]. Mortality rate 24 hours following the 2CLP procedure was approximately 10%. Alveolar Epithelial Type II Cell Isolation Twenty-four hours following the 2CLP or sham procedures, animals were sacrificed and alveolar epithelial type II cells isolated and cultured for 5 days as previously described [17]. Following 5 days in culture, the cells expressed markers of a type I alveolar epithelial phenotype (RT1-40 and plasminogen activator in- hibitor-1 (PAI-1)) and formed confluent monolayers with intact tight junctions [15]. Cell Monolayer Stretching Protocol Monolayers (4–5 days in culture) were designated as stretched or unstretched groups in one of four conditions: DMEM, DMEM and a MAPk inhibitor, DMEM and vehicle control (DMSO), or DMEM and an inhibitor to actin depolymerization (Jasplakino- lide, 1 mM) [19]. Monolayers were (1) stretched for 0 or 60 minutes to 12% or 25% DSA and used to test monolayer permeability; (2) stretched for 0, 10, or 60 minutes to 12% or 25% DSA, and then lysed for assessment of MAPk and myosin light chain kinase (MLCK) phosphorylation and TJ protein expression level; or (3) stretched for 0 or 60 minutes to 12% or 25% DSA and used to visualize actin organization. The stretch device has been previously described and was operated in an incubator maintained at 37uC [20]. Monolayer Permeability Assessment Paracellular permeability was assessed in 2CLP and sham monolayers by monitoring binding of the fluorescent tracer BODIPY-ouabain (approximate radius 20 A˚) to basal Na+/K + - ATPase pumps which are inaccessible to the apically applied tracer when the monolayer is intact [21]. The percent area stained on each image was compared for 2CLP and sham monolayers with and without stretch using an ANOVA and post-hoc Student’s t-test for individual comparisons (N$3 isolations). Significance was defined as p,0.05 with a Bonferonni correction (b = 4). Stretch of Septic Monolayers Triton Solubility of Cytoskeletal Associated Proteins Unstretched 2CLP and sham monolayers were lysed in incubation buffer (phosphate buffered saline, 140 mM NaCL, 5.3 mM KCL, 0.35 mM Na2PO4, 0.35 mM KH2PO4, 0.8 mM MgCl2, 2.7 mM HEPES) with protease inhibitors at 4 degrees C, homogenized, and the nucleus and large debris removed by centrifugation (10 min, 10 Kg). The supernatant was ultracentrifuged (1 hour 100 Kg) to separate the cytoskeletal fraction (pellet) from the cytosol. The pellet was re-suspended in incubation buffer containing 0.1% triton X-100 for 30 min at 4 degrees C. Following this incubation, the sample was re- centrifuged, the supernatant was collected (soluble fraction), and the pellet was re-suspended in incubation buffer and stored (insoluble fraction). Tight junction protein levels were analyzed by Western blot as described below and the ratio of soluble to insoluble protein was determined by densitometry. Western Analysis of MAPk and MLCK Phosphorylation and TJ Proteins Following stretch, whole cell lysate was collected from 2CLP and sham monolayers (N$3 isolations for each protein at each stretch condition and treatment) and MAPk phosphorylation and expression of TJ proteins was analyzed via western blot as previously described [15]. MLCK phosphorylation was assayed using antibody to MLCK pS1760 (Life Technologies, Grand Island, NY). Band intensity of actin was used to normalize between lanes. Significant differences across conditions were determined through individual ANOVAs for each signaling pathway and junctional protein, with post-hoc Student’s t-tests with a Bonfer- onni correction (b = 4) for multiple comparisons used to test between 2CLP and sham populations at each condition. Immunofluorescence Monolayers (N = 2 monolayers per group from each of 2 isolations per group) were fixed and actin fibers were stained with rhodamine-phalloidin and imaged as previously described [22]. Na+/K+-ATPase pump distribution on the basal surface was assessed using antibody to mouse Na+/K+-ATPase alpha-1 (Millipore, Billerica, MA). Results Epithelial Permeability Following Stretch Cultured 2CLP and sham monolayers were stretched for 60 minutes to magnitudes of 0%, 12%, and 25% DSA, after which epithelial permeability was immediately analyzed (Figure 1). Without stretch, we observed no significant differences between 2CLP and sham permeability to BODIPY-ouabain (20 A˚ tracer). Following 60 minutes of stretch to 12% or 25% DSA, sham monolayer permeability was unchanged compared to unstretched. Permeability of 2CLP monolayers was significantly greater than unstretched sham and stretched sham controls at both stretch magnitudes. These data show that the stretch magnitude required to alter permeability is lower in 2CLP monolayers compared to sham. Importantly, the percentage of the field stained with BODIPY in sham monolayers is similar to the values previously reported in unstretched and stretched healthy monolayers, while values from stretched 2CLP monolayers are more comparable to a healthy monolayer stretched to 37% DSA [21]. Expression of Na+/K+-ATPase pumps was not different between sham and 2CLP at unstretched or stretched (25% DSA for 60 min) conditions indicating that an increase in BODIPY binding sites was not responsible for increased staining (Figure 1). PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38748