DiPaolo, Brian Christopher
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Publication RAC1-Mediated Actin Cytoskeleton Remodeling and Monolayer Barrier Properties of Stretched Alveolar Epithelial Cells(2010-12-22) DiPaolo, Brian ChristopherMechanical ventilation is vital for supporting patients with pulmonary insufficiency. However, experimental data suggests that pulmonary injury can occur during mechanical ventilation as a result of elevated regional lung volumes resulting in excessive alveolar epithelial cell (AEC) stretch due to basement membrane deformation and reduced barrier function. AECs maintain integrity of the blood-gas barrier with gasket like intercellular tight junctions (TJ) which are anchored internally to the actin cytoskeleton. Our goal was to evaluate the effect of physiologically relevant, high magnitude biaxial stretch on the actin cytoskeleton, a principal regulator of monolayer barrier properties, as a foundation to develop strategies to reduce mechanical injury in stretched AECs. We used primary rat type I-like AEC monolayers cultured on flexible membranes and stretched biaxially to mimic the alveolus during high volume mechanical ventilation. We hypothesized that the actin cytoskeleton underwent rapid remodeling initiated through activation of the Rac1/Rho signaling pathways and resulted in increased monolayer permeability. We have demonstrated that AEC monolayers stretched biaxially undergo rapid magnitude and frequency-dependent actin cytoskeletal remodeling to form perjunctional actin rings (PJARs). PJAR formation during stretch was mediated by the Rac1/Akt pathway but not by the Rho kinase and myosin light chain pathway. Inhibition of several Rac1 pathway targets, including PI3K, Rac1, and PAK-1, resulted in the attenuation of stretch-induced PJAR formation and preservation of barrier properties in stretched monolayers. Rac1 pathway agonists platelet derived growth factor and calyculin-A were shown to induce actin cytoskeleton remodeling to form PJAR in unstretched monolayers. Furthermore, exogenous PIP3 increased Akt phosphorylation in unstretched monolayers and reduced cell death, but increased monolayer permeability in stretched monolayers. In summary, stretch of primary rat AEC monolayers increased Rac1 pathway activity, cytoskeleton remodeling, and monolayer permeability, all of which could be attenuated with Rac1 pathway inhibitors. With this data we can begin to develop novel treatment strategies aimed at preventing mechanical ventilator associated barrier dysfunction in vivo. We anticipate these treatments will reduce the incidence of pulmonary injury and mortality in patients that require mechanical ventilation.Publication Stretch Magnitude and Frequency-Dependent Actin Cytoskeleton Remodeling in Alveolar Epithelia(2010-07-01) DiPaolo, Brian Christopher; Lenormand,, Guillaume; Margulies, Susan S; Fredberg, Jeffrey JAlveolar 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 (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min, or held tonic at 25% ΔSA for up to 60 min, or left unstretched. By 10 min of stretch PJARs were evident in 25% and 37% ΔSA at 0.25 Hz, but not for 12% ΔSA at 0.25 Hz, or at tonic 25% ΔSA, or with no stretch. Treatment with 1 μM jasplakinolide abolished stretch-induced PJAR formation, however. As a rough index of remodeling rate, we measured spontaneous motions of 5-μm microbeads bound to actin focal adhesion complexes on the apical membrane surfaces; within 1 min of exposure to ΔSA 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.Publication Rho Kinase Signaling Pathways During Stretch in Primary Alveolar Epithelia(2012-05-15) DiPaolo, Brian Christopher; Margulies, Susan SAlveolar 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.Publication Sepsis Enhances Epithelial Permeability With Stretch in an Actin Dependent Manner(2012-01-01) DiPaolo, Brian Christopher; Cohen, Taylor Sitarik; Margulies, Susan S; Lawrence, Gladys GrayVentilation 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.