title: Hyperoxia increases the elastic modulus of alveolar epithelial cells through Rho kinase.
abstract: Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and while both hyperoxia and mechanical ventilation are necessary, each can independently cause additional injury. However, the precise mechanisms that lead to injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be stiffer due to increased filamentous actin (f-actin) formation via the GTPase RhoA and its effecter Rho kinase (ROCK). We examined cytoskeletal structures in cultured murine lung alveolar epithelial cells (MLE-12) under normoxic and hyperoxic (48 h) conditions. We also measured cell elasticity (E) using an atomic force microscope in the indenter mode. Hyperoxia caused increased f-actin stress fibers and bundle formation, an increase in g- and f-actin, an increase in nuclear area and a decrease in nuclear height, and cells became stiffer (higher E). Treatment with an inhibitor (Y-27632) of ROCK significantly decreased E and prevented the cytoskeletal changes, while it did not influence the nuclear height and area. Pre-exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched cyclically, but the ROCK inhibitor prevented this effect. Hyperoxia caused thickening of vinculin focal adhesion plaques, and inhibition of ROCK reduced the formation of distinct focal adhesion plaques. Phosphorylation of focal adhesion kinase was significantly reduced by both hyperoxia and treatment with Y-27632. Hyperoxia caused increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK.
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, USA.
authors.forename: Kristina R
authors.initials: KR
authors.lastname: Wilhelm
authors.forename: Esra
authors.initials: E
authors.lastname: Roan
authors.forename: Manik C
authors.initials: MC
authors.lastname: Ghosh
authors.forename: Kaushik
authors.initials: K
authors.lastname: Parthasarathi
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Waters
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D053144:Cell Nucleus Shape / Q000187:drug effects; D003599:Cytoskeleton / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D055119:Elastic Modulus / Q000187:drug effects; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D051379:Mice; D018625:Microscopy, Atomic Force; D018384:Oxidative Stress / Q000187:drug effects; D010100:Oxygen / Q000009:adverse effects*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011650:Pulmonary Alveoli / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D016596:Vinculin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054460:rho-Associated Kinases; D010100:Oxygen
keywords: Rho kinase; RhoA; acute respiratory distress syndrome; alveolar epithelial cell; atomic force microscopy; elastic modulus; force maps; hyperoxia