Determinants of Plasma Membrane Wounding by Deforming Stress

Richard A Oeckler, Won-Yeon Lee, Mun-Gi Park, Othmar Kofler, Deborah L. Rasmussen, Heung-Bum Lee, Hewan Belete, Bruce J. Walters, Randolph W. Stroetz, and Rolf Dieter Hubmayr

Once excess liquid gains access to airspaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that inteventions, such as a hypertonic treatment, increase adhesive PM/cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells andfibroblasts and measured their retractive force as read-out of PM/CSK adhesive interactions using optical tweezers. A 50mOsm increase in media tonicity consistently increased the tether retractive force in epithelial cells, but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with Latrunculin, Cytochalasin D and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel and the fraction of wounded cells measured. Interventions which increased PM/CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex-vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleuralalveolus resident cells to baseline levels. Our observations support the hypothesis that PM/CSK adhesive interactions are important determinants of the cells response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury.

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