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A2978 - Protein Kinase R-Like Endoplasmic Reticulum Kinase Confers Ca Sensitive Mechanosensory Function in Ventilator-Induced Lung Injury
Author Block: T. Dolinay1, C. Aonbangkhen2, A. Stablow3, W. Zacharias1, B. Plucinsky3, G. G. Lawrence3, E. Cantu4, Y. Suzuki4, S. Singhal4, D. Chenoweth2, E. E. Morrisey5, J. D. Christie1, S. S. Margulies6; 1Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States, 2Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States, 3Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, United States, 4Department of Surgery, University of Pennsylvania, Philadelphia, PA, United States, 5Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, United States, 6Department of Biomedical Engineering, Georgia Institute of Technology and Emory University Medical School, Atlanta, GA, United States.
Background: Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome (ARDS). VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Lung protective ventilator strategies offer only modest benefit due to inability to prevent alveolar over-distension and concomitant epithelial barrier dysfunction. We have previously shown that chemical and genetic inhibition of the RNA-like endoplasmic reticulum kinase (PERK)-dependent integrated stress response (ISR) signaling protects the alveolar epithelium from stretch-induced damage (AJRCMB 2017;57:193-203). Hypothesis: Based on our findings, we hypothesized that PERK is a sensor of mechanical stretch in the alveolar epithelium. Methods: Human cadaver lung tissue was obtained from the PROPEL tissue repository and used to extract total protein (N=18). The expression of ISR signaling pathway was compared between samples with acute lung injury (ALI) and no injury using western blot. In a separate experiment, a human lung lobe was perfused with 3% albumin and ventilated 4 hours with high tidal volume (Vt=12ml/kg). The adjacent lung lobe served as a perfused but not ventilated control. The alveolar cell specific activation of PERK in ventilated and non-ventilated tissue was assessed with immunofluorescence using antibodies against phospho (p)-PERK, epithelial and endothelial cells. To study the mechanism of PERK-mediated lung injury signaling in the alveolar epithelium, cultured primary rat alveolar type-1 like epithelial cell (RAEC-I) monolayers were exposed to injurious biaxial cyclic mechanical stretch in the presence or absence of the endoplasmic reticulum (ER)-specific Ca-ion channel blocking agent Ryanodine (N=4 biological replicates/condition). Results: ALI resulted in the activation of ISR signaling measured with the phosphorylation of Eukaryotic initiation factor (EIF)-2α and the expression of its downstream transcription factors Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). In ventilated human lung tissue, p-PERK significantly increased and it localized primarily to alveolar epithelial cells. In RAEC-I monolayers, stretch increased p-PERK, which was mitigated by the blockade of ER Ca-ion efflux. Conclusion: PERK-mediated ISR pathway is activated by VILI in alveolar epithelial cells. PERK may serve as an ER Ca-sensitive signal sensor of mechanical stretch and the inhibition of PERK offers potential therapeutic utility in ARDS.