Cigarette or E‐cigarette content alters autophagy and permeability of lung endothelium

Abstract

Cigarette smoking (CS) is the major cause of Chronic Obstructive Pulmonary Disease (COPD). E-cigarettes are considered by some as a safer alternative; however, inhalation of nicotine-containing e-cigarettes can also cause pathophysiologic changes, and “vaping” of some substances has led to severe lung damage. Our group has previously identified cortactin (CTTN), a central regulator of the actin cytoskeleton, as an important modulator of lung endothelial cell (EC) function. Our present study seeks to characterize the role of CTTN in lung endothelial dysfunction upon CS and e-cigarette challenge. Human lung EC were exposed to CS (40 µg/ml, 24 h) and cell lysates were subjected to Western blotting for autophagy markers p62 and LC3B-II. CS exposure increased both p62 and LC3B-II expression, suggesting autophagic flux inhibition. Further CTTN down-regulation by siRNA altered CS-induced p62 and LC3B-II expression. Excess mitochondrial ROS (mitoROS) production is associated with mitochondrial dysfunction and altered autophagy. Here, the mitoROS production in lung ECs was assessed using the MitoSOX assay. CS (40 µg/ml, 2 h) challenge significantly increased mitoROS production, which was potentiated by cortactin siRNA. Blocking mitoROS with Mito-TEMPO (10 µM, 3 h), a cell-permeable antioxidant, decreased mitochondrial superoxide production. We next assessed the role of Mito-TEMPO on E-cigarette-induced endothelial permeability. Lung EC exposure to E-cigarette extract (50 µg/ml) resulted in disruption of endothelial permeability, assessed by Electric Cell-substrate Impedance Sensing system (ECIS). Pretreatment with Mito-TEMPO provided barrier protection upon e-cigarette challenge. These results suggest a key role of mitoROS in e-cigarette-induced endothelial permeability. In conclusion, this study identified new mechanisms by which CS/e-cigarette exposure induces EC dysfunction and provides evidence for a previously undescribed role for CTTN in these responses.