Inhibiting Connexin43/Zonula Occludens‐1 Interactions Improves Alcoholic Lung Barrier Function in vivo

Abstract

Chronic alcohol consumption is well known to contribute to an array of health complications and increases the incidence and severity of acute respiratory distress syndrome (ARDS), an acute lung injury marked by severe pulmonary edema and impairment of normal gas exchange. Despite advancements in understanding its pathogenesis, the mortality rate for ARDS remains over 40% and there are currently no effective pharmacological treatments for ARDS. Chronic alcohol consumption contributes to the pathogenesis of ARDS by altering the tight junction composition of the pulmonary epithelium which is essential to maintain normal barrier function. In this study, we investigate the use of a pharmacological peptide, aCT1, which mimics the cytoplasmic carboxy-terminal PDZ binding motif of the gap junction protein Connexin43 (Cx43) to disrupt endogenous Cx43 from binding scaffold proteins, including the tight junction protein Zonula Occludens-1 (ZO-1). By inhibiting ZO-1 binding to Cx43, we hypothesize that more ZO-1 is available to associate with tight junction proteins, including claudins, to enhance epithelial barrier function. We have previously shown that chronic alcohol use exacerbates acute endotoxin-mediated lung injury by compromising both the alveolar and pulmonary capillary epithelial barriers. Here, we tested whether treatment with aCT1 strengthens alveolar barrier function and mitigates the effects of alcohol-mediated impairment of tight junctions. Male C57BL/6J mice were pair-fed 20% ethanol or water for 14 weeks to establish a chronic alcohol model in vivo. Mice were sedated and given an intratracheal instillation of 5mg/kg of the bacterial endotoxin lipopolysaccharide (LPS) derived from E. coli O55:B5 to induce a second, direct insult in the alcohol-primed lung to impact the lung epithelium. Mice were administered via nebulizer 1h post-LPS instillation either 5mg/kg of the aCT1 peptide or a scrambled control. Evans Blue (EB) dye was injected into mouse tail veins 24h after LPS instillation. To measure alveolar flooding, bronchoalveolar lavage fluid (BAL) was collected 1h later and EB in BAL was measured and normalized to serum EB. We found that administration of aCT1 decreased the leakage of EB dye from the blood into the alveolar air space in both water-fed and alcohol-fed mice, thus improving lung barrier function. This improvement in pulmonary leak suggests that aCT1 has the capacity to mitigate ARDS. Further investigation will determine whether aCT1 has a positive impact on short-term mortality rates due to endotoxemia and whether it can improve pulmonary barrier dysfunction due to other causes of acute lung injury.