Acute respiratory distress syndrome (ARDS), is a devastating syndrome of respiratory failure that contributes to nearly 75,000 deaths annually in the United States. Therapy for acute lung injury is primarily supportive and currently no pharmacologic therapies are available. The pathophysiology of acute lung injury is characterized by inflammation and disruption of alveolar-capillary barrier. Recovery from acute lung injury requires re- establishment of the alveolar epithelial barrier, including the formation f low-permeability tight junctions between epithelial cells. To advance the field, a more complete understanding of alveolar epithelial barrier regulation and repair is needed. Claudin proteins are central to barrier function. Claudins are transmembrane proteins that are required for tight junction formation, but likely participate in other cellular processes important to repair. Therefoe we have investigated the functions and regulation of claudins in the alveolar epithelium. Our work has shown that two of the most abundantly expressed claudins in the alveolar epithelium are selectively regulated during lung injury. This proposal will investigate the specific functions and regulation of claudins-18 and -4 in mechanistic detail using an integrative approach that includes experimental models of lung injury, primary cell culture, and studies in ex vivo perfused human lungs.
In Aim 1 we will determine the unique functions of lung-specific claudin-18.1, and the mechanisms for its selective down regulation in acute lung injury. Will we test the hypothesis that claudin-18 is required for the macromolecule permeability barrier in the lung and that the loss of claudin-18 contributes to more severe lung injury. We propose that claudin-18 forms homotypic tight junction strands that uniquely limit macromolecule permeability. During injury, TNF-? mediates the selective loss of claudin-18 from intact tight junctions, in part through effects on the cytoskeleton.
In Aim 2 we will follow up on our previous finding that claudin-4 is consistently induced in acute lung injury. We will determine if claudin-4 is required for EGFR-mediated epithelial repair in lung injury. We hypothesize that claudin-4 is induced by EGFR in response to epithelial cell injury and the loss of cell-cell contact. We propose claudin-4 acts to accelerate repair through a previously unknown interaction with active Rap1 that speeds tight junction sealing and promotes cell spreading. Preliminary data show that claudin-4 is consistently induced in several models of lung injury and in human lungs in association with more preserved barrier function. Claudin- 4 is required for repair and preferentially associates with active Rap1.
In Aim 3 we will determine the clinical significance of differences in claudin expression to epithelial barrier function using the ex vivo perfused human lung model. We hypothesize that claudin-18 is the predominant claudin in human type 1 cells and the specific loss of claudin-18 results in increased alveolar epithelial permeability in the ex vivo model and greater lung injury in donors. We will investigate the mechanism for the regulation of claudin-18 and claudin-4 using a bacterial pneumonia model in the ex vivo system. These studies will provide novel insights into the contributions of claudin proteins to the loss and recovery of alveolar epithelial barrier function in acute lung injury.

Public Health Relevance

Acute respiratory distress syndrome (ARDS) is a common and devastating syndrome of respiratory failure with a high mortality rate. In ARDS, a breakdown of the normal barrier properties of the alveolar epithelium in the lung results in flooding of the airspaces with edema fluid. To gain insight into novel therapeutic strategies for lung injury, this proposal examines the function and regulation of claudin proteins during acute lung injury, as these proteins are required for epithelial barrier function and repair.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
High Priority, Short Term Project Award (R56)
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Special Emphasis Panel (ZRG1-CVRS-G (02))
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Moore, Timothy M
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Northern California Institute Research & Education
San Francisco
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Brune, Kieran; Frank, James; Schwingshackl, Andreas et al. (2015) Pulmonary epithelial barrier function: some new players and mechanisms. Am J Physiol Lung Cell Mol Physiol 308:L731-45
Hirsch, Jan; Chalkley, Robert J; Bentley, Trevor et al. (2014) Double impact of cigarette smoke and mechanical ventilation on the alveolar epithelial type II cell. Crit Care 18:R50
LaFemina, Michael J; Sutherland, Katherine M; Bentley, Trevor et al. (2014) Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice. Am J Respir Cell Mol Biol 51:550-8
Rokkam, Deepti; Lafemina, Michael J; Lee, Jae Woo et al. (2011) Claudin-4 levels are associated with intact alveolar fluid clearance in human lungs. Am J Pathol 179:1081-7
LaFemina, Michael J; Rokkam, Deepti; Chandrasena, Anita et al. (2010) Keratinocyte growth factor enhances barrier function without altering claudin expression in primary alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 299:L724-34