The roles of hyperoxia and mechanical deformation in alveolar epithelial injury and repair

The alveolar epithelium is a key functional component of the air-blood barrier in the lung. Comprised of two morphologically distinct cell types, alveolar epithelial type I (ATI) and type II (ATII) cells, effective repair of the alveolar epithelial barrier following injury appears to be an important determinant of clinical outcome. The prevailing view suggests this repair is achieved by the proliferation of ATII cells and the transdifferentiation of ATII cells into ATI cells. Supplemental oxygen and mechanical ventilation are key therapeutic interventions in the supportive treatment of respiratory failure following lung injury, but the effects of hyperoxia and mechanical deformation in the injured lung, and on alveolar epithelial repair in particular, are largely unknown. The clinical impression however, is that poor outcome is associated with exposure of injured (repairing) epithelium to such iatrogenic ‘hits’. This thesis describes studies investigating the hypothesis that hyperoxia & mechanical deformation inhibit normal epithelial repair. The in vitro data presented demonstrate that hyperoxia reversibly inhibits the transdifferentiation of ATII-like cells into ATI-like cells with time in culture. Whilst confirming that hyperoxia is injurious to alveolar epithelial cells, these data further suggest the ATII cell population harbours a subpopulation of cells resistant to hyperoxia-induced injury. This subpopulation of cells appears to generate fewer reactive oxygen species and express lower levels of the zonula adherens protein E-cadherin. Using a panel of antibodies to ATI (RTI40) and ATII (MMC4 & RTII70) cell-selective proteins, the effect of hyperoxia on the phenotype of the alveolar epithelium in a rat model of resolving S. aureus-induced lung injury was investigated. These in vivo studies support the view that, under normoxic conditions, alveolar epithelial repair occurs through ATII cell proliferation & transdifferentiation of ATII cells into ATI cells, with transdifferentiation occurring via a novel intermediate (MMC4/RTI40-coexpressing) immunophenotype. However, in S. aureus-injured lungs exposed to hyperoxia, the resolution of ATII cell hyperplasia was impaired, with an increase in ATII cell-staining membrane and a reduction in intermediate cell-staining membrane compared to injured lungs exposed to normoxia alone. As hyperoxia is pro-apoptotic and known to inhibit ATII cell proliferation, these data support the hypothesis that hyperoxia impairs normal epithelial repair by inhibiting the transdifferentiation of ATII cells into ATI cells in vivo. The effect of mechanical deformation on alveolar epithelial cells in culture was investigated by examining changes in cell viability following exposure of epithelial cell monolayers to quantified levels of cyclic equibiaxial mechanical strain. In the central region of monolayers, deformation-induced injury was a non-linear function of deformation magnitude, with significant injury occurring only following exposure to strains greater than those associated with inflation of the intact lung to total lung capacity. However, these studies demonstrate for the first time that different epithelial cell phenotypes within the same culture system have different sensitivities to deformation-induced injury, with spreading RTI40-expressing cells in the peripheral region of epithelial cell monolayers and in the region of ‘repairing’ wounds being injured even at physiological levels of mechanical strain. These findings are consistent with the hypothesis that alveolar epithelial cells in regions of epithelial repair are highly susceptible to deformation-induced injury