Mechanical ventilation worsens pre-existing lung injury in animals and contributes to mortality in patients with the acute respiratory distress syndrome (ARDS). Mechanical stretch of cultured cells increases oxidant generation, induces the expression of pro-inflammatory genes, inhibits epithelial wound healing and induces apoptosis. The mechanism(s) by which alveolar epithelial cells sense cyclic stretch, however, have not been elucidated.
 Preliminary experiments suggest that basement membrane proteins may play an important role in mediating mechanosignal transduction in the lung. In particular, cultured alveolar cells secrete a matrix rich in laminin containing an alpha 3 subunit. The alpha 3 subunit-containing laminin organizes into a fibrillar array, together with beta 1 and gamma 1 subunits. Moreover, a transient increase in mitogen activated protein kinase (MAPK) in rat alveolar cells induced by stretch is inhibited by an antibody against the alpha 3 laminin subunit globular domain and by antibodies that perturb the function of dystroglycan. These data provide support for a hypothesis that mechanosignaling in alveolar cells is mediated by a laminin/dystroglycan complex. We will test this hypotheis in three specific aims.
In aim 1, we will determine the functional consequences of alveolar epithelial cell adhesion to laminins in the extracellular matrix. Specifically, we propose to determine laminin isoforms expressed by alveolar cells in vitro and in vivo. In addition, we will assess the role of cell surface receptors in alveolar attachment, spreading and migration on different laminin isoforms. In aim 2, we will determine whether a laminin/dystroglycan interaction is responsible for mechanosignal transduction in cultured cells. We will examine the effects of inhibitors of the laminin/dystroglycan interaction in primary cultures of alveolar epithelial cells exposed to cyclic stretch focusing on MAP kinase activation. In aim 3 we will determine whether the alpha 3 laminin subunit is responsible for mechanosignal transduction in vivo. We will develop a transgenic lung-specific conditional knockout of alpha 3 laminin using Cre recombinase mediated excision. Lung function will be evaluated in adult animals lacking the alpha 3 laminin. These studies will provide new insight into the role of extracellular matrix in regulating lung cell physiology. 
Matrix organization of AECs. AECs at 2 and 4 days after isolation were processed for double-label indirect immunofluorescence microscopy using antibodies against perlecan (A,D) in combination with an antiserum against the ß1 laminin subunit (B,E). Images of cells were generated using confocal laser scanning microscopy. The focal plane was as close as possible to the substratum-attached surface of the cells. Areas of co-localization are indicated by the yellow color in the overlay images shown in C,F. Bar, 10 µm (click on picture for citation). |
