Acute oxidant lung injury results in damage to the alveolar epithelium. Repair of the epithelium is key to prevention of alveolar fibrosis. The overall hypothesis of this proposal is that the alveolar epithelial type II cell has a central role in alveolar repair, having both responder and effector functions. Two of these functions, which are the basis of this proposal, are production of fibronectin (FN) and expression of Vascular Endothelial Growth Factor (VEGF), which regulates endothelial cells. FN is an extracellular matrix (ECM) glycoprotein that is abundant in injured tissues and that has many putative roles in wound repair. FN strongly influence type II cells, promoting the transition to a type I cell-like phenotype. FN action is mediated by binding to cell integrins. Type II cell expression of FN mRNA and protein are induced by TGF-beta, which is abundant in lung injury. To examine a responder function of type II cells, this proposal investigates TGF-beta1 regulation of type II cell FN and the alpha5beta1 integrin in vitro. These studies focus on TGF-beta1 induction of alternatively spliced FN pre.mRNA, which are unique to cellular FN, and on expression of the alpha5 integrin subunit mRNA and protein. A second responder function of type II cells, altered phenotype in response to FN, will be analyzed by 1) blocking type II cell FN expression in vitro with antisense oligodeoxynucleotides, 2) culturing type Ii cells on a FN-rich matrix and 3) blocking FN binding to alpha5beta1. A panel of type II cell and type I cell differentiation markers will be measured. To correlate the in vitro experiments with lung injury in vivo, oxygen injured lung will be investigated for cell-specific expression of FN, the alternatively spliced variants and alpha5beta1. These studies will employ double in situ hybridization and combined in situ hybridization/immunohistochemistry to identify cell-specific expression. The second part of the overall hypothesis, that type II cells produce growth factors that modulate the behavior of other cell types, will be investigated by determining expression of VEGF. VEGF is expressed by epithelial cells and is a powerful regulator of endothelial cell growth and permeability, both important in lung injury. This proposal investigates the hypothesis that type Ii cells express VEGF in lung injury and the expression is modulated by TGF-beta and the ECM. The outcome of this research will be new information about regulation of novel type II cell functions that are key to repair of alveolar lung injury.
