Studies are propose to examine airway epithelial cell growth, migration and attachment to extracellular matrices. Denudation of the epithelium may lead to worsened airflow obstruction by loss of epithelium-derived factors which modulate airway tone, ineffective mucous transport and perturbations in airway fluid transport. Repair of the epithelium may be an important factor in restoring airway homeostasis, while inflammatory cells may perpetuate epithelial damage. Preliminary data suggest that neuropeptides secreted by airway sensory nerves and neuroendocrine bodies may enhance epithelium repair. Two major goals are identified in this proposal: 1) Determine the modulation of airway epithelial cell growth by neuropeptides derived from airway sensory afferent nerves and neuroendocrine cells. We will test the hypothesis that neuropeptides (such as substance P, bombesin, gastrin releasing peptide, calcitonin gene-related peptide, and neurokinin A) stimulate proliferation of epithelial cells. The mechanisms by which neuropeptides stimulate proliferation, including the events transducing the signal, will be examined. Additional experiments will examine the differentiation of epithelial cells after neuropeptide stimulation, and the potential physiologic significance of neuropeptide stimulation. Epithelial cells will be collected from human bronchial surgical specimens and guinea pig tracheas and grown in culture. Specific epithelial cell sub-types will be isolated by immunomagnetic separation. Epithelial cell proliferation will be measured by incorporation of bromodeoxyuridine into DNA and by direct cell count. Expression of nuclear transcription factors will be determined by northern blot assay. Activation of mitogen-associated protein kinase will be tested using a specific renaturation assay. Initial activation of epithelial cells will be measured by cyclic AMP assay and by inhibition of G-protein related signal transduction. Involvement of protein kinase C will be tested using specific inhibitors. 2) Determine the mechanisms by which neuropeptides derived from airway sensory afferent nerves and neuroendocrine cells modulate airway epithelial cell migration and adhesion. We will test the hypotheses that neuropeptides increase migration of epithelial cells and augment epithelial cell adhesion to extracellular matrix proteins. Specific mechanisms will be identified by which neuropeptides elicit migration and stimulate expression of specific adhesion receptors in airway epithelial cells, and studies will determine the sequence of events in airway epithelial cell migration through extracellular matrices. Migration will be measured by blindwell assay through matrix-impregnated porous filters. Cell attachment to extracellular matrix proteins will be measured by specific microplate assay. Specific adhesion receptor antagonists and antibodies will be used to determine mechanisms of attachment. The role of focal adhesion kinase in transducing the neuropeptide stimulus for migration will be examined. These experiments will clarify mechanisms by which sensory nerves promote the repair of airway epithelium in the recovery from injury or inflammation. Data derived from these studies should provide insight into mechanisms by which airway epithelium may be restored in human asthma.

R01HL051853

1995-04-05

1999-02-28