A deficiency of pulmonary surfactant at birth is a major contributing cause of lung injury and long-term lung disease such as bronchopulmonary dysplasia (BPD). The severe respiratory distress associated with inherited deficiency of surfactant protein-B (SP-B), in both mice pups and infants born at term, indicates a key role for the hydrophobic SPs in differentiation of type II cells. In the absence of SP-B there is a failure of normal lamellar body genesis as well as incomplete processing of SP-C. Recently, isolated deficiency of SP-C has been described in infants with interstitial lung disease. Respiratory distress also occurs in newborn term BWB calves which lack mature SP-C and have reduced SP-B, and in rodents respiratory distress and acquired deficiency of SP-B/-C occurs with lung injury secondary to bleomycin or infection (P. carinii and endotoxin). Based on these and other findings, this project proposes that synthesis of SP-B, SP-C and lamellar bodies are closely linked and that relative levels of both SP-B and SP-C influence surfactant function. The objectives of this proposal are to characterize the biosynthetic pathway for human SP-C, determine the roles of SP-B and SP-C in lamellar body genesis, and investigate SP-B and SP-C in lung disease.
Aim I will determine expression of mature SP-C during type II cell differentiation in vivo and in vitro in relationship to production of SP-B and lamellar bodies and also define targeting domains and cleavage events in SP-C processing. The studies will utilize antibody to mature SP-C and a recently developed culture system for hormonally induced type II cell differentiation in vitro.
Aim II will investigate the role and interactions of SP-B and SP-C in lamellar body genesis and trafficking of surfactant components using cell culture models of SP deficiency. The studies will examine the hypothesis that expression of mature SP-B is required for both lamellar body formation and final processing of SP-C intermediates. Experiments will be carried out in the cultured type II cell model and SP-B or -C gene expression will be selectively inhibited using adenovirus expressing antisense mRNAs. Processing and intracellular trafficking of each SP will be studied using epitope specific antibodies, pulse/chase labeling, and tagged recombinant proteins. In addition, processing and effects of alternatively spliced SP-B and mutated SP-C will be determined.
Aim III will investigate expression of SP-B and SP-C in surfactant from infants with lung disease and after treatment with inhaled nitric oxide. It is hypothesized that a deficiency of SP-B and/or SP-C occurs in infants with severe BPD, and that this process is modulated by anti-inflammatory effects of nitric oxide. In addition, the developmental pattern for alternative SP-B splicing in human lung and relationship of splicing variants to SP-B levels and newborn lung disease will be determined. The proposed studies will utilize both the Tissue Culture and Clinical Cores and involve collaboration with Projects 6, 4 and 7. The new information will provide further understanding of the role of the hydrophobic surfactant proteins in lung development and newborn lung diseases.
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