Morbidity and mortality due to lung immaturity in premature infants is a continuing major health problem. The role of endogenous neuropeptides derived from pulmonary neuroendocrine cells (PNECs) and pulmonary nerve fibers, in particular bombesin-like peptides (BLPs), in modulating normal fetal lung development has been essentially unexplored. BLPs are likely to participate in normal lung development. We previously demonstrated transient expression of BLP mRNAs in mid-gestation human fetal lung in parallel with growth of the airways. In utero BLP administration accelerated murine fetal lung growth and maturation and endogenous lung maturation was blocked by an anti-BLP monoclonal antibody (mAb). The cell surface enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) has been found to hydrolyze and inactivate BLPs, which are important mitogens for normal bronchial epithelial cells, pulmonary fibroblasts, and many small cell carcinomas of the lung (SCLCs). Specific inhibition of CD10/NEP by SCH32615 potentiated fetal lung growth in human fetal lung organ cultures and both growth and maturation in murine fetal lung in utero. All of these paracrine effects on fetal lung organ growth and maturation were blocked by bombesin receptor antagonists. The proposed study will focus on cellular and molecular events involved in the regulation of lung morphogenesis, growth and maturation by BLPs and by CD10/NEP. We plan to analyze effects of BLPs and/or CD10/NEP inhibition on lung development in fetal mice first in utero and second in lung organ cultures during early organogenesis (branching morphogenesis and growth) and later fetal lung growth and maturation. These effects will be compared with responded to glucocorticoids and growth factors, and potential synergism between diverse pharmacological agents will be explored. Third, we will localize and quantitate expression of genes regulating the observed effects on mouse lung development in vivo. BLP, BLP receptor and CD10/NEP transcripts and proteins will be localized using in situ hybridization and immunoperoxidase analyses, then correlated with BLP receptor autoradiography and NEP enzyme histochemistry. Transcript levels will be quantitated using RNAase protection and/or semi- quantitative RT-PCR. Fourth, cellular requirements for BLP effects will be assessed in isolated and reconstituted fetal lung cell cultures. If an intermediary cell such as the fibroblast is required, we will determine whether cell contact is required or whether a diffusible factor can mediate the effect. Fifth, we will determine whether homeobox genes are involved in BLP effects on branching and cell differentiation using RNA analyses and antisense strategies. Finally, we will study fetal lung development in transgenic mice with primary PNEC hyperplasia. If any effects observed in these mice can be blocked using BLP antisense strategies, this would confirm BLPs as the major PNEC-derived peptide(s) involved in lung development. Understanding mechanisms of paracrine/autocrine regulation by neuropeptides and CD10/NEP would facilitate a comprehensive application of these agents in clinical medicine.
