Abstract 1880 Pulmonary Development Platform, Saturday, 5/1

Fetal breathing movements (FBM) are necessary for normal lung growth and maturation. Absence of FBM or lung constriction results in lung hypoplasia and impaired alveolarization. The molecular mechanisms by which FBM and mechanical deformation stimulate lung growth and differentiation are not defined. We analyzed the effects of mechanical stretch in rat fetal lung saccular epithelial and mesenchymal cells (18-21 pca; term=21 days). Highly purified type II cells and co-cultures of type II cells and lung fibroblasts plated were on silastic membranes (Bioflex) precoated with Madin-Darby canine kidney (MDCK) or with purified placental extracellular matrix components and were maintained for up to two days in serumless Waymouth's medium. Cyclic deformation simulating FBM was achieved by mounting the culture plates in a F-3000 strain unit (Flexcell®) and applying an intermittent equibiaxial elongation of 6.5% across the culture surface at 50 cycles/min for 16-48 h. Mounted unstretched cells were used as controls. Surfactant protein A (SP-A), SP-B, SP-C and the constitutively expressed GAPDH mRNA abundance were assayed by Northern blot and parathyroid hormone-related protein (PTHrP) mRNA was measured by ribonuclease protection assay (RPA). We also analyzed the effect of stretch on cell cycle regulation and apoptosis entry using *FACS analysis. We found that cyclic cell deformation of isolated type II cells (>90% purity) increased steady-state mRNA levels of SP-B, SP-C and PTHrP by 2-4 fold. There was only a modest increase in SP-A message. In contrast, when d 18-19 type II cells were cultured with co-isolated fibroblasts and ratios of 1:1 and 3:1, cyclic strecth increased SP-B and SP-C expression robustly (>20 fold). Cyclic stretch also promoted pseudoglandular structure formation by phase contrast and DIC microscopy. Addition of dexamethasone and cyclic AMP analog (dbcAMP) or co-culture with fetal lung fibroblasts also increased new transcription of lung epithelial-specific genes in unstretched cultures and enhanced the effects of cyclic deformation on type II cell differentiation. Addition of a PTHrP competitive antagonist to the mixed cell cultures abolished >50% of the stretch-activated increases in SP-B and SP-C message. Cyclic deformation of isolated epithelial cells (d 18) inhibited cell cycle G1 to S phase progression and activated apoptosis (8-10 fold) compared to unstretched controls. Our findings confirm in an in vitro system that mechanical forces induce alveolar cytodifferentiation and type II cell maturation during late fetal development. Stretch-induced epithelial-mesenchymal cell signaling appears to play an important part in this process. PTHrP produced by respiratory epithelium may be an important paracrine regulator of stretch-mediated alveolar remodeling.

(Supported by NIH HL55268)