Abstract
Fetal lung growth is critically dependent on the degree to which the lungs are expanded by liquid, although the mechanisms involved are unknown. As thrombospondin-1 (TSP-1) can regulate cell proliferation, attachment, spreading and angiogenesis, we investigated the effects of alterations in fetal lung expansion on TSP-1 expression in sheep. TSP-1 mRNA levels were investigated using Northern blot analysis and in situ hybridization, whereas the protein levels were determined by immunohistochemistry. Early growth response 1 (EGR1) mRNA levels were measured by quantitative real-time PCR. TSP-1 was expressed in type-II alveolar epithelial cells and fibroblasts and its mRNA levels increased from 100.0 +/- 14.0% in control fetuses to 347.5 +/- 73.6% at 36 h of increased lung expansion (P < 0.05), and were reduced to 39.4 +/- 6.1% of control levels (100.0 +/- 20.4%) at 20 days of decreased lung expansion (P < 0.05). The percentage of cells positive for TSP-1 mRNA increased from 1.9 +/- 0.4% to 5.2 +/- 0.8% at 36 h of increased fetal lung expansion (P < 0.01). The proportion of tissue stained positive for TSP-1 protein doubled at 36 h of increased lung expansion (23.3 +/- 2.2%) compared to controls (11.7 +/- 3.2%; P < 0.05). Conversely, at 20 days of decreased lung expansion, the percentage of tissue that stained positive for TSP-1 was halved (25.7 +/- 3.2%) compared to controls (39.8 +/- 3.3%; P < 0.05). The increase in TSP-1 expression may be due to increased mRNA levels of the transcription factor EGR1 at 36 h of increased lung expansion (2.7 +/- 0.7-fold of control levels (1.0 +/- 0.2); P < 0.05). Given the known functions of TSP-1 and its localization within the lung, we speculate that TSP-1 may have a significant role in regulating fetal lung growth.