Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is a potent mitogen for several cell types. To investigate the possible role of TGF-alpha in the development of midgestation human fetal lung, we studied its distribution with immunohistochemistry and determined levels of steady-state TGF-alpha mRNA by Northern analysis of cellular RNA isolates from lung. Lung was obtained from fetuses at 10 to 22 wk of gestation (n = 14) and immunostained for TGF-alpha. TGF-alpha was localized in epithelial cells at all gestational ages examined. Immunostaining was particularly prominent in bronchiolar epithelial cells. TGF-alpha immunoreactivity was also associated with arterial smooth muscle cells, as well as with nerves. Occasional chondrocytes were also associated with TGF-alpha immunoreactivity. Total cellular RNA was isolated from lung tissue obtained from additional fetuses at gestational ages 10 to 24 wk (n = 22). TGF-alpha mRNA was present in RNA extracts of all fetal lungs studied. We conclude that TGF-alpha is probably produced in human fetal lung during mid-gestation. The prominent immunostaining of bronchiolar epithelial cells for TGF-alpha is consistent with its playing a role in distal airway formation.
To assess maturational changes in collagen synthesis, lung tissue was obtained from healthy Macaca nemestrina monkeys at different ages, ranging from 68% of term gestation to adulthood. We hypothesized that infants delivered prematurely have a greater rate of collagen synthesis than do older animals because of their greater rate of lung growth during gestation. Secondly, we hypothesized that lung repair in infants with hyaline membrane disease (HMD) is associated with an additional increase in lung collagen synthesis rate. Therefore, lung tissue was obtained during the first week of life from monkeys delivered at 82% of term gestation, a stage at which half of them developed HMD. The rate of total protein synthesis in lung samples was determined by measuring the incorporation of [3H]proline; the rate of collagen synthesis was determined by measuring the conversion of proline into hydroxyproline. Premature monkeys had a higher rate of collagen synthesis (9.9 +/- 2.7 nmol/mg DNA/h) than did term infants (5.3 +/- 1.1) or older animals (2.1 +/- 0.4, p less than 0.05). There was no additional increase in rate of collagen synthesis in animals with HMD from 3 h (14.3 +/- 6.9) to 7 days of age (15.1 +/- 6.1); control premature animals also had no significant change during the first week of life (10.9 +/- 3.0 at 3 h; 11.6 +/- 4.6 at 7 days). The early stage of recovery from HMD in premature monkeys does not appear to be associated with an increase in collagen production beyond the already increased synthesis rate associated with lung growth.
Perfluorochemical (PFC) liquids have been shown to improve gas exchange and lung compliance in models of lung injury. We reasoned they may also be useful as a vehicle for gene transfer by improving transgene distribution throughout the lung as well as increasing total transgene expression. We have developed a model for PFC liquid use in spontaneously breathing rodents that obviates the need for intubation and ventilation. Intratracheal instillation of the adenoviral vector Adlac-Z resulted in patchy distribution of beta-galactosidase (beta-gal) activity as demonstrated using X-gal histochemistry. In contrast, in rats instilled with Adlac-Z followed by instillation of PFC liquid, more uniformly distributed and increased beta-gal activity was observed. Activity in distal airway and alveolar epithelium was particularly increased. Quantitative measure of beta-gal activity in lung homogenates demonstrated a 3- to 6-fold increase in total activity in lungs of rats receiving Adlac-Z and PFC liquid compared to animals receiving Adlac-Z alone. These studies show that PFC liquids can enhance both the distribution and the total amount of transgene expressed following adenoviral-mediated vector transfer to lungs during spontaneous breathing. Use of PFC liquids may increase the efficacy of gene transfer strategies for treatment of cystic fibrosis and other lung diseases.
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