In situ hybridization of SP-A mRNA in adult human conducting airways.

In our study, surfactant protein (SP)-A was characterized in adult human trachea and bronchi. SP-A mRNA and protein were localized to serous cells in submucosal gland by in situ hybridization and immunohistochemistry, respectively. A 2.2 kb SP-A mRNA transcript was detected in tracheal tissues by Northern blot analysis. Primer extension analysis and gene-specific reverse transcriptase polymerase chain reaction (RT-PCR) revealed the predominance of SP-A2 mRNA. However, using nested PCR, we also detected low amounts of SP-A1 mRNA in the tracheal tissues. A approximately 35 kDa SP-A immunoreactive protein was detected in the tracheal tissues by immunoblot analysis and was shown to be modified by the addition of N-linked oligosaccharides. We conclude that submucosal glands in the conducting airways produce a novel SP-A protein with a molecular weight and post-translational modification similar to the SP-A produced in the distal lung. We speculate that this SP-A2 protein, like other serous secretions from airway submucosal glands, functions in local antimicrobial host defense mechanisms in the conducting airways.

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.

Vascular endothelial growth factor gene expression in human fetal lung in vitro.

Neonatal respiratory function depends on the development of a well-formed pulmonary capillary bed. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and angiogenesis. High levels of VEGF protein and messenger RNA (mRNA) have been detected in the developing lung, suggesting that VEGF plays a role in the development of the pulmonary capillary bed. To begin to understand the role of VEGF in human lung development, we explored the regulation of VEGF gene expression and the localization of VEGF protein and mRNA in a model of the developing human lung. VEGF protein and mRNA were detected in midtrimester human fetal lung tissue, and their levels increased with time in explant culture. VEGF protein and mRNA were increased by the maintenance of human fetal lung explants in 2% O2 environments compared with 20% O2 environments. VEGF mRNA levels were found to be increased by cyclic adenosine monophosphate (cAMP) in explants that were incubated in 20% O2, but not in those incubated in 2% O2. Immunostaining for VEGF protein demonstrated localization primarily in airway epithelial cells in midtrimester human fetal lung tissue. Immunostaining for VEGF increased with incubation of human fetal lung explants in 2% and 20% O2. Interestingly, VEGF protein was localized primarily in the basement membrane subjacent to airway epithelial cells after 4 d of incubation in 20% O2. Incubation of tissues in the presence of dibutyryl cAMP resulted in an increase in immunostaining for VEGF, primarily in the basement membranes of prealveolar ducts in 20% O2-treated tissues. In situ hybridization studies indicated that VEGF mRNA was present in both mesenchymal cells and airway epithelial cells. These data suggest that VEGF gene expression is regulated by both oxygen and cAMP in the developing human lung. The detection of VEGF mRNA and protein in distal airway epithelial cells and the detection of VEGF protein in the basement membrane subjacent to the airway epithelial cells suggest that translocation of VEGF protein occurs after its synthesis in the epithelium. Localization of VEGF to the basement membrane of airway epithelial cells may be important for directing capillary development in the human lung.

SP-A2 gene expression in human fetal lung airways.

In the present study, we characterized surfactant protein (SP)-A messenger RNA (mRNA) in mid-trimester human fetal trachea and bronchi. SP-A protein was localized by immunocytochemistry to scattered epithelial cells in the airway surface epithelium and in submucosal glands of the fetal trachea and bronchi. SP-A mRNA (2.2 kb) was detected by Northern blot analysis in human fetal trachea, as well as in primary and more distal bronchi. The levels of detectable SP-A mRNA were highest in the upper airways and were decreased in smaller bronchi in comparison. SP-A mRNA was barely detectable in the distal fetal lung tissue. In contrast, SP-A mRNA was abundant in cultured explants of distal human fetal lung tissue. SP-A1 and SP-A2 mRNA were detected by primer extension analysis in adult human lung tissue and in cultured human fetal lung explants. Only SP-A2 mRNA was detected in RNA isolated from human fetal trachea and bronchi. SP-A mRNA was localized by in situ hybridization in the fetal trachea and bronchi in scattered cells in the surface epithelium and, most prominently, in submucosal glands. Our results suggest that SP-A2, and not SP-A1, is produced in the human fetal tracheal and bronchial epithelium and in submucosal glands.

Mechanism of all trans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA.

The surfactant proteins (SPs) are required for the normal function of pulmonary surfactant, a lipoprotein substance that prevents alveolar collapse at end expiration. We characterized the effects of cortisol and all trans-retinoic acid (RA) on SP-A and SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 10(-6) M, caused a significant inhibition of SP-A mRNA to levels that were 60-70% of controls and a five- to sixfold increase in the levels of SP-B mRNA. RA alone (10(-6) M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol and RA both significantly increased SP-B mRNA levels. RA had no effect on the rate of SP-A gene transcription or on SP-A mRNA stability. Cortisol alone and the combination of cortisol and RA significantly inhibited the rate of SP-A gene transcription but had no effect on SP-A mRNA half-life. RA at 10(-6) M had no effect on the rate of SP-B gene transcription but prolonged SP-B mRNA half-life. Cortisol alone and the combination of cortisol and RA caused a significant increase in the rate of SP-B gene transcription and also caused a significant increase in SP-B mRNA stability. We conclude that RA has no effect on SP-A gene expression and increases SP-B mRNA levels by an effect on SP-B mRNA stability and not on the rate of SP-B gene transcription. In addition, the effects of the combination of RA and cortisol were generally similar to those of cortisol alone.