Abstract
Human Fetal Type Ii Epithelial Cells Maintain Differentiated Function In Monolayer Culture.
LW Gonzales, S Angampalli, A Matlapudi, SI Feinstein, KO Solarin, MF Beers, PL Ballard., Depts. of Peds and Medicine, Children's Hosp. of Phila, and Institute for Environmental Medicine, Univ. of Penn, Philadelphia, PA.
Studies of Type II (TII) cell differentiation and function require in vitro models expressing differentiated functions, and such models have not been readily available. Recently Alcorn et al. (Am J Respir Cell Molec Biol, 17:672, 1997) described TII cells, released from human fetal lung explants by collagenase digestion, which maintained differentiation when cultured in low volume media containing dibutyryl-cAMP. We have modified this method and further characterized the isolated cells. Explants were cultured in Dexamethasone [Dex]+8-Br-cAMP for 4 days, then digested with collagenase + trypsin and fibroblasts were removed by differential adherence. The TII cells were plated on either MDCK cell matrix or plastic in serum-free Waymouth's media. The TII cells on matrix develop confluent monolayers without epithelial cell clumping and maintain content of SP-B and SP-A mRNAs (110±14%, n=5 and 76±10%, n=3), respectively of original content) for at least 4 days in the presence of Dex (10 nM) + 8-Br-cAMP (0.1 mM) + isobutylmethylxanthine (0.1 mM). Cells on plastic showed similarly high retention of SP mRNAs in the presence of hormones. Addition of either Dex or cAMP alone produced lesser responses. Dibutyryl-cAMP and 8-Br-cAMP were equally effective. Responses were similar at low or high media volume and in the presence of 10% serum. In the presence of Dex+cAMP, proSP-C protein labeled with 35S-methionine was processed in cells on either matrix or plastic at a rate comparable to that for lung explants. We conclude that differentiated TII cell function can be maintained by hormones under usual cell culture conditions. This method provides a useful model for studying development and regulation of the surfactant system in an accessible in vitro model.
Organ Culture As A Model Of Alveolar Development.
M.J. Kresch, C. Christian, L Zhu, N. Hussain. Perinatal Research Laboratory, Dept. of Pediatrics, University of Connecticut School of Medicine, Farmington, CT.
Explants of fetal rat lung have been a useful model to study fetal rat lung development. The alveolar phase of rat lung development begins just prior to birth and continues for the first 3 weeks of life. The cellular and molecular mechanisms of alveolar formation have not been studied. We have previously shown that there is an abrupt increase in apoptosis of lung mesenchyme around the time of birth in rat lung. We formulated the hypothesis that the regulated balance between apoptosis and cell proliferation is a key mechanism by which the lung remodels during the alveolar phase of lung development. The goal of this study was to determine the usefulness of the organ culture model for the study of early alveolar lung development. Explants of 21 days' gestation (term=22 days) fetal rat lung were placed in explant culture for 0-7 days. At each day tissue was harvested for protein extraction, total RNA extraction and histologic analysis. Immunoblots using appropriate primary antibodies were used to measure changes in SP-A receptor (specific marker of type II cells) and p21 (marker of cell cycle arrest). Northern blots measured changes in steady state levels of mRNA for a-1 procollagen (marker of alveolar septal formation). Apoptosis was quantified using the TUNEL assay in histologic sections. We found increased apoptosis in explants after 0-2 days in culture corresponding to the increased levels of apoptosis measured in vivo. These changes in culture occurred concomitantly with increased levels of p21. We also found increased levels of SP-A receptor after 1-2 days in culture. Levels of a-1 procollagen mRNA increased after 5 days in culture. We conclude that the explant culture is a useful model for study of the early events during the alveolar phase of lung development: 1) increased apoptosis and cell cycle arrest; 2) increased alveolar septum formation; 3) increased specific markers of type II cells.
The Role Of Calcium In The Regulation Of Surfactant Secretion.
White, M.K. and Strayer, D.S. Dept. of Pathology, Anat., and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107.
Secretion of pulmonary surfactant by type II alveolar cells is stimulated by secretagogues that act either in a calcium-dependent (e.g. ionomycin) or calcium-independent manner (e.g. phorbol ester, PMA). Surfactant protein A (SP-A) acts through a cell surface receptor to prevent calcium mobilization and to inhibit secretion stimulation by both types of secretagogue. Several findings indicate that it is calcium release from stores rather than the consequent change in cytoplasmic calcium concentration ([Ca2+]i) that activates secretion. For example, treating cells with the calcium chelator BAPTA-AM elicited secretion without increasing [Ca2+]i and this was inhibited by SP-A or calcium depletion. However PMA induces secretion by a different mechanism that is not inhibited by calcium depletion and is additive to secretion induced by calcium-releasing secretagogues.
Temporospatial Expression Of Specific Regulators Of Angiogenesis During Alveolization In Rats.
RC Savani, E Wentz, Z Cui, PM Pooler, Z Zhou and HM DeLisser. Depts. of Peds & Med., Univ. of Pennsylvania School of Medicine, Philadelphia PA.
Normal rat alveolization occurs in the first two weeks of life. Concomitant steroid administration impedes normal alveolar septation. We hypothesized that normal angiogenesis during alveolization would require appropriate temporospatial expression of soluble factors, cell:cell and cell:matrix adhesion molecules, and that steroid treatment would disrupt this normal pattern. Since Vascular Endothelial Growth Factor (VEGF, soluble factor), Platelet-Endothelial Cell Adhesion Molecule (PECAM)-1 (cell:cell) and hyaluronan (HA, matrix) have previously been implicated in angiogenesis, we examined the expression of these molecules and the Receptor for HA-Mediated Motility (RHAMM) during normal and steroid-retarded alveolization in rats. Litter sizes were normalized to 10 pups/litter to equalize somatic growth. Untreated pups were compared to those receiving either 0.1 µg dexamethasone or an equivalent volume of saline daily subcutaneously. Immunostaining of normal lungs demonstrated the expression of VEGF (in epithelial cells) and PECAM-1 (on endothelium) at birth. These molecules became almost undetectable on days 5 and 10, and reappeared by day 15. In contrast, RHAMM and HA expression was restricted to epithelial and sub-epithelial areas of bronchioles at birth, increased in alveolar walls and developing septae on days 5 and 10, and returned to a more restricted pattern of expression by day 15 during normal development. Steroid treatment resulted in a delay in these changes as well as decreased septation at all timepoints examined. All changes in expression were confirmed by northern blot analysis of VEGF, PECAM-1 and RHAMM, and by HA content measured by radiometric assay. We conclude that steroid-induced changes in the normal temporospatial expression of angiogenic factors is associated with altered septation. We speculate that angiogenesis may be critical for alveolization.