Fibroblasts Expressing PDGF-Receptor-Alpha Diminish During Alveolar Septal Thinning in Mice

Abstract

In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-R␣), is required for expansion, but the receptor's relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfr␣-expressing LF (pdgfr␣-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-R␣-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (␣SMA) producing pdgfr␣-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-R␣ gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.

Platelet-derived growth factor-A and sonic hedgehog signaling direct lung fibroblast precursors during alveolar septal formation

“…These heterozygous mice are phenotypically identical to wild-type (GFPϪ) mice, except for nuclear GFP, which enables their identification (13). Lung fibroblasts were isolated from heterozygous mice on P4 to P12 by a previously reported method (30). In brief, the lungs were removed, avoiding the hilar structures, and digested for 1 h with 300 g/ml collagenase type I, 250 U trypsin (8,500 U/mg solid), 75 g/ml DNAse I, and 2 mM calcium chloride, with the dispersed cells removed from the undigested tissue at 15-min intervals (21).…”

“…At P12, epithelial, macrophage, and endothelial cells comprised ϳ8, 18, and 1.6%, respectively, of the adherent cells. Protocols for animal use were approved by the Iowa City Veterans Affairs Medical Center animal use committee (30). Total RNA was obtained from freshly isolated LF that had not been separated using TRI reagent (Sigma-Aldrich) according to pdgfr␣ expression.…”

“…For the Ki67, ␣-SMA, and cytokeratin 18, and in some instances for Shh, the LF were permeabilized prior to immunostaining (21). Flow cytometry (FACS) was conducted following previously published methods (30). More detailed information about the antibodies used for staining appears under Materials above.…”

Alterations in expression of elastogenic and angiogenic genes by different conditions of mechanical ventilation in newborn rat lung

“…expression has been shown to be upregulated prior to the development of fibroproliferative lung lesions (26), while a decrease in PDGFRA-expressing lung fibroblasts coincided with alveolar thinning (36). MMPs represent a family of zinc-dependent proteases that degrade the ECM.…”

Divergent fibroblast growth factor signaling pathways in lung fibroblast subsets: where do we go from here?

“…More recently, fibroblasts have been credited with additional roles in alveologenesis, including regulatory fibroblast-epithelial cell cross-talk (45,48), and this idea has gained prominence with the delineation of multiple lung fibroblast subsets, including lipofibroblasts (31) and myofibroblasts (8,47), and their respective precursor cells (29,33). Although the discrimination between, and markers of, these fibroblast subsets is complicated and not yet fully resolved, the platelet-derived growth factor (PDGF) receptor (PDGFR)-␣ is believed to mark a population of fibroblasts that plays a key role in postnatal lung maturation (7,26,28). This is in line with the critical role of PDGF/PDGFR-␣ signaling in lung development, where inactivation of PDGF-A or PDGFR-␣ in mice (6,7) blocks alveolarization.…”