Contribution of Epithelial-derived Fibroblasts to Bleomycin-induced Lung Fibrosis

Abstract

Rationale: Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. Objectives: We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. Methods: Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-b 1 and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with b-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, b-galactosidase, green fluorescent protein, and a-smooth muscle actin. Measurements and Main Results: In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-b 1 and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. Conclusions: Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Lung Pericytes and Resident Fibroblasts

“…9,10,25,29,32,84e88 In two independent experiments, a substantial population of myofibroblasts reported transgenic expression from an incomplete section of the human surfactant protein C promoter after lung injury. 32,86 Because transcription of endogenous surfactant protein C is specific for type II airway epithelial cells, these results support the epithelial-tomesenchymal transition hypothesis that pathogenic myofibroblasts originate from dedifferentiated epithelial cells. 79 However, a different reporter system inserted into the endogenous mouse Sftpc locus found epithelial cells did not become myofibroblasts in the bleomycin lung injury model.…”

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs

“…3D). In previous studies, we have used S100A4 as a fibroblast marker in the lungs (14)(15)(16)(17). Therefore, we evaluated lung sections by IHC for S100A4 expression and noted greater numbers of S100A4-positive fibroblasts in L188Q SFTPC mice compared with WT controls after bleomycin treatment ( Additionally, we wanted to determine whether lung mechanics were different at 3 wk after bleomycin treatment in L188Q SFTPC mice.…”

“…in mice by intubation as previously described (17). Lungs were harvested for histology, frozen tissue, bronchoalveolar lavage, or cell isolation as previously described (14)(15)(16)(17). L188Q SFTPC mice and controls were maintained on normal water ad libitum until transgene activation was desired.…”

“…Formalin-fixed lung sections were prepared as previously described (14)(15)(16). IHC (14,15), immunofluorescence (16,17), TUNEL (15,17), and bronchoalveolar lavage with cell counts (15,17) were performed as previously described. RNA isolation, real-time RT-PCR, and Western blot analysis were performed by using standard techniques (detailed in SI Materials and Methods).…”

Mechanical ventilation augments bleomycin-induced epithelial–mesenchymal transition through the Src pathway

“…15 The impaired gas exchange and lung fibrogenesis can occur in the first week of the fibroproliferative phase of ARDS, which is similar to that observed in the fibrogenesis model following bleomycin exposure. 5,29 A previous study of an acid-induced lung injury model in mice also showed the crucial role of mechanical ventilation in initiating lung fibrogenesis, which was associated with TGF-b1-induced EMT and lung fibroblast proliferation. 3 Recent studies using alveolar type II epithelial cells and rats have demonstrated that cyclic mechanical stretch can lead to disorganization and ECM remodeling of the lung by activating the Wnt/b-catenin pathway and inducing EMT.…”