SAECs inhibit TGF-β induced fibroblast proliferation without affecting cell viability.

<p>(A) HLFs were treated with 5ng/ml TGF-β in fresh SAGM or in SAEC conditioned medium for 24 hours. Cells were allowed to incorporate ³H-thymidine for another 18 hours. n = 6 per group. Data shown are mean ± SD. *** = p<0.001 by ANOVA. (B) HLFs were treated with 5ng/ml TGF-β in the presence or absence SAECs for 72 hours and HLF viability was measured by trypan blue dye exclusion method. n = 3 per group.</p

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E₂

<div><p>Introduction</p><p>Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.</p><p>Measurements and Main Results</p><p>In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.</p><p>Conclusions</p><p>We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.</p></div

Epithelial cells inhibit TGF-β induced α-SMA protein expression in fibroblasts irrespective of their tissue origin.

<p>Multiple strains of human epithelial cells and fibroblasts were treated with or without 5ng/ml TGF-β and co-cultured for 72 hours. (A) SAECs co-cultured with Graves’ orbital fibroblasts. (B) SAECs co-cultured with keloid fibroblasts. (C) Human epidermal keratinocytes-neonatal co-cultured with HLFs. (D) A549 cells co-cultured with HLFs. α-SMA protein expression was analyzed by western blot. Each lane represents a replicate culture. Representative blots are shown from at least two independent experiments per condition.</p

Epithelial cell PGE₂ production is COX-2 dependent and is reversed by a PGE₂ neutralizing antibody.

<p>(A) Conditioned medium was collected from SAECs treated with or without 70nM SC-58125 for 24 hours. HLFs were treated with 5ng/ml TGF-β either in the presence or absence of SC58125 alone, or in the presence of SAEC conditioned medium from control or SC58125-treated SAECs. α−SMA protein expression was analyzed after 48 hours by western blot and densitometric analysis. (B) Conditioned medium from SAECs was neutralized by addition of 10μg/ml of anti- PGE₂ antibody, 2B5. HLFs were treated with 5 ng/ml TGF-β in either fresh SAGM, SAEC conditioned medium, or PGE₂ neutralized SAEC conditioned medium for 48 hours. α-SMA protein expression was analyzed by western blot. A representative blot from multiple independent experiments is shown. Samples were resolved on the same gel, and intervening irrelevant lanes are not shown. Note that, Neut. Ab– 2B5, PGE₂ neutralizing antibody, CTRL Ab–control antibody, n.s.–not significant. Data shown are mean ± SEM for n = 3 replicates. *** = p<0.001 by ANOVA.</p

SAECs inhibit TGF-β induced α-SMA protein expression in multiple normal and fibrotic human lung fibroblast strains.

<p>(A) Two additional normal, and (B) three additional IPF fibroblast strains from different donors were treated with or without 5ng/ml TGF-β and co-cultured with SAECs for 72 hours. α-SMA protein expression was analyzed by western blot. Representative blots are shown. Note that in Fig 2A, the indicated samples were resolved on the same gel, and intervening irrelevant lanes are not shown. Densitometry of n = 3 replicates per cell strain, normalized to untreated control. Data shown are mean ± SD. *** = p<0.001 ** = p<0.01 and * = p<0.05 by ANOVA.</p

SAECs inhibit collagen gel contraction and fibroblast migration.

<p>(A) HLFs were seeded into collagen gels and floated in medium containing 5ng/ml TGF-β. SAECs were grown separately on Transwell inserts and added to the wells. After 72 hours gels were weighed and percent contraction was calculated. n = 3–4 per group. (B) HLFs were grown on 6-well plates until they formed a confluent monolayer. A scratch wound was made on HLF monolayer, cells were washed with PBS and co-cultured with SAECs. HLF migration was tracked over time and imaged by phase contrast microscopy. (C) The scratch assay was performed on 3 replicate cultures for each condition. Each culture was photographed at 3 locations, and the width of the scratch was determined at 3 positions in each photograph (total of 9 measurements per condition), and percentage of original width was calculated by measuring the width between the edges of the scratch wound in three distinctive areas of each image. Data shown are mean ± SEM. *** = p<0.001 by ANOVA.</p

SAECs inhibit TGF-β induced fibroblast proliferation without affecting cell viability.

<p>(A) HLFs were treated with 5ng/ml TGF-β in fresh SAGM or in SAEC conditioned medium for 24 hours. Cells were allowed to incorporate ³H-thymidine for another 18 hours. n = 6 per group. Data shown are mean ± SD. *** = p<0.001 by ANOVA. (B) HLFs were treated with 5ng/ml TGF-β in the presence or absence SAECs for 72 hours and HLF viability was measured by trypan blue dye exclusion method. n = 3 per group.</p

SAECs inhibit TGF-β induced pro-fibrotic protein expression in human lung fibroblasts.

<p>(A) A schematic of co-culture system of HLFs and SAECs. HLFs and SAECs were grown separately on lower wells and upper inserts, respectively, of a Transwell co-culture system. HLFs were washed with PBS, co-cultured with SAECs with or without TGF-β for 72 hours (unless otherwise indicated). (B) HLFs were treated with 5ng/ml TGF- β in the presence or absence SAECs and α-SMA protein expression was analyzed by western blot and densitometric analysis. (C) Soluble collagen in culture medium from co-cultures was measured by slot blot with densitometric analysis. (D, E) HLFs were co-cultured with SAECs from two additional (different donors). Blots are representative of at least three independent experiments. (F) Alveolar epithelial cells were co-cultured with HLFs, and HLF expression of α-SMA was determined by western blot. Note that in Fig 1F, the indicated samples were resolved on the same gel, and intervening irrelevant lanes are not shown. Densitometry of n = 3 replicates per cell strain, normalized to untreated control. Data shown are mean ± SD. *** = p<0.001 ** = p<0.01 and * = p<0.05 by ANOVA.</p

SAECs exert anti-fibrotic effects through the soluble mediator PGE₂.

<p>PGE₂ in culture medium from SAEC-HLF co-cultures was measured by competitive EIA at 24 hours (A) and 72 hours (B) after TGF-β treatment. (C) PGE₂ concentrations in culture medium from AEC-HLF co-cultures were measured at 72 hours. (D) PGE₂ in culture medium from SAECs that were treated with TGF-β was measured at 48 hours post treatment. Data shown are mean ± SEM for n = 3 replicates. *** = p<0.001, ** = p<0.01 and * = p<0.05 by ANOVA. ### = p<0.001 by student’s t-test. (E) COX-2 protein expression was analyzed by western blot in SAECs that were co-cultured with HLFs and treated with or without 5ng/ml TGF-β. Densitometry of n = 3 replicates, normalized to untreated control. Data shown are mean ± SD. *** = p<0.001 by student’s t-test. (F) HLFs were treated with or without 5ng/ml TGF-β, or with 5ng/ml TGF-β and increasing concentrations of exogenous PGE₂ for 24 hours in serum-free MEM. α-SMA protein expression was analyzed by western blot. A representative blot is shown.</p