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Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

MacKenzie B, Korfei M, Henneke I, Sibinska Z, Tian X, Hezel S, Dilai S, Wasnick R, Schneider B, Wilhelm J, El Agha E, Klepetko W, Seeger W, Schermuly R, Günther A, Bellusci S - Respir. Res. (2015)

Bottom Line: Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts.The FGFR inhibitor (PD173074) attenuated these effects.

View Article: PubMed Central - PubMed

Affiliation: German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center, Giessen, Hessen, Germany.

ABSTRACT

Background: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

No MeSH data available.


Related in: MedlinePlus

Expression and localization of FGF1 and FGF-receptors FGFR1, FGFR2, FGFR3 and FGFR4 in hyperplastic, overlying alveolar epithelium, fibroblastic foci and basal cell sheets in idiopathic pulmonary fibrosis (IPF) lungs. a. Representative immunohistochemistry for FGF1, FGFR1, FGFR2, FGFR3, and prosurfactant protein C (pro-SP-C) in serial sections of IPF lung tissue. Alveolar epithelial type-II cells (AECII, indicated by arrows) of IPF lungs express FGF1 (a1,2), FGFR2 (a4,5), FGFR3 (a6,7), and FGFR4 (a8,9), but not FGFR1 (a3,4). b. Representative immunohistochemistry for Cytokeratin-5 (KRT5) (b1,2), Fascin (b3,4), FGF1 (b5,6) α-SMA (b7,8), FGFR1 (b9,10), and FGFR2 (b11,12) in serial sections of IPF lung tissue. In IPF, immunostaining for FGF1, FGFR1 and FGFR2 was observed in myofibroblasts of fibroblast foci [FF] (indicated by arrowheads and α-SMA-staining) as well as in overlying hyperplastic bronchiolar basal cells (indicated by asterisks and KRT5-staining), and colocalized with expression of the migratory marker Fascin (2). Representative immunohistochemistry for α-SMA (b13), FGFR3 (b14) and FGFR4 (b15) and FGF1 (b16) in serial sections of IPF lung tissue. In general, α-SMA-positive myofibroblasts of FF (indicated by arrowheads) express FGF1, FGFR3 and FGFR4. Of note, FGFR3 expression appeared predominantly nuclear in AECII as well as myofibroblastic cells. Scale bars: a1-9 (100 μm); b1,3,5,7,9,11 (250 μm), b2,4,6,8,10,12 (100 μm), b13-16 (100 μm)
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Fig3: Expression and localization of FGF1 and FGF-receptors FGFR1, FGFR2, FGFR3 and FGFR4 in hyperplastic, overlying alveolar epithelium, fibroblastic foci and basal cell sheets in idiopathic pulmonary fibrosis (IPF) lungs. a. Representative immunohistochemistry for FGF1, FGFR1, FGFR2, FGFR3, and prosurfactant protein C (pro-SP-C) in serial sections of IPF lung tissue. Alveolar epithelial type-II cells (AECII, indicated by arrows) of IPF lungs express FGF1 (a1,2), FGFR2 (a4,5), FGFR3 (a6,7), and FGFR4 (a8,9), but not FGFR1 (a3,4). b. Representative immunohistochemistry for Cytokeratin-5 (KRT5) (b1,2), Fascin (b3,4), FGF1 (b5,6) α-SMA (b7,8), FGFR1 (b9,10), and FGFR2 (b11,12) in serial sections of IPF lung tissue. In IPF, immunostaining for FGF1, FGFR1 and FGFR2 was observed in myofibroblasts of fibroblast foci [FF] (indicated by arrowheads and α-SMA-staining) as well as in overlying hyperplastic bronchiolar basal cells (indicated by asterisks and KRT5-staining), and colocalized with expression of the migratory marker Fascin (2). Representative immunohistochemistry for α-SMA (b13), FGFR3 (b14) and FGFR4 (b15) and FGF1 (b16) in serial sections of IPF lung tissue. In general, α-SMA-positive myofibroblasts of FF (indicated by arrowheads) express FGF1, FGFR3 and FGFR4. Of note, FGFR3 expression appeared predominantly nuclear in AECII as well as myofibroblastic cells. Scale bars: a1-9 (100 μm); b1,3,5,7,9,11 (250 μm), b2,4,6,8,10,12 (100 μm), b13-16 (100 μm)

Mentions: In IPF lungs, FGF1 was present in SpC+, hyperplastic AECII cells, overlying regions of fibrosis (Fig. 3; a1-2). No or only weak FGFR1 staining was observed (Fig. 3; a3-4). FGFR2 was strongly expressed by proSP-C+ alveolar epithelium (Fig. 3; a5). as well as FGFR3 and FGFR4 (Fig. 3; a7 and a9, respectively). Alveolar macrophages also stained strongly for FGF1 as well as for FGFR2 and 4. FGFR1 and 3 staining of macrophages was faint (Additional file 3: Figure S2).Fig. 3


Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

MacKenzie B, Korfei M, Henneke I, Sibinska Z, Tian X, Hezel S, Dilai S, Wasnick R, Schneider B, Wilhelm J, El Agha E, Klepetko W, Seeger W, Schermuly R, Günther A, Bellusci S - Respir. Res. (2015)

Expression and localization of FGF1 and FGF-receptors FGFR1, FGFR2, FGFR3 and FGFR4 in hyperplastic, overlying alveolar epithelium, fibroblastic foci and basal cell sheets in idiopathic pulmonary fibrosis (IPF) lungs. a. Representative immunohistochemistry for FGF1, FGFR1, FGFR2, FGFR3, and prosurfactant protein C (pro-SP-C) in serial sections of IPF lung tissue. Alveolar epithelial type-II cells (AECII, indicated by arrows) of IPF lungs express FGF1 (a1,2), FGFR2 (a4,5), FGFR3 (a6,7), and FGFR4 (a8,9), but not FGFR1 (a3,4). b. Representative immunohistochemistry for Cytokeratin-5 (KRT5) (b1,2), Fascin (b3,4), FGF1 (b5,6) α-SMA (b7,8), FGFR1 (b9,10), and FGFR2 (b11,12) in serial sections of IPF lung tissue. In IPF, immunostaining for FGF1, FGFR1 and FGFR2 was observed in myofibroblasts of fibroblast foci [FF] (indicated by arrowheads and α-SMA-staining) as well as in overlying hyperplastic bronchiolar basal cells (indicated by asterisks and KRT5-staining), and colocalized with expression of the migratory marker Fascin (2). Representative immunohistochemistry for α-SMA (b13), FGFR3 (b14) and FGFR4 (b15) and FGF1 (b16) in serial sections of IPF lung tissue. In general, α-SMA-positive myofibroblasts of FF (indicated by arrowheads) express FGF1, FGFR3 and FGFR4. Of note, FGFR3 expression appeared predominantly nuclear in AECII as well as myofibroblastic cells. Scale bars: a1-9 (100 μm); b1,3,5,7,9,11 (250 μm), b2,4,6,8,10,12 (100 μm), b13-16 (100 μm)
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Fig3: Expression and localization of FGF1 and FGF-receptors FGFR1, FGFR2, FGFR3 and FGFR4 in hyperplastic, overlying alveolar epithelium, fibroblastic foci and basal cell sheets in idiopathic pulmonary fibrosis (IPF) lungs. a. Representative immunohistochemistry for FGF1, FGFR1, FGFR2, FGFR3, and prosurfactant protein C (pro-SP-C) in serial sections of IPF lung tissue. Alveolar epithelial type-II cells (AECII, indicated by arrows) of IPF lungs express FGF1 (a1,2), FGFR2 (a4,5), FGFR3 (a6,7), and FGFR4 (a8,9), but not FGFR1 (a3,4). b. Representative immunohistochemistry for Cytokeratin-5 (KRT5) (b1,2), Fascin (b3,4), FGF1 (b5,6) α-SMA (b7,8), FGFR1 (b9,10), and FGFR2 (b11,12) in serial sections of IPF lung tissue. In IPF, immunostaining for FGF1, FGFR1 and FGFR2 was observed in myofibroblasts of fibroblast foci [FF] (indicated by arrowheads and α-SMA-staining) as well as in overlying hyperplastic bronchiolar basal cells (indicated by asterisks and KRT5-staining), and colocalized with expression of the migratory marker Fascin (2). Representative immunohistochemistry for α-SMA (b13), FGFR3 (b14) and FGFR4 (b15) and FGF1 (b16) in serial sections of IPF lung tissue. In general, α-SMA-positive myofibroblasts of FF (indicated by arrowheads) express FGF1, FGFR3 and FGFR4. Of note, FGFR3 expression appeared predominantly nuclear in AECII as well as myofibroblastic cells. Scale bars: a1-9 (100 μm); b1,3,5,7,9,11 (250 μm), b2,4,6,8,10,12 (100 μm), b13-16 (100 μm)
Mentions: In IPF lungs, FGF1 was present in SpC+, hyperplastic AECII cells, overlying regions of fibrosis (Fig. 3; a1-2). No or only weak FGFR1 staining was observed (Fig. 3; a3-4). FGFR2 was strongly expressed by proSP-C+ alveolar epithelium (Fig. 3; a5). as well as FGFR3 and FGFR4 (Fig. 3; a7 and a9, respectively). Alveolar macrophages also stained strongly for FGF1 as well as for FGFR2 and 4. FGFR1 and 3 staining of macrophages was faint (Additional file 3: Figure S2).Fig. 3

Bottom Line: Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts.The FGFR inhibitor (PD173074) attenuated these effects.

View Article: PubMed Central - PubMed

Affiliation: German Center for Lung Research, Excellence Cluster Cardio-Pulmonary System, Universities of Giessen and Marburg Lung Center, Giessen, Hessen, Germany.

ABSTRACT

Background: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

No MeSH data available.


Related in: MedlinePlus