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Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice.

Dong Y, Geng Y, Li L, Li X, Yan X, Fang Y, Li X, Dong S, Liu X, Li X, Yang X, Zheng X, Xie T, Liang J, Dai H, Liu X, Yin Z, Noble PW, Jiang D, Ning W - J. Exp. Med. (2015)

Bottom Line: Pulmonary fibrosis is an epithelial-mesenchymal disorder in which TGF-β1 plays a central role in pathogenesis.Here we show that follistatin-like 1 (FSTL1) differentially regulates TGF-β and bone morphogenetic protein signaling, leading to epithelial injury and fibroblast activation.These data suggest that Fstl1 may serve as a novel therapeutic target for treatment of progressive lung fibrosis.

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Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China Cam-Su Genomic Resource Center, Soochow University, Suzhou 215123, China.

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Attenuated pulmonary fibrosis in Fstl1+/− mice.Fstl1+/− and their WT littermates were subjected to 5 U/kg bleomycin-induced lung injury, and lungs at the indicated time points were harvested for the following analyses. (A) Western blot analysis of FSTL1 expression in lung tissues 14 d after saline or bleomycin treatment (BLM; n = 5 per group). Genotypes are indicated (+/+, WT; +/−, Fstl1+/−). (B) Percentages of surviving mice were plotted over a 21-d period after bleomycin treatment. Log-rank test was used to compare the difference between Fstl1+/− and WT mice (n = 29 per group): P = 0.012. (C) Hydroxyproline contents in lung tissues from Fstl1+/− and WT mice 0, 7, 14, and 21 d after bleomycin treatment were measured (n = 7 per group; **, P < 0.01). (D) Masson trichrome staining of collagen on lung sections from Fstl1+/− and WT mice 7, 14, and 21 d after bleomycin treatment. Representative images of the staining are shown (n = 7 per group). (E) Lung fibrotic score analysis of the Masson trichrome staining D21 lung sections. The fibrotic area is presented as a percentage. Horizontal lines indicate the mean. *, P = 0.02. (F–H) Fstl1+/− and WT mice were treated with saline or bleomycin treatment for 14 d (n = 5 per group), and lung tissues were harvested for the following analyses. qRT-PCR analysis of Col1a1 (F) and fibronectin (Fn1; G) mRNA expressions (*, P < 0.05) and Western blot analysis of type I collagen (Col1) and fibronectin (Fn1; H). (A and H) β-Actin was used as a loading control. (I) Immunohistochemical analysis of collagen I in lung sections. Representative images of the staining are shown (n = 7 per group). (D and I) Bars, 100 µm. (A–D and F–I) The experiments were performed three times. (C, F, and G) Error bars indicate mean ± SEM.
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fig2: Attenuated pulmonary fibrosis in Fstl1+/− mice.Fstl1+/− and their WT littermates were subjected to 5 U/kg bleomycin-induced lung injury, and lungs at the indicated time points were harvested for the following analyses. (A) Western blot analysis of FSTL1 expression in lung tissues 14 d after saline or bleomycin treatment (BLM; n = 5 per group). Genotypes are indicated (+/+, WT; +/−, Fstl1+/−). (B) Percentages of surviving mice were plotted over a 21-d period after bleomycin treatment. Log-rank test was used to compare the difference between Fstl1+/− and WT mice (n = 29 per group): P = 0.012. (C) Hydroxyproline contents in lung tissues from Fstl1+/− and WT mice 0, 7, 14, and 21 d after bleomycin treatment were measured (n = 7 per group; **, P < 0.01). (D) Masson trichrome staining of collagen on lung sections from Fstl1+/− and WT mice 7, 14, and 21 d after bleomycin treatment. Representative images of the staining are shown (n = 7 per group). (E) Lung fibrotic score analysis of the Masson trichrome staining D21 lung sections. The fibrotic area is presented as a percentage. Horizontal lines indicate the mean. *, P = 0.02. (F–H) Fstl1+/− and WT mice were treated with saline or bleomycin treatment for 14 d (n = 5 per group), and lung tissues were harvested for the following analyses. qRT-PCR analysis of Col1a1 (F) and fibronectin (Fn1; G) mRNA expressions (*, P < 0.05) and Western blot analysis of type I collagen (Col1) and fibronectin (Fn1; H). (A and H) β-Actin was used as a loading control. (I) Immunohistochemical analysis of collagen I in lung sections. Representative images of the staining are shown (n = 7 per group). (D and I) Bars, 100 µm. (A–D and F–I) The experiments were performed three times. (C, F, and G) Error bars indicate mean ± SEM.

Mentions: To investigate the biological significance of the inducible expression of Fstl1 during fibrogenesis, we examined the fibrotic response to bleomycin-induced lung injury in Fstl1-deficient mice. Because homozygous Fstl1−/− mice die of respiratory failure shortly after birth (Geng et al., 2011), heterozygous Fstl1+/− mice were used to study the fibrotic response to bleomycin injury. Fstl1+/− mice made significant less FSTL1 protein in the lung tissue (∼59% decrease), and bleomycin-induced increase of FSTL1 protein was dramatically reduced in Fstl1+/− lungs (∼57% decrease; Fig. 2 A). Fstl1+/− mice were significantly less susceptible to bleomycin-induced lung injury and showed an increase in survival relative to WT littermates (Fig. 2 B). Importantly, Fstl1+/− mice exhibited reduced lung fibrosis. Collagen accumulation was significantly reduced in lung tissue of Fstl1+/− mice, as determined by hydroxyproline content (Fig. 2 C) and Masson’s trichrome staining (Fig. 2 D). Quantification of fibrotic lung sections by a blinded pathologist illustrated the attenuated fibrosis in Fstl1+/− mice (Fig. 2 E). The attenuated fibrosis in Fstl1+/− mice was further supported by decreased mRNA and protein levels for type I collagen and fibronectin (Fig. 2, F–I). These in vivo data indicate that Fstl1 is induced in response to lung injury and causally involved in driving pulmonary fibrogenesis as a profibrotic factor.


Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice.

Dong Y, Geng Y, Li L, Li X, Yan X, Fang Y, Li X, Dong S, Liu X, Li X, Yang X, Zheng X, Xie T, Liang J, Dai H, Liu X, Yin Z, Noble PW, Jiang D, Ning W - J. Exp. Med. (2015)

Attenuated pulmonary fibrosis in Fstl1+/− mice.Fstl1+/− and their WT littermates were subjected to 5 U/kg bleomycin-induced lung injury, and lungs at the indicated time points were harvested for the following analyses. (A) Western blot analysis of FSTL1 expression in lung tissues 14 d after saline or bleomycin treatment (BLM; n = 5 per group). Genotypes are indicated (+/+, WT; +/−, Fstl1+/−). (B) Percentages of surviving mice were plotted over a 21-d period after bleomycin treatment. Log-rank test was used to compare the difference between Fstl1+/− and WT mice (n = 29 per group): P = 0.012. (C) Hydroxyproline contents in lung tissues from Fstl1+/− and WT mice 0, 7, 14, and 21 d after bleomycin treatment were measured (n = 7 per group; **, P < 0.01). (D) Masson trichrome staining of collagen on lung sections from Fstl1+/− and WT mice 7, 14, and 21 d after bleomycin treatment. Representative images of the staining are shown (n = 7 per group). (E) Lung fibrotic score analysis of the Masson trichrome staining D21 lung sections. The fibrotic area is presented as a percentage. Horizontal lines indicate the mean. *, P = 0.02. (F–H) Fstl1+/− and WT mice were treated with saline or bleomycin treatment for 14 d (n = 5 per group), and lung tissues were harvested for the following analyses. qRT-PCR analysis of Col1a1 (F) and fibronectin (Fn1; G) mRNA expressions (*, P < 0.05) and Western blot analysis of type I collagen (Col1) and fibronectin (Fn1; H). (A and H) β-Actin was used as a loading control. (I) Immunohistochemical analysis of collagen I in lung sections. Representative images of the staining are shown (n = 7 per group). (D and I) Bars, 100 µm. (A–D and F–I) The experiments were performed three times. (C, F, and G) Error bars indicate mean ± SEM.
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fig2: Attenuated pulmonary fibrosis in Fstl1+/− mice.Fstl1+/− and their WT littermates were subjected to 5 U/kg bleomycin-induced lung injury, and lungs at the indicated time points were harvested for the following analyses. (A) Western blot analysis of FSTL1 expression in lung tissues 14 d after saline or bleomycin treatment (BLM; n = 5 per group). Genotypes are indicated (+/+, WT; +/−, Fstl1+/−). (B) Percentages of surviving mice were plotted over a 21-d period after bleomycin treatment. Log-rank test was used to compare the difference between Fstl1+/− and WT mice (n = 29 per group): P = 0.012. (C) Hydroxyproline contents in lung tissues from Fstl1+/− and WT mice 0, 7, 14, and 21 d after bleomycin treatment were measured (n = 7 per group; **, P < 0.01). (D) Masson trichrome staining of collagen on lung sections from Fstl1+/− and WT mice 7, 14, and 21 d after bleomycin treatment. Representative images of the staining are shown (n = 7 per group). (E) Lung fibrotic score analysis of the Masson trichrome staining D21 lung sections. The fibrotic area is presented as a percentage. Horizontal lines indicate the mean. *, P = 0.02. (F–H) Fstl1+/− and WT mice were treated with saline or bleomycin treatment for 14 d (n = 5 per group), and lung tissues were harvested for the following analyses. qRT-PCR analysis of Col1a1 (F) and fibronectin (Fn1; G) mRNA expressions (*, P < 0.05) and Western blot analysis of type I collagen (Col1) and fibronectin (Fn1; H). (A and H) β-Actin was used as a loading control. (I) Immunohistochemical analysis of collagen I in lung sections. Representative images of the staining are shown (n = 7 per group). (D and I) Bars, 100 µm. (A–D and F–I) The experiments were performed three times. (C, F, and G) Error bars indicate mean ± SEM.
Mentions: To investigate the biological significance of the inducible expression of Fstl1 during fibrogenesis, we examined the fibrotic response to bleomycin-induced lung injury in Fstl1-deficient mice. Because homozygous Fstl1−/− mice die of respiratory failure shortly after birth (Geng et al., 2011), heterozygous Fstl1+/− mice were used to study the fibrotic response to bleomycin injury. Fstl1+/− mice made significant less FSTL1 protein in the lung tissue (∼59% decrease), and bleomycin-induced increase of FSTL1 protein was dramatically reduced in Fstl1+/− lungs (∼57% decrease; Fig. 2 A). Fstl1+/− mice were significantly less susceptible to bleomycin-induced lung injury and showed an increase in survival relative to WT littermates (Fig. 2 B). Importantly, Fstl1+/− mice exhibited reduced lung fibrosis. Collagen accumulation was significantly reduced in lung tissue of Fstl1+/− mice, as determined by hydroxyproline content (Fig. 2 C) and Masson’s trichrome staining (Fig. 2 D). Quantification of fibrotic lung sections by a blinded pathologist illustrated the attenuated fibrosis in Fstl1+/− mice (Fig. 2 E). The attenuated fibrosis in Fstl1+/− mice was further supported by decreased mRNA and protein levels for type I collagen and fibronectin (Fig. 2, F–I). These in vivo data indicate that Fstl1 is induced in response to lung injury and causally involved in driving pulmonary fibrogenesis as a profibrotic factor.

Bottom Line: Pulmonary fibrosis is an epithelial-mesenchymal disorder in which TGF-β1 plays a central role in pathogenesis.Here we show that follistatin-like 1 (FSTL1) differentially regulates TGF-β and bone morphogenetic protein signaling, leading to epithelial injury and fibroblast activation.These data suggest that Fstl1 may serve as a novel therapeutic target for treatment of progressive lung fibrosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China Cam-Su Genomic Resource Center, Soochow University, Suzhou 215123, China.

Show MeSH
Related in: MedlinePlus