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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.

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.

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Reduced accumulation of myofibroblasts in Fstl1+/− mouse lungs.Fstl1+/− and their WT littermates were treated with saline or 2.5 U/kg bleomycin (BLM) for 14 d. (A) Immunofluorescence analysis of α-SMA expression in lung sections (nucleus, DAPI; n = 6 per group). Representative images of the staining are shown. Note that myofibroblast staining (green) is not co-located with blood vesicles (red). Insets show hematoxylin and eosin staining of the adjacent section. (B) qRT-PCR analysis of α-SMA mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (C) Western blot analysis of α-SMA expression in lung tissues 7 and 14 d after bleomycin treatment (n = 5 per group). The white line indicates intervening lanes have been spliced out. (D) qRT-PCR analysis of Fsp1 mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (E) Newly isolated lung fibroblasts from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment (n = 6 per group) were stained for goat anti–rabbit IgG control (red lines) or α-SMA–FITC (blue lines). The proportions of α-SMA–positive cells (myofibroblasts) were determined. Mean percentages of α-SMA+ cells in lung fibroblasts were summarized in the upper region of each panel. The experiments were performed twice. (F) Immunofluorescent staining of myofibroblasts in lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment. Representative images of the staining are shown (n = 5 per group). Arrows indicate myofibroblasts with α-SMA– and vimentin-positive staining. Nuclear, DAPI. (A and F) Bars, 100 µm. (G) Quantification of F presents percentage of α-SMA–positive cells (myofibroblasts). **, P < 0.01. (H) Western blot analysis of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in the medium (supernatant [SN]) of primary cultured lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after bleomycin treatment (n = 6 per group). (C and H) β-Actin was used as a loading control. (A–D, F, and H) The experiments were performed three times. (B, D, and G) Error bars indicate mean ± SEM.
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fig4: Reduced accumulation of myofibroblasts in Fstl1+/− mouse lungs.Fstl1+/− and their WT littermates were treated with saline or 2.5 U/kg bleomycin (BLM) for 14 d. (A) Immunofluorescence analysis of α-SMA expression in lung sections (nucleus, DAPI; n = 6 per group). Representative images of the staining are shown. Note that myofibroblast staining (green) is not co-located with blood vesicles (red). Insets show hematoxylin and eosin staining of the adjacent section. (B) qRT-PCR analysis of α-SMA mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (C) Western blot analysis of α-SMA expression in lung tissues 7 and 14 d after bleomycin treatment (n = 5 per group). The white line indicates intervening lanes have been spliced out. (D) qRT-PCR analysis of Fsp1 mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (E) Newly isolated lung fibroblasts from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment (n = 6 per group) were stained for goat anti–rabbit IgG control (red lines) or α-SMA–FITC (blue lines). The proportions of α-SMA–positive cells (myofibroblasts) were determined. Mean percentages of α-SMA+ cells in lung fibroblasts were summarized in the upper region of each panel. The experiments were performed twice. (F) Immunofluorescent staining of myofibroblasts in lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment. Representative images of the staining are shown (n = 5 per group). Arrows indicate myofibroblasts with α-SMA– and vimentin-positive staining. Nuclear, DAPI. (A and F) Bars, 100 µm. (G) Quantification of F presents percentage of α-SMA–positive cells (myofibroblasts). **, P < 0.01. (H) Western blot analysis of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in the medium (supernatant [SN]) of primary cultured lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after bleomycin treatment (n = 6 per group). (C and H) β-Actin was used as a loading control. (A–D, F, and H) The experiments were performed three times. (B, D, and G) Error bars indicate mean ± SEM.

Mentions: The accumulation of fibroblasts with an activated phenotype (named as myofibroblasts) in the fibrotic foci characterizes progressive pulmonary fibrosis (Scotton and Chambers, 2007; Phan, 2012). To examine whether the attenuated fibrotic phenotype in Fstl1+/− mice is associated with the changed accumulation of myofibroblasts, we performed immunohistochemistry on lung sections with anti–α-SMA antibodies, a marker for newly appearing myofibroblasts. As shown in Fig. 4 A, bleomycin-increased myofibroblast accumulation was apparent in WT mice, but not in Fstl1+/− mice. Consistently, qRT-PCR and immunoblotting analyses of lung tissues showed that bleomycin-induced expression of α-SMA in WT mice was significantly decreased in Fstl1+/− mice (Fig. 4, B and C). Meanwhile, we observed that the mRNA level of Fsp-1, a marker for fibroblasts, was lower in Fstl1+/− mice than that in WT (Fig. 4 D). These data suggest that Fstl1 deficiency is protective against fibrogenesis, possibly via limiting the bleomycin-induced accumulation of myofibroblasts.


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)

Reduced accumulation of myofibroblasts in Fstl1+/− mouse lungs.Fstl1+/− and their WT littermates were treated with saline or 2.5 U/kg bleomycin (BLM) for 14 d. (A) Immunofluorescence analysis of α-SMA expression in lung sections (nucleus, DAPI; n = 6 per group). Representative images of the staining are shown. Note that myofibroblast staining (green) is not co-located with blood vesicles (red). Insets show hematoxylin and eosin staining of the adjacent section. (B) qRT-PCR analysis of α-SMA mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (C) Western blot analysis of α-SMA expression in lung tissues 7 and 14 d after bleomycin treatment (n = 5 per group). The white line indicates intervening lanes have been spliced out. (D) qRT-PCR analysis of Fsp1 mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (E) Newly isolated lung fibroblasts from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment (n = 6 per group) were stained for goat anti–rabbit IgG control (red lines) or α-SMA–FITC (blue lines). The proportions of α-SMA–positive cells (myofibroblasts) were determined. Mean percentages of α-SMA+ cells in lung fibroblasts were summarized in the upper region of each panel. The experiments were performed twice. (F) Immunofluorescent staining of myofibroblasts in lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment. Representative images of the staining are shown (n = 5 per group). Arrows indicate myofibroblasts with α-SMA– and vimentin-positive staining. Nuclear, DAPI. (A and F) Bars, 100 µm. (G) Quantification of F presents percentage of α-SMA–positive cells (myofibroblasts). **, P < 0.01. (H) Western blot analysis of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in the medium (supernatant [SN]) of primary cultured lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after bleomycin treatment (n = 6 per group). (C and H) β-Actin was used as a loading control. (A–D, F, and H) The experiments were performed three times. (B, D, and G) Error bars indicate mean ± SEM.
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fig4: Reduced accumulation of myofibroblasts in Fstl1+/− mouse lungs.Fstl1+/− and their WT littermates were treated with saline or 2.5 U/kg bleomycin (BLM) for 14 d. (A) Immunofluorescence analysis of α-SMA expression in lung sections (nucleus, DAPI; n = 6 per group). Representative images of the staining are shown. Note that myofibroblast staining (green) is not co-located with blood vesicles (red). Insets show hematoxylin and eosin staining of the adjacent section. (B) qRT-PCR analysis of α-SMA mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (C) Western blot analysis of α-SMA expression in lung tissues 7 and 14 d after bleomycin treatment (n = 5 per group). The white line indicates intervening lanes have been spliced out. (D) qRT-PCR analysis of Fsp1 mRNA expression in lung tissues (n = 6 per group). *, P < 0.05. (E) Newly isolated lung fibroblasts from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment (n = 6 per group) were stained for goat anti–rabbit IgG control (red lines) or α-SMA–FITC (blue lines). The proportions of α-SMA–positive cells (myofibroblasts) were determined. Mean percentages of α-SMA+ cells in lung fibroblasts were summarized in the upper region of each panel. The experiments were performed twice. (F) Immunofluorescent staining of myofibroblasts in lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after saline or bleomycin treatment. Representative images of the staining are shown (n = 5 per group). Arrows indicate myofibroblasts with α-SMA– and vimentin-positive staining. Nuclear, DAPI. (A and F) Bars, 100 µm. (G) Quantification of F presents percentage of α-SMA–positive cells (myofibroblasts). **, P < 0.01. (H) Western blot analysis of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in the medium (supernatant [SN]) of primary cultured lung fibroblasts newly isolated from Fstl1+/− and WT mice 14 d after bleomycin treatment (n = 6 per group). (C and H) β-Actin was used as a loading control. (A–D, F, and H) The experiments were performed three times. (B, D, and G) Error bars indicate mean ± SEM.
Mentions: The accumulation of fibroblasts with an activated phenotype (named as myofibroblasts) in the fibrotic foci characterizes progressive pulmonary fibrosis (Scotton and Chambers, 2007; Phan, 2012). To examine whether the attenuated fibrotic phenotype in Fstl1+/− mice is associated with the changed accumulation of myofibroblasts, we performed immunohistochemistry on lung sections with anti–α-SMA antibodies, a marker for newly appearing myofibroblasts. As shown in Fig. 4 A, bleomycin-increased myofibroblast accumulation was apparent in WT mice, but not in Fstl1+/− mice. Consistently, qRT-PCR and immunoblotting analyses of lung tissues showed that bleomycin-induced expression of α-SMA in WT mice was significantly decreased in Fstl1+/− mice (Fig. 4, B and C). Meanwhile, we observed that the mRNA level of Fsp-1, a marker for fibroblasts, was lower in Fstl1+/− mice than that in WT (Fig. 4 D). These data suggest that Fstl1 deficiency is protective against fibrogenesis, possibly via limiting the bleomycin-induced accumulation of myofibroblasts.

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