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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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Fstl1 deficiency protects epithelial cells from injury. (A) Lung sections of Fstl1+/− and their WT littermates treated with 2.5 U/kg bleomycin for 7 d were stained with hematoxylin and eosin. Representative images of the staining are shown (n = 7 per group). (B and C) Primary AECs were isolated from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (BLM; n = 5 per group). After 2-d culture, mRNA expression levels of epithelial cell markers including Sftpc, E-cadherin (Cdh1), Occludin (Ocln), ZO-1+, and ZO-1– (B) and mesenchymal cell markers including vimentin (Vim), Fsp1, and α-SMA (C) in AECs were assessed by qRT-PCR. *, P < 0.05; **, P < 0.01. Error bars indicate mean ± SEM. (D) Representative flow cytometry plots of E-cadherin (E-cad)– and FSP1-positive populations in newly isolated AECs from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (n = 5 per group). The percentage of E-cad+FSP1+ AECs is indicated on the top right corner of each diagram. (E) Lung sections of Fstl1+/− and WT littermates 7 and 14 d after 2.5 U/kg bleomycin treatment were immunostained with pro-SPC, α-SMA, and DAPI as nuclear. Representative images of the staining are shown (n = 6 per group). Several pro-SPC+α-SMA+ AECs are shown (arrows). The percentage of pro-SPC+α-SMA+ AECs is presented on the top right corner of each diagram. Bars: (A) 100 µm; (E) 25 µm. (F and G) Primary AECs were newly isolated from Fstl1+/− and WT littermates 7 d after saline (black) or 2.5 U/kg bleomycin (red) treatment (n = 4 per group). Primary lung fibroblasts (Fb) were newly isolated from untreated Fstl1+/− and WT mice (n = 4 per group). (F) After 2-d culture, AECs in the upper chamber were co-cultured with fibroblasts in the lower plate of the transwell for 24 h. (G) AECs directly co-cultured with fibroblasts (AEC/Fb = 1:10) for 48 h. Protein expression levels of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in medium (supernatant [SN]) of lung fibroblasts were assessed by Western blot. β-Actin was used as a loading control. (A–G) The experiments were performed three times.
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fig5: Fstl1 deficiency protects epithelial cells from injury. (A) Lung sections of Fstl1+/− and their WT littermates treated with 2.5 U/kg bleomycin for 7 d were stained with hematoxylin and eosin. Representative images of the staining are shown (n = 7 per group). (B and C) Primary AECs were isolated from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (BLM; n = 5 per group). After 2-d culture, mRNA expression levels of epithelial cell markers including Sftpc, E-cadherin (Cdh1), Occludin (Ocln), ZO-1+, and ZO-1– (B) and mesenchymal cell markers including vimentin (Vim), Fsp1, and α-SMA (C) in AECs were assessed by qRT-PCR. *, P < 0.05; **, P < 0.01. Error bars indicate mean ± SEM. (D) Representative flow cytometry plots of E-cadherin (E-cad)– and FSP1-positive populations in newly isolated AECs from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (n = 5 per group). The percentage of E-cad+FSP1+ AECs is indicated on the top right corner of each diagram. (E) Lung sections of Fstl1+/− and WT littermates 7 and 14 d after 2.5 U/kg bleomycin treatment were immunostained with pro-SPC, α-SMA, and DAPI as nuclear. Representative images of the staining are shown (n = 6 per group). Several pro-SPC+α-SMA+ AECs are shown (arrows). The percentage of pro-SPC+α-SMA+ AECs is presented on the top right corner of each diagram. Bars: (A) 100 µm; (E) 25 µm. (F and G) Primary AECs were newly isolated from Fstl1+/− and WT littermates 7 d after saline (black) or 2.5 U/kg bleomycin (red) treatment (n = 4 per group). Primary lung fibroblasts (Fb) were newly isolated from untreated Fstl1+/− and WT mice (n = 4 per group). (F) After 2-d culture, AECs in the upper chamber were co-cultured with fibroblasts in the lower plate of the transwell for 24 h. (G) AECs directly co-cultured with fibroblasts (AEC/Fb = 1:10) for 48 h. Protein expression levels of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in medium (supernatant [SN]) of lung fibroblasts were assessed by Western blot. β-Actin was used as a loading control. (A–G) The experiments were performed three times.

Mentions: Evidence suggests that lung fibrosis is a dysregulated epithelial-mesenchymal disorder, in which epithelial injury is an initiating event (Selman and Pardo, 2002). Haplodeletion of Fstl1 showed a significant reduction of lung architectural damage 7 d after bleomycin injury (Fig. 5 A). Injured AECs likely undergo many cellular and molecular changes and may create a profibrotic environment in an effort to activate fibroblasts. Injured AECs may lose epithelial markers and gain mesenchymal markers at the same time (King et al., 2011; Kage and Borok, 2012). We isolated AECs from WT mice 7 d after bleomycin treatment. We found decreased mRNA expression of epithelial markers, such as Sftpc, E-cadherin (Cdh1), Occludin (Ocln), and ZO-1, and increased mRNA expression of mesenchymal markers, such as α-SMA, vimentin (Vim), and Fsp1 (Fig. 5, B and C). FACS analysis (Fig. 5 D) also showed increased numbers of E-cad+Fsp1+ AECs in response to bleomycin injury, supporting the notion that injured AECs are activated and may express mesenchymal genes. We performed double immunostaining for α-SMA and pro-SPC on WT lung sections and identified costaining of pro-SPC+α-SMA+ AECs on days 7 and 14 after bleomycin treatment (Fig. 5 E), confirming that epithelial cells with a mesenchymal phenotype serve as a marker of epithelial injury. Notably, Fstl1 haplodeficiency inhibited bleomycin-induced expression of mesenchymal markers and restored the expression of epithelial markers in Fstl1+/− AECs (Fig. 5, B and C). In addition, the bleomycin-increased numbers of mesenchymal markers expressing AECs were significantly decreased in Fstl1+/− epithelial cells (Fig. 5, D and E), indicating that Fstl1 deficiency in Fstl1+/− mice is protective against bleomycin-induced epithelial injury.


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)

Fstl1 deficiency protects epithelial cells from injury. (A) Lung sections of Fstl1+/− and their WT littermates treated with 2.5 U/kg bleomycin for 7 d were stained with hematoxylin and eosin. Representative images of the staining are shown (n = 7 per group). (B and C) Primary AECs were isolated from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (BLM; n = 5 per group). After 2-d culture, mRNA expression levels of epithelial cell markers including Sftpc, E-cadherin (Cdh1), Occludin (Ocln), ZO-1+, and ZO-1– (B) and mesenchymal cell markers including vimentin (Vim), Fsp1, and α-SMA (C) in AECs were assessed by qRT-PCR. *, P < 0.05; **, P < 0.01. Error bars indicate mean ± SEM. (D) Representative flow cytometry plots of E-cadherin (E-cad)– and FSP1-positive populations in newly isolated AECs from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (n = 5 per group). The percentage of E-cad+FSP1+ AECs is indicated on the top right corner of each diagram. (E) Lung sections of Fstl1+/− and WT littermates 7 and 14 d after 2.5 U/kg bleomycin treatment were immunostained with pro-SPC, α-SMA, and DAPI as nuclear. Representative images of the staining are shown (n = 6 per group). Several pro-SPC+α-SMA+ AECs are shown (arrows). The percentage of pro-SPC+α-SMA+ AECs is presented on the top right corner of each diagram. Bars: (A) 100 µm; (E) 25 µm. (F and G) Primary AECs were newly isolated from Fstl1+/− and WT littermates 7 d after saline (black) or 2.5 U/kg bleomycin (red) treatment (n = 4 per group). Primary lung fibroblasts (Fb) were newly isolated from untreated Fstl1+/− and WT mice (n = 4 per group). (F) After 2-d culture, AECs in the upper chamber were co-cultured with fibroblasts in the lower plate of the transwell for 24 h. (G) AECs directly co-cultured with fibroblasts (AEC/Fb = 1:10) for 48 h. Protein expression levels of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in medium (supernatant [SN]) of lung fibroblasts were assessed by Western blot. β-Actin was used as a loading control. (A–G) The experiments were performed three times.
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Related In: Results  -  Collection

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fig5: Fstl1 deficiency protects epithelial cells from injury. (A) Lung sections of Fstl1+/− and their WT littermates treated with 2.5 U/kg bleomycin for 7 d were stained with hematoxylin and eosin. Representative images of the staining are shown (n = 7 per group). (B and C) Primary AECs were isolated from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (BLM; n = 5 per group). After 2-d culture, mRNA expression levels of epithelial cell markers including Sftpc, E-cadherin (Cdh1), Occludin (Ocln), ZO-1+, and ZO-1– (B) and mesenchymal cell markers including vimentin (Vim), Fsp1, and α-SMA (C) in AECs were assessed by qRT-PCR. *, P < 0.05; **, P < 0.01. Error bars indicate mean ± SEM. (D) Representative flow cytometry plots of E-cadherin (E-cad)– and FSP1-positive populations in newly isolated AECs from Fstl1+/− and WT littermates 7 d after saline or 2.5 U/kg bleomycin treatment (n = 5 per group). The percentage of E-cad+FSP1+ AECs is indicated on the top right corner of each diagram. (E) Lung sections of Fstl1+/− and WT littermates 7 and 14 d after 2.5 U/kg bleomycin treatment were immunostained with pro-SPC, α-SMA, and DAPI as nuclear. Representative images of the staining are shown (n = 6 per group). Several pro-SPC+α-SMA+ AECs are shown (arrows). The percentage of pro-SPC+α-SMA+ AECs is presented on the top right corner of each diagram. Bars: (A) 100 µm; (E) 25 µm. (F and G) Primary AECs were newly isolated from Fstl1+/− and WT littermates 7 d after saline (black) or 2.5 U/kg bleomycin (red) treatment (n = 4 per group). Primary lung fibroblasts (Fb) were newly isolated from untreated Fstl1+/− and WT mice (n = 4 per group). (F) After 2-d culture, AECs in the upper chamber were co-cultured with fibroblasts in the lower plate of the transwell for 24 h. (G) AECs directly co-cultured with fibroblasts (AEC/Fb = 1:10) for 48 h. Protein expression levels of α-SMA in cell extracts (Cell), type I collagen (Col1), and fibronectin (Fn1) in medium (supernatant [SN]) of lung fibroblasts were assessed by Western blot. β-Actin was used as a loading control. (A–G) The experiments were performed three times.
Mentions: Evidence suggests that lung fibrosis is a dysregulated epithelial-mesenchymal disorder, in which epithelial injury is an initiating event (Selman and Pardo, 2002). Haplodeletion of Fstl1 showed a significant reduction of lung architectural damage 7 d after bleomycin injury (Fig. 5 A). Injured AECs likely undergo many cellular and molecular changes and may create a profibrotic environment in an effort to activate fibroblasts. Injured AECs may lose epithelial markers and gain mesenchymal markers at the same time (King et al., 2011; Kage and Borok, 2012). We isolated AECs from WT mice 7 d after bleomycin treatment. We found decreased mRNA expression of epithelial markers, such as Sftpc, E-cadherin (Cdh1), Occludin (Ocln), and ZO-1, and increased mRNA expression of mesenchymal markers, such as α-SMA, vimentin (Vim), and Fsp1 (Fig. 5, B and C). FACS analysis (Fig. 5 D) also showed increased numbers of E-cad+Fsp1+ AECs in response to bleomycin injury, supporting the notion that injured AECs are activated and may express mesenchymal genes. We performed double immunostaining for α-SMA and pro-SPC on WT lung sections and identified costaining of pro-SPC+α-SMA+ AECs on days 7 and 14 after bleomycin treatment (Fig. 5 E), confirming that epithelial cells with a mesenchymal phenotype serve as a marker of epithelial injury. Notably, Fstl1 haplodeficiency inhibited bleomycin-induced expression of mesenchymal markers and restored the expression of epithelial markers in Fstl1+/− AECs (Fig. 5, B and C). In addition, the bleomycin-increased numbers of mesenchymal markers expressing AECs were significantly decreased in Fstl1+/− epithelial cells (Fig. 5, D and E), indicating that Fstl1 deficiency in Fstl1+/− mice is protective against bleomycin-induced epithelial injury.

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