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Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Ghavami S, Cunnington RH, Gupta S, Yeganeh B, Filomeno KL, Freed DH, Chen S, Klonisch T, Halayko AJ, Ambrose E, Singal R, Dixon IM - Cell Death Dis (2015)

Bottom Line: In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown.Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells.These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [2] Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [3] Department of Physiology and Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada [4] Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transforming growth factor-β(1) (TGF-β(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

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TGFβ1 simultaneously induces fibrosis and autophagy in hATMyofbs. (a) Primary hATMyofbs (passages 2–5) were treated with TGF-β1 (10 ng/ml) for 0–120 h, and cell lysates were collected. Immunoblots were probed for the autophagy hallmark protein LC3β II, p62, as well as indicator proteins of the fibrogenic response in fibroblasts (i.e., collagen Iα2, fibronectin and the Smad signaling pathway). TGF-β1 induced LC3β II lipidation, with parallel increases in collagen Iα2, fibronectin protein expression and Smad2 and Smad3 phosphorylation. Data were normalized to GAPDH levels. Results are the means of three independent experiments from four different donors. (b) Densitometric analysis of LC3β II, p62, collagen Iα2 and fibronectin levels in hATMyofbs. Data are the means of three independent experiments from three different donors. For each experiment, LC3β II, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (c and d) TGF-β1 treatment does not affect cell viability of hATMyofbs, but it associated with their proliferation at 72 and 120 h. hATMyofbs were exposed to TGF-β1 (10 ng/ml) for the indicated time points (48, 72, 120 h), and cell viability and proliferation was measured as described in the Materials and Methods section in three different culture experiments (n=3). TGF-β1 treatment was not associated with any significant changes in cell viability (*P<0.01) (c) while it induced significant hATMyofb proliferation at 72 and 120 h compared with 48 h (P<0.01). (e and f) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 15 600 (e) and 6750 (f). Extracellular fiber deposition (collagen type I or fibronectin) was compared between the control and TGF-β1 treatment groups. TGF-β increased extracellular fiber deposition. (g) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for the indicated time points (0–120 h). The cell culture medium was collected and concentrated with filter tube (MESH 20 kDa). Collagen Iα2 was probed in concentrated cell culture media. TGF-β1 increased mature and immature collagen secretion at different time points. (h and i) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 3600 (control, top panel left), 7500 (control, top panel right) and for TGF-β1 treatment (right panel 2750, left panel 27 500 and (i) 127 000). An autophagosome is highlighted in panel (i). (j) hATMyofbs treated with TGF-β1 (10 ng/ml, 96 h) showed increased LysoTracker Red DND-99 staining (a marker of lysosomal activation) and an increase in punctuate staining for LC3β (green), a marker of autophagy, and LC3β lysosomal co-localization. (k) hATMyofbs were treated with TGF-β1 (10 ng/ml, 96 h) and were immunostained for LC3β (green) and lysosomes (red). Ten different fields (10 cells in each field) were randomly chosen in control and TGF-β1 treatment and were counted manually by an operator. The percentage of yellow cells (merged LC3 and lysosomes) were compared between control and TGF-β1 treatment. TGF-β1 significantly increased the percentage of yellow cells, which indicated LC3β II lysosomal co-localization (***P <0.001). NS, not significant
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fig1: TGFβ1 simultaneously induces fibrosis and autophagy in hATMyofbs. (a) Primary hATMyofbs (passages 2–5) were treated with TGF-β1 (10 ng/ml) for 0–120 h, and cell lysates were collected. Immunoblots were probed for the autophagy hallmark protein LC3β II, p62, as well as indicator proteins of the fibrogenic response in fibroblasts (i.e., collagen Iα2, fibronectin and the Smad signaling pathway). TGF-β1 induced LC3β II lipidation, with parallel increases in collagen Iα2, fibronectin protein expression and Smad2 and Smad3 phosphorylation. Data were normalized to GAPDH levels. Results are the means of three independent experiments from four different donors. (b) Densitometric analysis of LC3β II, p62, collagen Iα2 and fibronectin levels in hATMyofbs. Data are the means of three independent experiments from three different donors. For each experiment, LC3β II, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (c and d) TGF-β1 treatment does not affect cell viability of hATMyofbs, but it associated with their proliferation at 72 and 120 h. hATMyofbs were exposed to TGF-β1 (10 ng/ml) for the indicated time points (48, 72, 120 h), and cell viability and proliferation was measured as described in the Materials and Methods section in three different culture experiments (n=3). TGF-β1 treatment was not associated with any significant changes in cell viability (*P<0.01) (c) while it induced significant hATMyofb proliferation at 72 and 120 h compared with 48 h (P<0.01). (e and f) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 15 600 (e) and 6750 (f). Extracellular fiber deposition (collagen type I or fibronectin) was compared between the control and TGF-β1 treatment groups. TGF-β increased extracellular fiber deposition. (g) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for the indicated time points (0–120 h). The cell culture medium was collected and concentrated with filter tube (MESH 20 kDa). Collagen Iα2 was probed in concentrated cell culture media. TGF-β1 increased mature and immature collagen secretion at different time points. (h and i) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 3600 (control, top panel left), 7500 (control, top panel right) and for TGF-β1 treatment (right panel 2750, left panel 27 500 and (i) 127 000). An autophagosome is highlighted in panel (i). (j) hATMyofbs treated with TGF-β1 (10 ng/ml, 96 h) showed increased LysoTracker Red DND-99 staining (a marker of lysosomal activation) and an increase in punctuate staining for LC3β (green), a marker of autophagy, and LC3β lysosomal co-localization. (k) hATMyofbs were treated with TGF-β1 (10 ng/ml, 96 h) and were immunostained for LC3β (green) and lysosomes (red). Ten different fields (10 cells in each field) were randomly chosen in control and TGF-β1 treatment and were counted manually by an operator. The percentage of yellow cells (merged LC3 and lysosomes) were compared between control and TGF-β1 treatment. TGF-β1 significantly increased the percentage of yellow cells, which indicated LC3β II lysosomal co-localization (***P <0.001). NS, not significant

Mentions: TGF-β1 is but one of an array of factors shown to be involved in the induction of cardiac fibrosis, as demonstrated in overexpression and knockout models.28, 29, 30 As atrial fibrillation is a serious clinical problem with high incidence in society31 and is linked to fibrosis of atrial tissues,32 we investigated whether or not there was an association between TGF-β1-induced fibrosis and autophagy in hATMyofbs. Primary hATMyofbs constitute a clinically relevant model for the study of TGF-β1-induced fibrosis and autophagy. Our results show that TGF-β1 (10 ng/ml) induces significant increases in the synthesis of collagen type Iα2 and fibronectin in the presence of LC3β II lipidation and increases Smad2 and Smad3 phosphorylation and p62 degradation (Figures 1a and b). We also showed that TGF-β1 treatment significantly did not affect the viability of hATMyofb cells (Figure 1c) while conversely it is associated with a significant induction of their proliferation at 72 and 120 h after treatments when compared with 48-h cultures (Figure 1d; P≤0.01). Fibrillar collagen type I and fibronectin deposition was also confirmed using transmission electron microscopic (TEM) images from hATMyofbs, which were stimulated with TGF-β1 at 10 ng/ml for 96 h (Figures 1e and f). Figures 1e and f indicate an increase of fiber deposition after TGF-β1 treatment. TGF-β1 stimulation increased collagen Iα2 secretion from hATMyofb cells compared with time-matched control cells, which also proved TGF-β1-induced fibrosis (western blotting analysis in Figure 1g). Possible TGF-β1 autophagy induction was further investigated using TEM images from hATMyofbs stimulated with TGF-β1 at 10 ng/ml for 96 h. Figures 1h and i clearly show autophagosome and autophagolysosmes in hATMyofbs stimulated with TGF-β1. Moreover, our immunocytochemistry data indicate punctate LC3β II staining and lysosomal activation in hATMyofbs treated with TGF-β1 (Figure 1j), with a significant (P<0.001) increase in the number of cells. In this case, LC3β and lysotracker were co-localized (Figure 1k), which is consistent with the autophagy activation in these cells. To obtain more quantitative assessment of the induction of autophagy, we used bafilomycin-A1 (Baf-A1; 10 nM) to block the fusion of autophagosomes and lysosomes and assess the presence of autophagy flux.13, 33, 34 As shown in Figure 2a, we demonstrate autophagic flux, for example, autophagosome delivery to lysosomes and autophagolysosome formation, by co-treating hATMyofbs with TGF-β1 and Baf-A1 for 48 and 96 h. TGF-β1-induced accumulation of LC3-II was enhanced in the presence of Baf-A1, which supports the suggestion that TGF-β1 enhances autophagosome synthesis.


Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts.

Ghavami S, Cunnington RH, Gupta S, Yeganeh B, Filomeno KL, Freed DH, Chen S, Klonisch T, Halayko AJ, Ambrose E, Singal R, Dixon IM - Cell Death Dis (2015)

TGFβ1 simultaneously induces fibrosis and autophagy in hATMyofbs. (a) Primary hATMyofbs (passages 2–5) were treated with TGF-β1 (10 ng/ml) for 0–120 h, and cell lysates were collected. Immunoblots were probed for the autophagy hallmark protein LC3β II, p62, as well as indicator proteins of the fibrogenic response in fibroblasts (i.e., collagen Iα2, fibronectin and the Smad signaling pathway). TGF-β1 induced LC3β II lipidation, with parallel increases in collagen Iα2, fibronectin protein expression and Smad2 and Smad3 phosphorylation. Data were normalized to GAPDH levels. Results are the means of three independent experiments from four different donors. (b) Densitometric analysis of LC3β II, p62, collagen Iα2 and fibronectin levels in hATMyofbs. Data are the means of three independent experiments from three different donors. For each experiment, LC3β II, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (c and d) TGF-β1 treatment does not affect cell viability of hATMyofbs, but it associated with their proliferation at 72 and 120 h. hATMyofbs were exposed to TGF-β1 (10 ng/ml) for the indicated time points (48, 72, 120 h), and cell viability and proliferation was measured as described in the Materials and Methods section in three different culture experiments (n=3). TGF-β1 treatment was not associated with any significant changes in cell viability (*P<0.01) (c) while it induced significant hATMyofb proliferation at 72 and 120 h compared with 48 h (P<0.01). (e and f) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 15 600 (e) and 6750 (f). Extracellular fiber deposition (collagen type I or fibronectin) was compared between the control and TGF-β1 treatment groups. TGF-β increased extracellular fiber deposition. (g) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for the indicated time points (0–120 h). The cell culture medium was collected and concentrated with filter tube (MESH 20 kDa). Collagen Iα2 was probed in concentrated cell culture media. TGF-β1 increased mature and immature collagen secretion at different time points. (h and i) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 3600 (control, top panel left), 7500 (control, top panel right) and for TGF-β1 treatment (right panel 2750, left panel 27 500 and (i) 127 000). An autophagosome is highlighted in panel (i). (j) hATMyofbs treated with TGF-β1 (10 ng/ml, 96 h) showed increased LysoTracker Red DND-99 staining (a marker of lysosomal activation) and an increase in punctuate staining for LC3β (green), a marker of autophagy, and LC3β lysosomal co-localization. (k) hATMyofbs were treated with TGF-β1 (10 ng/ml, 96 h) and were immunostained for LC3β (green) and lysosomes (red). Ten different fields (10 cells in each field) were randomly chosen in control and TGF-β1 treatment and were counted manually by an operator. The percentage of yellow cells (merged LC3 and lysosomes) were compared between control and TGF-β1 treatment. TGF-β1 significantly increased the percentage of yellow cells, which indicated LC3β II lysosomal co-localization (***P <0.001). NS, not significant
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Related In: Results  -  Collection

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fig1: TGFβ1 simultaneously induces fibrosis and autophagy in hATMyofbs. (a) Primary hATMyofbs (passages 2–5) were treated with TGF-β1 (10 ng/ml) for 0–120 h, and cell lysates were collected. Immunoblots were probed for the autophagy hallmark protein LC3β II, p62, as well as indicator proteins of the fibrogenic response in fibroblasts (i.e., collagen Iα2, fibronectin and the Smad signaling pathway). TGF-β1 induced LC3β II lipidation, with parallel increases in collagen Iα2, fibronectin protein expression and Smad2 and Smad3 phosphorylation. Data were normalized to GAPDH levels. Results are the means of three independent experiments from four different donors. (b) Densitometric analysis of LC3β II, p62, collagen Iα2 and fibronectin levels in hATMyofbs. Data are the means of three independent experiments from three different donors. For each experiment, LC3β II, collagen Iα2 and fibronectin levels were compared with those from time-matched controls and normalized to GAPDH levels. (c and d) TGF-β1 treatment does not affect cell viability of hATMyofbs, but it associated with their proliferation at 72 and 120 h. hATMyofbs were exposed to TGF-β1 (10 ng/ml) for the indicated time points (48, 72, 120 h), and cell viability and proliferation was measured as described in the Materials and Methods section in three different culture experiments (n=3). TGF-β1 treatment was not associated with any significant changes in cell viability (*P<0.01) (c) while it induced significant hATMyofb proliferation at 72 and 120 h compared with 48 h (P<0.01). (e and f) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 15 600 (e) and 6750 (f). Extracellular fiber deposition (collagen type I or fibronectin) was compared between the control and TGF-β1 treatment groups. TGF-β increased extracellular fiber deposition. (g) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for the indicated time points (0–120 h). The cell culture medium was collected and concentrated with filter tube (MESH 20 kDa). Collagen Iα2 was probed in concentrated cell culture media. TGF-β1 increased mature and immature collagen secretion at different time points. (h and i) hATMyofbs were either untreated or treated with 10 ng/ml TGF-β1 for 96 h. Cells were then imaged by TEM at a magnification of 3600 (control, top panel left), 7500 (control, top panel right) and for TGF-β1 treatment (right panel 2750, left panel 27 500 and (i) 127 000). An autophagosome is highlighted in panel (i). (j) hATMyofbs treated with TGF-β1 (10 ng/ml, 96 h) showed increased LysoTracker Red DND-99 staining (a marker of lysosomal activation) and an increase in punctuate staining for LC3β (green), a marker of autophagy, and LC3β lysosomal co-localization. (k) hATMyofbs were treated with TGF-β1 (10 ng/ml, 96 h) and were immunostained for LC3β (green) and lysosomes (red). Ten different fields (10 cells in each field) were randomly chosen in control and TGF-β1 treatment and were counted manually by an operator. The percentage of yellow cells (merged LC3 and lysosomes) were compared between control and TGF-β1 treatment. TGF-β1 significantly increased the percentage of yellow cells, which indicated LC3β II lysosomal co-localization (***P <0.001). NS, not significant
Mentions: TGF-β1 is but one of an array of factors shown to be involved in the induction of cardiac fibrosis, as demonstrated in overexpression and knockout models.28, 29, 30 As atrial fibrillation is a serious clinical problem with high incidence in society31 and is linked to fibrosis of atrial tissues,32 we investigated whether or not there was an association between TGF-β1-induced fibrosis and autophagy in hATMyofbs. Primary hATMyofbs constitute a clinically relevant model for the study of TGF-β1-induced fibrosis and autophagy. Our results show that TGF-β1 (10 ng/ml) induces significant increases in the synthesis of collagen type Iα2 and fibronectin in the presence of LC3β II lipidation and increases Smad2 and Smad3 phosphorylation and p62 degradation (Figures 1a and b). We also showed that TGF-β1 treatment significantly did not affect the viability of hATMyofb cells (Figure 1c) while conversely it is associated with a significant induction of their proliferation at 72 and 120 h after treatments when compared with 48-h cultures (Figure 1d; P≤0.01). Fibrillar collagen type I and fibronectin deposition was also confirmed using transmission electron microscopic (TEM) images from hATMyofbs, which were stimulated with TGF-β1 at 10 ng/ml for 96 h (Figures 1e and f). Figures 1e and f indicate an increase of fiber deposition after TGF-β1 treatment. TGF-β1 stimulation increased collagen Iα2 secretion from hATMyofb cells compared with time-matched control cells, which also proved TGF-β1-induced fibrosis (western blotting analysis in Figure 1g). Possible TGF-β1 autophagy induction was further investigated using TEM images from hATMyofbs stimulated with TGF-β1 at 10 ng/ml for 96 h. Figures 1h and i clearly show autophagosome and autophagolysosmes in hATMyofbs stimulated with TGF-β1. Moreover, our immunocytochemistry data indicate punctate LC3β II staining and lysosomal activation in hATMyofbs treated with TGF-β1 (Figure 1j), with a significant (P<0.001) increase in the number of cells. In this case, LC3β and lysotracker were co-localized (Figure 1k), which is consistent with the autophagy activation in these cells. To obtain more quantitative assessment of the induction of autophagy, we used bafilomycin-A1 (Baf-A1; 10 nM) to block the fusion of autophagosomes and lysosomes and assess the presence of autophagy flux.13, 33, 34 As shown in Figure 2a, we demonstrate autophagic flux, for example, autophagosome delivery to lysosomes and autophagolysosome formation, by co-treating hATMyofbs with TGF-β1 and Baf-A1 for 48 and 96 h. TGF-β1-induced accumulation of LC3-II was enhanced in the presence of Baf-A1, which supports the suggestion that TGF-β1 enhances autophagosome synthesis.

Bottom Line: In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown.Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells.These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Physiology, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [2] Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada [3] Department of Physiology and Institute of Cardiovascular Sciences, St. Boniface Research Centre, University of Manitoba, Winnipeg, Manitoba, Canada [4] Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
Transforming growth factor-β(1) (TGF-β(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

Show MeSH
Related in: MedlinePlus