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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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Concomitant occurrence of autophagy, fibrosis and Smad2 phosphorylation in scar tissue from post-MI rats. (a–c) Western blotting analysis of infarct scar and non-infarcted (NI) control tissues from post-MI experimental animals confirmed the concomitant occurrence of fibrosis (i.e., elevation of fibronectin levels), autophagy (i.e., elevation of LC3β II and Atg5-12 levels) and Smad2 phosphorylation, with Smad phosphorylation (p-Smad) being a hallmark of TGF-β1 activation, in scar tissue 2 weeks after MI. This time point was chosen as it reflects the active healing phase after MI. (d) Densitometry analysis of fibronectin and LC3β-II of tissues from post-MI experimental animals showed that both proteins have increased in scar area compared with sham area at 2 and 4 weeks time point. (e–g) Immunofluorescence histochemical analysis of sham and scar tissue showed the co-localization of LC3β with vimentin (e), EDA-Fibronectin (f) and phospho Smad2 (g) in scar compared with sham area
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fig3: Concomitant occurrence of autophagy, fibrosis and Smad2 phosphorylation in scar tissue from post-MI rats. (a–c) Western blotting analysis of infarct scar and non-infarcted (NI) control tissues from post-MI experimental animals confirmed the concomitant occurrence of fibrosis (i.e., elevation of fibronectin levels), autophagy (i.e., elevation of LC3β II and Atg5-12 levels) and Smad2 phosphorylation, with Smad phosphorylation (p-Smad) being a hallmark of TGF-β1 activation, in scar tissue 2 weeks after MI. This time point was chosen as it reflects the active healing phase after MI. (d) Densitometry analysis of fibronectin and LC3β-II of tissues from post-MI experimental animals showed that both proteins have increased in scar area compared with sham area at 2 and 4 weeks time point. (e–g) Immunofluorescence histochemical analysis of sham and scar tissue showed the co-localization of LC3β with vimentin (e), EDA-Fibronectin (f) and phospho Smad2 (g) in scar compared with sham area

Mentions: MI is a major cause of congestive heart failure,36, 37, 38 and increased levels of TGF-β1 mRNA and protein expression are evident in the myocardium bordering the infarct region 2 days following MI.39 This observation is highly suggestive of a crucial role for TGF-β1 in cardiac wound healing and the fibrotic response. Thus, we investigated Smad2 phosphorylation, as well as the levels of autophagy and fibrosis markers, on scar and sham-operated tissues from our rat model of MI. As shown in Figure 3b, Smad2 phosphorylation, autophagy markers (e.g., LC3β II lipidation and Atg5-12 conjugate) and fibronectin synthesis are all increased in scar tissue at 2 and 4 weeks post-MI (Figures 3b and d). These findings positively correlate with the elevated expression levels of dimeric TGF-β1 (e.g., the 25-kDa band) that we have observed at 2 and 4 weeks post-MI versus non-infarcted control and sham-operated left ventricular muscle.40 However, we found no evidence of Smad2 phosphorylation or changes in the levels of autophagy markers and fibronectin synthesis at 24 or 48 h or at 8 weeks post-MI (Figures 3a and c). We also showed that LC3β punctate staining co-localized with vimentin (a mesenchymal marker – see Figure 3e), ED-A fibronectin (Figure 3f) and phosphorylated Smad2 (Figure 3g – wherein Smad2 phosphorylation is a marker of canonical TGF-β1 activation), which supports our hypothesis about the role of autophagy and TGF-β1 in fibrosis induction in MI tissue.


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)

Concomitant occurrence of autophagy, fibrosis and Smad2 phosphorylation in scar tissue from post-MI rats. (a–c) Western blotting analysis of infarct scar and non-infarcted (NI) control tissues from post-MI experimental animals confirmed the concomitant occurrence of fibrosis (i.e., elevation of fibronectin levels), autophagy (i.e., elevation of LC3β II and Atg5-12 levels) and Smad2 phosphorylation, with Smad phosphorylation (p-Smad) being a hallmark of TGF-β1 activation, in scar tissue 2 weeks after MI. This time point was chosen as it reflects the active healing phase after MI. (d) Densitometry analysis of fibronectin and LC3β-II of tissues from post-MI experimental animals showed that both proteins have increased in scar area compared with sham area at 2 and 4 weeks time point. (e–g) Immunofluorescence histochemical analysis of sham and scar tissue showed the co-localization of LC3β with vimentin (e), EDA-Fibronectin (f) and phospho Smad2 (g) in scar compared with sham area
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385916&req=5

fig3: Concomitant occurrence of autophagy, fibrosis and Smad2 phosphorylation in scar tissue from post-MI rats. (a–c) Western blotting analysis of infarct scar and non-infarcted (NI) control tissues from post-MI experimental animals confirmed the concomitant occurrence of fibrosis (i.e., elevation of fibronectin levels), autophagy (i.e., elevation of LC3β II and Atg5-12 levels) and Smad2 phosphorylation, with Smad phosphorylation (p-Smad) being a hallmark of TGF-β1 activation, in scar tissue 2 weeks after MI. This time point was chosen as it reflects the active healing phase after MI. (d) Densitometry analysis of fibronectin and LC3β-II of tissues from post-MI experimental animals showed that both proteins have increased in scar area compared with sham area at 2 and 4 weeks time point. (e–g) Immunofluorescence histochemical analysis of sham and scar tissue showed the co-localization of LC3β with vimentin (e), EDA-Fibronectin (f) and phospho Smad2 (g) in scar compared with sham area
Mentions: MI is a major cause of congestive heart failure,36, 37, 38 and increased levels of TGF-β1 mRNA and protein expression are evident in the myocardium bordering the infarct region 2 days following MI.39 This observation is highly suggestive of a crucial role for TGF-β1 in cardiac wound healing and the fibrotic response. Thus, we investigated Smad2 phosphorylation, as well as the levels of autophagy and fibrosis markers, on scar and sham-operated tissues from our rat model of MI. As shown in Figure 3b, Smad2 phosphorylation, autophagy markers (e.g., LC3β II lipidation and Atg5-12 conjugate) and fibronectin synthesis are all increased in scar tissue at 2 and 4 weeks post-MI (Figures 3b and d). These findings positively correlate with the elevated expression levels of dimeric TGF-β1 (e.g., the 25-kDa band) that we have observed at 2 and 4 weeks post-MI versus non-infarcted control and sham-operated left ventricular muscle.40 However, we found no evidence of Smad2 phosphorylation or changes in the levels of autophagy markers and fibronectin synthesis at 24 or 48 h or at 8 weeks post-MI (Figures 3a and c). We also showed that LC3β punctate staining co-localized with vimentin (a mesenchymal marker – see Figure 3e), ED-A fibronectin (Figure 3f) and phosphorylated Smad2 (Figure 3g – wherein Smad2 phosphorylation is a marker of canonical TGF-β1 activation), which supports our hypothesis about the role of autophagy and TGF-β1 in fibrosis induction in MI tissue.

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