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Mesenchymal-endothelial transition contributes to cardiac neovascularization.

Ubil E, Duan J, Pillai IC, Rosa-Garrido M, Wu Y, Bargiacchi F, Lu Y, Stanbouly S, Huang J, Rojas M, Vondriska TM, Stefani E, Deb A - Nature (2014)

Bottom Line: We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate.Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function.These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

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Effect of adding TGFβ to serum starved cardiac fibroblasts, or adding TGFβ, serum or Pifithrin-α on tubes that have already formed(a,b) Tube formation of cardiac fibroblasts subjected to serum starvation in the (a) absence or (b) presence of TGFβ. (TGFβ was added at the onset of serum starvation) Scale bar 50μm. (c–f) Effect of adding TGFβ or serum to tubes that had already formed. (c,e) twenty four hours following serum starvation (after tubes had already formed), PBS was added to tubes shown in c and e and photographs taken after another 24 hours. (d,f) After tubes had already formed (24 hours of serum starvation), (d) TGFβ or (f) serum was added and photographs taken after another 24 hours (note clumping of cells and regression of tubes in d and f). Scale bar: c–f: 50μm (g) Effect of adding TGFβ or serum to tubes that had already formed, expressed as a percentage decrease in tube length. (h–n) Effect of adding Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes. (h,k) Tube formation in cardiac fibroblasts serum starved for 24 hours in the absence of PBS or Pifithrin- α. Scale bar: 50μm (i,j) PBS was then added to cardiac fibroblasts shown in (h) and photographs were taken after another (i) 24 hours or (j) 48 hours of serum starvation. Scale bar: 50μm (l, m) Pifithrin-α was added to cardiac fibroblasts shown in (k) and photographs were taken after another (l) 24 hours or (m) 48 hours of serum starvation in the presence of Pifithrin-α. Scale bar: 50μm (n) Tube length in (j) and (m) was expressed as a percent change from their respective control (h and k) (mean±S.E.M. *p<0.05 compared to control, n=3).
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Figure 12: Effect of adding TGFβ to serum starved cardiac fibroblasts, or adding TGFβ, serum or Pifithrin-α on tubes that have already formed(a,b) Tube formation of cardiac fibroblasts subjected to serum starvation in the (a) absence or (b) presence of TGFβ. (TGFβ was added at the onset of serum starvation) Scale bar 50μm. (c–f) Effect of adding TGFβ or serum to tubes that had already formed. (c,e) twenty four hours following serum starvation (after tubes had already formed), PBS was added to tubes shown in c and e and photographs taken after another 24 hours. (d,f) After tubes had already formed (24 hours of serum starvation), (d) TGFβ or (f) serum was added and photographs taken after another 24 hours (note clumping of cells and regression of tubes in d and f). Scale bar: c–f: 50μm (g) Effect of adding TGFβ or serum to tubes that had already formed, expressed as a percentage decrease in tube length. (h–n) Effect of adding Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes. (h,k) Tube formation in cardiac fibroblasts serum starved for 24 hours in the absence of PBS or Pifithrin- α. Scale bar: 50μm (i,j) PBS was then added to cardiac fibroblasts shown in (h) and photographs were taken after another (i) 24 hours or (j) 48 hours of serum starvation. Scale bar: 50μm (l, m) Pifithrin-α was added to cardiac fibroblasts shown in (k) and photographs were taken after another (l) 24 hours or (m) 48 hours of serum starvation in the presence of Pifithrin-α. Scale bar: 50μm (n) Tube length in (j) and (m) was expressed as a percent change from their respective control (h and k) (mean±S.E.M. *p<0.05 compared to control, n=3).

Mentions: Using this ex vivo model, we next asked whether MEndoT was reversible. Transforming growth factor β(TGFβ) enhances EndMT5 and TGFβ added to cardiac fibroblasts at the onset of serum starvation prevented tube formation (Extended Data Fig. 8a,b) and induction of VECAD expression (0.98±0.03 fold change in VECAD with TGFβ, mean± S.E.M. p>0.05, n=3). Moreover, when TGFβ was added to serum starved cardiac fibroblasts after they had already formed tubes, it led to 99% regression of tube formation (Extended Data Fig. 8c,d,g). A similar effect was observed with adding back serum (Extended Fig. 8e,f,g). VECAD expression also decreased by 56.4±2.4% (mean±S.E.M. p<0.05, n=3). Addition of Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes demonstrated significant disruption of formed tubes compared to PBS treated controls (Extended Fig. 8h–n). These observations suggest that p53 is required for maintaining the endothelial phenotype of the fibroblast derived endothelial like cell. Notably, the fraction of fibroblast derived endothelial cells was maintained at 14 days after cardiac injury in vivo (Fig. 1d, Fig. 2c) despite declining p53 levels suggestive of other factors stabilizing the endothelial phenotype.


Mesenchymal-endothelial transition contributes to cardiac neovascularization.

Ubil E, Duan J, Pillai IC, Rosa-Garrido M, Wu Y, Bargiacchi F, Lu Y, Stanbouly S, Huang J, Rojas M, Vondriska TM, Stefani E, Deb A - Nature (2014)

Effect of adding TGFβ to serum starved cardiac fibroblasts, or adding TGFβ, serum or Pifithrin-α on tubes that have already formed(a,b) Tube formation of cardiac fibroblasts subjected to serum starvation in the (a) absence or (b) presence of TGFβ. (TGFβ was added at the onset of serum starvation) Scale bar 50μm. (c–f) Effect of adding TGFβ or serum to tubes that had already formed. (c,e) twenty four hours following serum starvation (after tubes had already formed), PBS was added to tubes shown in c and e and photographs taken after another 24 hours. (d,f) After tubes had already formed (24 hours of serum starvation), (d) TGFβ or (f) serum was added and photographs taken after another 24 hours (note clumping of cells and regression of tubes in d and f). Scale bar: c–f: 50μm (g) Effect of adding TGFβ or serum to tubes that had already formed, expressed as a percentage decrease in tube length. (h–n) Effect of adding Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes. (h,k) Tube formation in cardiac fibroblasts serum starved for 24 hours in the absence of PBS or Pifithrin- α. Scale bar: 50μm (i,j) PBS was then added to cardiac fibroblasts shown in (h) and photographs were taken after another (i) 24 hours or (j) 48 hours of serum starvation. Scale bar: 50μm (l, m) Pifithrin-α was added to cardiac fibroblasts shown in (k) and photographs were taken after another (l) 24 hours or (m) 48 hours of serum starvation in the presence of Pifithrin-α. Scale bar: 50μm (n) Tube length in (j) and (m) was expressed as a percent change from their respective control (h and k) (mean±S.E.M. *p<0.05 compared to control, n=3).
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Figure 12: Effect of adding TGFβ to serum starved cardiac fibroblasts, or adding TGFβ, serum or Pifithrin-α on tubes that have already formed(a,b) Tube formation of cardiac fibroblasts subjected to serum starvation in the (a) absence or (b) presence of TGFβ. (TGFβ was added at the onset of serum starvation) Scale bar 50μm. (c–f) Effect of adding TGFβ or serum to tubes that had already formed. (c,e) twenty four hours following serum starvation (after tubes had already formed), PBS was added to tubes shown in c and e and photographs taken after another 24 hours. (d,f) After tubes had already formed (24 hours of serum starvation), (d) TGFβ or (f) serum was added and photographs taken after another 24 hours (note clumping of cells and regression of tubes in d and f). Scale bar: c–f: 50μm (g) Effect of adding TGFβ or serum to tubes that had already formed, expressed as a percentage decrease in tube length. (h–n) Effect of adding Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes. (h,k) Tube formation in cardiac fibroblasts serum starved for 24 hours in the absence of PBS or Pifithrin- α. Scale bar: 50μm (i,j) PBS was then added to cardiac fibroblasts shown in (h) and photographs were taken after another (i) 24 hours or (j) 48 hours of serum starvation. Scale bar: 50μm (l, m) Pifithrin-α was added to cardiac fibroblasts shown in (k) and photographs were taken after another (l) 24 hours or (m) 48 hours of serum starvation in the presence of Pifithrin-α. Scale bar: 50μm (n) Tube length in (j) and (m) was expressed as a percent change from their respective control (h and k) (mean±S.E.M. *p<0.05 compared to control, n=3).
Mentions: Using this ex vivo model, we next asked whether MEndoT was reversible. Transforming growth factor β(TGFβ) enhances EndMT5 and TGFβ added to cardiac fibroblasts at the onset of serum starvation prevented tube formation (Extended Data Fig. 8a,b) and induction of VECAD expression (0.98±0.03 fold change in VECAD with TGFβ, mean± S.E.M. p>0.05, n=3). Moreover, when TGFβ was added to serum starved cardiac fibroblasts after they had already formed tubes, it led to 99% regression of tube formation (Extended Data Fig. 8c,d,g). A similar effect was observed with adding back serum (Extended Fig. 8e,f,g). VECAD expression also decreased by 56.4±2.4% (mean±S.E.M. p<0.05, n=3). Addition of Pifithrin-α to serum starved cardiac fibroblasts that had already formed tubes demonstrated significant disruption of formed tubes compared to PBS treated controls (Extended Fig. 8h–n). These observations suggest that p53 is required for maintaining the endothelial phenotype of the fibroblast derived endothelial like cell. Notably, the fraction of fibroblast derived endothelial cells was maintained at 14 days after cardiac injury in vivo (Fig. 1d, Fig. 2c) despite declining p53 levels suggestive of other factors stabilizing the endothelial phenotype.

Bottom Line: We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate.Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function.These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial-cell-like phenotype after acute ischaemic cardiac injury. Fibroblast-derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast-derived endothelial cells, reduces post-infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal-to-endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair.

Show MeSH
Related in: MedlinePlus