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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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RITA decreases inflammatory infiltrate after cardiac injury, does not increase apoptosis in myocytes and does not enhance MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice(a–d) Sections of hearts harvested at 3 days following cardiac injury were stained for the monocyte/macrophage marker CD68 (green, arrowheads) in (a) Col1a2CreERT:R26RtdTomato, (b) Col1a2CreERT:R26RtdTomato:p53CKO and (c) RITA injected Col1a2CreERT:R26RtdTomato Scale bar: 10μm. (d) quantification of the number of CD68 cells/high power field in the injury region (mean±S.E.M. *p<0.05 versus Col1a2CreERT:R26RtdTomato, ** p<0.05 versus Col1a2CreERT:R26RtdTomato, n=3) (e–g) p53 expression in myocytes after cardiac injury and effect of RITA on apoptosis in injury region. (e,f) Col1a2CreERT:R26RtdTomato mice were subjected to ischemic cardiac injury, hearts harvested at 3 days and sections stained for p53 and cardiomyocyte marker Troponin. (e) p53 (green, arrowheads) staining is observed in tdTomato expressing cells (red, arrowheads) but not in cardiomyocytes (blue), merged image shows arrowheads pointing to tdTomato labeled cells expressing p53. Scale bar: 10μm. (f) Higher magnification in injury region demonstrating tdTomato cells (arrowheads) expressing p53 (merged, yellow, arrowheads) but p53 staining is not visible in cardiomyocytes (blue). Scale bar: 10μm. (g) TUNEL staining and quantification to determine p53+ apoptotic cells after RITA injection (arrowheads point to p53+TUNEL+ cells in Col1a2CreERT:R26RtdTomato mice (left panel) and RITA injected Col1a2CreERT:R26RtdTomato mice (right panel), inset shows p53+TUNEL+ cell in higher magnification (data shown as mean±S.E.M, ns=not significant, n=3). Hearts in both cases were examined 3 days after injury. Scale bar: 10μm. (h,i) Effect of RITA on MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice after cardiac injury. (h) tdTomato labeled cardiac fibroblasts expressing VECAD in Col1a2CreERT:R26RtdTomato mice treated with/without RITA, Col1a2CreERT:R26RtdTomato:p53CKO mice treated with/without RITA. Scale bar: 10μm. (i) Quantitation of the percentage of labeled fibroblasts undergoing MEndoT for each treatment group (mean ±S.E.M., *p<0.05, n.s= not significant, n=4 animals/group).
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Figure 13: RITA decreases inflammatory infiltrate after cardiac injury, does not increase apoptosis in myocytes and does not enhance MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice(a–d) Sections of hearts harvested at 3 days following cardiac injury were stained for the monocyte/macrophage marker CD68 (green, arrowheads) in (a) Col1a2CreERT:R26RtdTomato, (b) Col1a2CreERT:R26RtdTomato:p53CKO and (c) RITA injected Col1a2CreERT:R26RtdTomato Scale bar: 10μm. (d) quantification of the number of CD68 cells/high power field in the injury region (mean±S.E.M. *p<0.05 versus Col1a2CreERT:R26RtdTomato, ** p<0.05 versus Col1a2CreERT:R26RtdTomato, n=3) (e–g) p53 expression in myocytes after cardiac injury and effect of RITA on apoptosis in injury region. (e,f) Col1a2CreERT:R26RtdTomato mice were subjected to ischemic cardiac injury, hearts harvested at 3 days and sections stained for p53 and cardiomyocyte marker Troponin. (e) p53 (green, arrowheads) staining is observed in tdTomato expressing cells (red, arrowheads) but not in cardiomyocytes (blue), merged image shows arrowheads pointing to tdTomato labeled cells expressing p53. Scale bar: 10μm. (f) Higher magnification in injury region demonstrating tdTomato cells (arrowheads) expressing p53 (merged, yellow, arrowheads) but p53 staining is not visible in cardiomyocytes (blue). Scale bar: 10μm. (g) TUNEL staining and quantification to determine p53+ apoptotic cells after RITA injection (arrowheads point to p53+TUNEL+ cells in Col1a2CreERT:R26RtdTomato mice (left panel) and RITA injected Col1a2CreERT:R26RtdTomato mice (right panel), inset shows p53+TUNEL+ cell in higher magnification (data shown as mean±S.E.M, ns=not significant, n=3). Hearts in both cases were examined 3 days after injury. Scale bar: 10μm. (h,i) Effect of RITA on MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice after cardiac injury. (h) tdTomato labeled cardiac fibroblasts expressing VECAD in Col1a2CreERT:R26RtdTomato mice treated with/without RITA, Col1a2CreERT:R26RtdTomato:p53CKO mice treated with/without RITA. Scale bar: 10μm. (i) Quantitation of the percentage of labeled fibroblasts undergoing MEndoT for each treatment group (mean ±S.E.M., *p<0.05, n.s= not significant, n=4 animals/group).

Mentions: To determine whether p53 mediates MEndoT in vivo, we subjected Col1a2CreERT:R26RtdTomato:p53fl/fl mice to ischemic cardiac injury 5 days following cessation of tamoxifen. Mice with intact p53 exhibited 6±0.64 fold increase (mean±S.E.M.) in cardiac fibroblast p53 levels 3 days after cardiac injury, but in mice with fibroblast specific p53 deletion [referred to as Col1a2CreERT:R26RtdTomato:p53CKO (conditional knock out)], the mean p53 expression in border zone cardiac fibroblasts failed to increase significantly (Fig. 3a–c). The degree of MEndoT at 3 days post-injury in Col1a2CreERT:R26RtdTomato:p53CKO animals decreased by 57% (Fig. 3d–f) and was associated with a decrease in capillary density in the injury region (Fig. 3g). Increase in vessel density in the injured heart is a critical post-ischemic repair mechanism. Diminished neovascularization can lead to rapid decline in cardiac function after cardiac injury and is associated with worsened scarring34. Echocardiography on hearts of mice 7 days after cardiac injury showed significant worsening of cardiac function in p53CKO mice (Fig. 3h,i). Masson Trichrome staining demonstrated a greater amount of collagen deposition in the hearts of Col1a2CreERT:R26RtdTomato:p53CKO animals (Fig. 3j,k). The degree of inflammatory infiltrate measured by the number of CD68 expressing macrophages was also significantly higher in the Col1a2CreERT:R26RtdTomato:p53CKO animals (Extended Data Fig. 9a,b,d). These observations demonstrate that p53 is necessary for MEndoT to occur after ischemic cardiac injury and that disruption of MEndoT is associated with diminished post-injury vascularity and cardiac function.


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)

RITA decreases inflammatory infiltrate after cardiac injury, does not increase apoptosis in myocytes and does not enhance MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice(a–d) Sections of hearts harvested at 3 days following cardiac injury were stained for the monocyte/macrophage marker CD68 (green, arrowheads) in (a) Col1a2CreERT:R26RtdTomato, (b) Col1a2CreERT:R26RtdTomato:p53CKO and (c) RITA injected Col1a2CreERT:R26RtdTomato Scale bar: 10μm. (d) quantification of the number of CD68 cells/high power field in the injury region (mean±S.E.M. *p<0.05 versus Col1a2CreERT:R26RtdTomato, ** p<0.05 versus Col1a2CreERT:R26RtdTomato, n=3) (e–g) p53 expression in myocytes after cardiac injury and effect of RITA on apoptosis in injury region. (e,f) Col1a2CreERT:R26RtdTomato mice were subjected to ischemic cardiac injury, hearts harvested at 3 days and sections stained for p53 and cardiomyocyte marker Troponin. (e) p53 (green, arrowheads) staining is observed in tdTomato expressing cells (red, arrowheads) but not in cardiomyocytes (blue), merged image shows arrowheads pointing to tdTomato labeled cells expressing p53. Scale bar: 10μm. (f) Higher magnification in injury region demonstrating tdTomato cells (arrowheads) expressing p53 (merged, yellow, arrowheads) but p53 staining is not visible in cardiomyocytes (blue). Scale bar: 10μm. (g) TUNEL staining and quantification to determine p53+ apoptotic cells after RITA injection (arrowheads point to p53+TUNEL+ cells in Col1a2CreERT:R26RtdTomato mice (left panel) and RITA injected Col1a2CreERT:R26RtdTomato mice (right panel), inset shows p53+TUNEL+ cell in higher magnification (data shown as mean±S.E.M, ns=not significant, n=3). Hearts in both cases were examined 3 days after injury. Scale bar: 10μm. (h,i) Effect of RITA on MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice after cardiac injury. (h) tdTomato labeled cardiac fibroblasts expressing VECAD in Col1a2CreERT:R26RtdTomato mice treated with/without RITA, Col1a2CreERT:R26RtdTomato:p53CKO mice treated with/without RITA. Scale bar: 10μm. (i) Quantitation of the percentage of labeled fibroblasts undergoing MEndoT for each treatment group (mean ±S.E.M., *p<0.05, n.s= not significant, n=4 animals/group).
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Figure 13: RITA decreases inflammatory infiltrate after cardiac injury, does not increase apoptosis in myocytes and does not enhance MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice(a–d) Sections of hearts harvested at 3 days following cardiac injury were stained for the monocyte/macrophage marker CD68 (green, arrowheads) in (a) Col1a2CreERT:R26RtdTomato, (b) Col1a2CreERT:R26RtdTomato:p53CKO and (c) RITA injected Col1a2CreERT:R26RtdTomato Scale bar: 10μm. (d) quantification of the number of CD68 cells/high power field in the injury region (mean±S.E.M. *p<0.05 versus Col1a2CreERT:R26RtdTomato, ** p<0.05 versus Col1a2CreERT:R26RtdTomato, n=3) (e–g) p53 expression in myocytes after cardiac injury and effect of RITA on apoptosis in injury region. (e,f) Col1a2CreERT:R26RtdTomato mice were subjected to ischemic cardiac injury, hearts harvested at 3 days and sections stained for p53 and cardiomyocyte marker Troponin. (e) p53 (green, arrowheads) staining is observed in tdTomato expressing cells (red, arrowheads) but not in cardiomyocytes (blue), merged image shows arrowheads pointing to tdTomato labeled cells expressing p53. Scale bar: 10μm. (f) Higher magnification in injury region demonstrating tdTomato cells (arrowheads) expressing p53 (merged, yellow, arrowheads) but p53 staining is not visible in cardiomyocytes (blue). Scale bar: 10μm. (g) TUNEL staining and quantification to determine p53+ apoptotic cells after RITA injection (arrowheads point to p53+TUNEL+ cells in Col1a2CreERT:R26RtdTomato mice (left panel) and RITA injected Col1a2CreERT:R26RtdTomato mice (right panel), inset shows p53+TUNEL+ cell in higher magnification (data shown as mean±S.E.M, ns=not significant, n=3). Hearts in both cases were examined 3 days after injury. Scale bar: 10μm. (h,i) Effect of RITA on MEndoT in Col1a2CreERT:R26RtdTomato:p53CKO mice after cardiac injury. (h) tdTomato labeled cardiac fibroblasts expressing VECAD in Col1a2CreERT:R26RtdTomato mice treated with/without RITA, Col1a2CreERT:R26RtdTomato:p53CKO mice treated with/without RITA. Scale bar: 10μm. (i) Quantitation of the percentage of labeled fibroblasts undergoing MEndoT for each treatment group (mean ±S.E.M., *p<0.05, n.s= not significant, n=4 animals/group).
Mentions: To determine whether p53 mediates MEndoT in vivo, we subjected Col1a2CreERT:R26RtdTomato:p53fl/fl mice to ischemic cardiac injury 5 days following cessation of tamoxifen. Mice with intact p53 exhibited 6±0.64 fold increase (mean±S.E.M.) in cardiac fibroblast p53 levels 3 days after cardiac injury, but in mice with fibroblast specific p53 deletion [referred to as Col1a2CreERT:R26RtdTomato:p53CKO (conditional knock out)], the mean p53 expression in border zone cardiac fibroblasts failed to increase significantly (Fig. 3a–c). The degree of MEndoT at 3 days post-injury in Col1a2CreERT:R26RtdTomato:p53CKO animals decreased by 57% (Fig. 3d–f) and was associated with a decrease in capillary density in the injury region (Fig. 3g). Increase in vessel density in the injured heart is a critical post-ischemic repair mechanism. Diminished neovascularization can lead to rapid decline in cardiac function after cardiac injury and is associated with worsened scarring34. Echocardiography on hearts of mice 7 days after cardiac injury showed significant worsening of cardiac function in p53CKO mice (Fig. 3h,i). Masson Trichrome staining demonstrated a greater amount of collagen deposition in the hearts of Col1a2CreERT:R26RtdTomato:p53CKO animals (Fig. 3j,k). The degree of inflammatory infiltrate measured by the number of CD68 expressing macrophages was also significantly higher in the Col1a2CreERT:R26RtdTomato:p53CKO animals (Extended Data Fig. 9a,b,d). These observations demonstrate that p53 is necessary for MEndoT to occur after ischemic cardiac injury and that disruption of MEndoT is associated with diminished post-injury vascularity and cardiac function.

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