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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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tdTomato expression in hearts of vehicle injected Col1a2CreERT:R26RtdTomato mice and immunostaining for Col1 and VECAD in tamoxifen injected Col1a2CreERT:R26RtdTomato mice after cardiac injury(a–c) Oil injected Col1a2CreERT:R26RtdTomato mice underwent ischemia reperfusion injury and 3 days after injury, hearts were harvested and sectioned. Area of injury (demarcated by white lines) was stained with (a) Alexa488 labeled wheat germ agglutinin (WGA, stains cell membranes) (b) rare tdTomato expressing cells in same field (arrowhead) and (c) merged image showing the presence of rare labeled cells in the injury region (arrowhead) (28 labeled cells out of 38,000 cells counted (0.07%), n=3 animals). Scale bar: 100 μm. (d,e) Col1a2Cre:R26RtdTomato (injected with tamoxifen as described in text) were subjected to ischemic cardiac injury and hearts harvested 3 days post injury and stained for Col1 and VECAD (d) Region of injury demonstrating a VECAD expressing cell (blue, arrow) staining positive for Col1 (green) but negative for the tdTomato label (merged arrow). Arrowheads in merged panel show tdTomato labeled cells expressing VECAD but not Col1. Scale bar: 10μm (e) tdTomato labeled cells expressing VECAD (arrowheads) that do not stain for Col1 (green) with merged image showing tdTomato+VECAD+ cells not staining with the Col1 antibody. Out of 225 cells counted (n=3 animals), we did not observe a single tdTomato+VECAD+ cell to stain for Col1. Conversely, not a single VECAD+Col1+ cell exhibited tdTomato fluorescence. Scale bar: 10μm.
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Figure 7: tdTomato expression in hearts of vehicle injected Col1a2CreERT:R26RtdTomato mice and immunostaining for Col1 and VECAD in tamoxifen injected Col1a2CreERT:R26RtdTomato mice after cardiac injury(a–c) Oil injected Col1a2CreERT:R26RtdTomato mice underwent ischemia reperfusion injury and 3 days after injury, hearts were harvested and sectioned. Area of injury (demarcated by white lines) was stained with (a) Alexa488 labeled wheat germ agglutinin (WGA, stains cell membranes) (b) rare tdTomato expressing cells in same field (arrowhead) and (c) merged image showing the presence of rare labeled cells in the injury region (arrowhead) (28 labeled cells out of 38,000 cells counted (0.07%), n=3 animals). Scale bar: 100 μm. (d,e) Col1a2Cre:R26RtdTomato (injected with tamoxifen as described in text) were subjected to ischemic cardiac injury and hearts harvested 3 days post injury and stained for Col1 and VECAD (d) Region of injury demonstrating a VECAD expressing cell (blue, arrow) staining positive for Col1 (green) but negative for the tdTomato label (merged arrow). Arrowheads in merged panel show tdTomato labeled cells expressing VECAD but not Col1. Scale bar: 10μm (e) tdTomato labeled cells expressing VECAD (arrowheads) that do not stain for Col1 (green) with merged image showing tdTomato+VECAD+ cells not staining with the Col1 antibody. Out of 225 cells counted (n=3 animals), we did not observe a single tdTomato+VECAD+ cell to stain for Col1. Conversely, not a single VECAD+Col1+ cell exhibited tdTomato fluorescence. Scale bar: 10μm.

Mentions: We next determined whether substantial recombination in endothelial cells (i.e. leaky promoter elements) could potentially confound our findings. Stochastic induction of Cre can occur after cardiac injury20. We injected Col1a2CreERT:R26RtdTomato animals with vehicle (corn oil) instead of tamoxifen and observed rare labeled cardiac fibroblasts (<0.07% at 3 and 7 days) after ischemic injury demonstrating that injury alone did not lead to significant Cre activation (Extended Data Fig. 3a–c).


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)

tdTomato expression in hearts of vehicle injected Col1a2CreERT:R26RtdTomato mice and immunostaining for Col1 and VECAD in tamoxifen injected Col1a2CreERT:R26RtdTomato mice after cardiac injury(a–c) Oil injected Col1a2CreERT:R26RtdTomato mice underwent ischemia reperfusion injury and 3 days after injury, hearts were harvested and sectioned. Area of injury (demarcated by white lines) was stained with (a) Alexa488 labeled wheat germ agglutinin (WGA, stains cell membranes) (b) rare tdTomato expressing cells in same field (arrowhead) and (c) merged image showing the presence of rare labeled cells in the injury region (arrowhead) (28 labeled cells out of 38,000 cells counted (0.07%), n=3 animals). Scale bar: 100 μm. (d,e) Col1a2Cre:R26RtdTomato (injected with tamoxifen as described in text) were subjected to ischemic cardiac injury and hearts harvested 3 days post injury and stained for Col1 and VECAD (d) Region of injury demonstrating a VECAD expressing cell (blue, arrow) staining positive for Col1 (green) but negative for the tdTomato label (merged arrow). Arrowheads in merged panel show tdTomato labeled cells expressing VECAD but not Col1. Scale bar: 10μm (e) tdTomato labeled cells expressing VECAD (arrowheads) that do not stain for Col1 (green) with merged image showing tdTomato+VECAD+ cells not staining with the Col1 antibody. Out of 225 cells counted (n=3 animals), we did not observe a single tdTomato+VECAD+ cell to stain for Col1. Conversely, not a single VECAD+Col1+ cell exhibited tdTomato fluorescence. Scale bar: 10μm.
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Figure 7: tdTomato expression in hearts of vehicle injected Col1a2CreERT:R26RtdTomato mice and immunostaining for Col1 and VECAD in tamoxifen injected Col1a2CreERT:R26RtdTomato mice after cardiac injury(a–c) Oil injected Col1a2CreERT:R26RtdTomato mice underwent ischemia reperfusion injury and 3 days after injury, hearts were harvested and sectioned. Area of injury (demarcated by white lines) was stained with (a) Alexa488 labeled wheat germ agglutinin (WGA, stains cell membranes) (b) rare tdTomato expressing cells in same field (arrowhead) and (c) merged image showing the presence of rare labeled cells in the injury region (arrowhead) (28 labeled cells out of 38,000 cells counted (0.07%), n=3 animals). Scale bar: 100 μm. (d,e) Col1a2Cre:R26RtdTomato (injected with tamoxifen as described in text) were subjected to ischemic cardiac injury and hearts harvested 3 days post injury and stained for Col1 and VECAD (d) Region of injury demonstrating a VECAD expressing cell (blue, arrow) staining positive for Col1 (green) but negative for the tdTomato label (merged arrow). Arrowheads in merged panel show tdTomato labeled cells expressing VECAD but not Col1. Scale bar: 10μm (e) tdTomato labeled cells expressing VECAD (arrowheads) that do not stain for Col1 (green) with merged image showing tdTomato+VECAD+ cells not staining with the Col1 antibody. Out of 225 cells counted (n=3 animals), we did not observe a single tdTomato+VECAD+ cell to stain for Col1. Conversely, not a single VECAD+Col1+ cell exhibited tdTomato fluorescence. Scale bar: 10μm.
Mentions: We next determined whether substantial recombination in endothelial cells (i.e. leaky promoter elements) could potentially confound our findings. Stochastic induction of Cre can occur after cardiac injury20. We injected Col1a2CreERT:R26RtdTomato animals with vehicle (corn oil) instead of tamoxifen and observed rare labeled cardiac fibroblasts (<0.07% at 3 and 7 days) after ischemic injury demonstrating that injury alone did not lead to significant Cre activation (Extended Data Fig. 3a–c).

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