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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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Phenotypic characterization of tdTomato labeled cells in hearts of Col1a2CreERT:R26RtdTomato mice(a–c) Hearts were digested to obtain a non-myocyte population and subjected to flow cytometry to determine expression of (b) DDR2 and (c) vimentin. (a) serves as a control with no primary antibody added (number in each quadrant represents fraction of total population of cells). (d,e) Immunofluorescent staining on sham injured hearts of Col1a2CreERT:R26RtdTomato mice to determine expression of (d) DDR2 (green) and (e) Vimentin (green) in tdTomato labeled cells (red) with merged image (right, arrowheads point to tdTomato labeled cells staining for DDR2 or vimentin. Scale bar: 10μm. (f–h) Non-myocyte cells from the heart were subjected to flow cytometry to determine expression of (f) VECAD (g) CD31 or (h) c-Kit (number in each quadrant represents fraction of total population of cells) (i–k) Immunofluorescent staining for (i) alpha-smooth muscle actin (j) CD68 or (k) podoplanin was performed on frozen heart sections prepared from 8 week old sham injured Col1a2CreERT:R26RtdTomato mice following tamoxifen injections and colocalization analysis performed to determine the number of labeled cardiac fibroblasts expressing alpha-smooth muscle actin (99.4% negative for alpha-SMA, 1500 cells examined), CD68 (100% negative for CD68, 1000 cells examined) or podoplanin (arrowheads). Scale bar: i–k: 10μm. (l,m) STED super-resolution microscopy demonstrating tdTomato labeled cells (arrows) not expressing (l) NG2 (green, arrows, STED channel) or (m) CD146 (green, arrows, STED channel). Scale bar: 10μm.
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Figure 5: Phenotypic characterization of tdTomato labeled cells in hearts of Col1a2CreERT:R26RtdTomato mice(a–c) Hearts were digested to obtain a non-myocyte population and subjected to flow cytometry to determine expression of (b) DDR2 and (c) vimentin. (a) serves as a control with no primary antibody added (number in each quadrant represents fraction of total population of cells). (d,e) Immunofluorescent staining on sham injured hearts of Col1a2CreERT:R26RtdTomato mice to determine expression of (d) DDR2 (green) and (e) Vimentin (green) in tdTomato labeled cells (red) with merged image (right, arrowheads point to tdTomato labeled cells staining for DDR2 or vimentin. Scale bar: 10μm. (f–h) Non-myocyte cells from the heart were subjected to flow cytometry to determine expression of (f) VECAD (g) CD31 or (h) c-Kit (number in each quadrant represents fraction of total population of cells) (i–k) Immunofluorescent staining for (i) alpha-smooth muscle actin (j) CD68 or (k) podoplanin was performed on frozen heart sections prepared from 8 week old sham injured Col1a2CreERT:R26RtdTomato mice following tamoxifen injections and colocalization analysis performed to determine the number of labeled cardiac fibroblasts expressing alpha-smooth muscle actin (99.4% negative for alpha-SMA, 1500 cells examined), CD68 (100% negative for CD68, 1000 cells examined) or podoplanin (arrowheads). Scale bar: i–k: 10μm. (l,m) STED super-resolution microscopy demonstrating tdTomato labeled cells (arrows) not expressing (l) NG2 (green, arrows, STED channel) or (m) CD146 (green, arrows, STED channel). Scale bar: 10μm.

Mentions: We used a genetic fate map strategy to label cardiac fibroblasts, by crossing transgenic mice harboring a tamoxifen inducible Cre recombinase driven by fibroblast specific regulatory sequence of the alpha2 (type 1) collagen gene (Col1a2CreERT)10–12 with the lineage reporter strain (Rosa26RtdTomato)13 to create Col1a2CreERT:Rosa26RtdTomato progeny mice. In these mice, administration of tamoxifen results in activation of Cre recombinase and cells expressing Col1a2 at the time of tamoxifen administration are irreversibly labeled by tdTomato fluorescence. We administered tamoxifen for 10 days to adult Col1a2CreERT:R26RtdTomato mice. Five days following cessation of tamoxifen, we observed that approximately 55% of all non-myocyte cells exhibited tdTomato fluorescence and greater than 96% and 99% of tdTomato fluorescent cells expressed the cardiac fibroblast markers Domain Discoidin Receptor 2 (DDR2) and vimentin (Extended Data Fig. 1a–c). Immunofluorescent staining showed that 87±9% and 99±0.5% (mean±S.E.M) of tdTomato labeled cells expressed DDR2 and vimentin respectively, supporting flow cytometry data (Extended Data Fig. 1d,e). tdTomato cells did not express endothelial markers VECAD and CD31 (99.9±0.06% and 99.8±0.02% negative respectively, mean±S.E.M.) (Extended Data Fig. 1f,g), did not express the cardiac progenitor marker C-Kit nor markers of smooth muscle, macrophages, and lymphatics (Extended Data Fig. 1h–k). Cardiac myocytes did not express Cre recombinase as previously shown10. Taken together these data strongly suggest that cells exhibiting tdTomato fluorescence in hearts of Col1a2CreERT:R26RtdTomato mice are cardiac fibroblasts and do not express canonical markers of other cardiovascular cell types.


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)

Phenotypic characterization of tdTomato labeled cells in hearts of Col1a2CreERT:R26RtdTomato mice(a–c) Hearts were digested to obtain a non-myocyte population and subjected to flow cytometry to determine expression of (b) DDR2 and (c) vimentin. (a) serves as a control with no primary antibody added (number in each quadrant represents fraction of total population of cells). (d,e) Immunofluorescent staining on sham injured hearts of Col1a2CreERT:R26RtdTomato mice to determine expression of (d) DDR2 (green) and (e) Vimentin (green) in tdTomato labeled cells (red) with merged image (right, arrowheads point to tdTomato labeled cells staining for DDR2 or vimentin. Scale bar: 10μm. (f–h) Non-myocyte cells from the heart were subjected to flow cytometry to determine expression of (f) VECAD (g) CD31 or (h) c-Kit (number in each quadrant represents fraction of total population of cells) (i–k) Immunofluorescent staining for (i) alpha-smooth muscle actin (j) CD68 or (k) podoplanin was performed on frozen heart sections prepared from 8 week old sham injured Col1a2CreERT:R26RtdTomato mice following tamoxifen injections and colocalization analysis performed to determine the number of labeled cardiac fibroblasts expressing alpha-smooth muscle actin (99.4% negative for alpha-SMA, 1500 cells examined), CD68 (100% negative for CD68, 1000 cells examined) or podoplanin (arrowheads). Scale bar: i–k: 10μm. (l,m) STED super-resolution microscopy demonstrating tdTomato labeled cells (arrows) not expressing (l) NG2 (green, arrows, STED channel) or (m) CD146 (green, arrows, STED channel). Scale bar: 10μm.
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Figure 5: Phenotypic characterization of tdTomato labeled cells in hearts of Col1a2CreERT:R26RtdTomato mice(a–c) Hearts were digested to obtain a non-myocyte population and subjected to flow cytometry to determine expression of (b) DDR2 and (c) vimentin. (a) serves as a control with no primary antibody added (number in each quadrant represents fraction of total population of cells). (d,e) Immunofluorescent staining on sham injured hearts of Col1a2CreERT:R26RtdTomato mice to determine expression of (d) DDR2 (green) and (e) Vimentin (green) in tdTomato labeled cells (red) with merged image (right, arrowheads point to tdTomato labeled cells staining for DDR2 or vimentin. Scale bar: 10μm. (f–h) Non-myocyte cells from the heart were subjected to flow cytometry to determine expression of (f) VECAD (g) CD31 or (h) c-Kit (number in each quadrant represents fraction of total population of cells) (i–k) Immunofluorescent staining for (i) alpha-smooth muscle actin (j) CD68 or (k) podoplanin was performed on frozen heart sections prepared from 8 week old sham injured Col1a2CreERT:R26RtdTomato mice following tamoxifen injections and colocalization analysis performed to determine the number of labeled cardiac fibroblasts expressing alpha-smooth muscle actin (99.4% negative for alpha-SMA, 1500 cells examined), CD68 (100% negative for CD68, 1000 cells examined) or podoplanin (arrowheads). Scale bar: i–k: 10μm. (l,m) STED super-resolution microscopy demonstrating tdTomato labeled cells (arrows) not expressing (l) NG2 (green, arrows, STED channel) or (m) CD146 (green, arrows, STED channel). Scale bar: 10μm.
Mentions: We used a genetic fate map strategy to label cardiac fibroblasts, by crossing transgenic mice harboring a tamoxifen inducible Cre recombinase driven by fibroblast specific regulatory sequence of the alpha2 (type 1) collagen gene (Col1a2CreERT)10–12 with the lineage reporter strain (Rosa26RtdTomato)13 to create Col1a2CreERT:Rosa26RtdTomato progeny mice. In these mice, administration of tamoxifen results in activation of Cre recombinase and cells expressing Col1a2 at the time of tamoxifen administration are irreversibly labeled by tdTomato fluorescence. We administered tamoxifen for 10 days to adult Col1a2CreERT:R26RtdTomato mice. Five days following cessation of tamoxifen, we observed that approximately 55% of all non-myocyte cells exhibited tdTomato fluorescence and greater than 96% and 99% of tdTomato fluorescent cells expressed the cardiac fibroblast markers Domain Discoidin Receptor 2 (DDR2) and vimentin (Extended Data Fig. 1a–c). Immunofluorescent staining showed that 87±9% and 99±0.5% (mean±S.E.M) of tdTomato labeled cells expressed DDR2 and vimentin respectively, supporting flow cytometry data (Extended Data Fig. 1d,e). tdTomato cells did not express endothelial markers VECAD and CD31 (99.9±0.06% and 99.8±0.02% negative respectively, mean±S.E.M.) (Extended Data Fig. 1f,g), did not express the cardiac progenitor marker C-Kit nor markers of smooth muscle, macrophages, and lymphatics (Extended Data Fig. 1h–k). Cardiac myocytes did not express Cre recombinase as previously shown10. Taken together these data strongly suggest that cells exhibiting tdTomato fluorescence in hearts of Col1a2CreERT:R26RtdTomato mice are cardiac fibroblasts and do not express canonical markers of other cardiovascular cell types.

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