. 2014 Oct 30;514(7524):585-90.

doi: 10.1038/nature13839. Epub 2014 Oct 15.

Mesenchymal-endothelial transition contributes to cardiac neovascularization

Affiliations

¹ Department of Cell Biology &Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
² 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Eli and Edythe Broad Institute of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California 90095, USA [4] Department of Molecular, Cell and Developmental Biology, College of Letters and Sciences, University of California, Los Angeles, California 90095, USA [5] Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA [6] Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
³ 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [4] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁴ Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁵ Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁶ Department of Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁷ 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Molecular Biology Institute, University of California, Los Angeles, California 90095, USA [4] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [5] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁸ 1] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.

PMID: 25317562
PMCID: PMC4214889
DOI: 10.1038/nature13839

Mesenchymal-endothelial transition contributes to cardiac neovascularization

Eric Ubil et al. Nature. 2014.

. 2014 Oct 30;514(7524):585-90.

doi: 10.1038/nature13839. Epub 2014 Oct 15.

Authors

Affiliations

¹ Department of Cell Biology &Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
² 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Eli and Edythe Broad Institute of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California 90095, USA [4] Department of Molecular, Cell and Developmental Biology, College of Letters and Sciences, University of California, Los Angeles, California 90095, USA [5] Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA [6] Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
³ 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [4] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁴ Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁵ Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁶ Department of Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
⁷ 1] Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Molecular Biology Institute, University of California, Los Angeles, California 90095, USA [4] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [5] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
⁸ 1] Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [2] Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA [3] Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.

PMID: 25317562
PMCID: PMC4214889
DOI: 10.1038/nature13839

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.

PubMed Disclaimer

Conflict of interest statement

The authors have no competing financial interests to disclose.

Figures

**Extended Figure 1. Phenotypic characterization of tdTomato labeled cells in hearts of Col1a2CreERT:R26R^tdTomato 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:R26R^tdTomato 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:R26R^tdTomato 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.

**Extended Figure 2. Expression of VECAD in fibroblast derived endothelial cells that take up DiO and expression of alpha-smooth muscle actin and VECAD in tdTomato labeled cells**
**(a)** Col1a2CreERT:R26R^tdTomato mice were subjected to ischemic injury, injected with DiO 7 days after cardiac injury and then harvested. High magnification of a wall of a blood vessel in the injured region demonstrates luminal cells staining for DiO (green, arrowheads), VECAD (blue, arrowheads), tdTomato (red, arrowheads) and merged image demonstrating colocalization of all three fluorophores (filled arrowheads); unfilled arrowhead points to VECAD+DiO+ cell that does not bear the tdTomato label. Scale bar: 10μm. **(b–f) Expression of alpha-smooth muscle actin and VECAD in tdTomato labeled cells (b–e)** Tamoxifen injected Col1a2CreERT:R26R^tdTomato mice underwent ischemia reperfusion injury and hearts were harvested 3 days after injury and immunofluorescent staining performed for alpha-smooth muscle actin and VECAD. Section shows **(b)** tdTomato labeled cardiac fibroblasts **(c)** VECAD **(d)** alpha smooth muscle actin expressing cells and **(e)** merged image showing colocalization of fluorophores. Arrowheads show labeled cardiac fibroblasts expressing alpha smooth muscle actin and arrow shows a labeled cardiac fibroblast expressing VECAD but not smooth muscle actin. Scale bar: b–e: 10μm **(f)** Fraction of labeled cardiac fibroblasts that are alpha-smooth muscle actin+ and VECAD+ or VECAD- (mean±S.E.M., *p<0.05, n=3).

Extended Figure 3. tdTomato expression in hearts of vehicle injected Col1a2CreERT:R26R^tdTomato mice and immunostaining for Col1 and VECAD in tamoxifen injected Col1a2CreERT:R26R^tdTomato mice after cardiac injury
**(a–c)** Oil injected Col1a2CreERT:R26R^tdTomato 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:R26R^tdTomato (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.

**Extended Figure 4. VECAD and Cre immunostaining in heart sections of Col1a2Cre:R26R^tdTomato:Col1GFP mice 3 days after injury**
**(a,b)** Col1a2Cre:R26R^tdTomato:Col1GFP mice were subjected to ischemic cardiac injury and hearts harvested at 3 days post injury and stained for VECAD **(a)** tdTomato labeled cells (red, arrowheads) expressing VECAD (blue, arrowheads) but not GFP (green), with merged image demonstrating co-localization of tdTomato and VECAD but not GFP (arrowheads). Scale bar: 10 μm. **(b)** High magnification of a blood vessel (asterisk) outlined by VECAD staining (blue) demonstrating rare cell that colocalizes all three fluorophores (tdTomato+VECAD+GFP+, white, arrowhead). Arrows point to tdTomato positive cells expressing VECAD but not GFP. Scale bar: 10 μm. **(c–f)** Immunostaining for Cre protein on hearts of Col1a2Cre:R26R^tdTomato:Col1GFP harvested 3 days following ischemic injury to detect Cre expression in the nucleus of tdTomato labeled cells expressing VECAD. **(c)** TdTomato labeled cells in area of injury (arrowheads) expressing **(d)** VECAD (arrowheads) **(e)** merged image showing co-localization of fluorophores (arrowheads) **(f)** nuclei stained for DAPI and Cre demonstrating absence of any detectable nuclear Cre protein. Scale bar: 10μm **(g–i)** positive control demonstrating section of heart of Wt-1Cre transgenic mouse heart 3 days after injury with region of injury stained for **(g)** DAPI **(h)** Cre (red) and **(i)** merged image demonstrating numerous cells in the injury region expressing nuclear Cre (arrowheads). Scale bar: 10μm **(j–m)** Cre immunostaining in tdTomato labeled cells expressing GFP. Area in region of injury with **(j)** nuclei stained for DAPI, **(k)** tdTomato expression (arrowhead) **(l)** GFP expression (arrowhead) and **(m)** merged image (DAPI, Cre and GFP) showing Cre staining (red channel) localized to the cytoplasm of GFP expressing cell (arrowhead). Scale bar: 10μm.

Extended Figure 5. Flow cytometry for endothelial progenitor markers on non-myocyte cells harvested from uninjured hearts of Col1a2CreERT:R26R^tdTomato mice and bone marrow cells isolated from the same animal
**(a,b)** Hearts of mice were digested, myocytes discarded and the entire non-myocyte population without any further selection was subjected to flow cytometry. Expression of tdTomato and **(a)** CD45 and Flk-1 (APC fluorophore) and **(b)** CD133, CD34 and combined expression of CD34 and Flk-1 in non-myocyte population. (c–f) Bone marrow cells were isolated from Col1a2CreERT:R26R^tdTomato mice and without further culture subjected to flow cytometry. Expression of tdTomato and (c) CD45 (d) Flk-1 (e) CD133 (f) CD34 and combined expression of Flk1 and CD34 in bone marrow cells (g) Expression of tdTomato in bone marrow derived mesenchymal stem cell colonies (black arrowhead points to rare tdTomato positive cell, white arrowheads point to mesenchymal stem cells). Scale bar: 50μm.

**Extended Figure 6. MEndoT in FSP1Cre:R26R^tdTomato mice**
FSP1Cre:R26R^tdTomato mice were subjected to (a) sham or (b) ischemia-reperfusion cardiac injury. Hearts were harvested 3 days after injury and stained for endothelial marker VECAD or isolectin. Injury region demonstrated tdTomato labeled cells expressing VECAD or isolectin (arrowheads, n=4). Scale bar: 10μm. (c) Quantitation of labeled fibroblasts that express VECAD or isolectin in sham injured animals and in the injury border zone (mean±S.E.M., *p<0.01, n=4)

**Extended Figure 7. Effect of serum starvation on p53 levels and effects of Pifithrin-α and RITA on tube formation of serum fed cardiac fibroblasts**
**(a)** Western blot for p53 in cardiac fibroblasts subjected to serum starvation for 24, 48 and 72 hours (representative sample from n=3) **(b)** densitometric quantitation of Western blot (mean±S.E.M., *p<0.05 compared to cells in 10% serum). **(c)** Effect on tube formation after adding Pifithrin-α or RITA to cardiac fibroblasts grown in 10% serum Scale bar: 250μm **(d)** quantitation of tube formation (mean±S.E.M., *p<0.05, n=3)

**Extended Figure 8. 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).

**Extended Figure 9. RITA decreases inflammatory infiltrate after cardiac injury, does not increase apoptosis in myocytes and does not enhance MEndoT in Col1a2CreERT:R26R^tdTomato: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:R26R^tdTomato, **(b)** Col1a2CreERT:R26R^tdTomato:p53CKO and **(c)** RITA injected Col1a2CreERT:R26R^tdTomato 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:R26R^tdTomato, ** p<0.05 versus Col1a2CreERT:R26R^tdTomato, n=3) (e–g) p53 expression in myocytes after cardiac injury and effect of RITA on apoptosis in injury region. **(e,f)** Col1a2CreERT:R26R^tdTomato 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:R26R^tdTomato mice (left panel) and RITA injected Col1a2CreERT:R26R^tdTomato 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:R26R^tdTomato:p53CKO mice after cardiac injury. **(h)** tdTomato labeled cardiac fibroblasts expressing VECAD in Col1a2CreERT:R26R^tdTomato mice treated with/without RITA, Col1a2CreERT:R26R^tdTomato: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).

**Extended Figure 10. γH2AX expression in cells expressing p53 after ischemic cardiac injury**
Col1a2CreERT:R26R^tdTomato mice were subjected to ischemic cardiac injury and immunostaining performed for γH2AX and p53. (a) Immunostaining for p53 (green), γH2AX (red) and DAPI (blue) in region of injury (arrowheads point to nuclei co-expressing γH2AX and p53). Scale bar: 10μm. (b) Immunostaining for p53 (green), γH2AX (blue) and tdTomato (red) to determine co-expression of p53 and γH2AX in tdTomato labeled cells. Arrowheads point to tdTomato positive cells co-expressing γH2AX and p53. Scale bar: 10μm. (c) quantitation of the fraction of tdTomato+p53+ cells expressing γH2AX (mean±S.E.M. *p<0.01, n=3).

**Figure 1. Cardiac fibroblasts adopt endothelial cell fates after cardiac injury**
**(a,b)** Hearts from Col1a2CreERT:R26R^tdTomato immunostained for endothelial markers (arrowheads) (c) tdTomato+ fibroblasts(%) expressing endothelial markers (*p<0.05 vs sham, † p<0.05 vs injury border zone). (d) Temporal expression of VECAD (* p<0.005 vs sham, †p<0.05 vs Day 1 and 2, $ p<0.05 vs Day 1,). (e) STED microscopy demonstrating tdTomato+VECAD+ cell (arrowhead; unfilled arrowhead shows tdTomato- endothelial cell) (**f–h**) DiO stained capillary in (**f,g**) longitudinal section (arrowheads show tdTomato+DiO+ cells) or (h) cross-section (cyan arrowheads show DiO stained inner and outer endothelial cell membranes, white arrowheads show tdTomato+ endothelial cell; unfilled arrowhead shows tdTomato- endothelial cell). Scale bar: 5μm. (i) Luminal surface area occupied by fibroblast derived endothelium (* p<0.005 vs sham, † p<0.05 vs Day 3). (j) AcLDL uptake by tdTomato+ endothelium (arrowheads). (n=3 animals /group/ time point, All graphs show mean±S.E.M., scale bar:10μm unless mentioned)

Figure 2. Cardiac fibroblasts upregulate p53 after injury and p53 mediates MEndoT *ex vivo*
**(a,b)** p53 immunostaining in injured hearts (arrowheads show tdTomato+P53+ cells) **(c)** Temporal p53 expression in labeled fibroblasts (*p<0.05 vs sham, n=3 animals/time point). **(d)** co-expression of p53, VECAD & tdTomato (arrowhead). **(e,f)** tdTomato+VECAD+ tubes and **(g,h)** AcLDL uptake after serum starvation (arrowheads, n=4). Scale bar: 250μm **(h, right panel)** Confocal image (XZ plane) showing AcLDL internalization (Scale bar: 20μm) (i–m) Tube formation of cardiac fibroblasts in **(i)**10% serum or 0% serum with **(j)** PBS **(k)** 100μM Pifithrin-α, **(m)** 0.1μM RITA, or **(l)** p53 deletion (bright field and fluorescence overlay). Scale bar: 250μm **(n)** Quantitation of tube length (** p<0.005 vs 10% serum. † p<0.005 and *p<0.05 vs starved cells, n=3). **(o)** Endothelial gene expression in cardiac fibroblasts (* p<0.005 vs 10% serum, † p<0.05 vs PBS, n=8). **(p)** ChIP with p53 (*p<0.05). (All graphs show mean±S.E.M., scale bar: 10μm unless mentioned).

**Figure 3. MEndoT after cardiac injury is p53 dependent**
**(a–c)** p53 immunostaining in Col1a2CreERT:R26R^tdTomato and Col1a2CreERT:R26R^tdTomato:p53CKO hearts (arrowheads) and (c) quantification of p53 expression in labeled fibroblasts (* p<0.005). (**d–f**) VECAD immunostaining in labeled fibroblasts (arrowheads) and (f) Labeled fibroblasts(%) expressing VECAD (* p<0.005). (g) Number of endothelial cells/high power field (* p<0.005, n=5 animals) (**h,i**) Cardiac function prior to and 7 days after injury. (*p<0.05, n=9 animals). (j) Masson’s Trichrome staining 14 days after injury. (k) Quantitation of fibrosis (* p<0.05). (n=4 animals unless mentioned, All graphs show mean±S.E.M., scale bar: 10μm).

**Figure 4. RITA enhances MEndoT after cardiac injury**
Hearts from PBS or RITA treated Col1a2CreERT:R26R^tdTomato mice (**a,b**) p53 immunostaining in labeled fibroblasts (arrowheads) and (c) quantitation of p53 expression (* p<0.005). (**d–e**) VECAD immunostaining in labeled fibroblasts (arrowheads). (f) Labeled fibroblasts(%) expressing VECAD (* p<0.05). (g) Endothelial cells/hpf (* p<0.05). (h) Masson’s Trichrome staining (blue, arrowheads) near (i) apex, (ii) between mid-ventricle and apex and (iii) mid-ventricle (n=14 animals) (i) Quantitation of fibrosis (* p<0.001, n=14 animals). (**j,k**) Cardiac function prior to and 7 days after injury. (* p<0.05, n=8 animals). (n=4 animals unless mentioned, All graphs show mean ± S.E.M., scale bar: 10μm).

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Comment in

Cardiac biology: Cell plasticity helps hearts to repair.
Miyake T, Kalluri R. Miyake T, et al. Nature. 2014 Oct 30;514(7524):575-6. doi: 10.1038/nature13928. Epub 2014 Oct 15. Nature. 2014. PMID: 25317554 No abstract available.

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Mesenchymal-endothelial transition contributes to cardiac neovascularization

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Mesenchymal-endothelial transition contributes to cardiac neovascularization

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