Mesenchymal-endothelial transition-derived cells as a potential new regulatory target for cardiac hypertrophy

Figures

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  Figure 1. Expression of endothelial markers in mesenchymal-endothelial transition (MEndoT)-derived cells in the heart tissue of Col1a2-CreERT: R26RtdTomato mice after cardiac hypertrophy. (a-d) Immunofluorescence staining for endothelial cell markers in heart tissue of Col1a2-CreERT: R26RtdTomato mice after transverse aortic constriction (TAC). (a) VECAD expression and the percentage of tdTomato labeled cells expressing VECAD. (b) Tek expression and the percentage of tdTomato labeled cells expressing Tek. (c) Isolectin B4 expression and the percentage of tdTomato labeled cells expressing isolectin B4. (d) CD31 expression and the percentage of tdTomato labeled cells expressing CD31. (e) Flow cytometry of isolectin B4 14 days post sham or TAC. (All graphs show mean ± S.E.M; n = 3 animals/group, *p < 0.05, using an unpaired t-test compared with sham. Colocalization of fluorophores is indicated by arrowhead. Scale bar: 10 µm).

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  Figure 2. Contribution of mesenchymal-endothelial transition (MEndoT)-derived cells to neovascularization of heart tissue after cardiac hypertrophy. Contribution to neovascularization was calculated based on immunofluorescence staining for endothelial cell markers in heart tissue of Col1a2-CreERT: R26RtdTomato mice for MEndoT-derived cells (a) and Tek-CreERT: R26RtdTomato mice for labeled coronary endothelial cells (b). (All graphs show mean ± S.E.M; n = 3 animals/group, *p < 0.05, using an unpaired t-test compared with sham).

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  Figure 3. RNA sequencing (RNA-seq) assay shows mesenchymal-endothelial transition (MEndoT)-derived cells are more endothelial-like after cardiac hypertrophy but may be a different cell sub-population. Non-myocytes were isolated from hearts of 14 day post-sham or -transverse aortic constriction (TAC) in Col1a2-CreERT: R26RtdTomato mice and native fibroblasts (tdTomato+-labeled fibroblasts after sham-injury), MEndoT-derived cells (tdTomato+CD31+-labeled cells) were collected using flow cytometry for RNA-seq. The tdTomato+-labeled endothelial cells from heart tissue of Tek-CreERT: R26RtdTomato mice after sham injury were collected and served as native coronary endothelial cells. (a) Principal component analysis clustering show MEndoT-derived cells are a subset cell population with characteristics that were closer to native endothelial cells than native fibroblasts. (b) p53 is specifically and significantly upregulated in MEndoT-derived cells. Heatmap show significantly expressed genes indicative of (c) fibroblast lineage commitment and (d) endothelial lineage commitment (all genes in b-d have a q-value <0.05 compared with native fibroblast, n = 3 animals/group).

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  Figure 4. Cardiac fibroblasts adopt endothelial cell characteristics after cardiac hypertrophy in a p53-dependent manner and conditional deletion of p53 in cardiac fibroblasts leads to temporary deterioration of cardiac function after cardiac hypertrophy. Hearts of Col1a2-CreERT: R26RtdTomato and Col1a2-CreERT: R26RtdTomato: p53CKO mice show (a) immunofluorescence staining of p53 7 days post-sham or -transverse aortic constriction (TAC) and (b) percentage of tdTomato-labeled cells expressing p53 in (a). (c) Immunofluorescence staining for endothelial markers vascular endothelial cadherin (VECAD) 14 days post-sham or -TAC. (d) Percentage of tdTomato-labeled cells expressing VECAD and vascular density per high-power field (hpf) in (c).(e,f) echocardiographic assessment of cardiac function prior to and after transverse aortic constriction (TAC, n = 8 animals/group, *p < 0.05 using ANOVA with post hoc tests). (g) Representative M-mode echocardiograms in (e,f). (h) Quantitation of fibrotic area in heart using Masson’s trichrome staining of sections isolated 5 weeks after TAC (n = 8 animals/group, Scale bar: 25 µm). All graphs show mean ± S.E.M.; n = 3 animals/group for a-d, *p < 0.05 using an…

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  Figure 5. p53 Activator administration (RITA) increase p53 expression and mesenchymal-endothelial transition (MEndoT) after transverse aortic constriction (TAC). Phosphate-buffered saline (PBS) and RITA-treated Col1a2-CreERT: R26RtdTomato mice 14 day post TAC (a) Immunostaining of p53 and percentage of tdTomato-labeled cells expressing p53. (b) Immunofluorescence staining for endothelial markers vascular endothelial cadherin (VECAD) and (c) vascular density per high-power field (hpf) in (b). (d) Immunofluorescence staining and percentage of tdTomato-labeled cells expressing endothelial markers Tek and isolectin B4. Graphs show mean ± S.E.M.; n = 3 animals/group, *p < 0.05, using an unpaired t-test compared with PBS control after TAC. Colocalization of fluorophores indicated by arrowhead. Scale bar: 25 µm.

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  Figure 6. RITA preserves heart function and attenuates cardiac hypertrophy after transverse aortic constriction (TAC). (a) Echocardiographic assessment of cardiac function in phosphate-buffered saline (PBS)- or RITA-treated mice prior to and post-TAC (n = 10 animals/group). (b) Representative M-mode echocardiograms in (a). (c) Ratio of heart to body weight in PBS- and RITA-treated mice 5 weeks TAC (n = 6 animals/group). (d) Quantitation of cardiomyocyte size in H&E stained heart tissue 5 weeks post-TAC (n = 10 animals/group, Scale bar: 50 µm). (e) Quantitation of fibrotic area in Masson’s trichrome stained heart tissue 5 weeks post-TAC (n = 10 animals/group). Graphs show mean ± S.E.M.; *p < 0.05, using ANOVA with post hoc tests, compared to PBS control after TAC. Scale bar: 25 µm.

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  Figure 7. Altered expression of paracrine factors in fibroblasts undergoing mesenchymal-endothelial transition (MEndoT) and a schematic for MEndoT regulation in cardiac hypertrophy. (a) RNA sequencing (RNA-seq) assay shows MEndoT-derived cells isolated from hearts of Col1a2-CreERT: R26RtdTomato mice 14 day post-TAC exhibit altered expression of paracrine factors demonstrated to be important for cardiac hypertrophy and heart failure (*q < 0.05, n = 3 animals/group). (b) Quantitative polymerase chain reaction (qPCR) analysis of expression of paracrine factors in fibroblasts undergoing MEndoT using in vitro serum starvation model. Expression level shown are average fold-change to fibroblasts in 10% serum.(n = 3 experiment repeat, *p < 0.05, using an unpaired t-test as indicated). (c) A schematic for MEndoT regulation in cardiac hypertrophy.