Modulation of cell fate by shock wave therapy in ischaemic heart disease

Figures

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  Figure 1. Shockwave therapy induces transdifferentiation via Toll-like receptor 3. (A) Expression of the innate immune receptor Toll-like receptor 3 on human cardiac fibroblasts. green = Toll-like receptor 3; blue = DAPI. Scale bar = 20 µm. (B) Toll-like receptor 3 activation of Toll-like receptor 3 reporter cells upon shockwave therapy or Poly(I:C) treatment (30 ng/mL). Data are means ± SEM. **P < 0.01. (C) The expression of the downstream adapter TIR-domain-containing adapter-inducing interferon-β after shockwave therapy or Poly(I:C) treatment (30 ng/mL). (D) mRNA levels of interleukin-6 after shockwave therapy or Poly(I:C) treatment (30 ng/mL). Data are means ± SEM. *P < 0.01. (E) Human fibroblasts were treated with shockwave therapy in the presence of endothelial induction medium containing bone morphogenetic protein 4, vascular endothelial growth factor, and fibroblast growth factor and subjected to reverse transcription polymerase chain reaction (RT-PCR) or fluorescence-activated cell sorting. (F) mRNA expression of CD31 after treatment with control medium, endothelial induction medium, shockwave therapy, or a specific dsRNA/Toll-like receptor 3 inhibitor. Data are means ± SEM. **P <…

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  Figure 2. Induced endothelial cells are functional in vitro and in vivo. (A) mRNA expression of CD31 after fluorescence-activated cell sorting of induced endothelial cells or fibroblasts. Data are means ± SEM. **P < 0.01. (B) mRNA expression of vascular endothelial growth factor receptor 2 after fluorescence-activated cell sorting of induced endothelial cells or fibroblasts. Data are means ± SEM. ****P < 0.0001. (C) CD31 expression of induced endothelial cells or fibroblasts after fluorescence-activated cell sorting. red = CD31; blue = DAPI. Scale bar = 20 µm. (D) Tube formation assay with induced endothelial cells or fibroblasts after fluorescence-activated cell sorting. Scale bar = 50 µm. (E) Number of nodes per high power field analysed in a tube formation assay in induced endothelial cells or fibroblasts. Data are means ± SEM. ****P < 0.0001. (F) Number of junctions per high power field analysed in a tube formation assay in induced endothelial cells or fibroblasts. Data are means ± SEM. ****P < 0.0001. (G) Nitric oxide concentration produced by induced endothelial cells or fibroblasts. Data are means ± SEM. ****P < 0.0001. (H) Induced endothelial cells, fibroblasts or human umbilical…

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  Figure 3. Shockwave therapy induces transdifferentiation in ischaemic myocardium and improves cardiac function. (A) To test whether we could induce transdifferentiation of cardiac fibroblasts within a myocardial scar towards endothelial cells, we created a transgenic mouse model of Fsp1-Cre/LacZ mice and performed a lineage tracing experiment after ligation of the left anterior descending and shockwave therapy. (B) Left ventricular fibrosis assessed via Masson trichrome staining after left anterior descending ligation with (shockwave therapy) or without shockwave therapy (control). Scale bar = 1 mm. (C) Quantification of left ventricular fibrosis after left anterior descending ligation with (shockwave therapy) or without shockwave therapy (control). Data are means ± SEM. *P < 0.05. (D) M-mode images after left anterior descending ligation with (shockwave therapy) or without shockwave therapy (control). (E) Left ventricular ejection fraction after left anterior descending ligation with (shockwave therapy) or without shockwave therapy (control) (%). Data are means ± SEM. **P < 0.01. (F) Left ventricular end-diastolic diameter after left anterior descending ligation with (shockwave therapy)…

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  Figure 4. Shockwave-treated hearts show larger endothelial-like fibroblast subcluster after myocardial infarction. (A) Uniform manifold approximation and projection plot showing all sequenced nuclei from all samples from different time points (control Day 1, control Day 14, shockwave (SWT) therapy Day 1, and shockwave therapy Day 14). (B) Uniform manifold approximation and projection plot showing fibroblast subclustering, revealing 10 fibroblast subclusters (Leiden clustering, resolution 0.5). (C) Gene expression of endothelial and fibroblast marker genes in the different fibroblast clusters shown in B. (D) Bar plots showing the relative fibroblast cell fraction of fibroblast Subcluster 8 at different time points. (E) Gene ontology analysis of differential expressed genes in fibroblast Cluster 8 at 14 days after shockwave therapy vs. control. (F) Partition-based graph abstraction showing cluster dynamics among fibroblast subclusters. Edges represent cluster connectivities; threshold: 0.1). CTRL, control; SWT, shockwave therapy; TLR, Toll-like receptor; UMAP, uniform manifold approximation and projection; VEGF, vascular endothelial growth factor.

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  Figure 5. Shockwave therapy induces epigenetic reprogramming. (A) Volcano plot displaying open chromatin loci as determined by Assay for Transposase-Accessible Chromatin sequencing. Shockwave therapy and PolyI:C induced up-regulation of chromatin accessibility. Average signal is displayed as log2 fold change (non-adjusted P < 0.05). (B) Gene ontology analysis of differentially accessible genomic regions upon treatment (P < 0.05). (C) Shockwave therapy and PolyI:C induced chromatin opening in the same manner. Statistically strong regulated genes are visualized as heatmap, revealing more accessible genes responsible for epigenetic regulation and angiogenesis (P < 0.01). CTRL, control; SWT, shockwave therapy.