Defining a therapeutic range for regeneration of ischemic myocardium via shock waves

Figures

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  Figure 1. Shock wave therapy induces necrosis only at very high energy levels. (a) To evaluate at which energy levels SWT would induce cellular damage, we performed an LDH assay upon therapy. SWT caused no necrosis induced until energy levels of 0.27 mJ/mm2 total flux density in T25 flasks. Means ± SEM. *p < 0.05, ****p < 0.0001. n = 3. (b) Scheme for the mechanical setup of SWT in vitro: To evaluate whether the occurrence of necrosis might depend on the size and form of the cell culture flask in which the cells undergo SWT, HUVECs were seeded in 6, 12, 24 and 96 well plates and subjected to SWT. (c) In microwell plates with a surface smaller than 9,5cm2 (12, 24 and 96 well plates) necrosis could be measured at a level as low as 0.15 mJ/mm. This might be attributed to phenomena of constructive interference. Means ± SEM. **p < 0.01; ***p < 0.001, ****p < 0.0001. n = 3–4. (d) Comparable levels of necrosis could be discovered in fibroblasts and cardiomyocytes upon shockwave therapy. (e) Within the targeted tissue, shock waves induce alternating positive and negative pressure due to their specific wave profile. Positive and negative pressure induced by SWs were measured by a hydrophone showing…

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  Figure 2. SWT induces angiogenesis in a dose-dependent manner in vitro. (a) To evaluate proliferation, HUVECs were subjected to SWT at different energy levels and analyzed via DAPI staining after 24 h. (b) SWT induced proliferation of HUVECs in a dose-dependent manner, beginning at an energy level of 0.07 mJ/mm2. Proliferation of cardio myocytes was enhanced upon shockwave therapy. In contrast no proliferative effect could be observed in fibroblasts. Means ± SEM. *p < 0.05, **p < 0.01. n = 6. (c) The highest proliferative effect of SWT was observed upon therapy at 3 Hz. (d) SWT enhanced the mRNA expression of VEGF in a dose dependent-manner. Means ± SEM. SWT resulted in phosphorylation of angiogenic AKT and ERK 30 min and 1 h after SW treatment. **p < 0.01; ***p < 0.001. n = 6. (e) To assess whether SW effects depended on VEGF signaling, a functional angiogenesis assay, the tube formation assay was performed. Angiogenic effect of SWT abolished upon treatment with VEGFR2 inhibitor SU 1498. **p < 0.01; ****p < 0.0001. n = 6. Statistical comparisons between multiple groups: one-way ANOVA with Tukey post hoc analysis. Blots are displayed in cropped format.

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  Figure 3. SWT improves limb perfusion dose-dependently. (a, b) To evaluate dose–response relationship in vivo, mice were subjected to hind limb ischemia and treated with different SW doses thereafter. Treatment with 0.07 and 0.15 mJ/mm2 improved limb perfusion 4 weeks after treatment compared to untreated controls, whereas treatment with 0.01 mJ/mm2 showed no beneficial effect . Means ± SEM. **p < 0.01; ***p < 0.001. n = 5–10. (c) To asses necrosis upon SWT in vivo, limbs of mice were treated with SWT. Upon treatment with different levels of energy flux density, no signs of necrosis could be observed in pathological examination. Statistical comparisons between multiple groups: one-way ANOVA with Tukey post hoc analysis.

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  Figure 4. SWT induces angiogenesis in vivo. (a) To evaluate angiogenesis in ischemic muscles, we performed immunostaining of endothelial cells and vascular smooth muscle cells. Quantification showed increased numbers of capillaries and arterioles in ischemic limbs subjected to SWT. Means ± SEM. ****p < 0.0001. n = 5–10. (b) qPCR revealed increased gene expression of VEGF and VEGFR2 in ischemic muscle 3 days after SW treatment. Means ± SEM. *p < 0.05. N = 5–10. Statistical comparisons between two groups: Student’s t test.

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  Figure 5. Central picture. These data define for the first time a therapeutic range of SWT. We provide evidence for a dose-dependent induction of angiogenesis after SWT, as well as the absence of cellular damage upon SWT within the therapeutic range.