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

Shockwave therapy (SWT) represents a promising regenerative treatment option for patients with ischemic cardiomyopathy. Although no side-effects have been described upon SWT, potential cellular damage at therapeutic energies has not been addressed so far. In this work, we aimed to define a therapeutic range for shock wave application for myocardial regeneration. We could demonstrate that SWT does not induce cellular damage beneath energy levels of 0.27 mJ/mm2 total flux density. Endothelial cell proliferation, angiogenic gene expression and phosphorylation of AKT and ERK are enhanced in a dose dependent manner until 0.15 mJ/mm2 energy flux density. SWT induces regeneration of ischemic muscle in vivo via expression of angiogenic gene expression, enhanced neovascularization and improved limb perfusion in a dose-dependent manner. Therefore, 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. These data define for the first time a therapeutic range of SWT, a promising regenerative treatment option for ischemic cardiomyopathy.


Scientific Reports
| (2021) 11:409 | https://doi.org/10.1038/s41598-020-79776-z www.nature.com/scientificreports/ effects on bone non-unions, chronic tendonitis (e.g. tennis-elbow) and diabetic wound healing disorders are well described and established in clinical routine 12,13 . The observed effects are mainly attributed to the induction of angiogenesis [14][15][16] . Our group could show recently that the mechanical stimulus of SWs cause release of (a) angiogenic growth factors from the extracellular matrix 17 and (b) specific extracellular vesicles containing angiogenic cargo 18 . Concomitant stimulation of inflammation enhances the angiogenic response 19 .
Over the past years, we showed a strong angiogenic effect of SWT improving left ventricular function in ischemic cardiomyopathy in small and large animal models. Treatment caused neovascularization and resulted in reduction of myocardial scar 17,20 . Thus, SWT emerged as promising treatment strategy for ischemic cardiomyopathy. As a consequence, we initiated a prospective-randomized clinical trial, the CAST (safety and efficacy of direct Cardiac Shockwave Therapy in patients with ischemic cardiomyopathy undergoing coronary artery bypass grafting) trial (ClinicalTrials.gov Identifier: NCT03859466). The trial is currently in the recruitment phase.
Despite the promising effects of SWT and their impact on the treatment of IHD, the biophysical principles behind the therapy are not well understood. Although no side-effects have been described upon SWT, potential cellular damage at angiogenic acting energy levels was never determined in studies. Dose-response studies regarding SWT are missing so far. Thus, it remains uncertain whether the underlying mechanism of angiogenesis upon shock-wave therapy is based on a "damage-repair" principle. For this purpose, we aimed to define a therapeutic range for SW application in vitro and in vivo.

Results
Shock wave therapy inflicts no cellular damage at therapeutic energies. Shock waves destroy kidney stones at high energy levels, but regenerate tissue at low energy levels [11][12][13] .To evaluate whether SWT induces cellular necrosis at commonly used energy levels, we subjected human endothelial cells to different energy levels of SWT in T25 flasks. Release of LDH was measured 1 h after treatment to evaluate necrosis. No signs of necrosis were detected upon SWT at energy flux density levels of 0.01 mJ/mm 2 , 0.07 mJ/mm 2 and 0.15 mJ/mm 2 (Fig. 1a). However, we found evidence of necrosis at 0.27 mJ/mm 2 .
Shock waves are physical pressure waves, their reflection can result either in positive interference amplifying the energy of the primary wave, or cause negative interference, extinguishing the wave. Like ultrasound waves, shock waves are reflected whenever the wave encounters a material with a different density (acoustical impedance) 21 .
Thus, reflection and potential consecutive interference depends on the size and form of the cell culture flask. 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, we repeated the experiment using different common cell culture flasks and well plates (Fig. 1b). In contrast to the first experiment, we could observe necrosis already at a level as low as 0.15 mJ/ mm 2 in plates with a surface smaller than 9.5 cm 2 (12, 24 and 96 well plates) (Fig. 1c). In contrast, in 6 well plates and T25 flasks necrosis only occurred at 0.27 mJ/mm 2 . These experiments suggest phenomena of constructive interference after SWT in smaller well plates. Cardiomyocytes and fibroblast inevitable receive SWT during direct epicardial application to the heart. To evaluate whether SWT induces necrosis in other cardiac domestic cells, we subjected cardio myocytes and cardiac fibroblasts to SWT. However, we could not observe any differences compared to human endothelial cells using therapeutic energy levels (Fig. 1d).
Positive pressure induces cellular damage. Within the targeted tissue, shock waves induce alternating positive and negative pressure due to their wave profile 22 . However, whether the positive squeezing or the negative pulling acting pressure induces the described effects remains unknown. Therefore, we evaluated pressure levels at different energy levels utilizing a hydrophone (Fig. 1e). The positive pressure increased concomitant with the total energy flux density and necrosis (Fig. 1f). The measured necrosis was thereby induced by the positive pressure of the SW, since negative peak pressure of SW treatment increased in a linear matter with corresponding energy levels, until reaching a plateau at 3.8 MPa at an energy level of 0.11 mJ/mm 2 (Fig. 1g). For all further experiments energy levels within the therapeutiv range were used.

SWT induces dose-dependent angiogenesis in vitro.
In a next set of experiments, we aimed to evaluate whether angiogenesis upon SWT was dose dependent. Proliferation of endothelial cells is crucial for the sprouting of newly formed vessels 23 . Therefore, human endothelial cells were subjected to SWT and analyzed for proliferation. We could demonstrate that beginning at an energy level of 0.07 mJ/mm 2 SWT induced dosedependent proliferation compared to untreated controls in human endothelial cells (Fig. 2a,b). In contrast, human fibroblasts showed no proliferation upon therapy. However, proliferation was enhanced after SWT in a human cardiomyocyte cell line at an energy flux density of 0.07 mJ/mm 2 (Fig. 2b). Moreover, effects depended on frequency, as SW treatment with 3 Hz induced proliferation of endothelial cells, whereas the effect was markedly reduced with 1 or 5 Hz (Fig. 2c). In line with these findings, we found a dose dependent increase of angiogenic Vascular Endothelial Growth Factor (VEGF) expression after SW treatment (Fig. 2d). VEGF activates its receptor VEGFR2, followed by phosphorylation of the kinases AKT and ERK both of which induce angiogenesis 24,25 . To evaluate angiogenic signaling upon SWT, we analyzed phosphorylation of both AKT and ERK after SWT. SWT induced phosphorylation of AKT and ERK 30 min and 1 h after SW treatment. Interestingly, although phosphorylation of AKT was independent of SW dose, activation of ERK was dose-dependent (Fig. 2d). These findings indicate a dose dependent angiogenic and proliferative effect of SWT. To assess whether SW effects depended on VEGF signaling, we performed a tube formation assay in the presence of the VEGFR2 inhibitor SU1498. Angiogenesis upon SWT was abolished upon pretreatment with VEGFR2 indicating a crucial role of the VEGF axis in the angiogenic response upon SWT (Fig. 2e).

Scientific Reports
| (2021) 11:409 | https://doi.org/10.1038/s41598-020-79776-z www.nature.com/scientificreports/ SWT improves limb perfusion. To assess dose-dependency in vivo, we performed SWT in a murine model of hind limb ischemia. We found improved limb perfusion compared to untreated mice 4 weeks after SW treatment (Fig. 3a). As anticipated from our in vitro findings, energy levels of 0.07 and 0.15 mJ/mm 2 had the most beneficial effects on hind limb regeneration (Fig. 3b). To evaluate possible cytotoxic effects in vivo, SW treated muscle samples underwent pathological examination. There was no evidence of necrosis or cellular damage, independent of dose ( Fig. 3c).

SWT induces angiogenesis in vivo.
To assess whether improved limb perfusion was due to angiogenesis we stained ischemic muscles for newly formed vessels. We found an increase of capillaries as well as arterioles in mice treated at an energy level of 0.07 mJ/mm 2 in comparison to untreated animals (Fig. 4a). In parallel, gene expression levels of VEGF and its receptor VEGFR2 were increased after SWT (Fig. 4b).

Discussion
The prevalence of ischemic heart failure rises due to constantly aging population. Frequent rehospitalizations and incapacity to work cause a severe socio-economic burden for Western health care systems 4-6 . Despite modern pharmacotherapy, ischemic heart failure is still associated with poor outcomes. Current treatments aim at symptom control rather than myocardial regeneration. Novel therapies as gene or stem cell therapy showed promising results in pre-clinical experiments. However, they have failed to gain routine clinical application due to unfavorable side effect profiles and ethical concerns [8][9][10] . Therefore, there is major need in novel and safe therapy for patients suffering from ischemic cardiomyopathy. Shockwave therapy has been used in clinical routine in various indications for more than 30 years without showing any form of side effects 11 . Beneficial effects of SWT in ischemic heart failure via induction of angiogenesis were shown before 18,20,[26][27][28] . On the other hand, SWT has been used at much higher energy levels for the destruction of kidney stones. Hence, depending on the used energy level, SW can both induce tissue regeneration as well as stone disintegration. However, the dose-response profile of SWT has not been defined so far.
In this work, we could define for the first time a therapeutic range of SWT. Within the therapeutic range, SWT did not cause cellular damage. Furthermore, we could show that necrosis beyond energy levels of 0.27 mJ/ mm 2 is attributed to positive pressure, since negative pressure released by SW plateaus already at an energy level of 0.11 mJ/mm 2 .
Other cellular responses to increased pressure upon SWT, as the formation of caveolae, have been described previously 29 . However, earlier works performed experiments only at one single level of energy flux density.
Since a therapeutic range for SWT has not been defined yet, we subject endothelial cells to SWT at different energy levels. We could demonstrate that the proliferative effect of SWT is dose dependent and plateaus at an energy level of 0.07 mJ/mm 2 . Concomitant with these findings we could observe a dose depended increase of angiogenic gene expression with subsequent phosphorylation of protein kinases AKT and ERK upon SWT. Therefore we could demonstrate in vitro an extended therapeutic range of the SWT, since experiments in earlier works were only performed at an energy flux density of 0.08 mJ/mm 219, 26 . www.nature.com/scientificreports/ In a murine model of hind limb ischemia we investigated the effects of SW treatment on ischemic tissue. As anticipated from earlier in vitro findings, we could show again a dose dependent increase in limb perfusion in animals subjected to SWT. Immunofluorescence staining revealed an increased amount of capillaries and arterioles in ischemic muscles after SWT. Combined with increased angiogenic gene expression in vivo after SWT, these findings underline the angiogenic effect of SWT. However, these experiments show a dose-response profile in soft tissue only. Whether the beneficial effects of SWT on bone non-unions is in the same therapeutic range has to be demonstrated in future works 12,13 . We found no damage after SWT in muscle tissue, previous studies found no evidence of cellular damage after SWT of ischemic myocardium 17,20 .
Overall, we provide evidence for a dose-dependent induction of angiogenesis after SWT (Fig. 5). VEGF signaling resulted in endothelial proliferation and neovascularization improving limb perfusion of ischemic muscle. We were able to define a therapeutic range of SWT from 0.07 to 0.15 mJ/mm 2 . Moreover, we could show the absence of necrosis upon SWT within the therapeutic range. Hence, the angiogenic properties are not attributed to a "damage-repair" mechanism, but rather due to a specific induction of angiogenic signaling pathways. Combined the results of this study and earlier works, suggest the SWT as a novel and side effect free treatment option for ischemic heart failure 17,18,20 . Translating our findings to a clinical setting, the currently recruiting CAST trial (ClinicalTrials.gov Identifier: NCT03859466) investigates the safety and efficacy of cardiac SWT during CABG surgery for myocardial regeneration.

Materials and methods
Cell culture. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords obtained by Caeseream sections. Informed consent was obtained from all participants and/or their legal guardians. Approval of this study protocol was given from the ethics committee of Innsbruck Medical University (no. UN4435) and complied to the Declaration of Helsinki. Isolation of endothelial cells was performed as described in detail before 19 . HUVECs were grown in endothelial growth medium (EGM) (Lonza, Basel, Switzerland) and cultured as described previously. Fibroblasts and cardio myocytes were purchased from Promo Cell (Heidelberg, Germany) and cultured in DMEM containing 10% FCS (Lonza, Basel, Switzerland) and Myocyte basal medium (Pan Bio-Tech, Aidenbach, Germany) respectively. Cells were used until passage 5.
Shock wave therapy. Electrohydraulic generated shockwave treatment was applied as described previously 15 . The Orthogold 180 device with applicator CE50 (MTS Europe GmbH, Konstanz, Germany) was used for all treatments. Respectively cells or animals were treated with 300 impulses with an energy flux density of 0.01, 0.07, 0.15 and 0.27 mJ/mm 2 at frequencies ranging from 1 to 5 Hz. Common ultrasonic gel Skintact (Leonhard Lang, Innsbruck, Austria) was used for coupling. Necrosis assay. Thermo Scientific Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific Waltham, MA) was used for quantitatively measure for cellular cytotoxicity and cytolysis. HUVECs, fibroblasts and cardio myocytes were seeded in T25 flasks, 96, 24, 12 and 6 well plates and cultivated in cell specific media (EGM (Lonza, 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.  18 . Briefly, 12-15 weeks old male C57/BL6 mice (Charles River, Sulzfeld, Germany) were administered to anesthesia via an intraperitoneal injection of ketamine hydrochloride (Ketanest, Graeub, Switzerland, 80 mg/kg body weight) and xylazine hydrochloride (Xylasol, ani-Medica, Germany, 5 mg/kg body weight). Popliteal artery and femoral artery proximal to the branching into saphenous were ligated with 7-0 polypropylene sutures (Ethicon, USA) and femoral artery was excited subsequently. Limb perfusion was measured using a laser Doppler perfusion image analyser (Moor Instruments, USA). Therefore, animals were kept on a 37 °C tempered heating plate. Limb perfusion was calculated as ration of left (operated, ischemic limb) to right (not operated, non-ischemic limb). For elevation of necrosis upon SWT in vivo, mice were administered to anesthesia as described above. Limbs were treated with SWT subsequently. 15 min after SW application skeletal muscle was harvested for histological processing.
Immunofluorescence staining. Immunofluorescence staining was performed as described previously to analyze number of vessels 19 . Histological sections of the heart were incubated with monoclonal rat anti-CD31 (Dianova, Hamburg, Germany) or rabbit polyclonal anti-alpha smooth muscle actin antibodies (Abcam,Cambridge, UK) over night at 4 °C. For WGA staining histological sections were incubated at room temperature with WGA (Invitrogen, Carlsbad, CA) for 15 min. Five areas per sample were analyzed. Sections were examined with a Zeiss Axioplan 2 (Zeiss, Oberkochen, Germany) and a Leica SP5 confocal microscope (Leica, Wetzlar, Germany). Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) and processed with Adobe Photoshop CS5.1 for Mac (Adobe Systems Inc., San Jose, CA, USA) 30 .
Statistic. Graphs are presented as dot plots. Experiments illustrating a time course or dose response are represented as line graphs. All results are expressed as mean + SEM. Statistical comparisons between two groups were Scientific Reports | (2021) 11:409 | https://doi.org/10.1038/s41598-020-79776-z www.nature.com/scientificreports/ performed by Student's t. Multiple groups were analyzed by one-way ANOVA with Tukey post hoc analysis to determine statistical significance. Probability values < 0.05 were considered statistically significant.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.