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Low Energy Shock Wave Therapy Induces Angiogenesis in Acute Hind-Limb Ischemia via VEGF Receptor 2 Phosphorylation

Objectives Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. Methods Hind-limb ischemia was induced in 10–12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery. Results Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. Conclusions Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.

Epicardial shock-wave therapy improves ventricular function in a porcine model of ischaemic heart disease.

Previously we have shown that epicardial shock‐wave therapy improves left ventricular ejection fraction (LVEF) in a rat model of myocardial infarction. In the present experiments we aimed to address the safety and efficacy of epicardial shock‐wave therapy in a preclinical large animal model and to further evaluate mechanisms of action of this novel therapy. Four weeks after left anterior descending (LAD) artery ligation in pigs, the animals underwent re‐thoracotomy with (shock‐wave group, n = 6) or without (control group, n = 5) epicardial shock waves (300 impulses at 0.38 mJ/mm2) applied to the infarcted anterior wall. Efficacy endpoints were improvement of LVEF and induction of angiogenesis 6 weeks after shock‐wave therapy. Safety endpoints were haemodynamic stability during treatment and myocardial damage. Four weeks after LAD ligation, LVEF decreased in both the shock‐wave (43 ± 3%, p < 0.001) and control (41 ± 4%, p = 0.012) groups. LVEF markedly improved in shock‐wave animals 6 weeks after treatment (62 ± 9%, p = 0.006); no improvement was observed in controls (41 ± 4%, p = 0.36), yielding a significant difference. Quantitative histology revealed significant angiogenesis 6 weeks after treatment (controls 2 ± 0.4 arterioles/high‐power field vs treatment group 9 ± 3; p = 0.004). No acute or chronic adverse effects were observed. As a potential mechanism of action in vitro experiments showed stimulation of VEGF receptors after shock‐wave treatment in human coronary artery endothelial cells. Epicardial shock‐wave treatment in a large animal model of ischaemic heart failure exerted a positive effect on LVEF improvement and did not show any adverse effects. Angiogenesis was induced by stimulation of VEGF receptors. Copyright

Toll-like receptor 3 signalling mediates angiogenic response upon shock wave treatment of ischaemic muscle

AIMS Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding double-stranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. METHODS AND RESULTS SWT induces TLR3 and IFN-β1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3(-/-) mice (n = 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. CONCLUSION Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT.

Alteration of inflammatory response by shock wave therapy leads to reduced calcification of decellularized aortic xenografts in mice

OBJECTIVES Tissue-engineered xenografts represent a promising treatment option in heart valve disease. However, inflammatory response leading to graft failure and incomplete in vitro repopulation with recipient cells remain challenging. Shock waves (SWs) were shown to modulate inflammation and to enhance re-epithelialization. We therefore aimed to investigate whether SWs could serve as a feasible adjunct to tissue engineering. METHODS Porcine aortic pieces were decellularized using sodium deoxycholate and sodium dodecylsulphate and implanted subcutaneously into C57BL/6 mice (n = 6 per group). The treatment (shock wave therapy, SWT) group received SWs (0.1 mJ/mm(2), 500 impulses, 5 Hz) for modulation of inflammatory response directly after implantation; control animals remained untreated (CTR). Grafts were harvested 72 h and 3 weeks after implantation and analysed for inflammatory cytokines, macrophage infiltration and polarization, osteoclastic activity and calcification. Transmission electron microscopy (TEM) was performed. Endothelial cells (ECs) were treated with SWs and analysed for macrophage regulatory cytokines. In an ex vivo experimental set-up, decellularized porcine aortic valve conduits were reseeded with ECs with and without SWT (0.1 mJ/mm(2), 300 impulses, 3 Hz), fibroblasts as well as peripheral blood mononuclear cells (all human) and tested in a pulsatile flow perfusion system for cell coverage. RESULTS Treated ECs showed an increase of macrophage migration inhibitory factor and macrophage inflammatory protein 1β, whereas CD40 ligand and complement component C5/C5a were decreased. Subcutaneously implanted grafts showed increased mRNA levels of tumour necrosis factor α and interleukin 6 in the treatment group. Enhanced repopulation with recipient cells could be observed after SWT. Augmented macrophage infiltration and increased polarization towards M2 macrophages was observed in treated animals. Enhanced recruitment of osteoclastic cells in proximity to calcified tissue was found after SWT. Consequently, SWT resulted in decreased areas of calcification in treated animals. The reseeding experiment revealed that fibroblasts showed the best coverage compared with other cell types. Moreover, SW-treated ECs exhibited enhanced repopulation compared with untreated controls. CONCLUSIONS SWs reduce the calcification of subcutaneously implanted decellularized xenografts via the modulation of the acute macrophage-mediated inflammatory response and improves the in vitro repopulation of decellularized grafts. It may therefore serve as a feasible adjunct to heart valve tissue engineering.

Vein graft thrombi, a niche for smooth muscle cell colonization - a hypothesis to explain the asymmetry of intimal hyperplasia.

Essentials Vein graft failure is the most frequent late onset complication of coronary artery bypass grafting. Cuff technique‐based interposition mouse model including new anticoagulation regime was conducted. Early vein graft thrombi may serve as a niche for smooth muscle cell colonization. The focal character of early thrombi may form the basis for the asymmetry of intimal hyperplasia.

Combined peri-ischemic administration of Bβ15-42 in treating ischemia reperfusion injury of the mouse kidney.

The disruption of endothelial integrity is a crucial step for the development of vascular leakage and consequently ischemia-reperfusion injury (IRI). Regarding the molecular cell-cell interaction, the fibrinopeptide Bβ15-42 prevents vascular leakage by stabilizing the inter-endothelial junctions via association with the vascular endothelial-cadherin. In a previous study we showed that a renoprotective effect in early IRI may be achieved by intravenous administration of Bβ15-42 at the time of reperfusion. We now aimed to investigate whether additional pre-ischemic application of Bβ15-42 could enhance this effect. Therefore C57BL/6 mice were subjected to 0.5h bilateral renal ischemia followed by reperfusion. The animals were randomized into 6 groups (n=6): two control groups treated with i.v. administration of NaCl at reperfusion for 0.5h (NaCl 1h) and 2.5h (NaCl 3h), two groups with Bβ15-42 at reperfusion for 0.5h (Bβ(rep) 1h) and 2.5h (Bβ(rep) 3h), and two groups with administration of Bβ15-42 immediately pre-ischemic as well as at reperfusion for 0.5h (Bβ(peri) 1h) and 2.5h (Bβ(peri) 3h). We found that both Bβ(rep) and Bβ(peri) mice displayed reduced early renal damage compared with NaCl treated mice. However, there was no further reduction of the IR damage through added pre-ischemic application of Bβ15-42. Overall, we detected significantly reduced endothelial activation, lower tissue infiltration of neutrophils as well as lower tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) in all mice treated with Bβ15-42 compared to mice treated with NaCl. Our data confirm the renoprotective effect of Bβ15-42 in the early therapeutic treatment of acute kidney injury due to ischemia and reperfusion. However, a combined pre-and post-ischemic administration of Bβ15-42 appears to provide no additional benefit compared with a sole administration at reperfusion.

Shock Wave Treatment Protects From Neuronal Degeneration via a Toll‐Like Receptor 3 Dependent Mechanism: Implications of a First‐Ever Causal Treatment for Ischemic Spinal Cord Injury

Background Paraplegia following spinal cord ischemia represents a devastating complication of both aortic surgery and endovascular aortic repair. Shock wave treatment was shown to induce angiogenesis and regeneration in ischemic tissue by modulation of early inflammatory response via Toll‐like receptor (TLR) 3 signaling. In preclinical and clinical studies, shock wave treatment had a favorable effect on ischemic myocardium. We hypothesized that shock wave treatment also may have a beneficial effect on spinal cord ischemia. Methods and Results A spinal cord ischemia model in mice and spinal slice cultures ex vivo were performed. Treatment groups received immediate shock wave therapy, which resulted in decreased neuronal degeneration and improved motor function. In spinal slice cultures, the activation of TLR3 could be observed. Shock wave effects were abolished in spinal slice cultures from TLR3−/− mice, whereas the effect was still present in TLR4−/− mice. TLR4 protein was found to be downregulated parallel to TLR3 signaling. Shock wave–treated animals showed significantly better functional outcome and survival. The protective effect on neurons could be reproduced in human spinal slices. Conclusions Shock wave treatment protects from neuronal degeneration via TLR3 signaling and subsequent TLR4 downregulation. Consequently, it represents a promising treatment option for the devastating complication of spinal cord ischemia after aortic repair.

Shock wave treatment after hindlimb ischemia results in increased perfusion and M2 macrophage presence.

Shock wave therapy (SWT) has been shown to induce angiogenesis in ischaemic muscle. However, the mechanism of action remains unknown. Macrophages are crucial for angiogenic responses after ischaemic injury. The M2 macrophage subset enables tissue repair and induces angiogenesis. It was hypothesized that the angiogenic effects of SWT are at least partly caused by enhanced macrophage recruitment. C57BL/6 mice were subjected to hind limb ischaemia with subsequent SWT or sham treatment. Muscles were analysed via immunofluorescence staining, reverse‐transcription polymerase chain reaction and western blot. Gene expression and proteins involved in macrophage recruitment were analysed and tissue sections were stained for macrophages, including subsets, capillaries and arterioles. Laser Doppler perfusion imaging was performed to assess functional outcome. Treated muscles showed increased expression of the pivotal macrophage recruiting factor monocyte chemotactic protein 1 (MCP‐1). Higher levels of macrophage marker CD14 were found. Increased numbers of macrophages after SWT could be confirmed by immunofluorescence staining. The expression of the M2 polarization promoting chemokine interleukin 13 was significantly elevated in the treatment group. Elevated mRNA expression of the M2 scavenger receptor CD163 was found after SWT. Immunofluorescence staining confirmed increased numbers of M2 macrophages after treatment. It was found that SWT resulted in higher number of capillaries and arterioles. Assessment of functional outcome revealed significantly improved limb perfusion in treated animals. Shock wave therapy causes increased macrophage recruitment and enhanced polarization towards reparative M2 macrophages in ischaemic muscle resulting in angiogenesis and improved limb perfusion and therefore represents a promising new treatment option for the treatment of ischaemic heart disease. Copyright

Shockwave therapy of the heart

Ischemic heart disease represents a collective term for a continuous disease pathophysiology, ranging from acute myocardial infarction to congestive and chronic heart failure. According to the World Health Organization (WHO) and its Global Burden of Disease study 2010 ischemic heart disease represents the most common cause of death and disability-adjusted life years (DALY) worldwide [1]. Due to demographic changes in developed countries with an increase in life expectancy and a changing, more Western lifestyle in threshold countries, the number of patients suffering from ischemic heart disease is expected to rise dramatically in the future, presenting a major challenge for health care systems [1].

Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes

Shock wave treatment (SWT) was shown to induce regeneration of ischaemic myocardium via Toll‐like receptor 3 (TLR3). The antimicrobial peptide LL37 gets released by mechanical stress and is known to form complexes with nucleic acids thus activating Toll‐like receptors. We suggested that SWT in the acute setting prevents from the development of heart failure via RNA/protein release. Myocardial infarction in mice was induced followed by subsequent SWT. Heart function was assessed 4 weeks later via transthoracic echocardiography and pressure–volume measurements. Human umbilical vein endothelial cells (HUVECs) were treated with SWT in the presence of RNase and proteinase and analysed for proliferation, tube formation and LL37 expression. RNA release and uptake after SWT was evaluated. We found significantly improved cardiac function after SWT. SWT resulted in significantly higher numbers of capillaries and arterioles and less left ventricular fibrosis. Supernatants of treated cells activated TLR3 reporter cells. Analysis of the supernatant revealed increased RNA levels. The effect could not be abolished by pre‐treatment of the supernatant with RNase, but only by a sequential digestion with proteinase and RNase hinting strongly towards the involvement of RNA/protein complexes. Indeed, LL37 expression as well as cellular RNA uptake were significantly increased after SWT. We show for the first time that SWT prevents from left ventricular remodelling and cardiac dysfunction via RNA/protein complex release and subsequent induction of angiogenesis. It might therefore develop a potent regenerative treatment alternative for ischaemic heart disease.