Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

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.

Long-Term Outcome of Bypass Surgery versus Endovascular Revascularization in Long Femoropopliteal Lesions.

Long-term follow-up data comparing surgical and endovascular revascularization of femoropopliteal lesions is rarely reported. This study presents 4-year results of revascularization for long femoropopliteal lesions (Trans-Atlantic Inter-Society Consensus Types C and D) with vein bypass (VBP), polytetrafluorethylene bypass (PTFE), and endovascular intervention with a nitinol stent (NS). Data from a randomized-controlled trial on VBP and NS was compared with a retrospective patient cohort using PTFE with the same inclusion and exclusion criteria. Primary, primary assisted, and secondary patency, as well as changes in Rutherford categories and limb salvage rates, are reported. Between 2016 and 2020, 332 femoropopliteal lesions underwent revascularization. The lesion lengths and basic patient characteristics were similar between the groups. 49% of the patients presented with chronic limb threatening ischemia at the time of revascularization. During the four-year follow-up, primary patency was comparable for all three groups. Primary assisted and secondary patency were significantly higher after VBP, while PTFE and NS had similar results. Clinical improvement was also significantly superior after VBP. After four years of follow-up, patency rates as well as the clinical outcome clearly favor VBP. If no vein is available, NS is as effective as PTFE bypass with regard to patency and clinical outcome.

[Establishment of a new research concept at a university location]. / Die Etablierung eines neuen Forschungskonzepts an einem universitären Standort.

Apart from daily clinical work and patient care, research and student teaching are essential assignments of a university hospital. The implementation of a completely new research laboratory at the Department of Vascular Surgery at the Medical University of Innsbruck led to the creation of a novel infrastructure for the design and set-up of a newly founded research focus. The cooperation with national as well as international research collaborators was essential for this process. The construction of a research network is not only important for the design and conception of a new research laboratory but also for the development of new study protocols and exchange and discussion of results. After the successful implementation of Experimental Vascular Surgery in Innsbruck, the realization of basic research projects as well as translational research projects is possible at this university location. Moreover, simulation training is an emerging field of research that aims to add more realistic surgical skills to residency programs, attract future residents and teach more complex novel techniques.

Neuronal Pre- and Postconditioning via Toll-like Receptor 3 Agonist or Extracorporeal Shock Wave Therapy as New Treatment Strategies for Spinal Cord Ischemia: An In Vitro Study.

Spinal cord ischemia (SCI) is a devastating and unpredictable complication of thoracoabdominal aortic repair. Postischemic Toll-like receptor 3 (TLR3) activation through either direct agonists or shock wave therapy (SWT) has been previously shown to ameliorate damage in SCI models. Whether the same applies for pre- or postconditioning remains unclear. In a model of cultured SHSY-5Y cells, preconditioning with either poly(I:C), a TLR3 agonist, or SWT was performed before induction of hypoxia, whereas postconditioning treatment was performed after termination of hypoxia. We measured cytokine expression via RT-PCR and utilized Western blot analysis for the analysis of signaling and apoptosis. TLR3 activation via poly(I:C) significantly reduced apoptotic markers in both pre- and postconditioning, the former yielding more favorable results through an additional suppression of TLR4 and its downstream signaling. On the contrary, SWT showed slightly more favorable effects in the setting of postconditioning with significantly reduced markers of apoptosis. Pre- and post-ischemic direct TLR3 activation as well as post-ischemic SWT can decrease apoptosis and proinflammatory cytokine expression significantly in vitro and might therefore pose possible new treatment strategies for ischemic spinal cord injury.

Cardiotoxic mechanisms of cancer immunotherapy - A systematic review.

Cancer immunotherapy is a success story of translational medicine that has led to improved survival in patients with different difficult-to-treat types of cancer, such as metastasized melanoma, non-small cell lung cancer or renal cell carcinoma. These novel therapeutic agents exert their antitumor effects by activating the patients' immune system against cancer cells. Immunotherapy can be divided into active agents, such as anti-tumour vaccines or adoptive T-cell transfer, and passive immunotherapies like monoclonal antibodies, checkpoint inhibitors, cytokine therapy, bispecific T-cell engagers. After initial experimental use, broad clinical application revealed a number of important cardiovascular side effects of immunotherapeutics, which limit treatment options and decrease patients' prognosis and quality of life. With the rising rate of new immunotherapeutics at a hand, the number of patients receiving cancer immunotherapy will constantly increase, resulting in improved long-term survival rates. This review aims to summarize available cancer immunotherapies, their mechanism of action, currently known cardiovascular toxicities and their treatment. Further optimization of patient care will depend on the combined efforts by oncologists, cardiologists and cardiac surgeons to identify patients at risk and the implementation of interdisciplinary screening and treatment strategies. It is therefore crucial to familiarize heart specialists with novel cancer therapeutics and their potential adverse effects.

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.

Safety and efficacy of direct Cardiac Shockwave Therapy in patients with ischemic cardiomyopathy undergoing coronary artery bypass grafting (the CAST-HF trial): study protocol for a randomized controlled trial.

BACKGROUND: Coronary artery diseases (CAD) remains a severe socio-economic burden in the Western world. Coronary obstruction and subsequent myocardial ischemia result in progressive replacement of contractile myocardium with dysfunctional, fibrotic scar tissue. Post-infarctional remodeling is causal for the concomitant decline of left-ventricular function and the fatal syndrome of heart failure. Available neurohumoral treatment strategies aim at the improvement of symptoms. Despite extensive research, therapeutic options for myocardial regeneration, including (stem)-cell therapy, gene therapy, cellular reprogramming or tissue engineering, remain purely experimental. Thus, there is an urgent clinical need for novel treatment options for inducing myocardial regeneration and improving left-ventricular function in ischemic cardiomyopathy. Shockwave Therapy (SWT) is a well-established regenerative tool that is effective for the treatment of chronic tendonitis, long-bone non-union and wound-healing disorders. In preclinical trials, SWT regenerated ischemic myocardium via the induction of angiogenesis and the reduction of fibrotic scar tissue, resulting in improved left-ventricular function. METHODS/DESIGN: In this prospective, randomized controlled, single-blind, monocentric study, 80 patients with reduced left-ventricular ejection fraction (LVEF≤ 40%) are subjected to coronary-artery bypass-graft surgery (CABG) surgery and randomized in a 1:1 ratio to receive additional cardiac SWT (intervention group; 40 patients) or CABG surgery with sham treatment (control group; 40 patients). This study aims to evaluate (1) the safety and (2) the efficacy of cardiac SWT as adjunctive treatment during CABG surgery for the regeneration of ischemic myocardium. The primary endpoints of the study represent (1) major cardiac events and (2) changes in left-ventricular function 12 months after treatment. Secondary endpoints include 6-min Walk Test distance, improvement of symptoms and assessment of quality of life. DISCUSSION: This study aims to investigate the safety and efficacy of cardiac SWT during CABG surgery for myocardial regeneration. The induction of angiogenesis, decrease of fibrotic scar tissue formation and, thus, improvement of left-ventricular function could lead to improved quality of life and prognosis for patients with ischemic heart failure. Thus, it could become the first clinically available treatment strategy for the regeneration of ischemic myocardium alleviating the socio-economic burden of heart failure. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03859466. Registered on 1 March 2019.

Shock waves promote spinal cord repair via TLR3.

Spinal cord injury (SCI) remains a devastating condition with poor prognosis and very limited treatment options. Affected patients are severely restricted in their daily activities. Shock wave therapy (SWT) has shown potent regenerative properties in bone fractures, wounds, and ischemic myocardium via activation of the innate immune receptor TLR3. Here, we report on the efficacy of SWT for regeneration of SCI. SWT improved motor function and decreased lesion size in WT but not Tlr3-/- mice via inhibition of neuronal degeneration and IL6-dependent recruitment and differentiation of neuronal progenitor cells. Both SWT and TLR3 stimulation enhanced neuronal sprouting and improved neuronal survival, even in human spinal cord cultures. We identified tlr3 as crucial enhancer of spinal cord regeneration in zebrafish. Our findings indicate that TLR3 signaling is involved in neuroprotection and spinal cord repair and suggest that TLR3 stimulation via SWT could become a potent regenerative treatment option.

miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.

AIMS: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. METHODS AND RESULTS: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. CONCLUSION: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Shock wave treatment after hindlimb ischaemia 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 © 2016 John Wiley & Sons, Ltd.

Shock Wave Therapy Improves Cardiac Function in a Model of Chronic Ischemic Heart Failure: Evidence for a Mechanism Involving VEGF Signaling and the Extracellular Matrix.

Background Mechanical stimulation of acute ischemic myocardium by shock wave therapy ( SWT ) is known to improve cardiac function by induction of angiogenesis. However, SWT in chronic heart failure is poorly understood. We aimed to study whether mechanical stimulation upon SWT improves heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis and to dissect underlying mechanisms. Methods and Results SWT was applied in a mouse model of chronic myocardial ischemia. To study effects of SWT on postnatal vasculogenesis, wild-type mice received bone marrow transplantation from green fluorescence protein donor mice. Underlying mechanisms were elucidated in vitro in endothelial cells and murine aortic rings. Echocardiography and pressure/volume measurements revealed improved left ventricular ejection fraction, myocardial contractility, and diastolic function and decreased myocardial fibrosis after treatment. Concomitantly, numbers of capillaries and arterioles were increased. SWT resulted in enhanced expression of the chemoattractant stromal cell-derived factor 1 in ischemic myocardium and serum. Treatment induced recruitment of bone marrow-derived endothelial cells to the site of injury. In vitro, SWT resulted in endothelial cell proliferation, enhanced survival, and capillary sprouting. The effects were vascular endothelial growth factor receptor 2 and heparan sulfate proteoglycan dependent. Conclusions SWT positively affects heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis. SWT upregulated pivotal angiogenic and vasculogenic factors in the myocardium in vivo and induced proliferative and anti-apoptotic effects on endothelial cells in vitro. Mechanistically, these effects depend on vascular endothelial growth factor signaling and heparan sulfate proteoglycans. SWT is a promising treatment option for regeneration of ischemic myocardium.

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-ß1 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.

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 © 2014 John Wiley & Sons, Ltd.

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.

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 1ß, 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.

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.