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 doublestranded (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-b1 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 2/2 mice (n ¼ 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. -

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

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

VEGF-A protein expression and stimulation of VEGF receptor 2.

<p>A, Pivotal growth factor VEGF-A protein was measured 4 weeks after left femoral artery excision and shock wave treatment by western blot analysis of muscle samples. VEGF-A protein was significantly increased in the treatment group (SWT) compared to untreated controls (CTR). *p<.05. B, Profiler assays were used to investigate the role of receptor tyrosine kinase after shock wave treatment. Results indicate that shock waves stimulate phosphorylation of VEGF receptor 2 whereas there was no effect observed on VEGFR1 phosphorylation. Increase in VEGFR2 phosphorylation is nearly five-fold compared to untreated controls. C, Western blot of HUVECs under hypoxic conditions showing clearly higher protein levels of VEGF-A after shock wave treatment. D, Western blot of phoshpo-p44/42 MAPK (ERK1/2) shows that VEGF signaling is abolished in shock wave treated HUVECs that were pre-incubated with VEGF receptor 2 inhibitor Vandetanib. This finding indicates that shock wave effects are VEGFR2-dependent. E, Relative protein levels of VEGF 24 hours after shock wave treatment of human umbilical vein endothelial cells (HUVECs) as assessed by angiogenesis profiler. F, Relative protein levels of PlGF 24 hours after treatment of HUVECs.</p

Higher numbers of capillary sprouts from aortic rings in the treatment group.

<p>A, Representative pictures from light microscope of untreated (CTR) and shock wave treated (SWT) mouse aortic rings depicting higher numbers of sprouts in the treatment group 7 days following SWT. B, Quantification of sprouts revealed a significant increase in the treatment group, *p<.05, n = 6–8 per group. C, Immunofluorescence confirmed that sprouts consist of endothelial cells (red: lectin endothelial staining, blue: DAPI nucleus counterstaining).</p

Increase of angiogenic factors in shock wave treated ischemic muscle.

<p>Significantly elevated mRNA levels of angiogenic growth factors A, VEGF-A and B, PlGF 72 hours after shock wave treatment (SWT). In line with these findings their main receptors C, Flt-1 and D, KDR were significantly upregulated in the treatment group compared to untreated controls (CTR). *p<.05.</p

Increase of capillary and arteriole density in ischemic muscle.

<p>A, Representative views of immunofluorescence stainings for DAPI (cell nuclei) and CD31 (endothelial cells). Magnification x400; B, Increase of capillary density in treated ischemic muscle. Immunofluorescence staining for CD31-positive cells revealed significantly higher numbers of capillaries per high power field (HPF) 4 weeks after shock wave therapy (SWT). *p<.05. C, Representative views of immunofluorescence stainings for arterioles (CD31– endothelial cells, alpha-SMA – smooth muscle cells). Magnification x200; D, Quantification of arteriole staining revealed significantly more arterioles per HPF in shock wave treated muscle. *p<.05.</p

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/β 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 &gt;300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/β 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