Background: Low energy shock waves (SW) have been shown to induce angiogenesis in ischemic myocardium. The mechanism translating the physical stimulus to a biological signal is unknown. Toll-like receptor (TLR)-3 is activated by RNA binding. It plays a key role in inflammation and angiogenesis. We therefore hypothesized that SW cause cellular cavitation, thus liberating cytoplasmic mRNA that activates TLR-3 as does the specific agonist Poly I:C. Effects are suppressed in TLR-3 silenced cells and in TLR-3 knock out mice.
Methods: The effect of SW was tested in human umbilical vein endothelial cells (HUVECs): untreated (control) vs. SW treated (SW group) vs. treated with 200 µg/ml Poly I:C (agonist). TLR-3 gene silencing was done with siRNA. Hind limb ischemia was performed in wild type and TLR-3 kock-out mice. Expression of mRNA and proteins of the TLR-3 signaling pathway as well as typical angiogenic genes and proteins were measured. Laser Doppler perfusion imaging and necrosis score were assessed for clinical outcome evaluation (n=6).
Results: Shock wave treatment of HUVECS shows increase of mRNA expression (% of control) as does Poly I:C after 2 hours: TLR-3 (SW group 123.8 ± 8.0 and agonist group 237.7 ± 14.1, p<0.0001), Tie-2 (SW group 154.3 ± 20.0 and agonist 125.7 ± 12.3, p<0.008). TLR-3 gene silencing in SW treated HUVECS causes loss of response for TLR-3 mRNA (107.0 ± 13.3) as compared to SW group (378.3 ± 14.2) or agonist (1261 ± 72.1), both p<0.0001.SW treated TLR-3 knock-out mice showed no improvement of perfusion ratio 4 weeks after hind limb ischemia (0.52 ± 0.07 vs. 0.53 ± 0.02 controls, p>0.05), whereas SW treated wild type animals improved significantly (0.78 ± 0.03 vs. 0.48 ± 0.08 controls, p=0.015). Pro angiogenic genes and proteins were up-regulated significantly. All known TLR-3 signaling pathways were involved as shown by significant increase of key proteins Trif, TRAF6 and IRF3.
Conclusion: Low energy shock waves induce angiogenesis in ischemic muscle by stimulation of Toll-like receptor 3 signaling in endothelial cells. Effects are suppressed in TLR-3 silenced cells and in TLR-3 knock-out mice.
Johannes Holfeld1, Can Tepeköylü1, Radoslaw Kozaryn1, Karin Albrecht-Schgoer², Christoph Krapf1, Rudolf Kirchmair2, Kai Zacharowski3, Anja Urbschat4, Michael Grimm1 and Patrick Paulus3
1Department of Cardiac Surgery. Innsbruck Medical University, Innsbruck, Austria 2Department of Internal Medicine I, Innsbruck Medical University, Innsbruck, Austria 3Clinic of Anesthesiology, Intensive Care Medicine and Pain Therapy, Goethe-University Hospital Frankfurt, Frankfurt am Main, Germany 4Clinic of Urology. Goethe-University Hospital Frankfurt am Main, Germany
Holfeld et al. 4th ISMST Basic Research Meeting in Vienna, Austria
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