Abstract
Background: Toll-like receptors (TLRs) are known as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiological and pathological processes has been reported. Since many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the specific TLR2/6 agonist MALP-2, a common bacterial lipopeptide. Methods and Results: MALP-2 induced angiogenesis in vivo (Matrigel implants, hemoglobin content, H&E staining, P<0.01, n=4–8) and tube formation in vitro (Matrigel, P<0.05, n=3–5) which could be inhibited with neutralizing antibodies against TLR2/6. Moreover, endothelial cells responded to MALP-2 with enhanced proliferation and migration (BrdU-incorporation, transwell, P<0.01, n=5–7). Protein array revealed a strong secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) from endothelial cells which was confirmed by ELISA (P<0.01, n=4) and could be inhibited with antibodies against TLR2/6. By contrast, smooth muscle cells and fibroblasts did not secrete GM-CSF in response to MALP-2 (ELISA, n=3–6). In addition, GM-CSF release from isolated vascular segments following MALP-2 stimulation was completely prevented when the endothelium was removed mechanically (ELISA, P<0.01, n=4). Capillaries generated by MALP-2 treatment contained erythrocytes as well as CD45 + cells (Matrigel implants, CD45 immunostaining, n=4). Following MALP-2 stimulation CD45 + cells showed likewise enhanced migration (transwell, P<0.05, n=5) and GM-CSF release (ELISA, P<0.01, n=5) however to a lower extent compared to endothelial cells. Finally, inhibition of GM-CSF by siRNA in vitro (Matrigel, P<0.05, n=4) or by antibodies in vivo (Matrigel implants, P<0.01, n=4–8) inhibited the MALP-2 induced angiogenesis. Conclusion: These results demonstrate a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2 which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.
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