Abstract 3601: Toll-Like Receptor-2/-6 Stimulation Promotes Angiogenesis and Reendothelialization
Introduction: Toll-like receptors (TLRs) are crucial for the recognition of pathogens which subsequently lead to the activation of inflammatory pathways. However, the mycoplasma lipopeptid and TLR-2/-6 ligand MALP-2 has been used in therapeutic applications, e.g. dermal wound healing. Since tissue regeneration requires the reestablishment of a functional vascular network we investigated the impact of the TLR-2/6 ligand MALP-2 on angiogenesis and reendothelialization.
Methods and results: Expression of TLR-2 and -6 in human endothelial cells was demonstrated by PCR, Western blot and immunofluorescence and significantly up regulated after stimulation with MALP-2 (100 ng/mL, qRT-PCR, Western blot). MALP-2 induced tube formation (Matrigel, P<0.05) and endothelial cell proliferation (BrdU-incorporation, P<0.01) in vitro which could be inhibited with neutralizing antibodies against TLR-2/-6. Furthermore, MALP-2 enhanced neovascularisation of matrigel implants in mice as determined by hemoglobin content (P<0.05) and H&E staining (P<0.05) which could again be inhibited with neutralizing antibodies against TLR-2/-6. In addition, MALP-2 accelerated reendothelialization of the carotid artery as measured by Evans blue staining after experimental injury of the endothelium (P<0.01). Protein array analysis revealed a remarkable MALP-2-dependent release of granulocyte/macrophage-colony stimulatory factor (GM-CSF) from ECs which was confirmed by ELISA (P<0.001). Likewise, MALP-2 induced GM-CSF release in murine aortic segments ex vivo and in the plasma of mice after i.v. injection (40μg/kg/bw). MALP-2-induced GM-CSF release and tube formation in vitro could be blocked with neutralizing antibodies against TLR-2/6 and with siRNA against GM-CSF. In addition, MALP-2 injection significantly induced colony forming units (MethoCult, P<0.01) and the mobilization of Sca-1+;Flk-1+ endothelial progenitor cells (FACS, P<0.05)
Conclusion: The TLR-2/-6 ligand MALP-2 exhibits highly regenerative properties. MALP-2 induces the release of GM-CSF as well as the mobilization of progenitor cells and promotes angiogenesis and reendothelialization.