Abstract 12518: Novel Interaction of Toll-Like Receptor 2/6 and the Vascular NADPH-Oxidase in Endothelial Cells
Background: Stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns leads to the activation of the NADPH-oxidase (NOX) in immune cells and the subsequently generated reactive oxygen species (ROS) are crucial for oxidative burst during pathogen defense. In contrast, NOX-dependent ROS-formation in vascular cells is crucial for intracellular signaling. However, little is known about an interaction of TLRs and NOX in endothelial cells and was therefore the aim of the present study.
Methods and Results: Human endothelial cells (HUVEC, HUAEC, HMVEC) were stimulated with the bacterial lipopeptid and TLR2/6 agonist MALP−2. We observed a fast ROS-induction (DCF-fluorescence, ESR-spectroscopy, P<0.05) and a strong gene expression and protein release of the granulocyte macrophage colony-stimulating factor (GM-CSF) (DNA/Protein-array, qRT-PCR, ELISA). MALP-2 injection (i.v.) not only increased GM-CSF plasma levels (ELISA, P<0.05) but also initiated progenitor cell mobilization (CFU, FACS, P<0.05). ROS scavenger (Tempol, Tiron) as well as NOX inhibitors (DPI, apocynin, VAS2870) completely blocked GM-CSF release from endothelial cells following TLR2/6 stimulation by MALP-2 (ELISA, P<0.01). Furthermore, explanted aortic rings stimulated by MALP-2 revealed significantly reduced GM-CSF release from mice deficient for the NOX subunit p47phox (P<0.05). Interestingly, inhibition of other radical-producing systems (NO-synthase, xanthine oxidase, cyclooxygenase, mitochondria) did not show any effect underlining the specific function of NOX in this context. However, neither NOX activators (PMA, angiotensin II, thrombin) nor supply of intracellular radicals (H2O2, menadione) alone were able to induce GM-CSF independent of TLR2/6 stimulation. Since plasma membrane topology is of great importance in endothelial cells it is noteworthy that inhibition of caveolae/lipid rafts (filipin III) as well as blockage of dynamin-dependent endocytosis (dynasore) prevented GM-CSF release (P<0.01) in response to TLR2/6 stimulation by MALP−2.
Conclusions: Our findings identify a novel interaction of a TLR2/6-dependent pathway with the NADPH-oxidase during the induction of the growth factor GM-CSF in endothelial cells.
- © 2010 by American Heart Association, Inc.