Extracellular RNA has been shown to induce vascular endothelial growth factor (VEGF)-dependent hyperpermeability in vivo as well as in vitro. Studies were performed to investigate the mechanism of these effects. For permeability studies primary cultures of porcine brain-derived microvascular endothelial cells (BMECs) and for all other analytical studies the human brain endothelial cell line HCMEC/D3 or human umbilical vein endothelial cells (HUVECs) were used. RNA, but not DNA, initiated signaling events by binding of VEGF to neuropilin-1, followed by VEGF-R2 phosphorylation, activation of phospholipase C (PLC), and intracellular release of Ca(2+). Activation of these pathways by RNA also resulted in the release of von Willebrand Factor from Weibel-Palade bodies. Pretreatment of cells with heparinase totally abrogated the RNA-induced permeability changes, whereas RNA together with VEGF completely restored VEGF-R2-mediated hyperpermeability. Although poly:IC increased the interleukin-6 release via activation of toll-like receptor-3 (TLR-3), permeability changes mediated by poly:IC or RNA remained unchanged after blocking TLR-3 or NF-kB activation. These results indicate that extracellular RNA serves an important cofactor function to engage VEGF for VEGF-R2-dependent signal transduction, reminiscent of the coreceptor mechanism mediated by proteoglycans, which might be of relevance for the mobilization and cellular activities of RNA-binding cytokines in general.