Formyl Peptide Receptor 1 Modulates Endothelial Cell Functions by NADPH Oxidase-Dependent VEGFR2 Transactivation.

Abstract

In the vasculature, NADPH oxidase is the main contributor of reactive oxygen species (ROS) which play a key role in endothelial signalling and functions. We demonstrate that ECV304 cells express p47phox, p67phox, and p22phox subunits of NADPH oxidase, as well as formyl peptide receptors 1 and 3 (FPR1/3), which are members of the GPCR family. By RT-PCR, we also detected Flt-1 and Flk-1/KDR in these cells. Stimulation of FPR1 by N-fMLP induces p47phox phosphorylation, which is the crucial event for NADPH oxidase-dependent superoxide production. Transphosphorylation of RTKs by GPCRs is a biological mechanism through which the information exchange is amplified throughout the cell. ROS act as signalling intermediates in the transactivation mechanism. We show that N-fMLP stimulation induces the phosphorylation of cytosolic Y951, Y996, and Y1175 residues of VEGFR2, which constitute the anchoring sites for signalling molecules. These, in turn, activate PI3K/Akt and PLC-γ1/PKC intracellular pathways. FPR1-induced ROS production plays a critical role in this cross-talk mechanism. In fact, inhibition of FPR1 and/or NADPH oxidase functions prevents VEGFR2 transactivation and the triggering of the downstream signalling cascades. N-fMLP stimulation also ameliorates cellular migration and capillary-like network formation ability of ECV304 cells.