Direct activation of NADPH oxidase 2 by 2-deoxyribose-1-phosphate triggers nuclear factor kappa B-dependent angiogenesis

Abstract

Aims: Deoxyribose-1-phosphate (dRP) is a pro-angiogenic paracrine stimulus released by cancer cells, platelets and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance (EPR), pull-down of dRPinteracting proteins followed by immunoblotting, gene silencing of different NOXs and their regulatory co-subunits by siRNA transfection, and experiments with inhibitors of the sugar transporter GLUT1 were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NADPH oxidase 2 (NOX2) complex, but not NOX4. Increased reactive oxygen species (ROS) generation in response to NOX2 activity leads to redoxdependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces VEGF receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and the NOX2-dependent stimulation of angiogenesis by dRP was confirmed in excisional wound and matrigel plug vascularisation assays in vivo using NOX2-/- mice.

Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex.

Conclusions: This study describes a novel molecular mechanism underlying the pro-angiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and the upregulation of VEGFR2