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 proangiogenic 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, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor 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 NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization 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 proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130.

Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth

“…Overproduction of ROS by NOXs results in oxidant stress and tumor progression [24]. Tumoral NOXs display many functions in cancer including promoting proliferation, apoptosis resistance, angiogenesis, glycolysis, EMT and metastasis [25], [26], [27], [28]. NOXs may be closely involved in inflammatory cancer microenvironment wherein oxidant species act as positive signaling intermediates for tumor growth [29], however, the interaction between tumoral microenvironment and NOXs remains still largely undentified.…”

Exploration of 2-Deoxy-D-Ribose and 17β-Estradiol as Alternatives to Exogenous VEGF to Promote Angiogenesis in Tissue-Engineered Constructs

“…They found 2dDR induces angiogenesis by paracrine signalling. The 2dDR directly activates NADPH oxidase 2 (NOX2) which in turn triggers nuclear factor Kappa B (NF-B (VEGFR2) and vascular endothelial growth factor-dependent angiogenesis [71]. Irrespective of the mechanism of action and dose response, our initial results looking at the response to hydrogels releasing 2dDR were very encouraging.…”

Assessment of the Angiogenic Potential of 2-Deoxy-D-Ribose Using a Novel in vitro 3D Dynamic Model in Comparison With Established in vitro Assays

“…In the first proposed mechanism, researchers suggest the endogenous production of 2dDR by enzymatic degradation of thymidine to thymine promotes oxidative stress and consecutively stimulates the secretion of angiogenic factors such as VEGF and interleukin-8 (IL-8) which can be internalized by ECs and promote angiogenesis (Brown et al, 2000;Sengupta et al, 2003;Nakajima et al, 2012). In the second mechanism, which has been recently studied by Vara et al (2018), the 2dDR-1phosphate (2dDRP) is produced either internally by cells which express TP such as macrophages, platelets and cancer cells and then secrete this extracellularly, or TP may be released from injured cells to act on enzymatic degradation of thymidine and generation of 2dDR extracellularly. 2dDRP then can be taken up by ECs, and this then activates NADPH oxidase 2 (NOX2) which later acts on nuclear factor kappa B (NF-κB).…”