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

Dina Vara, Joanna M Watt, Tiago Fortunato, Harry Mellor, Matthew Burgess, Kate Wicks, Kimberly Mace, Shaun Reeksting, Anneke Lubben, Caroline P D Wheeler-Jones, Giordano Pula

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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.

Original languageEnglish
Pages (from-to)110-130
Number of pages21
JournalAntioxidants & Redox Signaling
Volume28
Issue number2
Early online date18 Sep 2017
DOIs
Publication statusPublished - 10 Jan 2018

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Deoxyribose
NF-kappa B
NADPH Oxidase
Chemical activation
Phosphates
Vascular Endothelial Growth Factor Receptor
Up-Regulation
Endothelial cells
Gene Silencing
Small Interfering RNA
Assays
Endothelial Cells
Genes
2-deoxyribose 1-phosphate
Macrophages
Electron Spin Resonance Spectroscopy
Platelets
Immunoblotting
Sugars
Superoxides

Keywords

  • Cell Line
  • Endothelial Cells/drug effects
  • Humans
  • NADPH Oxidase 2/metabolism
  • NF-kappa B/metabolism
  • Neovascularization, Physiologic/drug effects
  • Oxidative Stress
  • Reactive Oxygen Species/metabolism
  • Ribosemonophosphates/pharmacology
  • Vascular Endothelial Growth Factor A/metabolism

Cite this

Direct activation of NADPH oxidase 2 by 2-deoxyribose-1-phosphate triggers nuclear factor kappa B-dependent angiogenesis. / Vara, Dina; Watt, Joanna M; Fortunato, Tiago; Mellor, Harry; Burgess, Matthew; Wicks, Kate; Mace, Kimberly; Reeksting, Shaun; Lubben, Anneke; Wheeler-Jones, Caroline P D; Pula, Giordano.

In: Antioxidants & Redox Signaling, Vol. 28, No. 2, 10.01.2018, p. 110-130.

Research output: Contribution to journalArticle

Vara, Dina ; Watt, Joanna M ; Fortunato, Tiago ; Mellor, Harry ; Burgess, Matthew ; Wicks, Kate ; Mace, Kimberly ; Reeksting, Shaun ; Lubben, Anneke ; Wheeler-Jones, Caroline P D ; Pula, Giordano. / Direct activation of NADPH oxidase 2 by 2-deoxyribose-1-phosphate triggers nuclear factor kappa B-dependent angiogenesis. In: Antioxidants & Redox Signaling. 2018 ; Vol. 28, No. 2. pp. 110-130.
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AU - Fortunato, Tiago

AU - Mellor, Harry

AU - Burgess, Matthew

AU - Wicks, Kate

AU - Mace, Kimberly

AU - Reeksting, Shaun

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AB - 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.

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KW - Ribosemonophosphates/pharmacology

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