ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis

Am J Physiol Cell Physiol. 2017 Jun 1;312(6):C749-C764. doi: 10.1152/ajpcell.00346.2016. Epub 2017 Apr 19.

Abstract

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H2O2) or overexpression of Nox4, which produces H2O2, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H2O2 to promote mtROS production. Mechanistically, H2O2 increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H2O2 in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

Keywords: NADPH oxidase; angiogenesis; mitochondria; reactive oxygen species; vascular endothelial growth factor.

MeSH terms

  • Biosensing Techniques
  • Catalase / genetics
  • Catalase / metabolism
  • Cell Movement / drug effects
  • Cell Proliferation / drug effects
  • Feedback, Physiological*
  • Gene Expression Regulation
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Membrane Glycoproteins / antagonists & inhibitors
  • Membrane Glycoproteins / genetics*
  • Membrane Glycoproteins / metabolism
  • Microscopy, Fluorescence
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • NADPH Oxidase 2
  • NADPH Oxidase 4
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics*
  • NADPH Oxidases / metabolism
  • Neovascularization, Physiologic / drug effects
  • Neovascularization, Physiologic / genetics
  • Oxidation-Reduction
  • Phosphorylation / drug effects
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction*
  • Src Homology 2 Domain-Containing, Transforming Protein 1 / genetics
  • Src Homology 2 Domain-Containing, Transforming Protein 1 / metabolism
  • Time-Lapse Imaging
  • Vascular Endothelial Growth Factor A / pharmacology
  • Vascular Endothelial Growth Factor Receptor-2 / genetics
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism

Substances

  • Membrane Glycoproteins
  • RNA, Small Interfering
  • SHC1 protein, human
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Hydrogen Peroxide
  • Catalase
  • CYBB protein, human
  • NADPH Oxidase 2
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX4 protein, human
  • KDR protein, human
  • Vascular Endothelial Growth Factor Receptor-2