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

Young Mee Kim, Seok Jo Kim, Ryosuke Tatsunami, Hisao Yamamura, Tohru Fukai, Masuko Ushio Fukai

Research output: Contribution to journalArticlepeer-review

151 Scopus citations


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 H2O2to promote mtROS production. Mechanistically, H2O2increases 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 H2O2in 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.

Original languageEnglish (US)
Pages (from-to)C749-C764
JournalAmerican Journal of Physiology - Cell Physiology
Issue number6
StatePublished - Jun 7 2017


  • Angiogenesis
  • Mitochondria
  • NADPH oxidase
  • Reactive oxygen species
  • Vascular endothelial growth factor

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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