The release of nitric oxide from S-nitrosothiols promotes angiogenesis

Bahjat Al-Ani, Peter W. Hewett, Suborna Ahmed, Melissa Cudmore, Takeshi Fujisawa, Shakil Ahmad, Asif Ahmed

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Free nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis.
NO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process.
Both DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis.
Original languageEnglish
Article numbere25
Number of pages8
JournalPLoS ONE
Issue number1
Publication statusPublished - 20 Dec 2006

Bibliographical note

© 2006 Al-Ani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


  • angiogenesis
  • antioxidants
  • cell membrane
  • endothelial cells
  • growth facotrs
  • nitiric acid
  • phosphorylation
  • vitamin c


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