The release of nitric oxide from S-nitrosothiols promotes angiogenesis.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Al-Ani
Bahjat
B
Department of Reproductive and Vascular Biology, Centre for Cardiovascular Sciences, Institute of Biomedical Research, Medical School, University of Birmingham, United Kingdom.
Hewett
Peter W
PW
Ahmed
Suborna
S
Cudmore
Melissa
M
Fujisawa
Takeshi
T
Ahmad
Shakil
S
Ahmed
Asif
A
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D010766:Phosphorylation; D011810:Quinoxalines / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000378:metabolism*; D013552:Swine
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D002227:Carbazoles; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D010069:Oxadiazoles; D011810:Quinoxalines; D026403:S-Nitrosothiols; C073601:KT 5823; D019335:Nitroarginine; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D009952:Ornithine; D052250:Nitric Oxide Synthase Type III; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D001205:Ascorbic Acid; D004229:Dithiothreitol