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Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a crucial role in the regulation of vascular tone and angiogenesis. NO reacts with sulfhydryl species to form S-nitrosothiols, which act as reservoirs for NO. We sought to determine whether thiol-preserving agents and antioxidants such as dithiothreitol (DTT) and vitamin C increase NO release and promote angiogenesis. Stimulation of porcine aortic endothelial cells (PAE) with DTT or vitamin C significantly increased NO release in a concentration-dependent manner as measured using a Sievers NOA 280 chemiluminescence analyzer (mean NO ± SEM: 90 ± 10 nM with 1 mM DTT; 80 ± 10 nM with 1 mM vitamin C; 18 ± 5 nM with vehicle; p<0.05 versus control, n=3). However, the eNOS inhibitor L-NNA did not block the release of NO induced by DTT or vitamin C nor was there a significant change in eNOS phosphorylation at the site Ser-1177 upon cell stimulation suggesting that the increase in free NO came from NO reservoirs. Vitamin C-induced NO release was inhibited by the PI 3-Kinase inhibitor, LY294002, but not DTT-mediated NO production. Endothelial cells plated on growth factor-reduced Matrigel formed capillary tube-like structures in response to DTT or vitamin C. Increasing concentrations of DTT significantly increased both total tube length (9-fold with 1 mM DTT) and total number of capillary connections (6-fold with 1 mM DTT). Similarly, vitamin C also caused an increase in capillary network structures but to a much lesser extent, and addition of copper doubled the effect of vitamin C possibly due to an increase in nitrosothiol reduction which in turn caused more NO release and hence more tube formation. Tube formation stimulated by DTT or vitamin C was inhibited by LY294002 and also by an inhibitor of guanylate cyclase LY83583, but not by L-NNA. These data indicate that DTT and vitamin C are capable of generating NO independent of eNOS activation to promote angiogenesis.
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J. Vasc. Biol. 42, Sup:2 (2005) p35