Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells
Rees, D. Aled
MetadataShow full item record
Introduction Extracellular vesicles (EVs) are small, spherical particles enclosed by a phospholipid bilayer (∼30–1000 nm) released from multiple cell types, and have been shown to have pathophysiological roles in a plethora of disease states. The transcription factor hypoxia-inducible factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. Methods Endothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 h) and compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium nitrite (NaNO2) (30 μM). Allopurinol (100 μM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein content. Results Endothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic exposure prevented the enhancement of EV release. Conclusion These data provide evidence that hypoxia enhances the release of EVs in endothelial cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2− to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production.
Burnley-Hall, N., Willis, G., Davis, J., Rees, D.A. and James, P.E. (2017) 'Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells', Nitric Oxide, 63, pp.1-12.
This article was published in Nitric Oxide on 23 December 2016 (online), available at http://dx.doi.org/10.1016/j.niox.2016.12.005
Cardiff Metropolitan University (Grant ID: Cardiff Metropolian (Internal))
Showing items related by title, author, subject and abstract.
Nitrite directly vasodilates hypoxic vasculature via nitric oxide-dependent and -independent pathways Pinder, A.G.; Pittaway, E.; Morris, Keith; James, Philip (2009)Background and purpose: It is postulated that nitrite requires reduction to nitric oxide in order to exert its relaxant effect upon isolated hypoxic vessels. Herein, we evaluate the relative contribution of nitric oxide ...
The effect of alpha1-adrenergic blockade on post-exercise brachial artery flow-mediated dilatation at sea-level and high-altitude Tymko, Michael; Tremblay, Joshua; Howe, Connor; Willie, Christopher; Stembridge, Mike; Green, Daniel; Hoiland, Ryan; Subedi, Prajan; Anholm, James; Ainslie, Philip (Wiley, 2016-11-07)We examined the hypotheses that 1) at rest, endothelial function would be impaired at high- altitude compared to sea-level, 2) endothelial function would be reduced to a greater extent at sea-level compared to high-altitude ...
Clopidogrel results in favourable changes in nitric oxide metabolism in patients undergoing percutaneous coronary intervention Bundhoo, Shantu; Sagan, Ewelina; James, Philip; Anderson, Richard (Thieme, 2017-11-27)Clopidogrel has been shown to have a morbidity and mortality benefit in the prevention of ischaemic complications in patients with acute coronary syndrome and/or after percutaneous coronary intervention (PCI)1. 600mg of ...