title: Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells.
abstract: BACKGROUND
  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 NaNO2to 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 NaNO2attenuated 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Medicine, Cardiff University, Cardiff, CF24 4HQ, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Burnley-Hall
authors.affiliation: Division Newborn Medicine, Boston Children's Hospital, Harvard Medical School, Harvard University, Boston, 02115, MA, USA.
authors.forename: Gareth
authors.initials: G
authors.lastname: Willis
authors.affiliation: Institute of Cancer & Genetics, Cardiff University, Cardiff, CF14 4XN, UK.
authors.forename: Jessica
authors.initials: J
authors.lastname: Davis
authors.affiliation: Neurosciences and Mental Health Research Institute, School of Medicine, Cardiff University, Cardiff, CF24 4HQ, UK.
authors.forename: D Aled
authors.initials: DA
authors.lastname: Rees
authors.affiliation: Cardiff School of Health Sciences, Cardiff Metropolitan University, Cardiff, CF5 2SG, UK. Electronic address: PJames@cardiffmet.ac.uk.
authors.forename: Philip E
authors.initials: PE
authors.lastname: James
mesh_terms: D000493:Allopurinol / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000067128:Extracellular Vesicles / Q000737:chemistry / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D010316:Particle Size; D012977:Sodium Nitrite / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000037:antagonists & inhibitors; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000493:Allopurinol; D051792:Basic Helix-Loop-Helix Proteins; D003676:Deferoxamine; D004791:Enzyme Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009569:Nitric Oxide; D012977:Sodium Nitrite; D014968:Xanthine Dehydrogenase; D000099162:Endothelial PAS Domain-Containing Protein 1
keywords: Extracellular vesicles; Hypoxia; Hypoxia-inducible factor; Nitric oxide; Nitrite