Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells

Abstract

factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced 34 release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, 35 and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to 36 selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial 37 EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. 38Methods: Endothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 hours) and 39 compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium 40 nitrite (NaNO2) (30 µM). Allopurinol (100 µM), an inhibitor of xanthine oxidoreductase, was added 41 both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations 42 were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western 43 blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow 44 cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein 45 content. 46Results: Endothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs 47 compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic 48 agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic 49 enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor 50 allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated 51 EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic 52 exposure prevented the enhancement of EV release. 53Conclusion: These data provide evidence that hypoxia enhances the release of EVs in endothelial 54 cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2 -to NO 55 via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production. 56

Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency

“…Indeed, “hypoxia” (1% O 2 ) has been shown to increase exosome production in numerous cell types in vitro . Previous studies have shown that the hypoxia-induced elevation in exosome secretion is chiefly governed by hypoxia-inducible factor-1 alpha (HIF-1α) and is independent of apoptosis ( 60 ). Often, laboratory cell culture conditions are at atmospheric oxygen levels (21% O 2 , corresponding to a PO2 of ~159 mmHg); following adjustment to 5% CO 2 , this equates to ~19.95% O 2 (~150 mmHg).…”

Engineered Extracellular Vesicles: Tailored-Made Nanomaterials for Medical Applications

“…Numerous approaches have been investigated to boost EVs production, such as altering the cells immediate environmental conditions. For instance, Burnley-Hall et al induced hypoxia (1% O 2 ) which increased EV synthesis 8-fold from human vascular endothelial cells when compared to normoxic conditions (21% O 2 ), which was found to be mediated by the transcription factor hypoxia-inducible factor-1α ( Figure 3 B) [ 55 ]. Similarly, serum-deprivation increased EVs yield in human myeloma (2.5-fold) and neuroblastoma cells (10-fold) [ 56 , 57 ].…”

“…( B ) Hypoxic conditions enhanced the yield of EVs derived from endothelial cells compared to normoxic conditions. Reproduced with permission from [ 55 ], Elsevier, 2017. ***, p < 0.001.…”

The role of tumor-derived exosomes in tumor angiogenesis and tumor progression

“…EVs produced by hypoxic tumor cells have been shown to have a more pronounced effect on ECs in promoting angiogenesis than those derived from normoxic cells [102,113]. Hypoxia increases the production of tumor and stromal cell-derived EVs, and alters their cargo [98,102,103,114,115]. For example, miR-23a is found in the EVs of hypoxic, but not normoxic, lung cancer cells [103].…”

“…For example, miR-23a is found in the EVs of hypoxic, but not normoxic, lung cancer cells [103]. Increased EVs production by hypoxic endothelial cells was abrogated by siRNA targeting hypoxia-inducible factor 1, thus providing a clear link between cell response to hypoxia and EVs production [114].…”