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
Extracellular vesicles (EVs) are implicated in the pathogenesis of cardiovascular disease (CVD). Specifically, platelet-derived EVs are highly pro-coagulant, promoting thrombin generation and fibrin clot formation. Nitrate supplementation exerts beneficial effects in CVD, via an increase in nitric oxide (NO) bioavailability. Clopidogrel is capable of producing NO-donating compounds, such as S-nitrosothiols (RSNO) in the presence of nitrite and low pH. The aim of this study was to assess the effect of nitrate supplementation with versus without clopidogrel therapy on circulating EVs in coronary artery disease (CAD) patients. In this randomized, double-blind, placebo-controlled study, CAD patients with ( = 10) or without ( = 10) clopidogrel therapy received a dietary nitrate supplement (SiS nitrate gel) or identical placebo. NO metabolites and platelet activation were measured using ozone-based chemiluminescence and multiple electrode aggregometry. EV concentration and origin were determined using nanoparticle tracking analysis and time-resolved fluorescence. Following nitrate supplementation, plasma RSNO was elevated (4.7 ± 0.8 vs 0.2 ± 0.5 nM) and thrombin-receptor mediated platelet aggregation was reduced (-19.9 ± 6.0 vs 4.0 ± 6.4 U) only in the clopidogrel group compared with placebo. Circulating EVs were significantly reduced in this group (-1.183e ± 3.15e vs -9.93e± 1.84e EVs/mL), specifically the proportion of CD41+ EVs (-2,120 ± 728 vs 235 ± 436 RFU [relative fluorescence unit]) compared with placebo. In vitro experiments demonstrated clopidogrel-SNO can reduce platelet-EV directly (6.209e± 4.074e vs 3.94e ± 1.91e EVs/mL). In conclusion, nitrate supplementation reduces platelet-derived EVs in CAD patients on clopidogrel therapy, increasing patient responsiveness to clopidogrel. Nitrate supplementation may represent a novel approach to moderating the risk of thrombus formation in CAD patients.
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