The procoagulant effects of extracellular vesicles derived from hypoxic endothelial cells can be selectively inhibited by inorganic nitrite.

BACKGROUND: Extracellular vesicles (EVs) derived from endothelial cells are elevated in cardiovascular disease and promote inflammation and coagulation. Hypoxia is often a key feature and is itself a potent stimulator of increased EV production. Inorganic nitrite (NO2-) has beneficial and protective effects that are enhanced in hypoxia. OBJECTIVES: Investigate the impact of hypoxia on the functional capacity of EV derived from endothelial cells under hypoxia, and assess whether pre-treatment of endothelial cells with NO2- can alter EV function. METHODS: Differential ultracentrifugation was used to isolate EV from the cultured endothelial cell line HECV (CEV), and from primary human umbilical cord derived endothelial cells (PEV), with time-resolved fluorescence used to assess EV protein composition. Clot formation was induced by thrombin and calcium in two assays; using an Alexa Fluor 594 human fibrinogen conjugate assay and standard turbidometry. Platelet aggregation was determined using multiple electrode aggregometry. Scanning electron microscopy was used to visualise fibrin clots. RESULTS: Hypoxia exposure (1% O2) significantly increased CEV production in comparison to normoxia (21% O2) (1825 ± 72 EVs/cell vs 117 ± 9 EVs/cell, p < 0.001, respectively) but had no effect on CEV mean size (221 ± 6 nm vs 203 ± 4 nm, p > 0.05). Hypoxia-derived PEVs contained significantly more tissue factor than normoxia-derived EVs (Relative Fluorescence Units (RFU) = 7666 ± 1698 vs 5958 ± 1644, p < 0.001, respectively) and less tissue factor pathway inhibitor (RFU = 9799 ± 2353 vs 19723 ± 2698, p < 0.05). Hypoxia significantly increased CEV induced fibrin polymer formation compared to normoxia (% area = 46.98 ± 0.97 vs 36.36 ± 0.72, p < 0.05). Pre-treatment of endothelial cells with NO2- in hypoxia abrogated this effect (% area = 15.70 ± 1.99, p < 0.001). Hypoxia derived CEV non-significantly increased the maximum clot formed, shortened time to max clot, and increased time to clot lysis by turbidometry. ADP-mediated platelet aggregation was significantly elevated with PEV derived from hypoxia compared to normoxia (888.0 ± 32.2 AU*min vs 671.5.2 ± 28.3 AU*min, p < 0.01). This was abrogated by pre-treatment of hypoxic endothelial cells with NO2- (716.5 ± 744.3 AU*min, p < 0.001). CONCLUSIONS: Hypoxia-derived PEVs and CEVs exhibit increased procoagulant activity compared to normoxia-derived EVs, which we confirm to be mediated by an imbalance of TF/TFPI. Pre-treatment of endothelial cells with NO2- reduces the pro-coagulant activity of EVs via a mechanism that is Hypoxia-inducible factor 1 (HIF-1) dependent, but independent of TF/TFPI.

Dietary Nitrate Supplementation Reduces Circulating Platelet-Derived Extracellular Vesicles in Coronary Artery Disease Patients on Clopidogrel Therapy: A Randomised, Double-Blind, Placebo-Controlled Study.

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 (n = 10) or without (n = 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 (-1.183e11 ± 3.15e10 vs -9.93e9 ± 1.84e10 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 (6.209e10 ± 4.074e9 vs 3.94e11 ± 1.91e10 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.

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

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.