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

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posted on 2023-10-23, 15:26 authored by Cass Whelan, Nicholas Burnley-Hall, Keith Morris, D. Aled Rees, Philip James

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.





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Whelan, Cass, Burnley-Hall, Nicholas, Morris, Keith, Rees, D. Aled and James, Philip E. 2022. The procoagulant effects of extracellular vesicles derived from hypoxic endothelial cells can be selectively inhibited by inorganic nitrite. Nitric Oxide: Biology and Chemistry 122-12 , pp. 6-18. 10.1016/j.niox.2022.02.002

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Cardiff Met Affiliation

  • Cardiff School of Sport and Health Sciences

Cardiff Met Authors

Cass Whelan Keith Morris Philip James

Cardiff Met Research Centre/Group

  • Cardiovascular Metabolism and Inflammation

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  • © The Publisher


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