亚硝酸盐衍生的一氧化氮可减少内皮细胞缺氧诱导因子 1α 介导的细胞外囊泡产生。
Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells.
机构信息
School of Medicine, Cardiff University, Cardiff, CF24 4HQ, UK.
Division Newborn Medicine, Boston Children's Hospital, Harvard Medical School, Harvard University, Boston, 02115, MA, USA.
出版信息
Nitric Oxide. 2017 Feb 28;63:1-12. doi: 10.1016/j.niox.2016.12.005. Epub 2016 Dec 23.
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 (NaNO) 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% O, 24 h) and compared to endothelial cells exposed to normoxia (21% O) with and without the presence of sodium nitrite (NaNO) (30 μM). Allopurinol (100 μM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO 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% O) 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 (NaNO) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO attenuated the NaNO-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 NO to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production.
简介
细胞外囊泡（EVs）是由多种细胞类型释放的小的、球形的、由磷脂双层（约 30-1000nm）包裹的颗粒，已被证明在多种疾病状态中具有病理生理学作用。转录因子缺氧诱导因子-1（HIF-1）允许细胞生理学在缺氧时适应，并可能允许在这种情况下增强 EV 的释放。一氧化氮（NO）在血管稳态中起着关键作用，并通过防止 HIF-1 积累来调节细胞对缺氧的反应。我们旨在通过添加亚硝酸钠（NaNO）选择性靶向 HIF-1，并检查其对内皮细胞 EV 大小、浓度和功能的影响，并阐明 HIF-1 在 EV 发生中的作用。
方法
将内皮（HECV）细胞暴露于低氧条件（1%O，24 小时），并与暴露于常氧（21%O）的内皮细胞进行比较，同时存在或不存在亚硝酸钠（NaNO）（30μM）。加入别嘌呤醇（100μM），一种黄嘌呤氧化还原酶抑制剂，单独加入和与 NaNO 一起加入到缺氧细胞中。通过纳米颗粒跟踪分析和电子显微镜确认 EV 和细胞制剂的定量。使用 Western 印迹和 siRNA 确认 HIF-1α 和 HIF-2α 在 EV 发生中的作用。使用流式细胞术和时间分辨荧光法评估表面和囊内蛋白含量。
结果
与暴露于常氧的细胞相比，暴露于低氧（1%O）的内皮（HECV）细胞产生的 EV 水平更高。使用缺氧模拟剂去铁胺证实了这一点。用亚硝酸钠（NaNO）处理细胞可降低缺氧对 EV 产生的增强作用。在用黄嘌呤氧化还原酶抑制剂别嘌呤醇处理细胞的同时，用 NaNO 处理细胞可减弱 NaNO 引起的缺氧介导的 EV 释放抑制作用。在低氧暴露前用 HIF-1α siRNA 转染细胞，但不用 HIF-2α siRNA 转染，可防止 EV 释放增强。
结论
这些数据提供了证据，表明低氧增强内皮细胞中 EV 的释放，并且这是由 HIF-1α介导的，而不是 HIF-2α。此外，在缺氧期间，黄嘌呤氧化还原酶将 NO 还原为 NO 似乎抑制了 HIF-1α 介导的 EV 产生。
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