Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, 1967 Riverside Drive, Room 535, Ottawa, ON, K1H 7W9, Canada.
Department of Cellular and Molecular Medicine, University of Ottawa, 2513-451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
Diabetologia. 2017 Sep;60(9):1791-1800. doi: 10.1007/s00125-017-4331-2. Epub 2017 Jun 10.
AIMS/HYPOTHESIS: Individuals with diabetes exhibit increases in circulating endothelial microparticles (eMPs, also referred to as endothelial microvesicles), which are associated with endothelial dysfunction and a heightened risk of cardiovascular complications. We have shown that eMPs are markers and mediators of vascular injury although their role in diabetes is unclear. We hypothesised that the composition and biological activity of eMPs are altered in response to high glucose exposure. We assessed the effects of high glucose on eMP formation, composition and signalling in cultured HUVECs.
eMPs were isolated from the media of HUVECs cultured under conditions of normal glucose (eMP), high glucose (eMP) or osmotic control of L-glucose (eMP). eMP size, concentration and surface charge were assessed by nanoparticle tracking analysis and flow cytometry. eMP protein composition was assessed by liquid chromatography-tandem mass spectrometry, and eMP-mediated effects on coagulation, reactive oxygen species (ROS) production and vessel function were assessed.
Exposure of HUVECs to high glucose for 24 h caused a threefold increase in eMP formation, increased mean particle size (269 ± 18 nm vs 226 ± 11 nm) and decreased surface charge. Compared with eMP or eMP, eMP possessed approximately threefold greater pro-coagulant activity, stimulated HUVEC ROS production to a greater extent (~250% of eMP) and were more potent inhibitors of endothelial-dependent relaxation. Proteomic analysis of eMPs identified 1212 independent proteins of which 68 were exclusively found in eMP. Gene ontology analysis revealed that eMP-exclusive proteins were associated with signalling pathways related to blood coagulation, cell signalling and immune cell activation.
CONCLUSIONS/INTERPRETATION: Our results indicate that elevated glucose is a potent stimulus for eMP formation that also alters their molecular composition leading to increased bioactivity. Such effects may contribute to progressive endothelial injury and subsequent cardiovascular complications in diabetes.
目的/假设：患有糖尿病的个体表现出血液中环内皮微颗粒（也称为内皮微囊泡，eMPs）的增加，这与内皮功能障碍和心血管并发症的风险增加有关。我们已经表明，eMPs 是血管损伤的标志物和介质，尽管它们在糖尿病中的作用尚不清楚。我们假设 eMPs 的组成和生物学活性会因高葡萄糖暴露而发生改变。我们评估了高葡萄糖对培养的 HUVECs 中 eMP 形成、组成和信号转导的影响。
从在正常葡萄糖（eMP）、高葡萄糖（eMP）或 L-葡萄糖渗透压控制（eMP）条件下培养的 HUVECs 培养基中分离 eMPs。通过纳米颗粒跟踪分析和流式细胞术评估 eMP 的大小、浓度和表面电荷。通过液相色谱-串联质谱法评估 eMP 蛋白组成，并评估 eMP 对凝血、活性氧（ROS）产生和血管功能的影响。
将 HUVECs 暴露于高葡萄糖 24 小时会导致 eMP 形成增加三倍，平均粒径增加（269±18nm 比 226±11nm），表面电荷降低。与 eMP 或 eMP 相比，eMP 具有约三倍的促凝血活性，刺激 HUVEC 产生的 ROS 增加了约 250%（比 eMP 多 250%），并且对内皮依赖性松弛的抑制作用更强。eMP 的蛋白质组学分析鉴定出 1212 种独立蛋白质，其中 68 种仅存在于 eMP 中。GO 分析表明，eMP 特有的蛋白质与血液凝固、细胞信号转导和免疫细胞激活相关的信号通路有关。
结论/解释：我们的结果表明，升高的葡萄糖是 eMP 形成的强烈刺激因素，也改变了它们的分子组成，导致生物活性增加。这种作用可能导致糖尿病中内皮损伤的进行性加重和随后的心血管并发症。
