BACKGROUND:Neutrophil extracellular traps (NETs) are composed of nDNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction.METHODS:NETosis-deficient Padi4^−/− mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative RT-PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence.RESULTS:Padi4^−/− mice exhibited attenuated hypertension in response to Ang II, reduced aortic inflammation, and improved EC-dependent vascular relaxation. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis.CONCLUSIONS:These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

中文翻译：

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NETosis 导致高血压患者血压升高和血管功能障碍

背景：中性粒细胞胞外陷阱（NET）由nDNA、酶和瓜氨酸组蛋白组成，在NETosis过程中被中性粒细胞排出。高血压患者的 NET 会积聚在主动脉和肾脏中。 PAD4（蛋白质-精氨酸脱亚胺酶-4）是一种钙依赖性酶，对于 NETosis 至关重要。 TRPV4（瞬时受体电位阳离子通道亚家族 V 成员 4）是中性粒细胞中表达的机械敏感钙通道。因此，我们假设 NETosis 通过 NET 介导的内皮细胞 (EC) 功能障碍导致高血压。方法：用 Ang II（血管紧张素 II）治疗NETosis 缺陷的Padi4 ^−/−小鼠。通过无线电遥测测量血压，并通过线肌动描记术测量血管反应性。在有或没有 EC 的情况下培养中性粒细胞，并暴露于正常血压或高血压单轴拉伸。通过流式细胞术测量NETosis。 ECs 用瓜氨酸组蛋白 H3 处理，并通过定量 RT-PCR 测量基因表达。主动脉环与瓜氨酸组蛋白 H3 一起孵育，并进行钢丝肌动描记术以评估 EC 功能。使用 TRPV4 激动剂 GSK1016790A 处理中性粒细胞。使用 Fluo-4 染料测量钙流入，并通过免疫荧光测量 NETosis。 结果：Padi4 ^−/−小鼠表现出对 Ang II 的反应减轻的高血压、减少主动脉炎症和改善 EC 依赖性血管舒张。中性粒细胞与 EC 的共培养以及暴露于高血压单轴拉伸会增加 NETosis 和中性粒细胞瓜氨酸组蛋白 H3 的积累。组蛋白 H3 和瓜氨酸组蛋白 H3 暴露会减弱 EC 依赖性血管舒张。用 TRPV4 激动剂 GSK1016790A 治疗中性粒细胞会增加细胞内钙和 NETosis。结论：这些观察结果确定了 NETosis 在高血压发病机制中的作用。此外，他们还定义了 EC 伸展和 TRPV4 作为 NETosis 引发剂的重要作用。最后，他们定义了瓜氨酸组蛋白作为高血压 EC 功能障碍驱动因素的作用。