<br>抗 PR3 阳性血管炎中内皮细胞对中性粒细胞来源的细胞外囊泡 miRNA 的反应,Clinical & Experimental Immunology

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抗 PR3 阳性血管炎中内皮细胞对中性粒细胞来源的细胞外囊泡 miRNA 的反应
Clinical & Experimental Immunology ( IF 3.4 ) Pub Date : 2021-02-02 , DOI: 10.1111/cei.13581
M Surmiak ₁ , J Kosałka-Węgiel ₁ , S Polański ₂ , M Sanak ₁

Affiliation

在血管炎疾病中，炎症会影响血管。肉芽肿性多血管炎 (GPA) 是一种慢性系统性血管炎，其特征是存在抗蛋白酶 3 自身抗体（抗 PR3）。在这项研究中，我们分析了人脐带内皮细胞 (HUVEC) 响应中性粒细胞来源的细胞外囊泡 (EV) 的分子特征。 EV 是从抗 PR3 激活的中性粒细胞中获得的，通过流式细胞术、纳米颗粒追踪和 miRNA 筛选进行纯化和表征。用 EV 刺激 HUVEC 并测量 miRNA/mRNA 表达。通过抗体微阵列鉴定细胞培养基蛋白质并测量选定的细胞因子。未刺激和 EV 刺激的 HUVEC 之间差异表达的 miRNA/mRNA 的比较揭示了两种调节模式。 14 个 mRNA 转录本（包括CXCL8 、 DKK1 、 IL1RL1 、 ANGPT-2 、 THBS1和VCAM-1 ）的显着上调伴随着 11 个 miRNA 沉默（包括 miR-661、miR-664a-3p、miR-377-3p、 miR-30d-5p）。观察到 9 种 mRNA 转录物（包括FASLG 、 CASP8 、 STAT3 、 GATA3 、 IRAK 1 和IL6 ）显着下调，并伴有 10 种 miRNA（包括 miR-223-3p、miR-142-3p、miR- 211-5p）。刺激的 HUVEC 释放 IL-8、Dickkopf 相关蛋白 1 (DKK-1)、可溶性白细胞介素 (IL)-1 样受体-1 (ST2)、生长分化因子 15 (GDF-15)、血管生成素-2、内皮糖蛋白、血小板反应蛋白‐1 和血管粘附分子-1 (VCAM-1)。此外，用EV中高表达的miR-223-3p或miR-142-3p模拟物转染HUVEC，刺激IL-8、ST2和内皮糖蛋白的产生。 GPA 患者血液中 HUVEC 释放的细胞因子也升高。增加最多的是 IL-8、DKK-1、ST2、血管生成素-2 和 IL-33。中性粒细胞来源的 EV 对 HUVEC 的体外刺激概括了内皮细胞在自身免疫性血管炎中的作用。释放的细胞因子的促炎表型与包含 EV miRNA 和内皮细胞转录物的 miRNA/mRNA 调节网络相对应。

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Endothelial cells response to neutrophil‐derived extracellular vesicles miRNAs in anti‐PR3 positive vasculitis

In vasculitis disorders, inflammation affects blood vessels. Granulomatosis with polyangiitis (GPA) is a chronic systemic vasculitis distinguished by the presence of anti‐proteinase‐3 autoantibodies (anti‐PR3). In this study we analyzed the molecular signature of human umbilical endothelial cells (HUVECs) in response to neutrophil‐derived extracellular vesicles (EVs). EVs were obtained from anti‐PR3‐activated neutrophils, purified and characterized by flow cytometry, nanoparticle tracking and miRNA screening. HUVECs were stimulated with EVs and miRNA/mRNA expression was measured. Cell culture media proteins were identified by antibody microarrays and selected cytokines were measured. Comparison of differentially expressed miRNAs/mRNAs between non‐stimulated and EV‐stimulated HUVECs revealed two regulatory patterns. Significant up‐regulation of 14 mRNA transcripts (including CXCL8, DKK1, IL1RL1, ANGPT‐2, THBS1 and VCAM‐1) was accompanied by 11 miRNAs silencing (including miR‐661, miR‐664a‐3p, miR‐377‐3p, miR‐30d‐5p). Significant down‐regulation was observed for nine mRNA transcripts (including FASLG, CASP8, STAT3, GATA3, IRAK1 and IL6) and accompanied by up‐regulation of 10 miRNAs (including miR‐223‐3p, miR‐142‐3p, miR‐211‐5p). Stimulated HUVECs released IL‐8, Dickkopf‐related protein 1 (DKK‐1), soluble interleukin (IL)‐1 like receptor‐1 (ST2), growth differentiation factor 15 (GDF‐15), angiopoietin‐2, endoglin, thrombospondin‐1 and vascular adhesion molecule‐1 (VCAM‐1). Moreover, transfection of HUVECs with mimics of highly expressed in EVs miR‐223‐3p or miR‐142‐3p, stimulated production of IL‐8, ST2 and endoglin. Cytokines released by HUVECs were also elevated in blood of patients with GPA. The most increased were IL‐8, DKK‐1, ST2, angiopoietin‐2 and IL‐33. In‐vitro stimulation of HUVECs by neutrophil‐derived EVs recapitulates contribution of endothelium in autoimmune vasculitis. Proinflammatory phenotype of released cytokines corresponds with the regulatory network of miRNAs/mRNAs comprising both EVs miRNA and endothelial cell transcripts.

更新日期：2021-02-02

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