Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland.
Department of Biochemical and Molecular Diagnostics, University Hospital, Kraków, Poland.
Clin Exp Immunol. 2021 May;204(2):267-282. doi: 10.1111/cei.13581. Epub 2021 Feb 28.
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.
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，我们分析了人脐静脉内皮细胞（HUVEC）对中性粒细胞衍生的细胞外囊泡（EV）反应的分子特征。从抗-PR3 激活的中性粒细胞中获得 EV，通过流式细胞术、纳米颗粒跟踪和 miRNA 筛选进行纯化和表征。用 EV 刺激 HUVEC，测量 miRNA/mRNA 的表达。通过抗体微阵列鉴定细胞培养物上清液中的蛋白质，并测量选定的细胞因子。非刺激和 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）的显著上调伴随着 11 个 miRNA 的沉默（包括 miR-661、miR-664a-3p、miR-377-3p、miR-30d-5p）。9 个 mRNA 转录本（包括 FASLG、CASP8、STAT3、GATA3、IRAK1 和 IL6）的显著下调伴随着 10 个 miRNA 的上调（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 和血管细胞粘附分子-1（VCAM-1）。此外，用 EV 中高表达的 miRNA 模拟物转染 HUVEC，可刺激 IL-8、ST2 和内皮糖蛋白的产生。GPA 患者血液中的细胞因子也升高。增加最多的是 IL-8、DKK-1、ST2、血管生成素-2 和 IL-33。中性粒细胞衍生的 EV 对 HUVEC 的体外刺激再现了自身免疫性血管炎中内皮的贡献。释放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 调控网络相对应。
