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

Summary 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 , IRAK 1 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.