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

Abstract

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.