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Updated: Nov 19, 2025
Stimulation of Vascular Endothelial Cells Using Neutrophil Extracellular Traps in the Presence of Low-Density Lipoprotein
Published on: August 12, 2025
Endothelial cells response to neutrophil-derived extracellular vesicles miRNAs in anti-PR3 positive vasculitis
M Surmiak1, J Kosałka-Węgiel1, S Polański2
1Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland.
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.
Insights
Neutrophil extracellular vesicles (EVs) from Granulomatosis with polyangiitis (GPA) patients induce inflammation in endothelial cells. This study reveals molecular changes in human umbilical endothelial cells (HUVECs) mimicking autoimmune vasculitis.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Granulomatosis with polyangiitis (GPA) is a systemic vasculitis characterized by anti-proteinase-3 autoantibodies (anti-PR3).
- The role of neutrophil-derived extracellular vesicles (EVs) in mediating endothelial cell activation in GPA is not fully understood.
Purpose of the Study:
- To investigate the molecular mechanisms by which anti-PR3-activated neutrophil EVs influence human umbilical endothelial cells (HUVECs).
- To identify specific molecular signatures and cytokine profiles associated with endothelial cell activation in response to EVs.
Main Methods:
- Neutrophils from anti-PR3-positive GPA patients were activated to generate EVs.
- EVs were purified and characterized using flow cytometry, nanoparticle tracking, and miRNA screening.
- HUVECs were stimulated with EVs, and subsequent changes in miRNA/mRNA expression and protein/cytokine release were analyzed.
- Antibody microarrays and cytokine measurements were employed to profile secreted factors.
Main Results:
- EV stimulation of HUVECs resulted in distinct miRNA and mRNA expression patterns, including upregulation of pro-inflammatory transcripts (e.g., CXCL8, VCAM-1) and downregulation of others (e.g., FASLG, IL6).
- Stimulated HUVECs released elevated levels of cytokines such as IL-8, Dickkopf-related protein 1 (DKK-1), and soluble interleukin-1 like receptor-1 (ST2).
- Transfection with specific EV-derived miRNAs (miR-223-3p, miR-142-3p) mimicked the cytokine release profile, implicating these miRNAs in endothelial activation.
- Elevated cytokine levels (IL-8, DKK-1, ST2) were observed in the blood of GPA patients, correlating with in-vitro findings.
Conclusions:
- Neutrophil-derived EVs from GPA patients can induce a pro-inflammatory phenotype in endothelial cells.
- The study elucidates a molecular crosstalk between neutrophils and endothelial cells involving EVs and their miRNA cargo.
- These findings provide insights into the contribution of endothelial cells to the pathogenesis of autoimmune vasculitis and identify potential therapeutic targets.
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