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Updated: Aug 12, 2025
A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation
Published on: December 9, 2021
Microscopic polyangiitis plasma-derived exosomal miR-1287-5p induces endothelial inflammatory injury and neutrophil
Yan Zhu1,2, Liu Liu1, Liepeng Chu1
1Department of Nephrology, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
Background:
An inflammatory environment around the vessel wall caused by leukocyte infiltration is one of the characteristic histopathological features of microscopic polyangiitis (MPA); however, the pathogenic mechanisms are not fully understood. Studies have found that circulating microRNA (miRNA) can be used as potential biomarkers for the diagnosis and classification of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), and the E3 ubiquitin ligase casitas B-lineage lymphoma (CBL) seems to be associated with inflammation. In addition, evidence indicates that miRNA can be tracked into exosomes and transferred into recipient cells to mediate the process of vascular endothelial injury. Herein, we aimed to identify the profiles of exosomal miRNA, and determine the effect of exosomal miR-1287-5p and its target gene CBL on vascular endothelial cells in MPA.
Method:
We isolated plasma exosomes from patients with MPA (MPA-exo) and healthy controls (HC-exo) by ultracentrifugation and conducted exosome small-RNA sequencing to screen differential miRNA expression in MPA-exo (n = 3) compared to HC-exo (n = 3). We measured the expression levels of miR-1303, miR-1287-5p, and miR-129-1-3p using quantitative reverse transcription-polymerase chain reaction (qRT-PCR, n = 6) and performed dual luciferase reporter gene assays to confirm the downstream target gene of miR-1287-5p. In addition, we treated human umbilical vein endothelial cell (HUVEC) with MPA-exo, or transfected them with miR-1287-5p mimic/inhibitor or with CBL-siRNA/CBL-siRNA+ miR-1287-5p inhibitor. After cell culture, we evaluated the effects on vascular endothelial cells by examining the mRNA levels of IL-6, IL-8, MCP-1, ICAM-1 and E-selectin using qRT-PCR and performed neutrophil adhesion assay with haematoxylin staining.
Result:
Transmission electron microscopy, Western blot and nanoparticle tracking analysis showed that we successfully purified exosomes and MPA-exo could be absorbed into HUVEC. We screened a total of 1,077 miRNA by sequencing and observed a high abundance of miR-1287-5p in the exosomes obtained from MPA plasma. The dual luciferase reporter assay identified CBL as a downstream target gene of miR-1287-5p, and the results revealed that MPA-exo decreased CBL protein expression in HUVEC. In addition, treatment with MPA-exo, up-regulating miR-1287-5p or silencing of CBL in HUVEC significantly increased the mRNA expression of inflammatory factors (including IL-6, IL-8, and MCP-1) and adhesion molecules (including ICAM-1 and E-selection) and promoted the adhesion of neutrophils to HUVEC. However, down-regulating miR-1287-5p had the opposite effect.
Conclusion:
Our study revealed that MPA-exo was involved in the intercellular transfer of miR-1287-5p and subsequently promote the development of acute endothelial injury in MPA. MiR-1287-5p and CBL agonists may be promising therapeutic approach for MPA-induced vascular inflammatory injury.
Insights
Microscopic polyangiitis (MPA) involves inflammation and endothelial injury, driven by exosomal miR-1287-5p transfer. This microRNA targets CBL, promoting vascular inflammation and potentially offering therapeutic targets for MPA.
Area of Science:
- Immunology
- Molecular Biology
- Vascular Biology
Background:
- Microscopic polyangiitis (MPA) is characterized by leukocyte infiltration and inflammation around vessel walls.
- The precise pathogenic mechanisms of MPA are not fully understood.
- Circulating microRNAs (miRNAs) are potential biomarkers for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), and CBL may play a role in inflammation.
Purpose of the Study:
- To identify exosomal miRNA profiles in MPA.
- To determine the effect of exosomal miR-1287-5p and its target gene CBL on vascular endothelial cells in MPA.
Main Methods:
- Plasma exosomes were isolated from MPA patients and healthy controls.
- Exosome small-RNA sequencing and qRT-PCR were used to screen differential miRNA expression.
- Dual luciferase reporter assays confirmed CBL as a target of miR-1287-5p.
- Human umbilical vein endothelial cells (HUVECs) were treated with MPA exosomes or transfected with miR-1287-5p mimic/inhibitor or CBL-siRNA.
Main Results:
- MPA exosomes showed a high abundance of miR-1287-5p and were absorbed by HUVECs.
- miR-1287-5p targets CBL, leading to decreased CBL protein expression in HUVECs.
- MPA exosomes, increased miR-1287-5p, or silenced CBL significantly increased inflammatory factors and adhesion molecules, promoting neutrophil adhesion.
Conclusions:
- MPA exosomes facilitate intercellular transfer of miR-1287-5p, promoting endothelial injury in MPA.
- miR-1287-5p and CBL modulation represent potential therapeutic strategies for MPA-induced vascular inflammation.
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