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Updated: Jul 16, 2025
Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Ox-LDL promotes M1-like polarization of macrophages through the miR-21-5p/SKP2/EP300 pathway
Jinlei Wu1, Tingting Liu2, Wenjie Xie1
1Department of Cardiology, He Xian Memorial Affiliated Hospital of Southern Medical University, Guangzhou, China.
Abstract:
Oxidized low-density lipoprotein (ox-LDL) mediated inflammatory damage, which possibly induces atherosclerosis (AS); however, the role of miRNA in this process has rarely been reported. In this paper, we study the ox-LDL-related endothelial cell damage and changes of macrophages. The bioinformatics method was used to analyze the expression changes of miRNA in AS patients, luciferase assay was used to study the interaction of protein and miRNA, and co-IP and ubiquitination experiments were used to analyze protein interaction. Flow cytometry was used to detect the polarization of macrophages. Database analysis showed that the expression of miR-21-5p was upregulated in AS patients. Luciferase assay showed that miR-21-5p can bind to SKP2 and subsequently influence ubiquitination of EP300. Overexpression of EP300 strengthens the HMGB1-induced acetylation and subsequently mediates the dissociation of HMGB1 from SIRT1, and thus HMGB1 could be secreted outside the cell. The HMGB1 released from endothelial cells can promote macrophage M1 polarization. This study shows that ox-LDL activates the SKP2/EP300 pathway through promoting upregulation of miR-21-5p, thereby acetylating and secreting HMGB1 outside the endothelium, subsequently enhancing macrophage polarization to further stabilize the inflammation situation.
Insights
Oxidized low-density lipoprotein (ox-LDL) triggers inflammation in atherosclerosis by upregulating miR-21-5p. This process promotes macrophage M1 polarization, contributing to the inflammatory cycle.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Immunology
Background:
- Oxidized low-density lipoprotein (ox-LDL) is implicated in atherosclerosis (AS) pathogenesis.
- The role of microRNAs (miRNAs) in ox-LDL-induced endothelial damage and macrophage response remains underexplored.
Purpose of the Study:
- To investigate the role of miR-21-5p in ox-LDL-induced endothelial cell damage and macrophage polarization.
- To elucidate the molecular mechanisms linking ox-LDL, miR-21-5p, and inflammatory pathways in AS.
Main Methods:
- Bioinformatic analysis of miRNA expression in AS patients.
- Luciferase assays to confirm miRNA-protein interactions.
- Co-immunoprecipitation and ubiquitination assays for protein interaction analysis.
- Flow cytometry to assess macrophage polarization.
Main Results:
- miR-21-5p expression was significantly upregulated in AS patients.
- miR-21-5p directly targets and influences the ubiquitination of SKP2 and EP300.
- EP300 overexpression enhances HMGB1 acetylation, leading to its secretion and subsequent M1 polarization of macrophages.
- ox-LDL activates the SKP2/EP300 pathway via miR-21-5p, promoting HMGB1 secretion and macrophage M1 polarization.
Conclusions:
- miR-21-5p plays a critical role in ox-LDL-induced endothelial dysfunction and inflammation.
- The miR-21-5p/SKP2/EP300/HMGB1 axis is a key pathway mediating ox-LDL-induced macrophage M1 polarization.
- Targeting this pathway may offer a novel therapeutic strategy for atherosclerosis.
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