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Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro
Published on: May 10, 2021
M1 macrophages promote aortic valve calcification mediated by microRNA-214/TWIST1 pathway in valvular interstitial
Xiao-Fei Li1, Yan Wang1, Dong-Dong Zheng1
1Department of Cardiology, Affiliated Hospital of Nantong University Nantong 226001, China.
Objective:
The identification of the biological function of M1 macrophages and the mechanism underlying their role in valvular interstitial cell (VIC) calcification may provide therapeutic targets for the prevention of aortic valve calcification (AVC). This study investigated the mechanism by which M1 macrophages and macrophage-derived microvesicles (MVs) affected the calcification of VICs. An additional aim was to investigate the involvement of the miR-214 pathway in this process.
Methods:
The M1 or M2 macrophage phenotype in human calcific aortic valve was confirmed by gene expression analysis of M1 or M2 macrophage markers. Two macrophage cell lines (BMDMs and RAW 264.7 macrophages) were transformed into M1 macrophages by lipopolysaccharide (LPS) stimulation. To investigate the mechanism by which M1 macrophages promoted VIC calcification, the generated M1 macrophages and macrophage-derived MVs were co-cultured with VICs and VICs were then used for calcification or signals analysis. In addition, a hypercholesterolemic apoE-/- AVC murine model was used to evaluate the therapeutic efficacy of miR-214 specific-siRNA (miR-214 inhibitor).
Results:
Macrophages in calcific aortic valves showed M1-directed polarization. In the VICs co-cultured with LPS-stimulated M1 macrophages and macrophage-derived MVs, VIC calcification was enhanced, and the expression of TWIST1, a direct target of miR-214, was downregulated. We showed that knockdown of TWIST1 serves as a responding molecule for miR-214 and reversed the anti-calcification action of miR-214 inhibitor, mediating signal delivery by the M1 macrophage-derived MVs to VICs and promoting VIC calcification. When M1 macrophages co-cultured with VICs, TWIST1 overexpression in M1 macrophages had no effect on the expression of TWIST1 in VICs. As shown by intravenous therapy, knockdown of miR-214 in mice seemed to improve AVC in apoE-/- mice with high-cholesterol (HC)-diet induced AVC.
Conclusions:
These findings suggested that M1 macrophages promoted AVC by the delivery of miR-214 to valvular interstitial cells via macrophage-derived MVs and subsequent downregulation of TWIST1 of valvular interstitial cells.
Insights
M1 macrophages promote aortic valve calcification (AVC) by transferring miR-214 via microvesicles, downregulating TWIST1 in valvular interstitial cells. Inhibiting miR-214 may offer a therapeutic strategy for AVC.
Area of Science:
- Cardiovascular Biology
- Immunology
- Cell Biology
Background:
- Aortic valve calcification (AVC) is a progressive disease.
- Valvular interstitial cells (VICs) play a key role in AVC pathogenesis.
- Macrophage polarization, particularly M1 phenotype, is implicated in inflammatory processes contributing to AVC.
Purpose of the Study:
- To investigate the mechanism by which M1 macrophages and their derived microvesicles (MVs) influence VIC calcification.
- To elucidate the role of the miR-214 pathway in M1 macrophage-mediated VIC calcification.
- To evaluate the therapeutic potential of targeting the miR-214 pathway for AVC prevention.
Main Methods:
- Analysis of macrophage polarization markers in human calcific aortic valves.
- In vitro co-culture of M1 macrophages and their MVs with VICs.
- Gene expression analysis of TWIST1 and miR-214.
- In vivo studies using a hypercholesterolemic apoE-/- mouse model of AVC.
- Therapeutic intervention with miR-214 specific-siRNA.
Main Results:
- Macrophages in calcific aortic valves exhibited M1 polarization.
- M1 macrophages and their MVs enhanced VIC calcification in vitro.
- miR-214 delivered by MVs downregulated TWIST1 expression in VICs, promoting calcification.
- Knockdown of miR-214 in vivo showed a trend towards improving AVC in a mouse model.
Conclusions:
- M1 macrophages promote AVC by delivering miR-214 to VICs via MVs, leading to TWIST1 downregulation.
- The miR-214/TWIST1 axis is a key mediator of M1 macrophage-induced VIC calcification.
- Targeting the miR-214 pathway represents a potential therapeutic strategy for AVC.
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