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MiR-199a-5p-containing macrophage-derived extracellular vesicles inhibit SMARCA4 and alleviate atherosclerosis by reducing endothelial cell pyroptosis

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  • Published:
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Abstract

Background

Endothelial cell disturbance underpins a role in pathogenesis of atherosclerosis. Notably, accumulating studies indicate the substantial role of microRNAs (miRs) in atherosclerosis, and miR-199a-5p dysregulation has been associated with atherosclerosis and other cardiovascular disorders. However, the effect of miR-199a-5p on the phenotypes of endothelial cells and atherosclerosis remains largely unknown.

Methods

ApoE−/− male mice were fed with high-fat diet for detection of inflammation and aorta plaque area. Extracellular vesicles (EVs) were separated from THP-1-derived macrophage (THP-1-DM) that was treated by oxidized low-density lipoprotein, followed by co-culture with human aortic endothelial cells (HAECs). Ectopic expression and downregulation of miR-199a-5p were done in THP-1-DM-derived EVs to assess pyroptosis and lactate dehydrogenase (LDH) of HAECs. Binding relationship between miR-199a-5p and SMARCA4 was evaluated by luciferase activity assay.

Results

EVs derived from ox-LDL-induced THP-1-DM expedited inflammation and aorta plaque area in atherosclerotic mice. Besides, miR-199a-5p expression was reduced in EVs from ox-LDL-induced THP-1-DM, and miR-199a-5p inhibition facilitated HAEC pyroptosis and LDH activity. Moreover, miR-199a-5p targeted and restricted SMARCA4, and then SMARCA4 activated the NF-κB pathway by increasing PODXL expression in HAECs.

Conclusion

EV-packaged inhibited miR-199a-5p from macrophages expedites endothelial cell pyroptosis and further accelerates atherosclerosis through the SMARCA4/PODXL/NF-κB axis, providing promising targets and strategies for the prevention and treatment of atherosclerosis.

Graphical abstract

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Data availability

The datasets generated and/or analyzed during the current study are available in the manuscript and supplementary materials.

Code availability

Not applicable.

Abbreviations

miRs:

MicroRNAs

EVs:

Extracellular vesicles

THP-1-DM:

THP-1-derived macrophage

HAECs:

Human aortic endothelial cells

LDH:

Lactate dehydrogenase

EVs:

Extracellular vesicles

SMARCA4:

SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a, member 4

PODXL:

Podocalyxin like

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

un-EVs:

EVs from DMSO-treated THP-1-DMs

FBS:

Fetal bovine serum

PMA:

Phorbol-12-myristate-13-acetate

ox-LDL:

Oxidized low-density lipoprotein

PBS:

Phosphate-buffered saline

NC:

Negative control

GFP:

Green fluorescent protein

TEM:

Transmission electron microscope

DAPI:

4′,6-Diamidino-2-phenylindole

LDH:

Lactate dehydrogenase

ELISA:

Enzyme-linked immunosorbent assay

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Funding

This work was funded by Guangzhou City Health and Family Planning Science and Technology Project (No.20181A011114).

Author information

Authors and Affiliations

  1. Department of Cardiology, Cardiovascular Institute of Panyu District, Panyu Central Hospital, No. 8, Fuyu East Road, Qiaonan Street, Panyu District, Guangzhou, 511400, Guangdong Province, People’s Republic of China

    Weijie Liang, Jun Chen, Hongyan Zheng, Aiwen Lin, Jianhao Li & Qiang Jie

  2. Department of Endocrinology, Guangdong Geriatrics Institute, Guangdong Academy of Medical Sciences, Guangdong Provincial People’s Hospital, No. 106, Zhongshan Second Road, Yuexiu District, Guangzhou, 510080, Guangdong Province, People’s Republic of China

    Wen Wu

Authors
  1. Weijie Liang
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  2. Jun Chen
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  3. Hongyan Zheng
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  4. Aiwen Lin
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  5. Jianhao Li
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  6. Wen Wu
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Contributions

Weijie Liang, Jun Chen, and Hongyan Zheng wrote the paper; Aiwen Lin, Jianhao Li, and Wen Wu conceived the experiments; Qiang Jie and Weijie Liang analyzed the data; Jun Chen and Hongyan Zheng collected and provided the sample for this study. All the authors have read and approved the final submitted manuscript.

Corresponding authors

Correspondence to Wen Wu or Qiang Jie.

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Ethics approval

The experiments involved animals were implemented under approval of the animal ethics committee of Guangdong Provincial People's Hospital (Approval number: NO.GDREC2019275A).

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The authors declare no competing interests.

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Graphical Headlights

1. ox-LDL downregulates miR-199a-5p expression in macrophage-derived EVs.

2. EVs-miR-199a-5p inhibits endothelial pyroptosis.

3. EVs-miR-199a-5p targets SMARCA4 and inhibits PODXL/NF-κB axis to reduce endothelial pyroptosis.

4. EVs-miR-199a-5p inhibits endothelial pyroptosis and ultimately alleviates atherogenesis.

Supplementary Information

Below is the link to the electronic supplementary material.

10565_2022_9732_MOESM1_ESM.jpg

Supplementary file1 (JPG 333 kb) SUPPLEMENTARY FIGURE 1 Morphology of isolated mouse aortic ECs in microscopic observation.

10565_2022_9732_MOESM2_ESM.jpg

Supplementary file2 (JPG 1355 kb) SUPPLEMENTARY FIGURE 2 ox-LDL-hMDM-EVs and hMDM-EVs-miR-199a-5p participate in the atherosclerosis of ApoE-/- mice. A, The lesion area on the frontal plaque of mouse aorta in response to EVs derived from hMDMs treated with/without ox-LDL (un-EVs and ox-LDL-EVs), as detected by oil red O staining. B, The lesion area of mice aortic root plaque in response to un-EVs or ox-LDL-EVs, as detected by oil red O staining. C, ELISA assay showing the content of inflammation-related factors in mice serum in response to un-EVs or ox-LDL-EVs. D, The expression level of miR-199a-5p in hMDMs treated with/without ox-LDL, as determined with qRT-PCR. E, The expression level of miR-199a-5p in un-EVs or ox-LDL-EVs, as determined with qRT-PCR. F, qRT-PCR detection of the efficiency of lentivirus-mediated transduction of miR-199a-5p mimic/inhibitor. G, The expression level of miR-199a-5p in EVs derived from miR-199a-5p-overexpressing/inhibiting cells (EV-miR-199a-5p/EV-miR-199a-5p-inhibitor). H, The lesion area on the frontal plaque of mouse aorta in response to EV-miR-199a-5p/EV-miR-199a-5p-inhibitor, as detected by oil red O staining. I, The lesion area of mice aortic root plaque in response to EV-miR-199a-5p/EV-miR-199a-5p-inhibitor, as detected by oil red O staining. J, ELISA assay showing the content of inflammation-related factors in mice serum in response to EV-miR-199a-5p/EV-miR-199a-5p-inhibitor. n = 10. *p < 0.05, **p < 0.01, ***p < 0.001. Cell experiments were repeated 3 times.

10565_2022_9732_MOESM3_ESM.jpg

Supplementary file3 (JPG 291 kb) SUPPLEMENTARY FIGURE 3 Detection of serum lipids following treatment with THP-1-DMs and hMDMs and corresponding EVs. A, The level of ox-LDL in HP-1-DMs and hMDMs treated with/without ox-LDL and in corresponding EVs, as determined with an ox-LDL detection kit. B, Serum levels of TC, TG, LDL-C and HDL-C in mice treated with un-EVs/ox-LDL-EVs. n = 10. *p < 0.05, **p < 0.01, ***p < 0.001. Cell experiments were repeated 3 times.

10565_2022_9732_MOESM4_ESM.jpg

Supplementary file4 (JPG 368 kb) SUPPLEMENTARY FIGURE 4 miR-199a-3p expression following miR-199a-5p mimic/inhibitor and miR-199a-5p/miR-199a-3p sequence. A, The expression of miR-199a-3p in HAECs treated with miR-199a-5p mimic/inhibitor. B, Sequences of miR-199a-5p and miR-199a-3p. *p < 0.05. Cell experiments were repeated 3 times.

10565_2022_9732_MOESM5_ESM.jpg

Supplementary file5 (JPG 584 kb) SUPPLEMENTARY FIGURE 5 The expression of miR-199a-5p in atherosclerotic mice and HAECs and the EV internalization over LSS exposure time. A, The expression of miR-199a-5p in atherosclerotic mice and control mice. B, The expression of miR-199a-5p in HAECs in response to LSS treatment for 0, 30, 60 and 120 min. C, Laser scanning confocal microscopy to detect EV internalization by HAECs under exposure to LSS, as reflected by the fluorescence intensity of PKH26 (red), with DAPI-labeled nuclei in blue. n = 10. *p < 0.05, **p < 0.01.

Supplementary file6 (DOCX 17 kb)

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Liang, W., Chen, J., Zheng, H. et al. MiR-199a-5p-containing macrophage-derived extracellular vesicles inhibit SMARCA4 and alleviate atherosclerosis by reducing endothelial cell pyroptosis. Cell Biol Toxicol 39, 591–605 (2023). https://doi.org/10.1007/s10565-022-09732-2

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