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TAK1-AMPK Pathway in Macrophages Regulates Hypothyroid Atherosclerosis

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Abstract

Purpose

Hypothyroidism (HT) is associated with accelerated atherosclerosis (AS). The efficacy of traditional strategies of hypothyroid AS remains controversial. Here, we aimed to deepen the understanding of the HT-induced acceleration of AS, to decrease the residual risk of coronary artery disease (CAD) and to find a new therapeutic target.

Methods

We collected peripheral venous blood samples from 20 patients and divided them into 4 groups, namely, the normal group, the HT group, the CAD group and the HT + CAD group. Then we performed mRNA microarray analysis and bioinformatics analysis to screen the differentially expressed genes and pathways, and we also conducted validations on ApoE knockout mice models and Raw264.7 cell models.

Results

In short, (1) in the analysis between the CAD group and the HT + CAD group, we found a total of 1218 differentially expressed genes, 11 upregulated pathways and 40 downregulated pathways. (2) We validated that patients with HT and CAD had a significantly decreased expression of MAP3K7 (encoding transforming growth factor-β-activated kinase 1, TAK1) gene than normal subjects. (3) In animal and cell experiments, we found the decreased expression of TAK1 and the reduced phosphorylation of AMP-activated protein kinase (AMPK) under the hypothyroid and atherosclerotic condition. (4) Changes in the expressions of TAK1 may affect the progression of AS.

Conclusion

Taken together, these data suggest that the accelerated AS in hypothyroid patients may be due to the suppression of TAK1-AMPK pathway in macrophages. This new finding may become a novel therapeutic target in hypothyroid AS.

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Acknowledgements

The authors thank Weiping Tang (Cnkingbio Company Ltd., Beijing, China) for bioinformatics assistance.

Funding

The work was supported by the Natural Science Foundation of China (No. 81670322); the Beijing Natural Science Foundation (No. 7182047); and the Beijing Science and Technology Nova Program Interdisciplinary Fund (No. Z181100006218133).

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Authors and Affiliations

  1. Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, No.2, Anzhen Road, Chaoyang District, Beijing, 100029, China

    Yunxiao Yang, Yifan Jia, Yu Ning, Wanwan Wen, Mengling Huang & Ming Zhang

  2. Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China

    Yanwen Qin, Huina Zhang, Yunhui Du, Linyi Li, Xiaolu Jiao & Yunyun Yang

  3. Endocrinology Department, Tongji Hospital affiliated to School of Medicine, Tongji University, Shanghai, China

    Guanghui Liu

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Contributions

MZ, YXY, YN, YWQ, HNZ, YHD and LYL conceived the idea and designed the study. YN and WWW performed the literature search. YXY, YN, WWW, YFJ and MLH performed the statistical analysis. YXY wrote the first draft. MZ, YXY, YFJ, XLJ and YYY made substantial contributions to study conception and design, interpretation of results and revising the manuscript critically for intellectual content. GHL provided theoretical guidance. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ming Zhang.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Supplemental Figure S1

Microarray analysis and bioinformation analysis in human peripheral blood. Based on the KEGG database, pathways with p <0.05 and FDR < 0.05 were identified as significantly changed pathways. The y-axis shows significantly changed pathways and the x-axis, -Lg P. The larger -Lg P indicates a smaller p-value. ( a ) Significantly enriched pathways in human peripheral blood from all 4 groups (n = 5 in each group). ( b ) Significantly up-regulated pathways in human peripheral blood from the CAD group and the HT + CAD group. Fisher's exact test was used to choose the significant pathways. HT: hypothyroidism; CAD: coronary artery disease. (PNG 3681 kb)

High Resolution Image (TIF 15647 kb)

Supplemental Figure S2

Representative images for transfection of lentivirus targeting MAP3K7 (LV- MAP3K7 ) in Raw264.7 cells. Representative images of Raw264.7 cells after 10-15 days of LV- MAP3K7 infection are shown. A fluorescence microscope was used to measure GFP (green fluorescent protein) expression (Scale bars = 100 μm). (PNG 1430 kb)

High Resolution Image (TIF 7113 kb)

Supplemental Figure S3

Representative Oil Red O stained cells (left) and quantitative assessment (right) (n = 3). (a) 7 5Z-7-Oxozeaenol treated cells exhibited increased accumulation of lipid droplets. RAW264.7 cells were stimulated with 8 or without ox-LDL in the presence or absence of 5Z-7-Oxozeaenol. (b) After the stimulation of ox-LDL, MAP3K7 -9 overexpressing cells ( MAP3K7 -OE) showed reduced accumulation of lipid droplets than control cells (Ctrl-OE, Raw264.7 cells infected with LV-NC). ANOVA followed by Tukey test was used for statistical analysis between 11 experimental sets (Scale bars = 100 μm). ox-LDL: 100 μg/ml, 24 hours; 5Z-7-Oxozeaenol: TAK1 inhibitor, 5 μM, 1 hour; LV-NC: negative control lentivirus. (PNG 5358 kb)

High Resolution Image (TIF 25445 kb)

Supplemental Figure S4

The top 35 genes that appear most frequently in the down-regulated pathway between CAD patients and HT + CAD patients. The y-axis shows the number of involved pathways and the x-axis, genes. HT: hypothyroidism; CAD: coronary artery disease. (PNG 4486 kb)

High Resolution Image (TIFF 577 kb)

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Yang, Y., Jia, Y., Ning, Y. et al. TAK1-AMPK Pathway in Macrophages Regulates Hypothyroid Atherosclerosis. Cardiovasc Drugs Ther 35, 599–612 (2021). https://doi.org/10.1007/s10557-020-06996-w

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