Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis
Biqing Wang, … , Hongfeng Jiang, Ding Ai
Biqing Wang, … , Hongfeng Jiang, Ding Ai
Published April 7, 2022
Citation Information: J Clin Invest. 2022;132(10):e154217. https://doi.org/10.1172/JCI154217.
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Research Article Cardiology Immunology

Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis

Abstract

Subendothelial macrophage internalization of modified lipids and foam cell formation are hallmarks of atherosclerosis. Deubiquitinating enzymes (DUBs) are involved in various cellular activities; however, their role in foam cell formation is not fully understood. Here, using a loss-of-function lipid accumulation screening, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a factor that suppressed lipid uptake in macrophages. We found that USP9X expression in lesional macrophages was reduced during atherosclerosis development in both humans and rodents. Atherosclerotic lesions from macrophage USP9X-deficient mice showed increased macrophage infiltration, lipid deposition, and necrotic core content than control apolipoprotein E–KO (Apoe–/–) mice. Additionally, loss-of-function USP9X exacerbated lipid uptake, foam cell formation, and inflammatory responses in macrophages. Mechanistically, the class A1 scavenger receptor (SR-A1) was identified as a USP9X substrate that removed the K63 polyubiquitin chain at the K27 site. Genetic or pharmacological inhibition of USP9X increased SR-A1 cell surface internalization after binding of oxidized LDL (ox-LDL). The K27R mutation of SR-A1 dramatically attenuated basal and USP9X knockdown–induced ox-LDL uptake. Moreover, blocking binding of USP9X to SR-A1 with a cell-penetrating peptide exacerbated foam cell formation and atherosclerosis. In this study, we identified macrophage USP9X as a beneficial regulator of atherosclerosis and revealed the specific mechanisms for the development of potential therapeutic strategies for atherosclerosis.

Authors

Biqing Wang, Xuening Tang, Liu Yao, Yuxin Wang, Zhipeng Chen, Mengqi Li, Naishi Wu, Dawei Wu, Xiangchen Dai, Hongfeng Jiang, Ding Ai

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Figure 3

Macrophage USP9X deficiency promotes foam cell formation.

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Macrophage USP9X deficiency promotes foam cell formation. (A) Oil Red O ...
(A) Oil Red O staining of bone marrow–derived macrophages (BMDMs) isolated from Usp9xfl/fl and Mac Usp9xKO mice and incubated with or without ox-LDL (50 μg/mL) for 24 hours. Scale bar: 20 μm. Two-way ANOVA with Bonferroni’s post hoc test (n = 5). (B) BMDMs were elicited from Usp9xfl/fl and Mac Usp9xKO mice and treated with Dil-ox-LDL (30 μg/mL) for 4 hours. Scale bar: 10 μm. Unpaired 2-tailed Student’s t test (n = 5). (C) Oil Red O staining of HMDMs transfected with siCtrl or siUSP9X for 48 hours and incubated with ox-LDL for a further 24 hours. Scale bar: 20 μm. Unpaired 2-tailed Student’s t test (n = 5). (D) Representative images of Dil-ox-LDL uptake in HMDMs transfected with siCtrl or siUSP9X for 48 hours. Scale bar: 10 μm. Unpaired 2-tailed Student’s t test (n = 5). (E) Plaque macrophages were isolated from aorta of Usp9xfl/fl Apoe–/– or Mac Usp9xKO Apoe–/– mice fed a Western diet (WD) for 12 weeks. Quantitative PCR was performed to detect the mRNA levels of the indicated genes. Target gene expression was normalized to the level of Actb mRNA. Unpaired 2-tailed Student’s t test (n = 5). (F) Western blot analysis of indicated proteins in BMDMs from Usp9xfl/fl and Mac Usp9xKO mice. (G) Quantification of F. Unpaired 2-tailed Student’s t test (n = 5). (H) Cholesterol efflux assay of BMDMs isolated from Usp9xfl/fl and Mac Usp9xKO mice. Two-way ANOVA with Bonferroni’s post hoc test (n = 5).