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 1

USP9X is identified as a suppressor of foam cell formation and negatively correlated with atherosclerosis.

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USP9X is identified as a suppressor of foam cell formation and negativel...
(A) Schematic diagram of the process used to screen for DUBs regulating transformation of macrophages into foam cells and lipid uptake. (B) Oil Red O staining of macrophages transfected with DUB-specific siRNAs. Volcano plot showing fold-change in intensity of staining. Labels indicate significantly upregulated genes. (C) Human monocyte–derived macrophages (HMDMs) were transfected with DUB-specific siRNAs for 48 hours followed by treatment with Dil-ox-LDL (30 μg/mL) for 4 hours. Cellular fluorescence intensity was measured (n = 3). (D) Western blot analysis of the levels of USP9X proteins in mild and severe atherosclerotic lesions in human carotid arteries. Paired 2-tailed Student’s t test (n = 5). (E) ELISA of USP9X protein levels in macrophages of the aorta from Apoe–/– mice fed a Western diet (WD) for 8, 10, and 12 weeks. One-way ANOVA with Bonferroni’s multiple-comparison post hoc test (n = 5). (F) Apoe–/– mice were fed a WD for 10 and 16 weeks. Immunofluorescence analysis of USP9X and CD68 expression in aortic root sections (left). Quantification of the fluorescence intensity of USP9X in CD68+ areas (right). Scale bar: 50 μm. Unpaired 2-tailed Student’s t test (n = 5). (G) Immunofluorescence analysis of USP9X and CD68 expression in severe and mild atherosclerotic lesions in human carotid arteries (left). Data represent the fluorescence intensity of USP9X in CD68+ areas (right). Scale bar: 50 μm. Paired 2-tailed Student’s t test (n = 5).