ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

Yuejie Zhang; Cen Yan; Yuan Dong; Jiwei Zhao; Xuanyi Yang; Yalan Deng; Li Su; Jiming Yin; Yang Zhang; Fenghui Sun; Yingmei Feng

doi:10.1016/j.jare.2024.05.011

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

J Adv Res. 2024 May 11:S2090-1232(24)00201-7. doi: 10.1016/j.jare.2024.05.011. Online ahead of print.

Authors

Yuejie Zhang¹, Cen Yan¹, Yuan Dong¹, Jiwei Zhao¹, Xuanyi Yang¹, Yalan Deng¹, Li Su², Jiming Yin¹, Yang Zhang³, Fenghui Sun⁴, Yingmei Feng⁵

Affiliations

¹ Beijing Institute of Hepatology, Beijing Youan Hospital, Beijing 100069, China.
² Department of Science and Technology, Beijing Youan Hospital, Beijing 100069, China.
³ Neuroscience Research Institute, Peking University Center of Medical and Health Analysis, Peking University, Beijing 100191, China.
⁴ Department of Neurology, Beijing Youan Hospital, Beijing 100069, China.
⁵ Beijing Institute of Hepatology, Beijing Youan Hospital, Beijing 100069, China; Department of Science and Technology, Beijing Youan Hospital, Beijing 100069, China. Electronic address: yingmeif13@ccmu.edu.cn.

PMID: 38740260
DOI: 10.1016/j.jare.2024.05.011

Abstract

Introduction: The N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3.

Objectives: ANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression.

Methods: Ldlr^-/- mice on a high-fat diet and ApoE^-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE^-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3^-/-ApoE^-/- mice and ApoE^-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system.

Results: Angptl3 overexpression increased atherosclerotic progression and CD68⁺ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG⁺ cells were identified in plaque of gene transferred ApoE^-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05).

Conclusions: Targeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Keywords: ANGPTL3; Atherosclerosis; Macrophages; NF-κB; Toll-like receptor (TLR)4.