ILF3 is responsible for hyperlipidemia-induced arteriosclerotic calcification by mediating BMP2 and STAT1 transcription

https://doi.org/10.1016/j.yjmcc.2021.07.011Get rights and content

Highlights

  • ā€¢

    ILF3 promotes the osteogenic switch of VSMCs and macrophages in atherosclerotic calcification.

  • ā€¢

    ILF3 regulates BMP2 and STAT1 transcription by targeting their promoter regions.

  • ā€¢

    ILF3 mediates upregulation of BMP2 and suppression of STAT1 expression in atherosclerotic calcification.

  • ā€¢

    Inhibition of ILF3 may be a potential therapeutic target for preventing atherosclerotic calcification and lesion rupture.

Abstract

Calcification is common in atherosclerotic plaque and can induce vulnerability, which further leads to myocardial infarction, plaque rupture and stroke. The mechanisms of atherosclerotic calcification are poorly characterized. Interleukin enhancer binding factor 3 (ILF3) has been identified as a novel factor affecting dyslipidemia and stroke subtypes. However, the precise role of ILF3 in atherosclerotic calcification remains unclear. In this study, we used smooth muscle-conditional ILF3 knockout (ILF3SM-KO) and transgenic mice (ILF3SM-Tg) and macrophage-conditional ILF3 knockout (ILF3M-KO) and transgenic (ILF3M-Tg) mice respectively. Here we showed that ILF3 expression is increased in calcified human aortic vascular smooth muscle cells (HAVSMCs) and calcified atherosclerotic plaque in humans and mice. We then found that hyperlipidemia increases ILF3 expression and exacerbates calcification of VSMCs and macrophages by regulating bone morphogenetic protein 2 (BMP2) and signal transducer and activator of transcription 1 (STAT1) transcription. We further explored the molecular mechanisms of ILF3 in atherosclerotic calcification and revealed that ILF3 acts on the promoter regions of BMP2 and STAT1 and mediates BMP2 upregulation and STAT1 downregulation, which promotes atherosclerotic calcification. Our results demonstrate the effect of ILF3 in atherosclerotic calcification. Inhibition of ILF3 may be a useful therapy for preventing and even reversing atherosclerotic calcification.

Graphical abstract

Our findings illustrate the working schematic that hyperlipidemia-induced ILF3 activation mediates acceleration of atherosclerotic calcification. Hyperlipidemia-increased ILF3 expression mediates BMP2 and STAT1 transcription by directly binding to their promoter regions. ILF3 upregulates BMP2 level and activates Smads signaling to elevate Runx2 transcription. Meanwhile, ILF3 suppresses STAT1 transcription, which promotes Runx2 nuclear translocation and regulates osteogenic differentiation. In addition, ILF3-mediated hyperlipidemia induces a phenotypic switch of VSMCs from contractile to a dedifferentiated synthetic phenotype and macrophages to a pro-inflammatory M1 phenotype, which in turn aggravates VSMC calcification to promote atherosclerotic calcification.

Unlabelled Image
  1. Download : Download high-res image (314KB)
  2. Download : Download full-size image

Introduction

Vascular calcification is a common phenomenon in many physiological and pathological diseases including aging, end-stage renal disease, diabetes mellitus and cardiovascular diseases [1,2]. Vascular calcification can occur in different locations of the vessel wall including intima and media but exists mainly in intimal layers in atherosclerosis and can induce atherosclerotic plaque susceptibility and further lead to myocardial infarction, plaque rupture and stroke [3].

Recent studies suggested that vascular calcification is an active cell regulatory process characterized by the involvement of various cells such as vascular smooth muscle cells (VSMCs), pericytes, myofibroblasts, macrophages, vascular mesenchymal progenitors and endothelial cells [[4], [5], [6], [7]]. Under multiple pro-calcific stimuli, VSMCs can undergo a phenotype switch from a contractile to osteoblastic phenotype accompanied by loss of contractile markers (smooth muscle 22 alpha [SM22Ī±], calponin and alpha smooth muscle actin [Ī±-SMA]) and an increase in levels of bone-related genes (runt-related transcription factor 2 [Runx2], osteopontin [OPN], osteocalcin, bone morphogenetic protein 2 [BMP2], and Msx and become the main source of osteoblastic cells, which leads to vascular calcification. In addition, macrophages can undergo a phenotype shift and participate in atherosclerotic calcification [5,8]. Because of the diversity and complexity of calcification mechanisms, ideal drugs preventing or reversing atherosclerotic calcification are unavailable. The underlying molecular mechanisms of atherosclerotic calcification still need further study.

Interleukin enhancer-binding factor 3 (ILF3), as a double-stranded RNA (dsRNA)-binding protein, combines with other proteins, mRNAs, small noncoding RNAs, and dsRNAs to regulate transcription, translation, mRNA stability and noncoding RNA biogenesis [9]. In the cardiovascular system, ILF3 can inhibit myocardial hypertrophy [10]. Also, the association between ILF3 and myocardial infarction is affected by low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol metabolism, which indicates interactions between genes [11]. Recent studies have reported insights into the possible physiological roles of ILF3 in stroke, inflammation, and dyslipidaemia, but its role in vascular calcification has not been reported.

In this study, we used human samples and murine models with conditional ILF3 knockout and overexpression in VSMCs and macrophages to explore the roles of ILF3 in atherosclerotic calcification.

Section snippets

Body weight (BW) and serum indexes in mice

Mice BW and serum levels of TC, TG, BG, HDL-C, LDL-C, calcium and phosphorus are in Tables 1 and 2. Both ApoEāˆ’/-ILF3SM-KO and ApoEāˆ’/-ILF3M-KO mice showed lower BW and TC, TG and LDL-C levels but higher HDL-C level than ApoEāˆ’/āˆ’ mice (*PĀ <Ā 0.05). For all ILF3-overexpressed mice, BW and TC, TG and LDL-C levels were higher but HDL-C level was lower relative to ApoEāˆ’/āˆ’mice (*PĀ <Ā 0.05). BG, calcium and phosphorus levels did not differ between groups. The one that plays the most main role in

Discussion

ILF3 has been verified to play important roles in dyslipidemia and the cardiovascular system [10,11]. However, whether ILF3 is linked to dyslipidemia-induced atherosclerotic calcification has not been reported. In the present study, we used ILF3 conditional genetic deletion and transgenic mouse models to investigate the role of ILF3 in atherosclerotic calcification. Hyperlipidemia could augment ILF3 expression in calcified VSMCs and macrophages and in atherosclerotic calcification in humans and

Human coronary artery samples

Atherosclerotic and control epicardial coronary artery segments were from human specimens with atherosclerotic disease. The specimens were donated by the Shandong Red Cross Society.The experiment protocols were examined and approved by the review committee of Qilu Hospital, Jinan, China(ethics approval No. KYLL-2018(KS)-233).

Animals

The study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (#91839301 and #81970251), the National key R & D program of China (#2017YFC0908700, 2017YFC0908703) and the Taishan Scholar Project of Shandong Province of China (No. ts20190972).

References (34)

  • Y. Sun et al.

    Smooth muscle cell-specific runx2 deficiency inhibits vascular calcification

    Circ. Res.

    (2012)
  • Y. Fu et al.

    Shift of macrophage phenotype due to cartilage oligomeric matrix protein deficiency drives atherosclerotic calcification

    Circ. Res.

    (2016)
  • A.S.A. Bardeesi et al.

    A novel role of cellular interactions in vascular calcification

    J. Transl. Med.

    (2017)
  • P. Li et al.

    Correction: loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis

    Cell Death Dis.

    (2020)
  • S. Castella et al.

    Ilf3 and NF90 functions in RNA biology

    Wiley Interdiscip. Rev. RNA.

    (2015)
  • T. Yoshida et al.

    Association of polymorphisms of BTN2A1 and ILF3 with myocardial infarction in Japanese individuals with different lipid profiles

    Mol. Med. Rep.

    (2011)
  • D. Noel et al.

    Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells

    Stem Cells

    (2004)
  • Cited by (9)

    • Programmed prodrug breaking the feedback regulation of P-selectin in plaque inflammation for atherosclerotic therapy

      2022, Biomaterials
      Citation Excerpt :

      Therefore, LI NPs could alleviate arteriosclerosis by a cascade anti-inflammatory pathway around P-selectin. In addition, previous studies had shown that there was a causal relationship between hyperlipidemia and arteriosclerosis [35,36]. Increased blood lipid content would accelerate endothelial damage and cause inflammation.

    View all citing articles on Scopus
    View full text