{
  "abstract": "Introduction Vascular calcification (VC) driven by osteogenic switching of vascular smooth muscle cells (VSMCs) is associated with increased cardiovascular event risks, with no effective therapies. We have previously demonstrated that nuclear factor erythroid 2-related factor 3 (Nrf3) is a critical regulator in VSMC differentiation as well as injury-induced neointimal hyperplasia and pathological cardiac remodeling. However, little is known about its potential role in VC. Herein, we aimed to explore the functional implication of Nrf3 in VC and elucidate its associated molecular mechanism.Methods Publicly available single-cell RNA sequencing data from human atherosclerosis, along with atherosclerotic plaque and calcified arteries from chronic kidney disease patients, were used to assess the correlation of Nrf3 expression with calcification severity. VC was induced via vitamin D overload or an adenine plus high phosphorus diet in both global and VSMC-specific Nrf3 knockout mice. Nrf3 target genes were identified by integrated metabolomic and transcriptomic analyses, which were further validated by several functional assays. Mendelian randomization analysis was used to examine the causal effect of human Nrf3 genetic variants on serum eicosapentaenoic acid (EPA) levels. The potential effects of Nrf3 on VC were further validated in a human-relevant VC model using stem cell-derived vascular organoids.Results Nrf3 expression was significantly elevated in VSMCs of calcified human plaques and was positively correlated with the calcification burden. Both global and VSMC-specific Nrf3 knockout mice exhibited attenuated VC. RNA-seq analysis of calcified aortas from VSMC-specific Nrf3 knockout mice showed downregulation of fatty acid oxidation (FAO) enzymes. Seahorse assays and 13C-palmitate tracing confirmed that Nrf3 silencing attenuated FAO in calcified VSMCs. Subsequent qPCR and western blot analysis confirmed that carnitine palmitoyltransferase 1a (CPT1A), the rate-limiting enzyme for FAO, was a key mediator of Nrf3-accelerated FAO. ChIP-qPCR and dual-luciferase assays demonstrated that Nrf3 directly binds to and transactivates the CPT1A promoter, promoting VSMC calcification. Meanwhile, fatty acid metabolomics profiling showed that Nrf3 silencing elevated omega-3 unsaturated fatty acids, particularly EPA. The roles of CPT1A and EPA in Nrf3-mediated calcification were further validated in two independent in vivo models. Mendelian randomization analysis supported a causal association between Nrf3 locus variants and circulating EPA concentrations. Finally, doxycycline-inducible Nrf3 overexpression or knockdown in stem cell-derived human vascular organoids promoted and mitigated VC phenotypes, respectively.Conclusions Nrf3 is novel regulator in VC and it promotes VC by accelerating FAO and eicosapentaenoic acid degradation. Targeting the Nrf3–CPT1A–FAO axis in combination with EPA supplementation may constitute a promising treatment approach for VC.",
  "authors": [
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Xiaoyue Zhou"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Leyu Wang"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Ancheng Zheng"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Mei Yang"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Zhenning Shi"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom",
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Qishan Chen"
    },
    {
      "affiliations": [
        "Department of Cardiology, Institute for Developmental and Regenerative Cardiovascular Medicine, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China"
      ],
      "name": "Li Zhang"
    },
    {
      "affiliations": [
        "Centre for Clinical Pharmacology and Precision Medicine, William Harvey Research Institute, Faculty of Medicine and Dentistry, William Harvey Research, Queen Mary University of London, London, United Kingdom"
      ],
      "name": "Qingzhong Xiao"
    }
  ],
  "title": "169 Nrf3 promotes vascular calcification by accelerating fatty acid oxidation and eicosapentaenoic acid degradation",
  "uid": "49e028ee-d9ef-5748-8b89-1a5e45b2f4b2"
}