{
  "abstract": "Introduction Vascular smooth muscle cells (VSMCs) orchestrate atherosclerosis (AS) growth and plaque stability via their dynamic transitions in phenotype, extracellular matrix remodeling, and adaptation to inflammatory stress. Accumulating evidence indicates that dysregulated circadian clock circuitry and defective efferocytosis in VSMCs are key drivers of plaque expansion and vulnerability. Nevertheless, the upstream regulators that link VSMC stress signaling to clock integrity and apoptotic cell clearance remain poorly defined. Protein kinase N2 (PKN2) is a serine/threonine kinase with established roles in cytoskeletal and inflammatory pathways, but whether PKN2 governs VSMC clock programs and efferocytosis to modulate plaque stabilization is unknown.Methods Cellular-specific PKN2 knockout mice on ApoE-/- genetic background were fed a Western diet and subjected to carotid tandem stenosis to induce atherosclerosis and vulnerable plaque, respectively. Aortic RNA-seq and multiple mechanistic studies were performed to define potential downstream signaling programs. In vivo AAV-mediated PKN2 downstream gene overexpression was conducted to confirm their functional implication in PKN2-mediated atherosclerosis and plaque vulnerability. The potential effects PKN2 on plaque-like phenotypes and efferocytosis were further validated in a human-relevant AS model using stem cell-derived human vascular organoids.Results PKN2 phosphorylation was significantly decreased in AS. Smooth muscle specific Pkn2 deletion exacerbated atherosclerosis and promoted plaque destabilization. Aortic RNA-seq identified marked circadian pathway disruption, with significant suppression of core clock components including BMAL1, NPAS2, and CLOCK. Mechanistically, PKN2 deficiency decreased c-Myc Thr58 phosphorylation, reducing c-Myc turnover and perturbing clock regulatory circuitry, culminating in repression of BMAL1-centred transcriptional factors. VSMC-directed AAV–BMAL1 overexpression significantly ameliorated Pkn2 deficiency–driven lesion worsening and instability. Functionally, PKN2–BMAL1 impairment was linked to defective efferocytosis. Specifically, PKN2 loss increased the ‘don’t-eat-me’ checkpoint CD47, limiting phagocyte clearance of apoptotic VSMCs and promoting AS. Translationally, doxycycline-inducible PKN2 overexpression in stem cell-derived human vascular organoids mitigated atherosclerosis-like phenotypes (lesion/plaque-like area expansion, inflammatory activation, necrotic core–like features) and improved efferocytosis-associated readouts.Conclusions PKN2 preserves VSMC circadian clock circuitry (BMAL1/NPAS2/CLOCK) and promotes VSMC efferocytosis to limit atherosclerotic plaque growth and reduce plaque vulnerability. Targeting the PKN2–c-Myc–circadian clock–efferocytosis axis may offer a novel strategy to attenuate plaque progression and reinforce plaque stability.",
  "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": "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": "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": "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"
      ],
      "name": "Haotian Zhao"
    },
    {
      "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": "167 Protein kinase N2 preserves VSMC clock integrity and efferocytosis to limit atherosclerotic plaque growth and vulnerability",
  "uid": "24929d02-3b34-5c19-9fcc-4bb8f2ac5b18"
}