Role of Metabolic Reprogramming in Formation and Maintenance of the Acta2+ Atherosclerotic Lesion Protective Fibrous Cap

由不稳定的动脉粥样硬化斑块破裂或侵蚀引起的动脉粥样硬化血栓形成是动脉粥样硬化的主要原因。 全世界的死亡然而，调节晚期动脉粥样硬化病变稳定性的机制仍然存在 不太了解。我们实验室的最新研究表明，平滑肌细胞（SMC）条件性敲除 ApoE-/-小鼠中血小板衍生生长因子受体-β（PdgfrbSMC-Δ/Δ）与几乎完全衰竭相关 的SMC投资到病变或纤维帽。然而，令人惊讶的是，我们没有观察到病变大小的变化， 18周西方饮食（WD）后的斑块稳定性指数，包括Acta 2+纤维帽的厚度 喂食进一步的研究提供了关于这些变化的机制的新见解。关键 结果包括：1）与长期以来的教条相反，Acta 2+纤维帽细胞几乎完全来自 从SMC中，我们发现它们仅占晚期ApoE-/-头臂型（BCA）中Acta 2+细胞的60-70%。 或人冠状动脉病变，其余部分来自内皮细胞-间充质细胞-肌成纤维细胞 2）巨噬细胞向肌成纤维细胞转化（EndoMT-MFT，20-25%）和巨噬细胞向肌成纤维细胞转化（MMFT，10-15%）; SMC PDGFRB KO病变中SMC投资的损失与SMC PDGFRB KO的适应性增加有关。 EndoMT-MFT和MMFT; 3）当WD时，EndoMT-MFT增加，MMFT不维持稳定指数 饲养时间延长至26周，表明Acta 2+纤维帽细胞可能存在质的差异， 4）对18周WD喂养的PdgfrbSMC-Δ/Δ ApoE-/-小鼠的BCA进行RNA-seq分析， 对照同窝小鼠显示能量代谢途径是前十个上调的途径， SMC-MF、EndoMT-MF和MMF转换可能需要代谢重编程;和5）抑制 在培养的SMC中有氧糖酵解阻止了它们在用PDGF和TGFβ处理后向MF状态的转变。 本研究将验证SMC、EC和巨噬细胞对维持细胞增殖的适应性反应的假设。 斑块的稳定性需要能量代谢的重大转变，并且这些细胞的代谢状态可以 以刺激病变细胞表型的有益变化和斑块稳定性的总体增加。 目的1将确定是否基因敲除或药物抑制SMC和EC谱系的有氧糖酵解 追踪患有晚期动脉粥样硬化的ApoE-/-小鼠与斑块稳定性降低的证据相关。目标2将 确定SMC和EC谱系中有氧糖酵解的遗传或药理学促进是否追踪ApoE-/-谱系 追踪患有晚期动脉粥样硬化的小鼠与斑块稳定性增加有关。为这两个目标进行的研究将 包括使用scRNAseq的人类验证研究和来自稳定的无症状与不稳定的 症状性颈动脉内膜切除术样本。所有研究将评估晚期病变的性别依赖性决定因素 发病机制总之，拟议的研究将提供有关机制的新见解， 细胞的代谢重编程有助于纤维组织的细胞和细胞外基质（ECM）组成。 并可能导致开发新的药理学和饮食方法来增强斑块稳定性。

Atherothrombosis, resulting from rupture or erosion of unstable atherosclerotic plaques, is the leading cause of death worldwide. However, the mechanisms that regulate the stability of late stage atherosclerotic lesions remain poorly understood. Recent studies from our lab showed that smooth muscle cell (SMC) conditional knockout of the platelet-derived growth factor receptor-β (PdgfrbSMC-Δ/Δ) in ApoE-/- mice was associated with nearly complete failure of SMC to invest into lesions or the fibrous cap. However, surprisingly we observed no changes in lesion size or indices of plaque stability, including the thickness of the Acta2+ fibrous cap, following 18 weeks of Western diet (WD) feeding. Further investigation provided novel insights regarding the mechanisms underlying these changes. Key findings included: 1) contrary to long-standing dogma that Acta2+ fibrous cap cells are derived almost exclusively from SMC, we showed that they account for only 60-70% of Acta2+ cells in advanced ApoE-/- brachiocephalic (BCA) or human coronary artery lesions with the remainder coming from endothelial cell-to-mesenchymal-to-myofibroblast transitions (EndoMT-MFT, 20-25%) and macrophage-to-myofibroblast transitions (MMFT), 10-15%) respectively; 2) loss of SMC investment into lesions with SMC PDGFRB KO was associated with large adaptive increases in EndoMT-MFT and MMFT; 3) increased EndoMT-MFT, and MMFT did not sustain indices of stability when WD feeding was extended to 26 weeks suggesting there may be qualitative differences in Acta2+ fibrous cap cells depending on their origin; 4) RNA-seq analysis on the BCA of 18-week WD-fed PdgfrbSMC-Δ/Δ ApoE-/- mice versus control littermate mice showed that energy metabolism pathways were the top ten upregulated pathways suggesting that metabolic reprogramming may be required for SMC-MF, EndoMT-MF, and MMF transitions; and 5) inhibition of aerobic glycolysis in cultured SMC prevented their transition to a MF state following treatment with PDGF and TGFβ. Studies in this proposal will test the hypothesis that SMC, EC, and macrophage adaptive responses for maintaining plaque stability require major shifts in energy metabolism and that the metabolic state of these cells can be manipulated to stimulate beneficial changes in the phenotype of lesion cells and overall increases in plaque stability. Aim 1 will determine if genetic knockout or pharmacologic inhibition of aerobic glycolysis in SMC and EC lineage tracing ApoE-/- mice with advanced atherosclerosis is associated with evidence of reduced plaque stability. Aim 2 will determine if genetic or pharmacologic promotion of aerobic glycolysis in SMC and EC lineage tracing ApoE-/- lineage tracing mice with advanced atherosclerosis is associated with increased plaque stability. Studies for both aims will include human validation studies using scRNAseq and histological data from stable asymptomatic versus unstable symptomatic carotid endarterectomy samples. All studies will assess sex-dependent determinants of late-stage lesion pathogenesis. Taken together, the proposed studies will provide novel insights regarding mechanisms by which metabolic reprogramming of cells contribute to the cellular and extracellular matrix (ECM) composition of the fibrous cap and may lead to development of novel pharmacological and dietary approaches for enhancing plaque stability.