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

动脉粥样硬化血栓形成，由不稳定的动脉粥样硬化斑块破裂或侵蚀引起，是 世界范围内的死亡。然而，调节晚期动脉粥样硬化病变稳定性的机制仍然存在。 人们对此知之甚少。我们实验室最近的研究表明，平滑肌细胞(SMC)条件性基因敲除 载脂蛋白E-/-小鼠中的血小板衍生生长因子受体-β(PdgfrbSMC-Δ/Δ)与几乎完全失败相关 SMC投资于皮损或纤维帽。然而，令人惊讶的是，我们没有观察到病变大小或 西方饮食(WD)18周后斑块稳定性的指标，包括Acta2+纤维帽的厚度 喂食。进一步的研究为这些变化背后的机制提供了新的见解。钥匙 研究结果包括：1)与长期以来认为Acta2+纤维帽细胞几乎完全起源的教条相反 在SMC中，我们发现它们只占晚期载脂蛋白E-/-头臂(BCA)患者Acta2+细胞的60%-70%。 或人类冠状动脉病变，其余来自内皮细胞到间充质细胞到肌成纤维细胞 转化(EndoMT-MFT，20-25%)和巨噬细胞-肌成纤维细胞转化(MMFT，10-15%)； SMC对SMC PDGFRb KO病变的投资损失与以下方面的适应性增加有关 EndoMT-MFT和MMFT；3)当WD时，EndoMT-MFT增加，MMFT不维持稳定性指标 喂养延长到26周，这表明Acta2+纤维帽细胞可能存在质的差异 4)18周龄PdgfrbSMC-Δ/Δ载脂蛋白E-/-小鼠BCA的rna-seq分析 对照仔鼠显示能量代谢途径是十大上调途径，提示 SMC-MF、EndoMT-MF和MMF转变可能需要代谢重新编程；以及5)抑制 体外培养的SMC中的有氧糖酵解可阻止其在PDGF和转化生长因子β处理后转变为MF状态。 这项建议中的研究将检验SMC、EC和巨噬细胞维持适应性反应的假设 斑块的稳定性需要能量代谢的重大变化，这些细胞的代谢状态可能是 操作以刺激病变细胞表型的有益变化和斑块稳定性的整体增加。 目标1将确定SMC和EC谱系中是否存在基因敲除或药物抑制的有氧糖酵解 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 确定是否遗传或药物促进SMC和EC谱系追踪载脂蛋白E-/-谱系中的有氧糖酵解 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。对这两个目标的研究将 包括使用scRNAseq和来自稳定无症状与不稳定的组织学数据的人类验证研究 有症状的颈动脉内膜切除术样本。所有研究都将评估晚期病变的性别相关决定因素 发病机制。综上所述，拟议的研究将提供关于 细胞的代谢重编程有助于纤维的细胞和细胞外基质(ECM)的组成 并可能导致开发新的药理和饮食方法来增强斑块的稳定性。

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.