BACKGROUND:While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood.METHODS:Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in nearly 19 000 single cells isolated during atherosclerosis progression in Ldlr^−/−Apob^100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study.RESULTS:Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity (SYNTAX/Duke scores). The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM, in cultured human vascular SMCs.CONCLUSIONS:By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced-stage, symptomatic atherosclerosis.

中文翻译：

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晚期症状性动脉粥样硬化的单细胞基因调控网络

背景：虽然我们对动脉粥样硬化进展过程中血管细胞类型转变的单细胞基因表达模式的理解正在迅速提高，但这些变化的临床和病理生理学相关性仍然知之甚少。 方法：生成的单细胞 RNA 测序数据在具有类人血浆脂蛋白的Ldlr ^−/− Apob ^100/100小鼠以及具有无症状和有症状颈动脉斑块的人类中，在动脉粥样硬化进展过程中分离出的近 19 000 个单细胞中，使用 SmartSeq2（约 8000 个基因/细胞）将其分为多个亚型。对于临床和病理生理学背景，晚期和症状亚型簇与 135 个组织特异性（动脉粥样硬化主动脉壁、乳动脉、肝脏、骨骼肌、内脏和皮下、脂肪）基因调节网络（GRN）进行了整合，这些基因调节网络推断自STARNET（斯德哥尔摩-塔尔图动脉粥样硬化逆向网络工程任务）研究中有 600 名冠状动脉疾病患者。结果：动脉粥样硬化进展的晚期阶段和有症状的颈动脉斑块主要以 3 个平滑肌细胞 (SMC) 和 3 个具有细胞外细胞的巨噬细胞亚型簇为特征。基质组织/成骨 (SMC) 和 M1 型促炎/Trem2 高脂质相关（巨噬细胞）表型。通过 STARNET 对这 6 个簇进行综合分析，揭示了 3 个动脉壁 GRN 的显着富集：GRN33（巨噬细胞）、GRN39（SMC）和 GRN122（巨噬细胞），对冠状动脉疾病遗传性有重大贡献，并与冠状动脉粥样硬化严重程度的临床评分密切相关（语法/杜克大学分数）。 GRN39 的存在和病理生理学相关性在从人冠状动脉和主动脉以及原代 SMC 获得的 5 个独立 RNAseq 数据集中得到了验证，并通过在培养的人血管 SMC 中定位其最关键的驱动因素FRZB和ALCAM。结论：通过识别并将迄今为止基因最丰富的动脉粥样硬化单细胞亚群与GRN的冠状动脉疾病框架相整合，GRN39被鉴定并独立验证为对于将收缩性SMC转化为促进晚期症状性动脉粥样硬化的成骨表型至关重要。