Robert M. Berne Cardiovascular Research Center (G.F.A., K.M.O, S.K., A.N., C.M.W., S.S., C.A.H., R.H., R.A.B., C.A.M., E.R.Z., G.K.O.), University of Virginia, Charlottesville.
Department of Biochemistry and Molecular Genetics (G.F.A., K.M.O., C.A.H., R.A.B., S.B.), University of Virginia, Charlottesville.
Circulation. 2020 Nov 24;142(21):2045-2059. doi: 10.1161/CIRCULATIONAHA.120.046672. Epub 2020 Jul 17.
Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability.
We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis.
We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11, Lgals3 population with a chondrocyte-like gene signature that was markedly reduced with SMC- knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization.
Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
破裂和侵蚀的晚期动脉粥样硬化病变导致心肌梗死或中风，是全球范围内导致死亡的主要原因。然而，我们对构成晚期动脉粥样硬化病变的许多细胞的身份、来源和功能，以及它们控制斑块稳定性的机制，了解有限。
我们对人类颈动脉内膜切除术的晚期样本进行了全面的单细胞 RNA 测序，并将这些样本与经单细胞 RNA 测序鉴定的具有平滑肌细胞 (SMC) 和内皮谱系追踪的小鼠晚期动脉粥样硬化病变进行了比较，以全面检测所有斑块细胞类型，并严格确定其来源。我们进一步使用染色质免疫沉淀测序 (ChIP-seq)、批量 RNA 测序和一种创新的双重谱系追踪小鼠，以了解 SMC 表型转变如何影响病变发病机制。
我们提供的证据表明，SMC 特异性的 Klf4 与 Oct4 敲除显示出几乎相反的基因组特征，它们的假定靶基因在调节 SMC 表型变化中起着重要作用。单细胞 RNA 测序揭示了小鼠和人类病变之间转录组簇的惊人相似性，以及 SMC 和内皮细胞衍生细胞的广泛可塑性，包括 7 个不同的簇，这些簇大多数对传统标志物呈阴性。特别是，SMC 促进了具有软骨细胞样基因特征的 Myh11、Lgals3 群体，而 SMC 敲除则显著减少了该群体。我们观察到，激活 Lgals3 的 SMC 构成了病变中所有 SMC 的三分之二以上。然而，这些细胞中 Lgals3 的最初激活并不代表向终末分化状态的转化，而是代表这些细胞向独特的干细胞标记基因阳性、细胞外基质重塑的“先驱”细胞表型的转变，这些细胞是最早在病变内浸润的细胞，并随后在晚期病变中产生至少 3 种其他 SMC 表型，包括 Klf4 依赖性成骨表型，可能导致斑块钙化和斑块不稳定。
综上所述，这些结果提供的证据表明，晚期小鼠和人类动脉粥样硬化病变中的 SMC 衍生细胞表现出比普遍认为的更大的表型可塑性，Klf4 调节向包括 Lgals3 成骨细胞在内的多种表型的转变，这些细胞可能对晚期动脉粥样硬化斑块发病机制有害。
