文献学习073--[sc]scRNAseq揭示人和小鼠AS的SM

20年的一篇circulation

1. Single-Cell Genomics Combined With SMC-Lineage Tracing Reveals Multiple SMC-Derived Cell States During Atherosclerosis

Fig 1a：为了track 动脉粥样硬化形成过程中SMC转分化的过程。作者通过cross ROSA26Zs^Green1/+ mice 和 Myh11-CreER^T2构建了SMC-lineage tracing小鼠模型。在这个模型中，他莫昔芬诱导后，SMC和它的progenies会永久表达ZsGreen1。随后作者对ROSA26^ZsGreen1/+; Myh11-CreER^T2鼠和Ldlr^−/−进行了cross以追踪atherosclerosis过程中的SMC。
作者对0w, 8w, 16w, 26w高脂喂养ROSA26^ZsGreen1/+;Ldlr^−/−; Myh11-CreER^T2鼠的主动脉组织进行了消化，分选了ZsGreen⁺和ZsGreen^-的细胞，分别进行了单细胞测序。
Fig 1b-d：注释得到了12群细胞，且在进展期动脉粥样硬化中，SMC-derived intermediate cell state (ICS)在SMC lineages中占据了最大的比例（病程中逐渐增多）。

Fig 1

2. SMC Transition to Macrophage-Like Cells in Both Ldlr^−/− and ApoE^−/− Mouse Models

Data Supplement Figure II A, B：和预期的一致，作者检测到了SMC-derived macrophage- like cells。而且流式结果显示随着时间进展这种细胞的占比增多。
Data Supplement Figure II C-E：在ROSA26^ZsGreen1/+;ApoE^−/−; Myh11-CreER^T2模型中也鉴定出了SMC-derived macrophage- like cells。

Efferocytosis(胞葬作用) is a classic function of macrophages whereby they “eat” apoptotic cells and debris.

Data Supplement Figure II F, G：为了排除SMC- derived ZsGreen1⁺ macrophage-like cells其实是样本处理过程中Zs- Green1^-巨噬细胞吞噬了ZsGreen1⁺ SMC- lineage cells所致，作者分选了SMC-derived macrophage-like cells (ZsGreen1⁺CD11b⁺)，发现几乎所有分选的ZsGreen1⁺CD11b⁺细胞在3天的ex vivo培养之后依然是ZsGreen1⁺。而正常情况下，巨噬细胞的内吞物将在2.5d内被降解。

Data Supplement Figure II

These data suggest that ZsGreen1⁺ macrophage-like cells are derived from SMCs during atherosclerosis and are unlikely to be produced through efferocytosis of ZsGreen1⁺ cells by ZsGreen1⁻ macrophages.

3. SMC-Derived ICS, SEM Cell, Possesses Multipotent Molecular and Cellular Features

Fig 1e-h：为了进一步探究和明晰SMC-Derived ICS细胞的结构和功能特征，作者将其与SMC做了比较，发现其在转录上具有很大差异。SMC-derived intermediate cells高表达Ly6a (stem cell marker), Vcam1 (endothelial cell marker),和 Ly6c1 (monocytes/macrophage 分化marker)。因此作者将这群细胞命名为SEM (stem cell, endothelial cell, monocyte) 细胞。

Fig 1

Fig 2a, b：和预期一致，经典SMC marker在SEM中下调，提示在分化过程中细胞 逐渐丢失了SMC的表型。相反， fibrochondrocyte related genes (如, Fn1, Col1a1, Col1a2)则出现上调。此外MSCs的经典marker Nt5e (encoding CD73) 和 Eng (encoding CD105)则没有表达，提示SEM cells are a unique SMC- derived cell state instead of a MSC-like cell type.
Fig 2c：功能富集显示SEM的胞外基质沉积相关通路上调而平滑肌表型相关通路则出现下调。
Fig 2d：IPA结果也显示SEM参与免疫反应相关通路。
Fig 2e：作者通过免疫荧光定位了SEM细胞，发现在16-week WD-fed mice, ZsGreen1⁺Vcam1⁺细胞主要位于media of the lesion,还有一些定位于 intima overlying the media。
Fig 2f：随后作者通过体外试验表明：给予SEM细胞M-CSF刺激，SEM细胞可以分化成巨噬细胞，刺激SMC则不行（附件）。给予SEM细胞connective tissue growth factor刺激，则会分化成成纤维，表达成纤维的marker如Col1a1, Col3a1, Fn1, Fsp1, Tnc, 和 Vim，而刺激SMC则没有这种现象。
Fig 2g：而且在TGF-b的诱导下，SEM可以再重新分化成SMC，高表达ACTA2。
Fig 2h：Taken together, these data demonstrate that SMC-derived SEM cells, but not non- SEM cells, harbor the potential to differentiate into SMC- derived macrophage-like and fibrochondrocyte-like cells and revert toward the SMC-like phenotype.

Fig 2

4. Counterparts of Mouse Intermediate SEM Cells Exist in Human Atherosclerosis

Fig 3a-c：为了确定在小鼠中的SMC向SEM现象也存在于人的动脉斑块，作者联合分析了小鼠和人主动脉斑块(n=3)的结果。发现人的主动脉斑块中存在着同样的SEM群。
Fig 3d：人的ICS(SMC-derived intermediate cell state)同样存在着SMC基因如MYH11的下调和纤维成骨细胞基因FN1的上调。

5. Master Regulator Analysis of SMC Transition Reveals Multiple Signaling Pathways, Especially RA Signaling, in Atherogenesis

随后作者使用了Virtual Inference of Protein activity by Enriched Regulon (VIPER)和VIPER的单细胞版本metaVIPER进行了蛋白活性推测。

VIPER was designed to measure the differential activities of regulatory proteins between 2 cellular states by assessing the enrichment of their transcriptional targets in differentially expressed genes

Fig 4a：a图展示了和SMC相比，SEMs的top 50 master regulators (MRs)调控网络。其中有一些通路参与SMC分化比如TGFβ信号通路，还有一些调控动脉粥样硬化中巨噬细胞的功能如PI3K/Akt。These analyses suggest that they may also be prominent modulators of SMC transition during atherosclerosis. 一个新的发现是：在SMC to SEM转分化过程中的top MRs 包括了CRABP2，而两个SEM的marker: Vcam1和 Ly6c1 被鉴定为CRABP2的靶基因。
Fig 4b：由于RA signaling是生长发育的一条重要信号通路，而CRABP2是RA signaling的transducer。所以作者推测其可能在SMC 向 SEM转分化过程中起到重要作用。所以作者查看了RA信号通路的靶基因，发现它们在SCM向SEM转分化过程中发生了显著变化。
Fig 4c：在人的样本中，SA信号通路的靶基因在稳定和不稳定斑块中同样存在显著表达差异。
Fig 4d：此外，另一个RA signaling transducer: RA receptor beta (RARB)在人的进展期斑块中也显著下调。

6. Genetic Variations in RA Signaling– Modulated Genes Are Associated With Risk of Human Atherosclerotic CVD

为了进一步探究RA信号通路对人CAD的影响，作者使用了公开的meta-analysis数据库：CARDIoGRAMplusC4D中CAD相关SNP的数据。
Fig 5a：作者发现robust and consistent enrichment of CAD/myocardial infarction associated single nucleotide polymorphisms in multiple loci containing RA genes.
Fig 5b-e：Multiple RA genes were found at loci with moderate to strong, but sub– genome-wide-significant, signals for CAD, such as PRTG, ITGA1, SKI, and TAGLN2. 值得注意的是，和稳定斑块相比，人的不稳定斑块中TGA1, IGFBP3, 和 LTBP3也显著下降(Figure 4C)，提示这些基因的抑制和粥样硬化的严重程度有关。
Fig 5f-i：在Genotype-Tissue Expression database进行RA基因的eQTLs分析结果显示一些RA基因的CAD相关SNP，例如PRTG 和 IGFBP3，在CAD-relevant tissues中 (eg, aorta and tibial ar- tery) 也是eQTLs and the risk alleles associated with decreased expression。

Taken together, these integrative human genomic analyses provide multiple lines of evidence for association of RA signaling genes with atherosclerotic CVD and hint at a link between repression of RA signaling and increased CVD risk.

7. RA Signaling Modulates SMC to SEM Cell Transition and Atherosclerosis Progression

最后作者使用了ATRA (an activator of RA signaling) ，在in vitro and in vivo systems中验证了其治疗效果。
Fig 6a：使用ATRA预处理体外培养的SMC，阻断了促动脉粥样硬化刺激、胆固醇loading, 和TNF-α诱导SMC向SEM转分化的过程，SEM的marker Ly6a和Vcam1显著下降。
Fig 6b-c：给ROSA26^ZsGreen1/+; Ldlr^−/−; Myh11-CreER^T2小鼠ATRA治疗，流式结果也显示SEM细胞显著减少。
Fig 6d-g：但尽管给予ATRA治疗后总的SEM细胞数下降，纤维帽中的SEM细胞比例增加，内膜intima中的SEM比例则上升。

These findings suggest that ATRA inhibits SEM cell generation from SMCs and may block their differentiation toward more differentiated SMC-derived cell types in atherosclerosis.

Taken together, these findings suggest that ATRA reduces atherosclerosis progression and increases fibrous cap thick- ness coincident with suppression of SMC transition to SEM cells.

作者19年的时候还在nature medicine上发过一篇 news & views