Fig 1: Depletion of KLF4 in AdvSca1-SM cells modifies the fate of YFP+ cells in the setting of atherosclerosis.Arteries from 16-week WT and Klf4 KO AdvSca1-SM lineage atherogenic mice were processed for scRNA-Seq. A. [Left] UMAP projection of YFP+ and YFP- cells from WT and Klf4 KO AdvSca1-SM cell lineage tracing mice. [Right] UMAP projection showing the different fates of YFP+ cells in atherosclerosis as a consequence of depleting KLF4 in AdvSca1-SM cells. Arrows indicate major phenotypic shifts in KO mice. B. Stacked bar graph of YFP+ cells from WT and Klf4 KO mice after 16-week atherogenic diet. Arrows indicate increases (red) or decreases (blue) in cell populations as a function of Klf4 depletion. C. Violin plots showing higher expression of ECM related genes (Col1a1, Col1a2) in YFP+ Fib_2 cluster from Klf4 KO mice compared to WT mice after 16 weeks of atherogenic diet. Col1a1 is also higher in YFP+ Fib_3 cluster cells from Klf4 KO mice compared to WT mice, but there is no difference with Col1a2 in this cluster. D. Violin plots showing higher expression of SMC contractile genes (Acta2, Myh11, Cnn1) in YFP+ Transitional cluster cells from Klf4 KO mice compared to WT mice after 16 weeks of atherogenic diet.
Fig 2: KLF4 depletion in AdvSca1-SM cells alters the fate of YFP- non-AdvSca1-SM-derived cells.Arteries from 16-week WT and Klf4 KO AdvSca1-SM lineage atherogenic mice were processed for scRNA-Seq. A. [Left] UMAP projection of YFP+ and YFP- cells from WT and Klf4 KO AdvSca1-SM cell lineage tracing mice. [Right] UMAP projection showing the different fates of YFP- cells in atherosclerosis as a consequence of depleting KLF4 in AdvSca1-SM cells. Arrows indicate major phenotypic shifts in KO mice, including changes to the SMC and macrophage clusters. B. Stacked bar graph of YFP- cells from WT and Klf4 KO mice after 16-week atherogenic diet. Arrows indicate increases (red) or decreases (blue) in the cell population as a result of KLF4 depletion. C. Violin plot showing expression of SMC contractile genes (Acta2, Tagln, Myh11) in YFP- cells in the major SMC cluster from Klf4 KO mice compared to WT mice. D. Violin plot showing expression of Ccl2 in YFP- cells of the major macrophage cluster from Klf4 KO mice compared to WT mice. E. Flow cytometry analysis of single cell arterial digests from both WT and Klf4 KO mice after 24 weeks of atherogenic diet. F. CellChat analysis was performed on the fibroblast/AdvSca1-SM and Mac_1 clusters. Bubble plot showing elevated levels of COL1A1/COL1A2 from the fibroblast clusters signaling to SDC4 in Mac_1 in the setting of atherosclerosis. G. Violin plots show expression of Sdc4 and Abcg1 in Mac_1 from Klf4 KO mice in the setting of atherosclerosis. Abca1 is not significantly different between the genotypes.
Fig 3: Dynamic, bidirectional differentiation between AdvSca1-SM cells and SMCs in the setting of atherosclerosis.A. The Transitional cluster consists of YFP+ and YFP- cells and exhibits features of both SMCs and AdvSCa1-SM cells, roughly divided in half (bottom). Feature plots show that the upper portion of the transitional cluster expresses more contractile SMC genes (Acta2, Myh11, Cnn1), whereas the lower portion expresses more mesenchymal stem cell markers (Ly6a, Cd34, Pdgfra). B. KEGG Pathway differences between the Transitional cluster and the AdvSca1-SM/fibroblast clusters or SMC clusters. C. Representative image of 14-week atherogenic braciocephalic artery from SMC lineage tracing mice (Myh11-CreERT+/-/Rosa26-YFP+/+) stained for YFP (green; expression indicates SMCs, not AdvSca1-SM). Arrows indicate adventitial YFP+/Sca1+ cells; arrowheads indicate adventitial progenitors not expressing YFP (YFP-/Sca1+); orange arrows indicate DAPI positive cells in the vessel media that are negative for YFP.
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