Smooth muscle–derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner
Allison M. Dubner, Sizhao Lu, Austin J. Jolly, Keith A. Strand, Marie F. Mutryn, Tyler Hinthorn, Tysen Noble, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Allison M. Dubner, Sizhao Lu, Austin J. Jolly, Keith A. Strand, Marie F. Mutryn, Tyler Hinthorn, Tysen Noble, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
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Research Article Stem cells Vascular biology

Smooth muscle–derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner

Abstract

We previously established that vascular smooth muscle–derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM–derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non–AdvSca1-SM–derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.

Authors

Allison M. Dubner, Sizhao Lu, Austin J. Jolly, Keith A. Strand, Marie F. Mutryn, Tyler Hinthorn, Tysen Noble, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans

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Figure 6

KLF4 depletion in AdvSca1-SM cells alters the fate of YFP– non–AdvSca1-SM–derived cells.

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KLF4 depletion in AdvSca1-SM cells alters the fate of YFP– non–AdvSca1-S...
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 with WT mice. (D) Violin plot showing expression of Ccl2 in YFP– cells of the major macrophage cluster from Klf4-KO mice compared with 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. Statistical analysis done with 2-tailed Student’s t test.