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 7

Depletion of KLF4 in AdvSca1-SM cells does not alter overall plaque burden but reduces late-stage plaque complexity.

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Depletion of KLF4 in AdvSca1-SM cells does not alter overall plaque burd...
(A) The aortic arch and descending aorta from 24-week atherogenic WT (n = 6) and Klf4-KO (n = 4) mice were stained using Sudan IV to assess overall atherosclerotic plaque burden. Two-tailed Student’s t test analysis of staining is shown to the right. (B) Representative H&E images of aortic root cross sections from WT (n = 6) and Klf4-KO mice (n = 6). Insets show higher magnification of the plaque structure with significantly smaller necrotic cores in Klf4-KO mice compared with WT mice. Two-tailed Student’s t test analyses of plaque percent coverage and necrotic core area (P = 0.0221) are shown below. (C) Representative H&E images of stained aortic root cross sections from WT (n = 6) and Klf4-KO (n = 6) mice. Arrows indicate cholesterol clefts. (D) Representative immunofluorescence images of aortic root cross sections from WT (n = 5) and Klf4-KO (n = 6) mice for CD68 (red) and DAPI (blue). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.0117. (E) Representative immunofluorescence images of aortic root cross sections from WT (n = 5) and Klf4-KO (n = 6) mice for YFP (green), αSMA (red), and DAPI (blue). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.026. (F) Representative Masson’s trichrome images from WT (n = 6) and Klf4-KO (n = 6) mice aortic roots with collagen (blue), cytoplasm (pink), and cell nuclei (brown). Two-tailed Student’s t test analysis of staining is shown to the right; P = 0.0165. Scale bars: 50 μm, 200 μm (E).