Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
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Research Article Stem cells Vascular biology

Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis

Abstract

Resident vascular adventitial SCA1+ progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage-tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA-sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling were significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM–specific expression of GLI-Kruppel family member GLI1 (Gli1), we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell–specific Kruppel-like factor 4 (Klf4) depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a profibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach.

Authors

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

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Figure 4

AdvSca1-SM cells contribute to injury-induced adventitial remodeling.

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AdvSca1-SM cells contribute to injury-induced adventitial remodeling. Gl...
Gli1-CreERT-YFP mice were subjected to carotid arterial injury, and uninjured right and injured left carotids were harvested at 3 days, 7 days, and 3 weeks postinjury; fixed; and embedded in OCT. (A) Arterial sections were immunofluorescently stained for YFP (green), αSMA (red), and SCA1 (white). Representative images from N = 7 (3 days), N = 10 (7 days), and N = 11 (3 weeks). Scale bars: 50 μm. M, arterial media; A, arterial adventitia, NI, neointima. Dashed lines indicate the external and internal elastic laminae. Note a time-dependent expansion of AdvSca1-SM cells predominantly in the adventitia, with a concomitant loss of AdvSca1-SM cell SCA1 expression, but also migration into the arterial media. (B) Uninjured and 3- and 7-day postinjury carotid artery sections were immunofluorescently stained for YFP (green) to identify AdvSca1-SM cells. Sections were imaged for coexpression of YFP and label-free SHG for collagen deposition (red). Elastin autofluorescence is also observed on the green channel. Representative 40× images from N = 6 per condition. M, arterial media; A, arterial adventitia. (C–F) Quantification of stained images from A and B. (C) Total YFP+ cells. (D) Percentage of YFP+SCA1+ cells per total YFP+ cells. (E) Percentage of YFP+αSMA+ cells per total YFP+ cells. (F) Normalized SHG+ area (pixel); SHG signal was normalized the outer medial circumference. Data represent mean ± SEM; (C, E, and F) Kruskal-Wallis test followed by Dunn’s multiple-comparison test; (D) 1-way ANOVA with Bonferroni’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.