Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis
Sizhao Lu, … , Mark W. Majesky, Mary C.M. Weiser-Evans
Sizhao Lu, … , Mark W. Majesky, Mary C.M. Weiser-Evans
Published October 29, 2020
Citation Information: JCI Insight. 2020;5(23):e139445. https://doi.org/10.1172/jci.insight.139445.
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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.