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 6

AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventitial remodeling.

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AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventiti...
WT and KLF4-KO Gli1CreERT-YFP mice were injected with tamoxifen as described in Methods. Arterial tissues were harvested 4 weeks after the final tamoxifen injection. (A and B) Single-cell suspensions were isolated from carotid artery + aortic arch, stained for SCA1, and analyzed by flow cytometry for quantification of SCA1 expression in YFP+ AdvSca1-SM cells (A) and for quantification of total YFP+ AdvSca1-SM–derived cells (B). Each point represents a single mouse; N = 6 WT and N = 9 KO. (C) Carotid artery sections were immunofluorescently stained for SCA1 (red) and YFP (green). Arrows show YFP+SCA1+ AdvSca1-SM cells; block arrows show YFP+SCA1– AdvSca1-SM cell-derived cells; arrowheads show YFP–SCA1+ AdvSca1-MA cells. (D and E) Carotid artery sections were stained with hematoxylin, and adventitia-to-media ratio was measured using ImageJ 1.47v (NIH) (D). Each point represents a single artery. N = 9 WT; N = 8 KO. Representative image shown in E. (F and G) Carotid artery sections were stained with Masson’s trichrome stain, and the intensity of collagen expression (blue; G) was quantified with ImageJ 1.47v and normalized to outer medial circumference (F). Each point represents a single artery. N = 11 WT; N = 19 KO. Representative image shown in G. Original magnification, ×40. (H) CA and aortic (AO) sections from WT or AdvSca1-SM cell–specific KLF4-KO mice 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. (I) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (red). Representative images from N = 3 per time point. Arrows show YFP+CD68– AdvSca1-SM cells; arrowheads show YFP–CD68+ macrophages. Note that AdvSca1-SM cells do not coexpress CD68. M, arterial media; A, arterial adventitia. Scale bars: 50 μm. (A, B, D, and F) Data represent mean ± SEM; unpaired Student’s t tests (2 tailed); **P < 0.01; ****P < 0.0001.