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 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.